The discovery and characterization of lenaldekar: a selective compound for the treatment of T-Cell acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood, with approximately 2000 cases diagnosed annually in the US. Although cure rates for childhood ALL are currently ~80%, T-cell ALL (T-ALL) is still more difficult to treat than B-cell ALL, requiring harsher treatments with concomitant harsher side effects. The goal of this study was to identify more targeted therapies for treating T-ALL with the intent of reducing harsh treatment side effects, thus preserving both lives and long-term quality of life. To meet this goal, 26,400 compounds from the ChemBridge library were screened utilizing zebrafish larvae since they have the combined attributes of vertebrate physiology and small size. The transgenic lck:eGFP zebrafish line with T-cell specific GFP was chosen since compounds which eliminate immature T-cells in the thymus might also eliminate developmentally arrested leukemic blasts. The screen identified five "hit" compounds that cause reduction in GFP without sickening the larvae or causing general cell cycle effects. Of these five compounds, one compound, "Lenaldekar" (LDK), was effective in killing human Jurkat T-ALL without harming healthy lymphocytes. In vivo, LDK shows efficacy in treating leukemia in both zebrafish and mouse xenograft models of T-ALL without observable toxicity or endorgan damage. Furthermore, expanded leukemia testing showed that T-ALL, B-ALL, and CML are all largely LDK-sensitive, including most treatment-refractory relapsed Ph+ leukemias and primary patient samples. Moreover, some AML and multiple myeloma cell lines also show LDK sensitivity. Molecular characterization shows that LDK down-regulates the PI3K/AKT/mTOR (P/A/mT) pathway, which pathway is up-regulated in ~50% of T-ALL cases. Recent results suggest that LDK may achieve this effect via inactivation of the insulin-like growth factor 1 receptor (IGF1-R), which activates the P/A/mT pathway. In addition, LDK treatment elicits a second activity of G2/M arrest in most sensitive cell lines, which arrest appears to be independent of P/A/mT pathway inhibition. Future directions include identifying and modeling LDK's direct biochemical target(s) with the intent of utilizing structure-activity relationships to optimize LDK's chemical structure and efficacy. This study's ultimate goal is to bring LDK into clinical trials for the treatment of T-ALL in both monotherapy and combination therapy applications.

THE DISCOVERY AND CHARACTERIZATION OF LENALDEKAR: A SELECTIVE COMPOUND FOR THE TREATMENT OF T CELL ACUTE LYMPHOBLASTIC LEUKEMIA by Suzanne Ridges A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Oncological Sciences The University of Utah May 2013 Copyright © Suzanne Ridges 2013 All Rights Reserved T h e Un i v e r s i t y o f Ut a h Gr a d u a t e S c h o o l STATEMENT OF DISSERTATION APPROVAL The dissertation of Suzanne Ridges has been approved by the following supervisory committee members: Nikolaus S. Trede , Chair 7/30/2012 Date Approved David A. Jones , Member 7/30/2012 Date Approved Bryan E. Welm , Member 7/30/2012 Date Approved David J. Bearss , Member 7/30/2012 Date Approved Amy M. Barrios , Member 7/30/2012 Date Approved and by Bradley R. Cairns , Chair of the Department of Oncological Sciences and by Charles A. Wight, Dean of The Graduate School. ABSTRACT Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood, with approximately 2000 cases diagnosed annually in the US. Although cure rates for childhood ALL are currently ~80%, T-cell ALL (T-ALL) is still more difficult to treat than B-cell ALL, requiring harsher treatments with concomitant harsher side effects. The goal of this study was to identify more targeted therapies for treating T-ALL with the intent of reducing harsh treatment side effects, thus preserving both lives and long-term quality of life. To meet this goal, 26,400 compounds from the ChemBridge library were screened utilizing zebrafish larvae since they have the combined attributes of vertebrate physiology and small size. The transgenic lck:eGFP zebrafish line with T-cell specific GFP was chosen since compounds which eliminate immature T-cells in the thymus might also eliminate developmentally arrested leukemic blasts. The screen identified five "hit" compounds that cause reduction in GFP without sickening the larvae or causing general cell cycle effects. Of these five compounds, one compound, "Lenaldekar" (LDK), was effective in killing human Jurkat T-ALL without harming healthy lymphocytes. In vivo, LDK shows efficacy in treating leukemia in both zebrafish and mouse xenograft models of T-ALL without observable toxicity or endorgan damage. Furthermore, expanded leukemia testing showed that T-ALL, B-ALL, and CML are all largely LDK-sensitive, including most treatment-refractory relapsed Ph+ leukemias and primary patient samples. Moreover, some AML and multiple myeloma cell lines also show LDK sensitivity. Molecular characterization shows that LDK down-regulates the PI3K/AKT/mTOR (P/A/mT) pathway, which pathway is up-regulated in ~50% of T-ALL cases. Recent results suggest that LDK may achieve this effect via inactivation of the insulin-like growth factor 1 receptor (IGF1-R), which activates the P/A/mT pathway. In addition, LDK treatment elicits a second activity of G2/M arrest in most sensitive cell lines, which arrest appears to be independent of P/A/mT pathway inhibition. Future directions include identifying and modeling LDK's direct biochemical target(s) with the intent of utilizing structure-activity relationships to optimize LDK's chemical structure and efficacy. This study's ultimate goal is to bring LDK into clinical trials for the treatment of T-ALL in both monotherapy and combination therapy applications. iv This work is dedicated to my family without whom I could never have even started, let alone finished, this journey. I also dedicate it to the clinicians and staff at Huntsman Cancer Hospital and Huntsman Cancer Institute for their excellent care and support for me while being treated for breast cancer while pursuing my own graduate work in cancer research. TABLE OF CONTENTS ABSTRACT ...................................................................................................................... iii LIST OF FIGURES ........................................................................................................ viii LIST OF TABLES ............................................................................................................. x ACKNOWLEDGMENTS ............................................................................................... xii Chapter ......................................................................................................................... Page 1. INTRODUCTION TO T CELL ACUTE LYMPHOBLASTIC LEUKEMIA . 1 Definition of T-ALL ......................................................................................... 1 T-ALL demographics and statistics .................................................................. 2 T-ALL diagnosis and classification .................................................................. 3 T-cell development ........................................................................................... 5 Molecular genetics and translocations .............................................................. 8 T-ALL treatment regimens ............................................................................. 15 Need for more targeted therapies in treating T-ALL ...................................... 27 Goals of the dissertation ................................................................................. 28 References ....................................................................................................... 30 2. ABERRANT SIGNALING PATHWAYS IN T-ALL ................................... 52 Background ..................................................................................................... 52 Ikaros' contribution to T-ALL leukemogenesis ............................................. 53 NOTCH1 contribution to T-ALL ................................................................... 54 The PI3K/AKT/mTOR pathway in T-ALL .................................................... 58 The Ras/Raf/MEK/ERK (MAPK) pathway.................................................... 67 JAK/STAT ...................................................................................................... 69 PI3K/AKT/mTOR pathway inhibitors ............................................................ 70 References ....................................................................................................... 77 3. ZEBRAFISH AS A MODEL ORGANISM FOR NORMAL AND MALIGNANT HEMATOPOIESIS ............................................................... 96 Historical background ..................................................................................... 96 Early studies .................................................................................................... 96 Zebrafish disease model strengths .................................................................. 97 Zebrafish disease model limitations ............................................................. 101 Zebrafish cancer models ............................................................................... 102 Zebrafish as a model for human hematopoiesis ............................................ 105 Zebrafish as a model for human leukemia .................................................... 109 Zebrafish utility in drug screening ................................................................ 113 References ..................................................................................................... 121 4. ZEBRAFISH SCREEN IDENTIFIES NOVEL COMPOUND WITH SELECTIVE TOXICITY AGAINST LEUKEMIA ..................................... 137 Introduction ................................................................................................... 139 Methods ........................................................................................................ 140 Results ........................................................................................................... 140 Discussion ..................................................................................................... 146 Acknowledgements ....................................................................................... 148 References ..................................................................................................... 149 Supplemental figures .................................................................................... 150 Supplemental tables ...................................................................................... 164 Supplemental methods .................................................................................. 170 5. CONCLUSION ............................................................................................. 178 Summary and perspectives ........................................................................... 178 Future directions ........................................................................................... 180 References ..................................................................................................... 183 APPENDIX: SUPPLEMENTARY DATA ................................................................... 184 vii LIST OF FIGURES Figure Page 1.1 Five-year pediatric ALL survival rates over the past 60 years. ........................... 47 1.2 Comparison of normal and T-ALL leukemic bone marrow ................................ 47 1.3 Stages of haematopoiesis and T‑cell development and T‑cell-leukaemia-related oncogenes. ................................................................................................ 48 1.4 Functional classifications of common T-ALL mutations .................................... 49 1.5 Classical karyotyping and FISH technique .......................................................... 50 1.6 Schematic representation of molecular contributors to T-ALL ontogeny ........... 50 1.7 Chemical structures of cortisol and synthetic glucocorticoids prednisone, prednisolone, and dexamethasone .................................................... 51 2.1 Schematic representation of the NOTCH1signaling pathway and transcriptional networks promoting leukemic cell growth downstream of oncogenic NOTCH1 ................................................................... 93 2.2 Diagram of key features of the PI3K/AKT/mTOR signaling network ................ 95 3.1 Zebrafish larva at 3-7dpf .................................................................................... 135 3.2 The ontogeny of hematopoiesis in zebrafish ..................................................... 136 4.1 Zebrafish drug screen identifies anti-T-cell compounds ................................... 141 4.2 LDK is active against malignant lymphoblasts ................................................. 142 4.3 LDK treatment inhibits tumor progession in 2 in vivo models of T-ALL......... 144 4.4 LDK down-regulates phosphorylation of targets in the PI3K/AKT/mTOR pathway and causes late mitosis arrest in treated cells ...................................... 145 4.5 LDK is active against primary patient samples without toxicity to hematopoietic progenitors .................................................................................. 147 4.S1 LDK decreases viability of primary murine T-ALL cells ................................. 150 4.S2 LDK has selective activity against hematological malignancies and induces apoptosis in Jurkat cells ........................................................................ 151 4.S3 LDK-mediated reduction of AKT and mTOR phosphorylation ........................ 153 4.S4 LDK does not target the AKT pathway directly, but AKT inhibition is required for its toxicity in Jurkat cells ............................................................... 154 4.S5 Time-course of LDK treatment shows progressive accumulation of cells in G2/M .............................................................................................................. 155 4.S6 LDK treatment results in de-phosphorylation of AKT and G2/M delay in the T-ALL cell line CCRF-CEM ....................................................................... 156 4.S7 Pharmacokinetics and lack of toxicity of LDK in mice ..................................... 157 4.S8 LDK treatment shows no significant toxicity in complete blood count, thymus, or spleen cell counts ............................................................................. 158 4.S9 Response of a collection of primary patient B-ALL samples to LDK treatment ........................................................................................................... 160 A.1 LDK impact on AKT downstream phosphorylation target GSK-3β ................. 190 A.2 LDK impact on AKT downstream phosphorylation target p-BAD ................... 191 A.3 Constitutively active mTOR partially rescues LDK-treated Jurkat T-ALL ...... 192 A.4 Characteristics of Jurkat LDK-resistant cell line derivatives Res 3 and Res 6 .. 193 A.5 Spatiotemporal isolation of phospho-Aurora B kinase in HeLa cells ................ 194 A.6 LDK treatment results in increased autophagy in Jurkat cells ........................... 195 A.7 Ewing Sarcoma is LDK-sensitive and shows G2/M delay upon LDK treatment ................................................................................................... 196 A.8 MCF7 breast cancer derivatives Msp-Ron and sfRon are LDK-sensitive ......... 199 ix LIST OF TABLES Table Page 1.1 WHO 2008 classification of T-cell neoplasms ................................................. 42 1.2 Stages of T cell development correlate with specific locations in the thymus, distinct cell-surface phenotypes, requirements for Notch signals, and TCR rearrangement ....................................................................... 43 1.3 EGIL classification system for T-ALL ............................................................. 43 1.4 The TCR system for classifying T-ALL ........................................................... 44 1.5 Most frequent genetic abnormalities in T-ALL ................................................ 44 1.6 Common fusion proteins in T-ALL .................................................................. 45 1.7 Relative long-term side effect risks of adult survivors of childhood cancers............................................................................................................... 46 2.1 Summary of drugs targeting the PI3K pathway in clinical trials for cancer treatment .......................................................................................... 92 3.1 Limitations of the zebrafish model in hematology research ........................... 132 3.2 Zebrafish models of human leukemia ............................................................. 133 3.3 Summary of chemical library screens performed in zebrafish and Xenopus .................................................................................................... 134 4.S1 Cell cycle effects of 21 candidate compounds on zebrafish embryos ............ 164 4.S2 LDK has minimal impact on zebrafish embryonic and larval development .................................................................................................... 165 4.S3 LDK is not a general kinase inhibitor ............................................................. 166 4.S4 LDK treatment results in G2/M arrest for sensitive T-ALL and B-ALL cell lines ............................................................................................. 167 4.S5 Characteristics of primary BCR-ABL translocated B-ALL and CML patient samples and growth inhibition by LDK treatment .................... 168 4.S6 Characteristics of primary B-ALL patient samples and LDK-mediated inhibition of proliferation ...................................................... 169 xi ACKNOWLEDGMENTS I wish to thank Nikolaus Trede and David Jones for their guidance, support and encouragement during the time that I have spent in my thesis program. I also wish to thank all members of the Trede and Jones labs, both past and present, for their untiring support, without which this project could not have been completed. I especially want to acknowledge and thank Deepa Joshi and Will Heaton for their enormous contribution to this project and for their unconditional and invaluable friendship to me while working in the lab. I also wish to give special thank to all personnel at Huntsman Cancer Institute, and particularly to the Deininger, Engel, Lessnick, Cairns, and CIT Labs for their cooperation and collaboration in many aspects of this work. Special thanks also go to the CZAR staff and to all University of Utah CORE facilities and personnel for their constant support. Many thanks also go to the off-campus collaborating labs elsewhere in the country that supported and advised us and contributed data to this study. The clinicians and staff of Huntsman Cancer Hospital also must be thanked for their phenomenal kindness and skill in treating me for cancer myself while pursuing my own doctoral degree in Oncological Sciences. And finally, I wish to thank my family for their untiring love, support and encouragement during this chapter of my life, without which I could not have survived, let alone succeeded. CHAPTER 1 INTRODUCTION TO T CELL ACUTE LYMPHOBLASTIC LEUKEMIA Definition of T-ALL Acute lymphoblastic leukemia (ALL) is a clinically aggressive disease. It is defined as the uncontrolled clonal expansion of an immature lymphocytic precursor cell of either the T- or B-cell lineage, which overwhelms the bone marrow, causing cessation of normal hematopoiesis in the bone marrow. If left untreated, it is inevitably lethal. T-ALL (T-cell acute lymphoblastic leukemia) pathogenesis is a multistep process consisting of acquisition by a T cell precursor of a series of genetic abnormalities, which disturb its normal maturation process, leading to differentiation arrest and uncontrolled proliferation. T-ALL is a specific and rare subtype of ALL that arises from a precursor cell of the T-cell lineage which becomes developmentally arrested early within certain defined stages of intrathymic differentiation. T-cell acute lymphoblastic leukemias (T-ALL) and lymphomas (T-LBL) are often considered to be the same disease, differing only in burden of the leukemic disease in the bone marrow (over 25% of blasts in T-ALL and below 25% in T-LBL). Indeed, pathological inspection of blasts from both diseases manifests no difference in cell morphology and they are often treated in the same manner in the clinic setting.1 However, recent findings indicate that some molecular markers differ between T-LBL and T-ALL, 2 for example BCL2 expression is more elevated in the former.2 For diagnostic purposes, these two types of T-cell neoplasms can be segregated into two groups: precursor T-cell lymphoblastic neoplasms, derived from immature thymocytes, and peripheral T-cell lymphomas, arising from T-cells with varying stages of differentiation.3 Peripheral T-cell lymphomas can be further subclassified by specific clinical features (Table 1.1).4 T-ALL demographics and statistics Each year approximately 6,050 new cases of ALL are diagnosed in the United States alone (3,450 male and 2,600 female), of which an estimated 2,400 occur in children and adolescents, with an estimated 1440 total deaths from ALL.5 This represents an incidence of approximately 3 to 4 cases per 100,000 in children ages 0-14 and 1 per 100,000 for ages 15 and older.6 Altogether ALL constitutes 35% of new cancer cases diagnosed in childhood as well as almost 75% of all childhood leukemias (ages 0-19), making it the most common pediatric malignancy.7 Approximately 15% and 25% of new ALL cases in children and adults, respectively, are T cell ALL. It is primarily a pediatric disease, with diagnoses peaking between 2-5 years of age, but T-ALL can also affect adults and exhibits a second incidence peak in the elderly.5 In contrast to pediatric leukemias, the most common forms of leukemia in the adult population are acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL).8,9 T-ALL occurs primarily as de novo disease and rarely as a secondary neoplasm, but it has been correlated to certain genetic and environmental factors. It has a slightly higher occurrence in patients with Down syndrome, Bloom syndrome, neurofibromatosis type I, and ataxia-telangiectasia. It has also been linked to environmental exposure in utero to ionizing radiation, pesticides, and solvents.10 In all age groups, there is a slight 3 predominance of cases among males as well as notable excess incidence among Caucasian children.6 Overall, T-ALL is more difficult to treat and carries a worse prognosis than B-ALL due to the additional difficulties encountered in T-ALL, such as CNS infiltration as well as a worse risk of failure of initial induction therapy. As a result, in recent decades, treatment outcome improvements for T-ALL have lagged behind B-ALL. Nevertheless, treatment expectations for ALL patients have improved dramatically in recent years due to the application of intensified chemotherapy regimens. Consequently, 5-year relapse-free survival currently is ~80% in pediatric11 and ~50% in adult cases of ALL (Figure 1.1).12 The poorer outcome in adult T-ALL patients can be attributed to multiple factors, including decreased tolerance for intensive chemotherapy regimens, higher incidence of poor-prognosis cytogenetics, and lower incidence of favorable subtypes such as the t(12;21) translocation.13,14 In all groups of patients, relapsed cases of T-ALL still carry a dismal prognosis since they often correlate with development of chemoresistance in the refractory disease and still constitute a significant clinical problem.15,16 T-ALL diagnosis and classification Patient presentation and symptoms T-ALL usually presents as acute disease. However, in rare cases disease may evolve insidiously over several months.17 A patient may present with fever caused by either the leukemia or by leukemia-induced secondary infection. Patients may also experience fatigue and lethargy caused by anemia, bone and joint pain, and bleeding diathesis related to thrombocytopenia. Other symptoms may include loss of appetite and 4 weight, excessive unexplained bruising, enlarged lymph nodes, pitting edema, petechiae due to low platelet levels, and wheezing due to an enlarged thymus (in T-ALL and T-LBL). Because of similarity of symptoms to many common maladies, T-ALL patients may not realize at first that they have a serious disease until it progresses to the point where medical attention is mandatory. Furthermore, T-ALL may initially be misdiagnosed as asthma due to observed wheezing and because it often can be relieved by corticosteroid treatment. Clinical diagnosis and pathology Due to its acute nature, T-ALL patients usually exhibit large tumor burdens upon initial presentation. The examining physician may observe organomegaly, particularly mediastinal masses with or without pleural effusions leading to respiratory distress, and often infiltration of the central nervous system at the time of diagnosis. Leukemic blasts may also spread to lymph nodes or other common extramedullary sites of involvement, including lymph nodes, liver, spleen, and meninges. Less commonly, ALL may infiltrate orbital tissues, testes, tonsils, and adenoids. Pathological diagnosis includes a complete blood analysis and bone marrow smear. Normal bone marrow from a healthy patient will exhibit a mixture of several heterogeneous cell types (Figure 1.2A). By contrast, a bone marrow smear from a T-ALL patient will show a largely homogeneous cell population of small to medium-sized blasts overwhelming the bone marrow (Figure 1.2B). T-ALL blasts are typically cells with round to irregular or convoluted nuclei and high mitotic activity. T-ALL blasts also usually exhibit expression of nuclear terminal deoxynucleotidyl transferase (TdT). Most 5 commonly, ALL blasts have scanty cytoplasm, open nuclear chromatin, and sometimes presence of nucleoli. Hematological assessment will usually show high numbers of circulating lymphoblasts in peripheral blood. Other blood count abnormalities include anemia, thrombocytopenia, neutropenia, and leucopenia or leucocytosis with hyperleukocytosis (>100 x109/L) present in approximately 15% of the pediatric patients.17 Laboratory findings may also include elevated serum uric acid and lactose dehydrogenase levels because of excessive cell turnover. T-cell development A discussion on T-ALL leukemogenesis first requires an understanding of T-cell ontogeny since many of the molecular mistakes that result in T-ALL arise from this normal process gone awry. Normal lymphocyte development takes place in the central lymphoid organs: in the bone-marrow for B-cells and in the thymus for T-cells. The thymus provides the microenvironment essential for the development of T-cells from common lymphoid progenitors (CLP). When CLPs leave the bone marrow and enter into the thymus, they receive signals from the thymic microenvironment, particularly NOTCH signaling, engaging them definitively in the T-cell lineage (Figure 1.3).18 Immature committed lymphocytes, called thymocytes, undergo two crucial steps. First, they acquire a unique mature T-cell receptor (TCR) following the rearrangement of the genes coding for the different chains of the receptor. Second, they undergo selection steps that will retain only thymocytes with a functional receptor able to recognize specific antigens presented by thymic epithelial cells via the major histocompatibility complex (MHC). If thymocytes receive signals from the functional TCR, they are transmitted to 6 the nuclei through different signaling proteins, initiating the transcription of survival genes (positive selection), while T-cells without a functional TCR die by neglect.19 Toward the end of this maturation process T-cells undergo negative selection, or "clonal deletion", of T-cells with high affinity for self-antigens to avoid self-reactivity.19 During this maturation and selection process, T-cells undergo a series of transitions in their expression of CD4 and CD8 cell surface markers from double negative (DN,CD4-/CD8-) to double positive (DP, CD4+/CD8+), to finally single positive (SP, either CD4+ or CD8+) before leaving the thymus (Table 1.2).20 Thymocyte development is regulated by a series of transcription factors and regulators of hematopoiesis such as E2A proteins. Other proteins such as HOX, MYB, IKAROS, and especially NOTCH family proteins all play a role in T-cell commitment, differentiation, and proliferation. NOTCH proteins also play an important role in self-renewal of progenitors.21 In T-ALL, alterations of these key proteins are common findings responsible for leukemic transformation. At the time when rearrangement of the different segments, variable (V), diversity (D), and joining (J), of the TCR genes takes place, illegitimate recombinations between TCR receptor gene promoter elements and genes that are in an open chromatin conformation at this stage of T-cell development, and thus susceptible to the recombinase enzyme activity of RAG1/2, can occur. Hence, though rare, translocations between TCR and HOX genes or genes coding for proteins interacting with E2A are recurrently found in T-ALL and point to the importance of these proto-oncogenes that are frequently over-expressed in T-ALL. 19 7 T-ALL subtype classifications Historically, T-ALL and T-NHL were once considered to be the same disease, differing only in the degree of bone marrow infiltration (>25% for T-ALL and <25% for T-NHL). Indeed, leukemic blasts from both disease classifications are largely indistinguishable under the microscope and are treated much the same in many cases. However, due to advances in molecular diagnostic and sequencing technologies in recent years, it has become apparent that T-cell neoplasms, though relatively rare, are a much more heterogeneous group of diseases than previously thought. In addition, Feng et al demonstrated that BCL2 is more strongly expressed in T-NHL than in T-ALL.2 Likewise, differential response to identical therapy regimens in different patients with seemingly identical diseases has spurred investigation into the molecular causes of such disparate clinical outcomes. It was also anticipated that a more in-depth understanding of the molecular mechanisms driving T-ALL could better inform customized treatment regimen decisions. Two classification systems have been proposed for T-ALL. The first classification system is from the European Group for the Immunological Characterization of Leukaemias (EGIL).22 Briefly, in the EGIL classification system cellular expression of CD3 defines T-ALL. The system further separates T-ALL into four categories depending on the maturation status of the cells involved, ie. pro-T, pre-T, cortical-T, and mature T-cell stages, depending on the expression status of CD markers exhibited by the malignant cells (Table 1.3). The second and more recent T-ALL classification system, developed by Macintyre et al.,23 is based on T-cell receptor (TCR) status (Table 1.4). It contains 8 classifications of immature stage (IM), the pre-αß stage, and the TCRαß or TCRγδ stages.24 Based on actual TRD, TRG, and TRB gene rearrangements, the IM group can be further differentiated into the IM0, IMδ, IMγ, or IMβ subtypes. Both of these T-ALL stage classification systems are intended to correlate with normal maturation steps involved in healthy T-cell development. However, only about half of T-ALL cases characterized by them can be assigned to equivalent developmental stages by both classification systems simultaneously.25 Molecular genetics and translocations Modern advances in chemotherapy regimen intensification, although currently successful in curing ~80% of pediatric patients, are not without liability in the form of short- and long-term detrimental side effects. Furthermore, 20% of pediatric patients still cannot be cured of T-ALL. Clearly novel treatment strategies are needed beyond mere chemotherapy intensification and/or hematopoietic stem cell transplant to improve treatment outcomes. These observations as well as advances in molecular diagnosis technology have spurred much research in recent years concerning cytogenetic abnormalities in T-ALL with the intent of identifying druggable T-ALL-specific biochemical targets. Many of the mutations in T-ALL (Summarized in Figure 1.4) can be attributed to chromosomal aberrations, which can be observed in ~50% of T-ALL cases by utilizing standard karyotyping techniques.26 Other cytogenetic abnormalities are far more subtle or "cryptic", requiring FISH (Fluorescence In Situ Hybridization) for detection (Figure 1.5). The genetic abnormalities observed in T-ALL may be grouped into four general categories as follows (summarized in Table 1.5): 1) Juxtaposition of a T cell receptor 9 (TCR) promoter or enhancer next to a strong transcription factor, 2) The creation of fusion proteins, 3) Deletions and amplifications, and 4) Mutations. Each of these will be discussed in turn. TCR-transcription factor gene juxtaposition T-cell progenitors rearrange their TCRD, TCRG, TCRB, and TCRA loci, which if successful, will lead to expression of a mature TCR. In T-ALL, alterations of these key proteins are common findings responsible for leukemic transformation. At the time when rearrangement of the different segments (V(D)J) of the TCR genes takes place, illegitimate recombinations between TCR receptor genes' promoter and enhancer elements and genes that are active and therefore in an open chromatin conformation at this stage of T-cell development can occur. Hence, translocations between TCR and HOX genes or genes coding for proteins interacting with E2A are recurrently found in T-ALL. 19 Transcription factor genes are the preferred targets of these rare pathogenetic chromosomal translocations in the acute T-cell leukemias. Notable examples include the bHLH genes MYC, TAL1, and LYL1 which are essential for the development of other lineages such as erythroid cells, but with the exception of MYC, they are not normally expressed in T-lymphoid cells. Hence, over-expression or mutation of these proteins are pathogenetic mechanisms in T-ALL.27 Fusion proteins The MLL (Mixed Lineage Leukemia) protein methylates histone H3 at lysine 4 (H3K4) and is the gene most associated with the formation of fusion oncoproteins in T-ALL. Under healthy circumstances it regulates gene expression, especially HOX gene 10 expression, to control skeletal patterning and HSC and early hematopoietic progenitor cell development.28 Over 40 different balanced chromosomal translocations have been identified as fusion partners for MLL in T-ALL, all of which produce a fusion protein possessing the NH2-terminus of MLL fused to the COOH-terminus of the fusion partner.27 The most common MLL-partner fusion genes are listed in Table 1.6. An 8.3KB breakpoint cluster region between exons 8 and 13 is the site for most MLL rearrangements, which always produce in-frame fusion proteins with fusion partners.29 The SIL-TAL1 fusion is also very common in T-ALL and results from a t(1;14) translocation (3% of cases) or from an interstitial deletion of the SIL-TAL locus (~25% of cases).30 Fusion proteins including ABL1 also occur recurrently in T-ALL, such as NUP214-ABL1 in 6% of T-ALL31 or EML1-ABL1.32 Genomic deletions and amplifications Often cryptic, genomic deletions and amplifications lead to the loss of tumor suppressor genes such as CDKN2A (p14/p16), which encodes a cell cycle regulator. Loss of CDKN2A is the most common genetic abnormality in T-ALL and is found in the vast majority of patients.33 Other deletions may fall upstream or downstream of transcription factor genes and thus activate the ectopic expression as in the cases of LMO2 in T-ALL. 34,35 Duplications or amplifications of genes in T-ALL may include MYB or the amplification of the ABL1 variant fusion gene NUP214-ABL1 on amplified episomes.31 Mutations Mutations are commonly found with or without chromosomal abnormalities in T-ALL. The most commonly mutated gene in T-ALL is NOTCH1, which exhibits 11 activating mutations in over 50% of T-ALL. Mutations in the Notch1 gene occur in the homodimerization (HD) and PEST domains, resulting in the constitutive activation of the Notch pathway or in an increase of its half-life, respectively. The NOTCH1-associated gene, FBXW7, which is responsible for ubiquitinylating NOTCH1 as well as cyclin E1, c-Myc, and c-Jun protein, is also often mutated in T-ALL, resulting in lack of proteosomal degradation of NOTCH1 protein.36,37 In addition to these types of aberrations, T-ALL may also exhibit aneuploidy in the form of either hyperdiploidy or hypodiploidy. Hyperdiploidy (51-65 chromosomes) usually indicates a good prognosis, whereas hypodiploidy (45 or fewer chromosomes) is usually accompanied by a worse prognosis (Reviewed in Harrison 200238). Unfortunately, with the exception of haploid/diploid status, cytogenetics in T-ALL are not as well understood as they are in B-ALL and have not been as informative in risk group classification or treatment planning for T-ALL as desired.39 Despite ~50% occurrence in T-ALL, cytogenetic abnormalities are not entirely responsible for all aberrant gene regulation that contributes to T-ALL ontogeny. In fact, gene expression analysis has demonstrated that up-regulation of oncogene expression in T-ALL can happen in the complete absence of such abnormalities. For instance, the Notch1 gene undergoes translocation in less than 1% of T-ALL. This translocation results in lack of the extracellular portion but retains the transmembrane and intracellular subunits, resulting in a constitutively active form of intracellular NOTCH1 protein.40 However, gain-of-function mutations in Notch1 have been observed in over 50% of T-ALL which are not caused by this translocation.37 If one includes mutations in Fbxw7, 12 which is responsible for ubiquitinylating and degrading NOTCH1 protein, mutations in Notch1/Fbxw7 may be observed in 80% of T-ALL.41,42 Micro RNA contributions More recent investigations indicate that there may be other gene misregulation phenomena which may also contribute to T-ALL. For instance, Mavrakis et al. (2011)43 demonstrated that specific micro RNAs (miRNA) targeted towards T-ALL tumor suppressor genes were upregulated in T-ALL. Five specific miRNAs with relevance to T-ALL were identified including MIR19B, MIR20A, MIR26A, MIR92, and MIR223, which were predicted to silence T-ALL tumor suppressor genes including IKZF1, PTEN, BCL2L11, PHF6, NF1, and FBXW7. Epigenetic contributions Epigenetic gene misregulation may also play a role in T-ALL development, as has been found to apply to other forms of cancer. Abnormal levels of DNA methylation in T-ALL can result in silencing of WNT pathway elements as well as the cell cycle regulators P15INK4B and P16INK4A.44,45 Furthermore, in two separate studies, Kraszewska et al. (2011)46 and Roman-Gomez et al. (2005)47 found that DNA methylation status of particular genes is different in T-ALL patients, healthy children, and normal thymic cell populations. However, it seems that the hypermethylation status of particular genes is not as important as assessing the general pattern of methylation of cancer cells, or the CpG island methylator phenotype (CIMP status).48 Both of these studies defined CIMP- status as two or less hypermethylated genes and CIMP+ status as three or more hypermethylated genes. In both studies, strong 13 positive genomic methylation (CIMP+) status correlated with poor outcome, whereas lack of high methylation status (CIMP-) correlated with good outcome. Specifically, in the Roman-Gomez et al. study (2005),47 relapse rates for CIMP- and CIMP+ were 0% vs 58%, mortality rates were 20% and 59%, and 13-year overall survival were 91% and 17%, respectively. For both studies, 12-year disease-free survival for patients who achieved complete remission was significantly different at 100% and 20% for CIMP- and CIMP+, respectively. Furthermore, 13-year overall survival was 91% for CIMP- and 17% for CIMP+ patients.49 However, although these data seem significant, larger high-powered studies need to be done to confirm these findings. Moreover, pre-existing germline mutations in enzymatic processing genes may result in pharmacogenetic inhibition of chemotherapeutic drug activity. These germline polymorphisms and mutations affect drug metabolizing genes, which may influence negatively the response of leukemic blasts to specific chemotherapy agents.50,51 Such genes may include thiopurine methyltransferase, glutathione S-transferase, cytochrome P450 3A4, and methylene-tetrahydrofolate reductase. All molecular contributors to T-ALL leukemogenesis are summarized graphically in Figure 1.6. T-ALL risk level assessment One of the most critical determinations that must be made before treating a patient for T-ALL is the relative risk level of the disease. Assessment of the risk category ensures that patients with high-risk T-ALL receive treatment of appropriate life-saving intensity and that low-risk patients are spared unnecessary toxic effects of treatment. A fine balance must be struck between these two considerations. 14 Many factors have been found to be contributory to T-ALL relative risk level. The first risk factors taken into consideration are patient characteristics, such as age and sex. The second set of considerations relate to the T-ALL disease features, such as WBC count, immunophenotype, and genetic abnormalities. Additional factors considered are the presence or absence of central nervous system (CNS) or testicular involvement, and early therapy response as determined in the initial 1-2 weeks of therapy.17,52 Within the last 10 years, pediatric T-ALL risk stratification classifications have expanded from two classes (standard and high risk) to four classes currently. The prior two-class risk stratification system relied only on patient age and WBC at the time of diagnosis. Currently the Children's Oncology Group (COG) defines four classes of T-ALL risk as low risk, standard risk, high risk, and very high risk.53 The estimated 4-year event-free survival (EFS) for these groups is 91%, 86%, 76%, and 46%, respectively.54 These groups were defined after examining clinical and cytogenetic data from more than 6000 patients enrolled on previous studies. However, risk level assessment can be much more complicated due to several contributing factors beyond those mentioned above. High risk factors include older age (with the exception of infancy), MLL rearrangements, hypodiploidy, CNS involvement, drug metabolizing enzyme polymorphisms, hepatosplenomegaly, mediastinal mass, male gender, Caucasian race, an immature T-ALL immunophenotype, and hypermethylation of tumor suppressor genes. A few of these factors will be discussed further here. Gene expression profiling With the recent development of microarray platforms for global gene expression assessment, it has become possible to assess comprehensively differential T-ALL gene 15 expression patterns. Lugthart et al.55 determined in vitro drug sensitivity of ALL cells in 441 patients. They used a genome-wide approach to identify 45 genes differentially expressed in ALL, exhibiting cross-resistance to multiple chemotherapeutic agents. The expression of these genes discriminated treatment outcome in two independent patient populations, identifying a subset of patients with a markedly inferior outcome. Furthermore, Golub et al.56 demonstrated the feasibility of using microarrays to accurately distinguish subtypes of leukemia using a set of genes as class predictors. T-ALL treatment regimens Once T-ALL has been definitively diagnosed, a course of clinical action is chosen. The primary clinical goal of treatment is to achieve a lasting remission, defined as the absence of detectable cancer cells in the body and a negative minimal residual disease (MRD) reading as determined by FACS. Treatment includes high-dose chemotherapy administered both intravenously and intrathecally (IV and IT). Chemotherapy regimens typically include three steps: 1) remission induction 2) intensification, and 3) maintenance therapy. The purpose of remission induction is to rapidly kill most leukemic blasts and achieve an initial remission regardless of non-debilitating toxicity. This allows the patient to achieve sufficient hematological recovery to proceed to further therapy as promptly as possible. CNS prophylactic (or therapeutic, in the case of CNS positive disease) chemotherapy is also administered at this point. Glucocorticoid therapy Glucocorticoids (prednisone/prednisolone and dexamethasone) play a fundamental role in the treatment of all lymphoid tumors because of their capacity to 16 induce apoptosis in lymphoid progenitor cells.57,58 For this reason, they will be discussed more in-depth as an example of T-ALL treatment. The importance of glucocorticoid therapy in leukemias and lymphomas is underscored by the strong association of glucocorticoid response with prognosis in childhood ALL. Thus, the initial response to 7 days of glucocorticoid therapy is a strong independent prognostic factor in this disease.59,60 Resistance to glucocorticoids in vitro is associated with an unfavorable prognosis.61,62 Moreover, the majority of patients with ALL in relapse show increase resistance to glucocorticoid therapy, identifying glucocortocid resistance as a major contributor to treatment failure.61,63 The glucocorticoids used in T-ALL therapy are predisone (or its derivative prednisolone) and dexamethasone. Though structurally very similar as derivatives of cortisol (Figure 1.7), these compounds have different efficacy and toxicity.64 Relative strengths of dexamethasone in comparison to prednisone include a longer plasma half-life (200 min vs 60 min),65,66 reduced sodium retention,66 and lower IC50 of ALL cells in vitro (0.2μM vs 3.5μM).67 Furthermore, treatment regimens which include dexamethasone have higher CNS penetration with a concomitant reduction in CNS relapse (14.3% vs 25.6%, p=0.017),68,69 and are associated with overall better event-free survival (81% vs 49%).70,71 However encouraging dexamethasone treatment outcomes may be in comparison to those of prednisone treatment, care must still be taken to consider the detrimental side effects of dexamethasone use. Prior studies have shown a consistent increase in side effects with dexamethasone use over prednisone, including bacterial and fungal infections,72,73 bone fractures and osteonecrosis (especially for patients age > 10 17 years),74,75 steroid psychosis,76,77 neurocognitive dysfunction,78,79 and proximal myopathy.76,80 Tyrosine kinase inhibitors With the recent trend toward more targeted therapies for cancer, tyrosine kinase inhibitors (TKIs) have become a useful treatment modality for leukemia. The first TKI to be used in treating leukemia was imatinib mesylate (STI-571, brand name Gleevec®), a small molecule inhibitor designed to inhibit a small group of tyrosine kinases, including BCR-ABL, also referred to as the Philadelphia (Ph+) chromosome.81 Imatinib acts as a competitive inhibitor for ATP cofactor binding and locks the Abelson kinase portion of BCR-ABL in an inactive conformation.82,83 Imatinib has been highly successful as frontline therapy for treating chronic myeloid leukemia (CML). In CML, imatinib induces complete hematologic remission in ~95% of cases, with complete cytogenetic responses in ~75% of patients with chronic phase CML.84 Philadelphia chromosome-positive B cell ALL Ph+ B-ALL usually carries a poor prognosis even with modern intensive chemotherapy regimens. Historically, bone marrow transplant had been recommended for children with this subtype of B-ALL, especially those with poor early response to induction therapy.85 Imatinib also serves as therapy for treating BCR-ABL B-cell ALL (Ph+ B-ALL) and has constituted a revolutionary development in treating this disease.86 In Ph+ B-ALL, remission is frequently achieved with imatinib alone.87 Unfortunately, unlike CML, these responses in Ph+ B-ALL are often transient and in most cases relapse within months. Hence, subsequent studies evaluated combinations of imatinib with 18 chemotherapy.86,88 Concurrent administration of imatinib with a multiagent regimen in an adult cohort led to molecular remission in 54% of patients compared with 19% in those who received an alternative chemotherapy regimen.89 Imatinib resistance CML in remission, maintained on imatinib, can become treatment resistant over time.90,91 Imatinib resistance in patients with Ph+ ALL also develops in most cases. A common mechanism of resistance is the development of point mutations within the kinase binding domain of BCR-ABL, most often in the P-loop or at codon 315 (T315I). These mutations can be detected in a small subclone of leukemic cells in 40% of newly diagnosed patients. But it becomes the dominant clone in 90% of cases at relapse, thus implying selective pressure of the resistant clone after treatment with tyrosine kinase inhibitors.92 Other mechanisms leading to imatinib resistance in Ph+ B-ALL include gene amplification and overexpression of BCR-ABL, and activation of the SRC family of kinases.93 The development of resistance to imatinib in CML and Ph+ B-ALL spawned the development of a second generation of tyrosine kinase inhibitors, including dasatinib and nilotinib, which are both effective in treating imatinib-resistant ALL with the exception of cases harboring the T315I mutation. Dasatinib is a multikinase inhibitor targeting several tyrosine kinases, including BCR-ABL and SRC kinases. It is 325 times more potent than imatinib, binds to both the active and inactive forms of BCR-ABL, and has excellent CNS penetration.94,95 Nilotinib is a derivative of imatinib in which modification of the aminopyrimidine backbone resulted in improved binding and a 30-fold increase in potency.96 19 Third generation TKIs A third generation of tyrosine kinase inhibitors, including ponatinib, are being actively developed, especially to overcome the problematic T315I mutation that confers resistance to all existing BCR-ABL-specific tyrosine kinase inhibitors. Though not found as often as in cases of B-ALL, ABL1 translocations can also be found in T-ALL in the form of NUP214-ABL1 and EML1-ABL1 translocations. TKIs have been found to be effective in treating these cases of T-ALL. Dasatinib inhibits NUP214-ABL1 (Nuclear pore complex protein 214-V-abl Abelson murine leukemia viral oncogene homolog 1) cell proliferation while EML1-ABL1 (Echinoderm microtubule-associated protein-like 1- V-abl Abelson murine leukemia viral oncogene homolog 1) cells show sensitivity to imatinib.97 However, despite encouraging results seen in utilizing second and third-generation TKIs, it is still likely that these drugs will have to be used in combination with other standard chemotherapies, as resistance to single kinase inhibitors is becoming an expected outcome in cancer.90 Maintenance therapy The goal of maintenance therapy is to eradicate any residual leukemia cells that were not eliminated by remission induction or intensification regimens. Maintenance therapy typically includes daily oral intake of mercaptopurine as well as weekly oral intake of methotrexate and a monthly injection of IV vincristine and a 5-day course of oral corticosteroids. The duration of maintenance therapy is typically 2 years for girls and adults and 3 years for boys.98 20 T-ALL relapse therapy Relapsed cases of T-ALL are the most difficult to treat and carry the worst prognosis since the disease has already undergone selection for chemoresistance characteristics. Overall, ALL relapses occur in 25% of pediatric and 50% of adult cases, respectively, with the rate of relapse being correlated with the immunophentotype and genetic subtype of the disease.17,52 The majority of ALLs relapse in the initial 3 to 5 years following initial diagnosis with a small percentage relapsing more than 5 years from diagnosis and up to as many as 10 to 20 years later in a small minority of patients.99 Relapsed ALL may involve the bone marrow or extramedullary tissues, often at "sanctuary sites," such as the CNS, gonads, or both. Statistically, relapses with isolated bone marrow involvement seem to correlate with a poorer prognosis than those of isolated extramedullary or combined bone marrow and extramedullary relapses.17 The morphologic and immunophenotypic features of relapsed ALL are often similar to that found at initial diagnosis and treatment. However, due to genomic instability coupled with drug-induced selection, imunophyenotypic variations may be present at relapse, wherein some cell surface antigens present at initial diagnosis may increase or decrease in intensity or may have been lost altogether at the time of relapse.100,101 The primary goal of T-ALL relapse therapy is to obtain a remission and to move to allogeneic transplantation as quickly as possible. Allogeneic transplantation must proceed quickly since most relapsed patients die shortly after relapsing. One study of 607 relapsed patients showed a 5-year survival of only 7%. In the same study, transplanted patients only experienced 14-16% survival for sibling and unrelated donors, respectively, a poignant reminder of how much progress has yet to be made in this field.102 In a French 21 study, 44% of relapsed patients achieved a second remission (CR2), but only 12% survived.103 These statistics clearly indicate a need to focus on optimizing initial therapy since salvage therapy in relapsed cases rarely succeeds long-term. These data also underscore a need to develop a much better understanding of the molecular mechanisms involved in relapsed T-ALL with the intent of using this knowledge to develop better targeted T-ALL therapies. Minimal residual disease (MRD) determination More than 80% of childhood and 35% of adult ALL patients can be cured with modern chemotherapy supplemented with HSCT (hematopoietic stem cell transplant) in high risk patients.104 Still, a substantial number of ALL patients relapse and the prediction of relapse with conventional prognostic factors as above and classical clinical risk group assignment is far from optimal. Mortality rates for relapsed patient cases of ALL are a particularly sobering reminder of such treatment limitations. For instance, in the FRALLE-93 study, ~80% of patients with induction failures achieved a complete second remission utilizing various salvage therapies. However, only 30% were long-term survivors, emphasizing the need for early treatment success via complete initial elimination of all remaining residual disease.105 Prior techniques for determination of early induction therapy success relied on morphological examination of bone and peripheral blood specimens to detect the presence of residual disease. However, the bulk of residual tumor burden is below this limit of visual detection. For that reason, molecular techniques for detection of residual disease are now available that can detect "submicroscopic disease" not previously 22 identifiable. Techniques are now available that can detect 1 tumor cell in a background of 10,000 (using FACS) to more than 1 million normal cells (using PCR). Correlation of MRD with treatment outcome has obvious applicability to treatment-making decisions in T-ALL. As the treatment side-effects in T-ALL can be very harsh, the overriding goal in risk level assessment is to assure that the patient receives only as intense a treatment regimen as necessary to eradicate the disease without excessive and undue toxicity. Patients who achieve MRD negativity after 2 to 3 weeks of remission induction, and therefore have an excellent prognosis, are good candidates for treatment de-intensification. At the very least they should not be subjected to further treatment intensification.106,107 Conversely, MRD determination has the prognostic ability to inform treatment decisions when treatment intensification is warranted in resistant cases of T-ALL. Augmentation of subsequent therapy for patients who demonstrate a slow early response can significantly improve cure rates as extended induction and consolidation are used to deepen morphologic remission.108,109 Relapsed cases of T-ALL are the most difficult to treat and carry the worst prognosis. In such cases, MRD determination can be utilized to herald impending relapse, thus accelerating the planning of salvage therapy and/or HSCT. In patients who relapse but manage to achieve a second remission, MRD assays can be used to guide the selection of optimal post-remission treatment (i.e., chemotherapy versus HSCT). MRD measurements can also be used to determine the optimal timing of HSCT. MRD measurements post HSCT can also be used to guide the administration of donor lymphocyte infusions or other agents.110 23 Detection of MRD can be achieved through utilization of three different molecular techniques, including flow-cytometry, PCR detection of leukemia-associated fusion genes, and PCR detection of Ig/TCR gene arrangements. PCR detection of Ig/TCR gene re-arrangements is the third MRD methodology. Since individual T-cell receptor and immunoglobulin genes undergo a unique clonal rearrangement, they can be used as specific targets for residual tumor detection.107 Although this MRD strategy is the most laborious, expensive, and time-consuming, it is reproducible not only within the same laboratory but also between different laboratories. Furthermore, it is the most sensitive technique. The junctional regions of clonal Ig and TCR gene rearrangements are fingerprint-like sequences for each lymphoid malignancy and can be identified in the vast majority of ALL patients using the standardized primer sets established through the European collaboration within the BIOMED-1 and -2 frameworks.111,112 Consensus primers can be used to amplify junctional sequences and the length of the product will allow discrimination from a background of normal cells. The sensitivity of this approach can be as high as 1 cell in 10,000 with sequencing of the initial product and the creation of allele-specific primers.113 Due to oligoclonality and clonal evolution of Ig/TCR gene rearrangements between diagnosis and relapse, it is recommended that at least two Ig/TCR targets be followed per patient.114,115 Regardless of the technique used, the following broad conclusions can be made. Patients with no detectable MRD at the end of induction have an exceedingly good outcome (EFS >90% at 3 years). Those children with a high MRD (>10-2) have a poor prognosis (3-year EFS of approximately 25%). Patients with intermediate levels (10-4 to 10-3) make up one-third to one-half of all patients depending on the technique used. 24 These patients can be further subdivided based on analysis of a second time point. For example, in the study by Coustan-Smith, of 32 patients who were MRD+ by flow cytometry at the end of remission induction who then became MRD- at week 14, only one relapsed. In comparison, 10 relapses occurred among 18 patients who remained MRD+ at the second time point.116 It is probable that MRD diagnostics will be included in all T-ALL treatment protocols, as MRD data provide so far the most optimal reflection of the in vivo response to treatment, which gives the clinician a better knowledge and control of the best clinical course of action for individual patients. Hematopoietic stem cell transplant Hematopoietic stem cell transplant (HSCT) is utilized as a treatment modality of last resort for patients who have failed chemotherapy. It is also often used as first-line therapy for patients who have high-risk T-ALL that is not expected to respond well to chemotherapy. The role of HSCT in treating T-ALL is expected to increase as alternative donor sources become available. Alternative donor sources, including cord blood, are increasingly used for transplantation in children and adults with leukemia. A higher degree of mismatch is acceptable with cord blood units, and outcomes are comparable with those of allele-matched transplants.117,118 Importantly, cord blood units can be obtained with a shorter waiting period and from a larger recipient pool, particularly for ethnic minorities who have a lower probability of having a suitably matched unrelated donor.119 HSCT is particularly applicable in cases of high-risk T-ALL. High-risk cases include those with poor initial response to induction therapy or high MRD, a stem cell-like immature T-ALL immunophenotype, high WBC count, or a t(4;11) translocation. 25 For example, in a large study of high-risk T-ALL in children comparing chemotherapy versus allogeneic HSCT, 5-year disease-free survival was 26.5% vs 56%, respectively.120 Cases of Ph+ B-ALL would also be considered high-risk cases of ALL and eligible for first-line HSCT therapy. For instance, in one study of 267 children with Ph+ B-ALL, 5- year disease-free survival was 25% with chemotherapy alone versus 72% for patients receiving HSCT from a matched related donor.121 In cases of successful remission induction followed by a subsequent relapse, HSCT would also be utilized if a second complete response (CR2) could be achieved using intensified salvage combination chemotherapy. HSCT is usually more successful when using an allele-matched donor (allogeneic transplant) in comparison to autologous transplant from the patient's own bone marrow. In the LALA-94 trial, patients with high-risk ALL were allocated to allogeneic bone marrow transplantation if they had an HLA identical sibling or were randomized to autologous BMT or chemotherapy if they did not. Disease-free survival was 45% in patients with a matched donor versus 18% in those without.122 A meta-analysis of this study and six others encompassing 1,274 patients showed a survival advantage for patients with an allogeneic stem cell donor that was augmented in high-risk patients. No benefit from autologous BMT was noted.123 An allogeneic transplant seeks to take advantage of the "graft versus leukemia" (GVL) effect, wherein if the patient experiences a relapse of T-ALL due to residual disease, the graft will recognize the relapsed T-ALL as foreign and destroy it. Allogeneic HSCT also carries with it the additional long-term benefit of being able to use T-cells harvested from the original donor in an infusion to "boost" the recipient's engrafted 26 immune system to destroy a refractory T-ALL relapse.124 Unfortunately, the GVL effect is also often accompanied by "graft versus host disease" (GVHD), in which the allogeneic HSCT graft may recognize other healthy recipient tissues as foreign and seek to destroy them, resulting in undesirable long-term side effects. However, despite GVHD side-effects, many studies have shown that the long-term benefits of using allogeneic HSCT are superior to those using autologous transplant. Children undergoing HSCT on the average fare better than do adults. The only exceptions are infants who do not show benefit from allogeneic HSCT over combination chemotherapy alone. In fact, some showed worse outcomes in infants transplanted in first remission even after adjusting for presenting clinical features and waiting time to transplantation.125,126 Although many adult T-ALL patients benefit from allogeneic HSCT, still many are unable to tolerate the harsh myeloablative (MA) treatments necessary to prepare for the transplant, resulting in many cases of treatment-related mortality (TRM). This lack of tolerance for the prerequisite myeloablative therapy and consequent TRM increases as age increases. In consequence of this observed lack of tolerance for harsh preparative MA, many treatment centers are implementing a reduced intensity conditioning (RIC) pre-treatment regimen for older patients with T-ALL. The EBMT group has reported on a group of 97 patients with adult ALL, including one third of the patients in first CR with the majority in higher levels of CR or with refractory or persistent disease. The patients received a variety of RIC regimens and, with nearly 3 years of follow-up, the OS for the first CR patients was 52%. OS was 27% and 20%, respectively, for patients in second/third CR or with a more advanced disease.127 Nevertheless, if a patient fails to 27 achieve a complete response after induction or salvage chemotherapy, HSCT is not likely to succeed and is not recommended in these cases. Need for more targeted therapies in treating T-ALL Long-term sequelae of T-ALL treatment Thus far, most of the statistics cited in this document have focused on overall and disease-free survival in patients treated for T-ALL. However, these statistics do not take into consideration the long-term detrimental side effects of treatment for T-ALL. T-ALL is typically more difficult to treat than B-ALL, requiring harsher chemotherapy regimens with a concomitant increase in undesirable side-effects in both the short- and the long-term. These effects can be particularly detrimental for children treated for this disease. For instance, in comparison to their unaffected siblings, adult survivors of childhood cancers are 54 times more likely to have a major joint replacement, 15.1 times more likely to have congestive heart failure, and 14.8 times more likely to have a second malignancy, to name only a few effects (Table 1.7). Although cure rates for T-ALL have improved dramatically in the past four decades, the means by which it has been accomplished has been for the most part intensification of the chemotherapy regimens used. Although this improvement in cure rates is welcome, such intensive chemotherapy comes at the cost of significant side effects, often continuing for years after treatment. And despite recent dramatic statistical improvements in cure rates, ~20% of pediatric and >50% of adult cases of T-ALL still cannot be cured. So the search for more targeted therapies that treat T-ALL more effectively while simultaneously reducing detrimental side-effects must be a research priority. 28 Goals of the dissertation Although T-ALL treatment results have improved dramatically in the last 50 years, the improvement has come at the cost of increased side-effects of therapy. Hence, identification and development of more targeted therapies for treating T-ALL is the primary focus for this study. Questions to be asked and goals to be achieved by this study include the following: 1. Can zebrafish be utilized in an in vivo screen for compounds that show efficacy in treating T-ALL? As a vertebrate teleost, zebrafish (Danio rerio) have an immune system that is very similar to that of humans, including a thymus in which T-cells undergo a maturation process very similar to that of humans. Early zebrafish larvae are small enough to fit in a 96-well format, thus facilitating screening of thousands of compounds. In addition, transgenic zebrafish are available in which the T-cells have been genetically engineered to express eGFP, thus facilitating the observation of drug effects on T cell survival as well as T-ALL leukemogenesis and dissemination in vivo. 2. Can candidate compounds be identified that are effective in killing T-ALL while not exhibiting other toxic side-effects? To this end, candidate compounds identified will include those reproducibly exhibiting selective elimination of developing T-cells in larval zebrafish thymus while not sickening or killing the larvae. Furthermore, any candidate identified from the screen must not exhibit general cell cycle effects, as do traditional chemotherapy agents. Candidate compounds must also show lethality toward human T-ALL while still demonstrating a sufficient therapeutic window between the IC50 in human T-ALL cells compared to the IC50 determined for normal healthy peripheral 29 lymphocytes. Candidate compounds will also show efficacy in treating primary T-ALL patient samples. 3. If compounds can be identified from the screen that exhibit T-ALL specific lethality, what is/are the biochemical mechanism(s) of action employed by them? Numerous aberrantly activated molecular pathways have been implicated in T-ALL, including MAPK, JAK/STAT, NOTCH, Wnt, and PI3K/AKT/mTOR. Any T-ALL specific compounds identified in this screen may down-regulate one or more of these pathways. In addition, it is also possible that a candidate compound's activity may reveal a novel molecular mechanism heretofore unrecognized for its contribution to T-ALL leukemogenesis. In such a case, experiments would need to be designed and executed to characterize the biochemical behavior of such a pathway with the intent of identifying all druggable targets functioning within it. 4. Are the candidate compounds safe and effective in treating T-ALL in vivo? Multiple zebrafish models of spontaneous and induced (i.e., c-Myc) T-ALL development exist which could be utilized for this purpose. In addition, NOD-SCID mice may be utilized in a human xenograft model of T-ALL to determine mammalian response to drug candidate treatment. In addition to therapeutic response to treatment, maximum tolerated dose will be determined for candidate compounds along with endorgan toxicity and impact on hematological parameters in vivo. 30 References 1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th edn. (2008) IARC Press, Lyon. 2. Feng H, Stachura DL, White RM, et al. T-lymphoblastic lymphoma cells express high levels of BCL2, S1P1, and ICAM1, leading to a blockade of tumor cell intravasation. Cancer Cell. 2010; 18(4):353-66. 3. Uyttebroeck A, et al. Is there a difference in childhood T-cell acute lymphoblastic leukaemia and T-cell lymphoblastic lymphoma? 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N Engl J Med. 2006; 355(15):1572-82. 42 Table 1.1 WHO 2008 classification of T-cell neoplasms. Adapted from de Leval et al. (2009).4 Category Classifications Subclassifications Precursor T-cell neoplasms Precursor T acute lymphoblastic leukemia (T-ALL/lymphoma, T-LBL) Mature T-cell neoplasms Leukemic or disseminated T_cell prolymphocytic leukemia T-cell large granular lymphocytic leukemia Adult T-cell lymphoma/leukemia (HTLV1- positive) Systemic EBV-positive T-cell lymphoproliferative disorders of childhood Extranodal-cutaneous Mycosis fungoides Sezary syndrome Primary cutaneous CD30+ lymphoproliferative disorders Primary cutaneous anaplastic large cell lymphoma Lymphomatoid papulosis Subcutaneous panniculitis-like T-cell lympooma Primary cutaneous gamma-delta T-cell lymphoma Primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma Primary cutaneous small/medium CD4+ T-cell lymphoma Nodal Angioimmunoblastic T-cell lyumphoma (AITL) Anaplastic large cell lymphoma, ALK-positive Anaplastic large cell lymphoma, ALK-negative Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) 43 Table 1.2 Stages of T cell development correlate with specific locations in the thymus, distinct cell-surface phenotypes, requirements for Notch signals, and TCR rearrangement. Adapted from Pui et al. (2002).125 Developmental stage Cell surface phenotype Location Notch signal TCRβ rearrangement TCRα rearrangement ETP DN1 CD117hiCD44hiCD25− CD24−/loCD27hi CMJ +++ Germline Germline DN2a CD117hiCD44hiCD25+ CD24hiCD27hi Cortex + Germline Germline DN2b CD117intCD44hiCD25+ CD24hiCD27int Cortex + DJH Germline DN3a CD117−/loCD44−/loCD25+ CD24hiCD27−/lo SCZ +++ DJH,VDJH Germline DN3b CD117−/loCD44−/loCD25int CD24hiCD27hi SCZ +++ VDJ+ Germline DN4 CD117−/loCD44−/loCD25−/lo CD24hiCD27hi SCZ + VDJ+ Germline DP CD4+CD8+TCRβint Cortex - VDJ+ VJ Abbreviations: CMJ, corticomedullary junction; D, diverse; DN, double negative; DP, double positive; ETP, early thymic progenitor; H, heavy chain; J, joining; SCZ, subcapsular zone; TCR, T cell receptor; V, variable. The single + for Notch signal indicates Notch1 receptor expression; however, a specific function or requirement has not been described. The triple +++ for Notch signal indicates a requirement for Notch. Table 1.3 EGIL classification system for T-ALL Adapted from Bene et al. (1995).22 T-ALL Subtype CD1a CD2 cCD3 sCD3 CD4 CD5 CD7 CD8 CD34 Pro-T - - + - - - + - + Pre-T - + + - - ± - - ± Cortical T + + + - + ± + + - Medullary T - + + + ± ± + ± - 44 Table 1.4 The TCR system for classifying T-ALL Adapted from Asnafi et al. (2004).23 Stage: M arkers: Immature (IM) Cytoplasmic-beta- (Cytß-), sCD3-, TCRαß- or TCRγδ- Pre-αß Cytß+, sCD3-, TCRαß- or TCRγδ- TCRαß sCD3+, TCRαß+ TCRγδ sCD3+, TCRαß+ Table 1.5 Most frequent genetic abnormalities in T-ALL Adapted from Graux (2011).126 (Cyto) genetic changes: Genes: Remarks/prognosis: TCR receptor genes translocations (14q11 & 7q34) t(10;14)(q24;q11) TLX1;TCRA/D Favorable t(11;14)(p15;q11) LMO1;TCR t(11;14(p13;q11) LMO2;TCR t(1;14)(p32;q11) TAL1;TCR inv(7)(p15q32) HOXA;TCRB t(7;19)(q34;p13) LYL1;TCRB t(5;14)(q35;q32) TLX3;BCL11B Poor? Fusion genes del1(p32) interstitial SIL-TAL1 9(q34) episomes NUP214-ABL1 t(10;11)(p13;q14) CALM-AF10 t(11;19)(q23;p13) MLL-ENL Deletion/amplification del(9)(p21) CDKN2A del(6q) Unknown dup(6)(q23) MYB Mutations NOTCH1/FBW7 Very frequent (>80%) JAK1, PTEN, RAS, FLT3, PHF6 45 Table 1.6 Common fusion proteins in T-ALL Adapted from Graux et al (2006).26 . 46 Table 1.7 Relative long-term side effect risks of adult survivors of childhood cancers Adapted from Oeffinger et al. (2006).130 Condition %Survivors (N = 10,397) %Siblings (N = 3034) Relative Risk (95% CI) Major joint replacement 1.61 0.03 54.0 (7.6-386.3) Congestive heart failure 1.24 0.10 15.1 (4.8-47.9) Second malignant neoplasm 1.24 0.33 14.8 (7.2-30.4) Cognitive dysfunction, severe 0.65 0.10 10.5 (2.6-43.0) Coronary artery disease 1.11 0.20 10.4 (4.1-25.9) Cerebrovascular accident 1.56 0.20 9.3 (4.1-21.2) Renal failure or dialysis 0.52 0.07 8.9 (2.2-36.6) Hearing loss not corrected by aid 1.96 0.36 6.3 (3.3-11.8) Legally blind or loss of an eye 2.92 0.69 5.8 (3.5-9.5) Ovarian failure 2.79 0.99 3.5 (2.7-5.2) 47 Figure 1.1. Five-year pediatric ALL survival rates over the past 60 years. Adapted from Koch et al. (2011).128 A. Normal healthy bone marrow B. Bone marrow from a T-ALL patient Figure 1.2. Comparison of normal and T-ALL leukemic bone marrow. Adapted from Graux et al. (2006).129 48 Thymus DN2 DN3 NOTCH1 NOTCH1 γδ T-cell Pre-TCR DN4 DP NOTCH1 E2A ETP Blood vessel E2A, MYC TCR+ DP CD4+ SP CD8+ SP Sub-capsular zone Cortex Cortico-medullary junction Medulla Pre-TCR Signaling Figure 1.3. Stages of haematopoiesis and T‑cell development and T‑cell-leukaemia-related oncogenes. Bone-marrow haematopoietic stem cells (HSCs) exit the quiescent ‘niche' and differentiate to become multipotent progenitors (MPPs). MPPs further commit to the lymphoid lineage generating common lymphoid progenitors (CLPs). Several progenitor subsets (including MPPs and CLPs) have been suggested to represent the progenitor of thymic pro‑T cells. These subsets migrate to the thymus (as early T‑cell-lineage progenitors (ETPs) and commit to the T‑cell lineage, progressing through the double negative (DN; CD4-CD8-) stages, DN2, DN3 and DN4. Upon successful recombination at the T‑cell receptor β (TCRB) locus, pre‑T cells acquire surface expression of the pre-TCR that promotes differentiation to the DN4 stage. Pre-TCR-selected cells reach the double positive (DP; CD4+CD8+) stage, at which point they are subjected to the processes of positive and negative selection. Selected cells then exit the thymus as single positive (SP) CD4+ or CD8+ T cells. The stages of differentiation at which oncogenes that are known to be associated with T‑cell acute lymphoblastic leukaemia and required in the bone marrow and thymus are also depicted. LMO2, LIM-only 2; TAL1, T-cell acute lymphocytic leukaemia 1. Adapted from Aifantis et al. (2008).24 49 Figure 1.4. Functional classifications of common T-ALL mutations. Molecular analysis of T-ALL shows that four major classes of mutations are involved in the molecular pathogenesis of T-ALL. These four classes are represented by the four diagrams, in which the frequency of each of the different mutations is given. Adapted from Armstrong and Look (2005).88 50 (A) (B) (C) Figure 1.5. Classical karyotyping and FISH technique. (A) A typical G-banded karyotype. (B) Deletion of SIL resulting in SIL-TAL1 fusion gene, demonstrated with the SIL-TAL1 DNA probe (DAKOcytomation). The SIL probe (in red) is absent on del(1). Adapted from Graux et al. (2006).132 (C) Multiple colour FISH (MFISH) of a highly abnormal metaphase taken from a child with ALL. The colour changes along the chromosomes indicate the locations and origins of chromosomal translocations. Adapted from Harrison et al. (2002).38 Figure 1.6. Schematic representation of molecular contributors to T-ALL ontogeny. Adapted from Kraszewska et al. (2012).39 51 Cortisol Prednisone Prednisolone Dexamethasone Figure 1.7. Chemical structures of cortisol and synthetic glucocorticoids prednisone, prednisolone, and dexamethasone. Adapted from Inaba and Pui (2010).64 CHAPTER 2 ABERRANT SIGNALING PATHWAYS IN T-ALL Background The last 50 years have seen remarkable progress in success rates for treating T-ALL. However, this remarkable progress has come about primarily through efforts in the clinic to intensify chemotherapy regimens. As a result, detrimental side effects have increased concomitantly with long-term remission percentages. Since intensity of untargeted chemotherapy intensity has been maximized in T-ALL therapy, further progress in treating refractory and relapsed cases must be achieved through alternative means. As a result, a primary focus in the field of T-ALL research has been to identify and characterize the biochemical pathways that are aberrantly activated in T-ALL leukemogenesis, and to discover mechanisms of resistance to chemotherapy. These research endeavors have the principal goal of identifying druggable targets, with the expectation that targeted therapy will achieve remission more efficiently and with fewer side effects than those observed with traditional chemotherapy. Many of the chromosomal translocations and fusion proteins responsible for T-ALL have been characterized within the last 15 years. However, with rare exceptions such as p16 inactivation/deletion, no single chromosomal aberration has been found to be dominant in most cases of the disease. As a result, much research has focused on the common biochemical pathways downstream of these sporadic cytogenetic abnormalities 53 with the intent of finding common molecular pathways involved in a majority of T-ALL cases. Ikaros' contribution to T-ALL leukemogenesis Ikaros (Ik) is a transcriptional regulator expressed exclusively in the lymphoid system that is required for the development of all lymphoid lineages.1 Ikaros is a member of the Kruppel transcription factor family characterized by the presence of zinc-finger domains located at their N- and C- termini. The N-terminal domain is involved in DNA binding, whereas the C-terminal domain is important for protein dimerization and stability. The Ikaros gene (ZNFN1A1) transcript is processed into a number of isoforms due to alternative splicing that regulates the expression of exons coding for the N-terminal DNA binding domain. Thus, all IKAROS proteins share a common C-terminal domain with two zinc-fingers attached to a domain with different combinations of one to four N-terminal zinc-fingers, each with distinct DNA binding capabilities and specificities. At least three N-terminal zinc-fingers are required for high affinity DNA binding to the motif GGGAA/T.2 Only the longer IKAROS isoforms that contain at least three N-terminal zinc fingers present high affinity to their specific DNA sequence, whereas the shorter IKAROS versions with fewer than three N-terminal zinc fingers cannot bind DNA with high affinity. Rather, they behave as dominant negative isoforms upon heterodimerization with IKAROS isoforms that have an intact DNA-binding domain.3 The shorter, non DNA-binding IKAROS isoforms are normally expressed at low levels with respect to predominant long isoforms that are abundantly expressed throughout lymphocyte development. A mutation that results in the deletion of the N-terminal zinc-finger DNA 54 binding domain from the IKAROS proteins completely blocks lymphocyte development.4,5 Aberrant Ikaros function has been correlated with T-ALL leukemogenesis. Mice heterozygous for Ikaros mutations at birth show an apparently normal lymphoid cell distribution at birth. However, within a few months they develop a very aggressive form of lymphoblastic leukemia with a concomitant loss of heterozygosity, resulting in predominant synthesis of short IKAROS isoforms.1,6 These data demonstrate that IKAROS functions as a tumor suppressor in the lymphoid system. In support of this model, loss of IKAROS activity has been associated with human leukemia. Loss of DNA-binding IKAROS isoforms and/or overexpression of the shorter dominant negative form of IKAROS was observed in nearly 100% of childhood and adolescent T-ALL cases examined in one study.7-9 A current model suggests that the increase in expression of dominant negative IKAROS isoforms is a result of alternative splicing and not as a result of genomic alterations. NOTCH1 contribution to T-ALL NOTCH1 activity in T-cell hematopoiesis The fundamental components of the NOTCH pathway include the Delta and Serrate family of ligands (Delta-like 1, 3, and 4; and Jagged 1 and 2), four distinct NOTCH receptors (NOTCH1-4) and the CBF1, Su(H), Lag-1(CSL) DNA-binding proteins.10 All NOTCH receptors are class I transmembrane glycoproteins expressed at the cell surface as heterodimers. They consist of an N-terminal extracellular fragment and a C-terminal transmembrane-intracellular subunit. Of all the NOTCH receptors, only NOTCH1 has been found to play a role in T-ALL. The NOTCH1 receptor functions as a 55 ligand-activated transcription factor that directly transduces extracellular signals at the cell surface into changes in gene expression in the nucleus.10 Activation of NOTCH receptors typically occurs via cell-cell contact and interaction of a NOTCH protein with a Delta-like or Jagged ligand expressed on the surface of a neighboring cell, typically a thymic epithelial cell (TEC) in T-cell ontogeny. Two proteolytic events activate intracellular NOTCH1(ICN). First, an extracellular metalloprotease, typically ADAM10 or ADAM17, cleaves the receptor in the C-terminal portion of the heterodimerization (HD) domain (S2 cleavage). This generates a truncated protein with a short extracellular stub that is recognized and subsequently processed by the γ-secretase complex (S3 cleavage). Once released from the cell membrane, the ICN rapidly translocates to the nucleus, where it forms a transcriptional complex with the CSL DNA-binding protein. The ICN-CSL complex then activates gene transcription via recruitment of coactivators of the mastermind-like (MAML) family, whose transcriptional gene targets include hairy and enhancer of split 1 (HES1) and c-Myc (Figure 2.1).11 Importantly, transcriptional activation of NOTCH target genes is coupled with an active mechanism that ensures the rapid termination of NOTCH signaling. Recruitment of RNA polymerase II to the SCL-DNA binding transcription complex triggers phosphorylation of the PEST domain of the receptor, and its proteasomal degradation via the FBXW7-SCF ubiquitin ligase complex.10 NOTCH plays a critical role in T-cell ontogeny at multiple stages.12 T-cell lineage specification is dependent on NOTCH, since ablation of NOTCH1 results in a complete block at the earliest stages of T-cell lymphopoiesis and lack of specification of the T-cell 56 lineage. Mice harboring a conditional deletion of NOTCH1 fail to develop T-cells and show ectopic B-cell development in the thymus. Conversely, mice expressing a constitutively active form of NOTCH1 show ectopic T-cell development in the bone marrow and fail to produce B lymphocytes.12 Furthermore, NOTCH1 plays a role in progression through DN1, DN2, and DN3 stages of thymocyte maturation, regulates in part TCRB gene rearrangement and β-selection, and regulates the lineage decision between αβ and γδ fate.12 NOTCH1 pathway role in T-ALL leukemogenesis Aberrant activation of NOTCH1 in T-ALL was first identified in 1991 in rare leukemia cases harboring the t(7;9)(q34;q34.3) translocation, which juxtaposes a truncated NOTCH1 gene next to the TCRB locus (originally termed the TAN1 oncogene).13,14 Thereafter occasional additional cases of the NOTCH1 translocation were found in a small percentage of patients. However, because of the rarity of these translocations, the significance of these findings for the pathogenesis of T-ALL remained elusive until 2004, when Weng et al.15 published a landmark study in which they identified activating mutations in NOTCH1, leading to high levels of NOTCH1 signaling in over 60% of human T-ALL cases. The most frequent hot spots for mutations in NOTCH1 are exons 26 and 27, which encode the N- and C-terminal components of the heterodimerization (HD) domains, respectively.15 These activating HD mutations are found in ~40% of human T-ALL. These HD mutations result in ligand hypersensitivity or ligand-independent NOTCH1 cleavage and thus activation by ADAM proteases and γ- secretase.16 57 A second type of NOTCH1 mutation found in 20-25% of T-ALL is found in the intracellular PEST (Proline, Glutamate, Serine and Threonine rich) degron domain. The loss of the PEST domain results in increased levels of activated NOTCH1 due to impaired degradation of the activated receptor by the proteasome.15,17 Similarly, 15% of T-ALL cases show mutations in FBXW7, which normally ubiquitylates the PEST degron domain. Either mutation can result in aberrant accumulation of intracellular NOTCH1 protein and hence over-activation of target genes' transcription.17-19 Furthermore, FBXW7 is also responsible for ubiquitylating additional oncoproteins such as C-MYC, JUN, Cyclin E, and mTOR, for proteosomal degradation. As such, the biological consequences of FBXW7 inactivation may be broader than merely those of accumulation of NOTCH1 ICN, and may include increased cell metabolism, cell growth, and cell cycle progression.20 Gamma-secretase inhibitor treatment of T-ALL The high frequency with which activating mutations in NOTCH1 were observed initially generated much enthusiasm regarding the therapeutic potential of targeting this pathway in T-ALL. Given the requirement of γ-secretase cleavage for NOTCH1 activation, γ-secretase inhibitors (GSIs), already in use for treatment of Alzheimer's disease, were found to block oncogenic NOTCH1 signaling in T-ALL in vitro. 5,6,21 These results stimulated a phase I clinical trial of GSI MK-0752 for the treatment of relapsed T-ALL. However, despite some correlative data showing downreguation of NOTCH1 target genes in T-cell lymphoblasts, there were no objective clinical responses, and the effect on T-ALL blasts appeared to be cytostatic rather than cytolytic.5,6 58 Furthermore, many patients showed dose-limiting gastrointestinal toxicities from GSI treatment.22 These dose-limiting gastrointestinal toxicities were not unexpected since inhibition of NOTCH signaling has been shown to induce cell cycle arrest and accumulation of mucus-secreting goblet cells in the intestinal epithelium.23 However, the combination of glucocorticoid treatment with GSIs was much more efficient and exhibited a marked protective effect against GSI-induced intestinal toxicity in a mouse model of T-ALL, and may suggest that this may be a desirable and effective treatment combination modality for human T-ALL.23 The PI3K/AKT/mTOR pathway in T-ALL Introduction With the disappointing results seen in GSI treatment of T-ALL, research efforts turned to determining the reason(s) for T-ALL's resistance to GSI treatment. In a landmark study by Palomero et al. in 2007,24 it was discovered that loss of PTEN in T-ALL conferred resistance to GSIs. Furthermore, it was determined that loss of PTEN resulted in upregulation of the PI3K/AKT/mTOR (P/A/mT) pathway, thus effectively transferring T-ALL's oncogene addiction from NOTCH1 to P/A/mT. The P/A/mT pathway is known to be upregulated in many forms of cancer, not solely in hematological malignancies. Upregulation of this pathway is correlated with poor prognosis in many cancers as well as premature aging and resistance to cancer therapy (Figure 2.2).25 PI3K is a heterodimeric protein with an 85KDa regulatory subunit and a 110KDa catalytic subunit.26,27 Upon activation of a surface receptor tyrosine kinase, PI3K phosphorylates membrane phospholipids phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-diphosphate (PIP2) to generate phosphatidylinositol 3,4- 59 diphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosphate (PIP3), respectively.27-29 Through its pleckstrin homology (PH) domain, AKT interacts with PIP3, translocates to the cell membrane, and undergoes a conformational change that allows phosphorylation of AKT at T308 by PDK1, which itself is recruited to the membrane by PIP3 via its PH domain.30,31 Full activation of AKT also requires phosphorylation at S473 by mTORC2.32 However, mTORC1 is also activated by AKT indirectly by inhibition of the tuberous sclerosis complex 2 (TSC2).33 Thus, AKT is both upstream and downstream of mTOR. Activation of the P/A/mT pathway affects cell survival, growth, proliferation, and transformation.34,35,36 Negative regulation of P/A/mT activity is mediated by PTEN, a phosphatase that removes phosphate groups from PIP2 and PIP3, thus antagonizing activity of the P/A/mT pathway.25 Each of these molecular entities in the P/A/mT pathway will be discussed in turn. Phosphatidylinositide 3-kinase The phosphatidylinositide 3-kinases (PI3Ks) are a family of lipid kinases that catalyze the phosphorylation of phosphotidylinositides at the 3'-hydroxyl group. Although three classes make up this family, only Class I PI3Ks have been shown to be coupled to extracellular stimuli.35,37-39 This class can be subdivided in to Class IA kinases, which are activated by receptor-tyrosine kinases (RTKs), and Class IB kinases, which are regulated by G-protein-coupled receptors.40 To date only Class IA enzymes have been clearly implicated in human cancers.41,42 Class IA PI3Ks are heterodimers consisting of a catalytic subunit (p110) and a regulatory subunit (p85). Three isoforms (p110α, p110β, and p110δ) are known for the catalytic subunit, which are encoded by the PIK3CA, PIK3CB, and PIK3CD genes, 60 respectively. Seven proteins (p85α, p85β, and p55γ and their splicing variants) comprise the regulatory subunits.40,43 P85 keeps p110 in a stable but inactive state until, upon growth factor stimulation, p110 is recruited to the membrane. p100 is then activated at the membrane via the interaction of SH2 domains on p85 and phosphotyrosine motifs on the stimulated RTKs. The activated p110 catalytic subunit subsequently phosphorylates the D3 hydroxyl position of the inositol ring of PIP2, generating PIP3 at the membrane. PIP3 provides docking sites for activation of signaling proteins containing pleckstrin-homology (PH) domains,44 including AKT and PDK1. Activating mutations in the PIK3CA gene, which codes for the specific PI3K p110α catalytic subunit, have been found in many forms of cancer, making it one of the most frequently mutated oncoproteins in human cancer, along with K-Ras and p53.45 Mutations in PIK3CA cluster in two "hotspot" regions: exon 9 encoding the beginning of the helical domain and exon 20 encoding the tail of the kinase domain.46,47 The mutations found in these two regions of p110α encode single amino acid changes of E542K and E545K in the helical domain and H1047R in the kinase domain.45,48 The E542K and E545K mutations in the helical domain are postulated to affect interactions with regulatory proteins, including p85, whereas the H1047R mutation in the kinase domain is postulated to affect specificity or affinity of p110α towards its substrates.49 PI3K activity can also be upregulated by gene amplification as has been found to be the case in 40% of ovarian cancers.50 Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) PTEN, located on 10q23.3, is a dual lipid and protein phosphatase. Its primary function is to hydrolyze the 3' phosphate on PIP3, the product of PI3K activity, thus 61 producing PIP2,51 and antagonizing the activity of PI3K.52 Incidentally, SHIP-1 can also mimic the PIP3 phosphatase function of PTEN but does so by hydrolyzing the 5'phosphate of PIP3, whereas PTEN hydrolyzes the 3' phosphate.53 Loss of PTEN function results in accumulation of PIP3, mimicking the effect of PI3K activation and triggering the activation of its downstream effectors, PDK1 and AKT.54,55 PTEN is a major tumor suppressor, frequently inactivated in human cancers.56 However, PTEN does not fully conform to the formal definition of a tumor suppressor as it has been shown to be haploinsufficient in its tumor suppressor function.57 PTEN gene mutations or deletions are associated with poor prognosis in T-ALL. Two mutations in PTEN's phosphatase domain abrogate tumor suppressor function: C124S abrogates both lipid and protein phosphatase activity, while G129E abrogates only lipid phosphatase activity.58 However, relatively few primary T-ALL patient samples harbor PTEN gene mutations (5-27%), and they occur almost exclusively within exon 7.59 Likewise, PTEN deletions are observed in no more than 9% of T-ALL cases.59 These data suggest the existence of other mechanisms whereby PTEN function is abrogated in T-ALL, as well as in other types of cancer, which has been verified in recent studies. Roman-Gomez et al. showed that PTEN promoter hypermethylation and consequent decrease in mRNA expression was reported in ~20% of T-ALL.60 NOTCH1 also transcriptionally inhibits PTEN expression in T-ALL via up-regulation of HES1.61 Furthermore, PTEN can also be regulated by miRNAs, and both miR-19 and miR-21 have been shown to be T-cell oncogenes by downregulating PTEN in T-ALL.62,63 PTEN can also be inactivated by several posttranslational mechanisms, including phosphorylation, ubiquitination, oxidation, and acetylation.64 Specifically, RAK and CK2 62 have been shown to phosphorylate the C-terminus of PTEN and consequently both inactivate and stabilize PTEN protein. Thus, it is still entirely possible to have overactivation of the P/A/mT pathway despite the presence of even high levels of PTEN in T-ALL.65,66 AKT The primary effector of the P/A/mT pathway is AKT (v-AKT thymoma retrovirus), also known as protein kinase B (PKB). It was originally discovered as the cellular homologue of the transforming retrovirus AKT8 and as a kinase with properties similar to protein kinases A and C.26,28,29,67 AKT is a serine-threonine protein kinase that is expressed as three isoforms-AKT1, AKT2, and AKT3, encoded by the genes PKBα, PKBβ, and PKBγ, located at chromosomal loci 14q32, 19q13, and 1q44, respectively.41,68 AKT1 and AKT2 are the isoforms with the highest expression in thymocytes.69 The three isoforms share a similar structure. AKT contains an amino-terminal PH domain that serves to target the protein to the membrane for activation. Within its central region, AKT has a large serine-threonine kinase domain and is flanked on the C-terminus regulatory domain by hydrophobic and proline-rich regions.26,67 AKT is one of the most frequently upregulated kinases in human cancers,70 and its over-expression is associated with a poor prognosis. AKT regulates cell survival, cell cycle progression, migration, proliferation, metabolism, tumor growth, and angiogenesis,71,72 and as a result, AKT activity is strongly correlated with invasion and metastasis.73 Furthermore, AKT activation is also found to correlate with resistance to chemotherapy and radiation therapy, and conversely, addition of AKT inhibitors to antitumor regimens renders cancer cells sensitive to chemotherapy.74,75 AKT protects 63 cells from apoptosis by phosphorylation of pro-apoptotic substrates which are subsequently sequestered by the chaperone 14-3-3, which then sequesters them away from their target sites of action.76 In addition, AKT has been shown to regulate HIF-1α and VEGF expression, which are both critical mediators of angiogenesis in cancer.72,77 Aberrant over-activation of AKT can occur by several different mechanisms, including amplification, over-expression, mutations in AKT, and/or alterations in AKT upstream regulators.78 Upstream regulators of AKT activity include cytokines and growth factors such as VEGF, FGF, EGF, HGF, IGF and angiopoietin, along with their respective RTK cell surface receptors.79,80 A rare mutation in AKT that increases AKT activity is E17K. Though this mutation is seen in only a small percentage of cancers,81 it has a significant impact on cellular function. The E17K mutation falls in the PH domain of the AKT protein, and hence alters the electrostatic interactions of AKT that allows it to form new hydrogen bonds with its natural phosphatidylinositol ligands.81 This confers many different aberrant properties to the AKT protein, including an altered PH domain conformation, constitutive activation and constitutive association with the cell membrane. In confirmation of these observations, mutated AKT (E17K) has been shown to interact with c-Myc to induce leukemia in Eμ-Myc mice.81 AKT can also be over-activated via gene amplification, though this phenomenon is not often observed in human cancers.82,83 Recruitment of AKT by PIP3 to the cell membrane results in a conformational change that exposes two crucial residues for phosphorylative activation. Full AKT activation requires phosphorylation of both of these two sites in the kinase domain, one in the "T-loop" at T308 (phosphorylated by PDK1) and the other in a hydrophobic motif 64 near the COOH terminus at S473.84,85 Several kinases have been proposed to phosphorylate AKT at the S473 residue, including ILK, PKC, DNA-PK, and ATM. However, it is now generally thought that mTORC2 is primarily responsible for phosphorylation of AKT at S473 under most circumstances.86 In vitro studies have established that AKT phosphorylated at only T308 is only 10% as catalytically active as AKT phosphoryated at both T308 and S473. Thus, phosphorylation of the hydrophobic motif controls both the activity and substrate specificity of AKT.87,88 After activation at the cell membrane, AKT is able to translocate to the cytoplasm and nucleus28,29,89 where it phosphorylates a number of downstream targets for regulation of various cellular functions (Figure 2.2). So far over 100 AKT substrates have been identified,33 of which about 40 have been characterized. AKT affects the activity of a number of critical transcriptional regulators including FOXO, CREB, E2F, NF-κB, Iκ-K, FKHR, HDM2, mTOR and MDM2.90,91 These are all either direct or indirect substrates of AKT and each can regulate proliferation, survival, and epithelial-to-mesenchymal transition.25 Besides transcription factors, AKT is able to target a number of other molecules to affect the survival state of the cell including BAD and GSK-3β.92 GSK-3β regulates β-catenin protein stability. Thus, the P/A/mT pathway is connected to the Wnt/β-catenin pathway through GSK-3β. AKT also activates mTORC1 by phosphorylating and inactivating PRAS40, an inhibitor of mTORC1 activity.28,29,93 AKT promotes the G1-S phase transition by blocking FOXO-mediated transcription of the cell-cycle inhibitor p27Kip1, and/or phosphorylating and inactivating p27Kip1 directly.90,94 AKT can also be down-regulated by means other than the expected PTEN- or SHIP-mediated dephosphoryation and inactivation of PIP3. The tumor suppressors PP2A 65 and PHLPP1/2 are known to inactivate AKT by dephosphorylating residues T308 and S473, respectively.95 Loss of either PHLPP1 or PHLPP2 results in a striking 30-fold increase in the amplitude of AKT phosphorylation after agonist stimulation. There also is a striking increase in the duration of AKT phosphorylation after agonist-induced activation.96 Inactivation of AKT results in G1 arrest since AKT normally phosphorylates and inactivates GSK-3β, a kinase that restricts G1 cell cycle progression through phosphorylation of cyclin D1 and cyclin E, targeting them for degradation.97,98 Mammalian target of rapamycin The mammalian target of rapamycin (mTOR) is a 289KDa serine/threonine kinase.99,100 It is considered to be a member of the PI3K-kinase-related kinase (PIKK) superfamily since its C-terminus has strong homology to the catalytic domain of PI3K.101,102 mTOR is considered to be a master switch of cellular anabolic and catabolic processes since it regulates the rate of cell growth and proliferation via its ability to sense mitogen, energy, and nutrient levels.103,104 Deregulation of the mTOR pathway is frequently observed in cancer as well as diabetes, and as a result, approximately 28% of human cancers are expected to be sensitive to mTOR inhibition.105 mTOR is found in two different signaling complexes: mTORC1 and mTORC2 (Figure 2.2). In each complex, mTOR is grouped with a different and distinct set of signaling proteins that confer upon it a distinct set of functions. mTORC1 consists of mTOR, mLST8/GβL, and raptor along with two negative regulatory proteins, PRAS40 and DEPTOR.106 mTORC1's main functions are to regulate cell growth, proliferation, and survival by sensing mitogen, energy, and nutrient signals.107 mTORC2 consists of mTOR, mLST8, mSin1, rictor, and DEPTOR.108,109 mTORC2 regulates the actin 66 cytoskeleton by mediating the phosphorylation state of protein kinase PKCα, and modulates cell survival in response to growth factors by phosphorylating AKT at S473.86,110 mTOR can be activated by multiple signaling means. It can be activated by upstream signals, including growth factors, such as insulin and type I insulin-like growth factor (IGF-1), energy, stress, and nutrients.107 In response to growth factor ligand binding, IGFR is activated and in turn phosphorylates the insulin receptor substrates 1-4 (IRS1-4) which then trigger multiple downstream signal transduction pathways, such as PI3K and hence AKT.111 AKT in turn modulates mTOR activity by at least three means. First, AKT phosphorylates and inactivates TSC2, which combined with TSC1 antagonize mTOR activity through GTP hydrolysis and inactivation of RHEB, which is an mTOR activator.112 Second, AKT can activate mTOR through phosphorylation and inactivation of PRAS40, a negative regulator of mTOR activity.29 Lastly, AKT can activate mTOR directly upon insulin stimulation through phosphorylation of the S2448 residue of mTOR.113 In mammals, the two best-characterized downstream phosphorylation target