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Early T-cell acute lymphoblastic leukemia (ETP-ALL) is an aggressive hematologic malignancy associated with early relapse and poor prognosis that is genetically, immunophenotypically, and transcriptionally distinct from more mature T-cell acute lymphoblastic leukemia (T-ALL) tumors. Here, we leveraged global metabolomic and transcriptomic profiling of primary ETP- and T-ALL leukemia samples to identify specific metabolic circuitries differentially active in this high-risk leukemia group. ETP-ALLs showed increased biosynthesis of phospholipids and sphingolipids and were specifically sensitive to inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in the mevalonate pathway. Mechanistically, inhibition of cholesterol synthesis inhibited oncogenic AKT1 signaling and suppressed MYC expression via loss of chromatin accessibility at a leukemia stem cell–specific long-range MYC enhancer. In all, these results identify the mevalonate pathway as a druggable novel vulnerability in high-risk ETP-ALL cells and uncover an unanticipated critical role for cholesterol biosynthesis in signal transduction and epigenetic circuitries driving leukemia cell growth and survival.Significance:Overtly distinct cell metabolic pathways operate in ETP- and T-ALL pointing to specific metabolic vulnerabilities. Inhibition of mevalonate biosynthesis selectively blocks oncogenic AKT–MYC signaling in ETP-ALL and suppresses leukemia cell growth. Ultimately, these results will inform the development of novel tailored and more effective treatments for patients with high-risk ETP-ALL.This article is highlighted in the In This Issue feature, p. 587

Low-intensity maintenance therapy with 6-mercaptopurine (6-MP) limits the occurrence of acute lymphoblastic leukemia (ALL) relapse and is central to the success of multiagent chemotherapy protocols. Activating mutations in the 5′-nucleotidase cytosolic II (NT5C2) gene drive resistance to 6-MP in over 35% of early relapse ALL cases. Here we identify CRCD2 as a first-in-class small-molecule NT5C2 nucleotidase inhibitor broadly active against leukemias bearing highly prevalent relapse-associated mutant forms of NT5C2 in vitro and in vivo. Importantly, CRCD2 treatment also enhanced the cytotoxic activity of 6-MP in NT5C2 wild-type leukemias, leading to the identification of NT5C2 Ser502 phosphorylation as a novel NT5C2-mediated mechanism of 6-MP resistance in this disease. These results uncover an unanticipated role of nongenetic NT5C2 activation as a driver of 6-MP resistance in ALL and demonstrate the potential of NT5C2 inhibitor therapy for enhancing the efficacy of thiopurine maintenance therapy and overcoming resistance at relapse.Significance:Relapse-associated NT5C2 mutations directly contribute to relapse in ALL by driving resistance to chemotherapy with 6-MP. Pharmacologic inhibition of NT5C2 with CRCD2, a first-in-class nucleotidase inhibitor, enhances the cytotoxic effects of 6-MP and effectively reverses thiopurine resistance mediated by genetic and nongenetic mechanisms of NT5C2 activation in ALL.This article is highlighted in the In This Issue feature, p. 2483

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib has significantly prolonged progression-free survival (PFS) in patients with EGFR-mutant lung cancer, including those with brain metastases. However, despite striking initial responses, osimertinib-treated patients eventually develop lethal metastatic relapse, often to the brain. Although osimertinib-refractory brain relapse is a major clinical challenge, its underlying mechanisms remain poorly understood. Using metastatic models of EGFR-mutant lung cancer, we show that cancer cells expressing high intracellular S100A9 escape osimertinib and initiate brain relapses. Mechanistically, S100A9 upregulates ALDH1A1 expression and activates the retinoic acid (RA) signaling pathway in osimertinib-refractory cancer cells. We demonstrate that the genetic repression of S100A9, ALDH1A1, or RA receptors (RAR) in cancer cells, or treatment with a pan-RAR antagonist, dramatically reduces brain metastasis. Importantly, S100A9 expression in cancer cells correlates with poor PFS in osimertinib-treated patients. Our study, therefore, identifies a novel, therapeutically targetable S100A9–ALDH1A1–RA axis that drives brain relapse.Significance:Treatment with the EGFR TKI osimertinib prolongs the survival of patients with EGFR-mutant lung cancer; however, patients develop metastatic relapses, often to the brain. We identified a novel intracellular S100A9–ALDH1A1–RA signaling pathway that drives lethal brain relapse and can be targeted by pan-RAR antagonists to prevent cancer progression and prolong patient survival.This article is highlighted in the In This Issue feature, p. 873

Supplementary Figure from Pharmacologic Inhibition of NT5C2 Reverses Genetic and Nongenetic Drivers of 6-MP Resistance in Acute Lymphoblastic Leukemia

Supplementary Figure from Targeting S100A9–ALDH1A1–Retinoic Acid Signaling to Suppress Brain Relapse in EGFR-Mutant Lung Cancer

Supplementary Table from Pharmacologic Inhibition of NT5C2 Reverses Genetic and Nongenetic Drivers of 6-MP Resistance in Acute Lymphoblastic Leukemia

Supplementary Data from Intracellular Cholesterol Pools Regulate Oncogenic Signaling and Epigenetic Circuitries in Early T-cell Precursor Acute Lymphoblastic Leukemia

ALL patients with NSD2 mutations

PDF file, 69KB.

Differentially expressed genes in NSD2 p.E1099K RCH-ACV cells with the change of H3K27me3 peaks

Supplementary Figures 1-8

Lineage-ambiguous leukemias are high-risk malignancies of poorly understood genetic basis. Here, we describe a distinct subgroup of acute leukemia with expression of myeloid, T lymphoid, and stem cell markers driven by aberrant allele-specific deregulation of BCL11B, a master transcription factor responsible for thymic T-lineage commitment and specification. Mechanistically, this deregulation was driven by chromosomal rearrangements that juxtapose BCL11B to superenhancers active in hematopoietic progenitors, or focal amplifications that generate a superenhancer from a noncoding element distal to BCL11B. Chromatin conformation analyses demonstrated long-range interactions of rearranged enhancers with the expressed BCL11B allele and association of BCL11B with activated hematopoietic progenitor cell cis-regulatory elements, suggesting BCL11B is aberrantly co-opted into a gene regulatory network that drives transformation by maintaining a progenitor state. These data support a role for ectopic BCL11B expression in primitive hematopoietic cells mediated by enhancer hijacking as an oncogenic driver of human lineage-ambiguous leukemia.Significance:Lineage-ambiguous leukemias pose significant diagnostic and therapeutic challenges due to a poorly understood molecular and cellular basis. We identify oncogenic deregulation of BCL11B driven by diverse structural alterations, including de novo superenhancer generation, as the driving feature of a subset of lineage-ambiguous leukemias that transcend current diagnostic boundaries.This article is highlighted in the In This Issue feature, p. 2659

PDF file, 306KB, Interaction between PF-03084014 and chemotherapeutic agents.

PDF file, 252KB, PF-03084014 reverses glucocorticoid resistance in T-ALL cell lines.

This file contains Supplementary Tables 1-15 detailing clinical and genomic information about each patient analyzed in this study, along with the results of RNA-seq and gene set enrichment analyses.

List of genes bound and regulated by GR in NSD2 wild-type RCH-ACV and RMPI-8402 cells

Combined RNA-Seq analysis of 4 ALL cell lines (RCH-ACV, SEM, RPMI-8402 and CEM) in response to dexamethasone treatment

This file contains Supplementary Figures 1-23 with corresponding supplementary figure legends, as well as the legends for Supplementary Tables 1-15.

PDF file, 567KB, Absence of synergistic interaction between PF-03084014 and glucocorticoids in gamma-secretase inhibitor- and glucocorticoid-resistant cell lines.

Supplementary Methods and Figures

Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation.Significance:NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL.This article is highlighted in the In This Issue feature, p. 1

The full drug screen results

T-cell acute lymphoblastic leukemias (T-ALL) and lymphomas are aggressive hematologic cancers frequently associated with activating mutations in NOTCH1. Early studies identified NOTCH1 as an attractive therapeutic target for the treatment of T-ALL through the use of γ-secretase inhibitors (GSI). Here, we characterized the interaction between PF-03084014, a clinically relevant GSI, and dexamethasone in preclinical models of glucocorticoid-resistant T-ALL. Combination treatment of the GSI PF-03084014 with glucocorticoids induced a synergistic antileukemic effect in human T-ALL cell lines and primary human T-ALL patient samples. Mechanistically PF-03084014 plus glucocorticoid treatment induced increased transcriptional upregulation of the glucocorticoid receptor and glucocorticoid target genes. Treatment with PF-03084014 and glucocorticoids in combination was highly efficacious in vivo, with enhanced reduction of tumor burden in a xenograft model of T-ALL. Finally, glucocorticoid treatment effectively reversed PF-03084014–induced gastrointestinal toxicity via inhibition of goblet cell metaplasia. These results warrant the analysis of PF-03084014 and glucocorticoids in combination for the treatment of glucocorticoid-resistant T-ALL. Mol Cancer Ther; 11(7); 1565–75. ©2012 AACR.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL.Significance:Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL.This article is highlighted in the In This Issue feature, p. 305

Glucocorticoid response of PDX Cells with NSD2 mutations

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib has significantly prolonged progression-free survival (PFS) in patients with EGFR-mutant lung cancer, including those with brain metastases. However, despite striking initial responses, osimertinib-treated patients eventually develop lethal metastatic relapse, often to the brain. Although osimertinib-refractory brain relapse is a major clinical challenge, its underlying mechanisms remain poorly understood. Using metastatic models of EGFR-mutant lung cancer, we show that cancer cells expressing high intracellular S100A9 escape osimertinib and initiate brain relapses. Mechanistically, S100A9 upregulates ALDH1A1 expression and activates the retinoic acid (RA) signaling pathway in osimertinib-refractory cancer cells. We demonstrate that the genetic repression of S100A9, ALDH1A1, or RA receptors (RAR) in cancer cells, or treatment with a pan-RAR antagonist, dramatically reduces brain metastasis. Importantly, S100A9 expression in cancer cells correlates with poor PFS in osimertinib-treated patients. Our study, therefore, identifies a novel, therapeutically targetable S100A9–ALDH1A1–RA axis that drives brain relapse. Significance: Treatment with the EGFR TKI osimertinib prolongs the survival of patients with EGFR-mutant lung cancer; however, patients develop metastatic relapses, often to the brain. We identified a novel intracellular S100A9–ALDH1A1–RA signaling pathway that drives lethal brain relapse and can be targeted by pan-RAR antagonists to prevent cancer progression and prolong patient survival. This article is highlighted in the In This Issue feature, p. 873

Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation. Significance: NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL. This article is highlighted in the In This Issue feature, p. 1

Supplementary figure legends

Supplementary Table S1: Immunohistochemical staining of Notch1-IC and HIPK2 in serial tissue arrays of 61 breast cancer samples. Supplementary Table S2: Semi-Quantitative analysis of Notch1-IC, phosphorylated Notch1-IC T2512, HIPK2, and Fbw7 expression in breast cancer tissues.

Description of additional methods and procedures used in the study.

Supplementary Figure 1: ADR suppresses Notch1 signaling. Supplementary Figure 2: ADR inhibits the transcriptional activity of Notch1 by inducing HIPK2. Supplementary Figure 3: HIPK2 regulates the Notch1-IC protein stability through proteasomal degradation pathways. Supplementary Figure 4: HIPK2 facilitates Notch1-IC degradation by Fbw7 ubiquitin ligase. Supplementary Figure 5: HIPK2 physically interacts with Notch1-IC. Supplementary Figure 6: HIPK2 phosphorylates Notch1-IC at the T2512 residue. Supplementary Figure 7: HIPK2-mediated downregulation of Notch1-IC suppresses the tumorigenesis of breast cancer. Supplementary Figure 8: Somatic mutations in the CPD motif of Notch1-IC make it resistant to HIPK2. Supplementary Figure 9: Physiological effects of somatic mutations in the CPD motif of Notch1-IC. Supplementary Figure 10: A propose model of Notch1 signaling pathway regulation by HIPK2 in cancer during chemotherapy.

The receptor Notch1 plays an important role in malignant progression of many cancers, but its regulation is not fully understood. In this study, we report that the kinase HIPK2 is responsible for facilitating the Fbw7-dependent proteasomal degradation of Notch1 by phosphorylating its intracellular domain (Notch1-IC) within the Cdc4 phosphodegron motif. Notch1-IC expression was higher in cancer cells than normal cells. Under genotoxic stress, Notch1-IC was phosphorylated constitutively by HIPK2 and was maintained at a low level through proteasomal degradation. HIPK2 phosphorylated the residue T2512 in Notch1-IC. Somatic mutations near this residue rendered Notch1-IC resistant to degradation, as induced either by HIPK2 overexpression or adriamycin treatment. In revealing an important mechanism of Notch1 stability, the results of this study could offer a therapeutic strategy to block Notch1-dependent progression in many types of cancer. Cancer Res; 76(16); 4728–40. ©2016 AACR.

Lineage-ambiguous leukemias are high-risk malignancies of poorly understood genetic basis. Here, we describe a distinct subgroup of acute leukemia with expression of myeloid, T lymphoid, and stem cell markers driven by aberrant allele-specific deregulation of BCL11B, a master transcription factor responsible for thymic T-lineage commitment and specification. Mechanistically, this deregulation was driven by chromosomal rearrangements that juxtapose BCL11B to superenhancers active in hematopoietic progenitors, or focal amplifications that generate a superenhancer from a noncoding element distal to BCL11B. Chromatin conformation analyses demonstrated long-range interactions of rearranged enhancers with the expressed BCL11B allele and association of BCL11B with activated hematopoietic progenitor cell cis-regulatory elements, suggesting BCL11B is aberrantly co-opted into a gene regulatory network that drives transformation by maintaining a progenitor state. These data support a role for ectopic BCL11B expression in primitive hematopoietic cells mediated by enhancer hijacking as an oncogenic driver of human lineage-ambiguous leukemia. Significance: Lineage-ambiguous leukemias pose significant diagnostic and therapeutic challenges due to a poorly understood molecular and cellular basis. We identify oncogenic deregulation of BCL11B driven by diverse structural alterations, including de novo superenhancer generation, as the driving feature of a subset of lineage-ambiguous leukemias that transcend current diagnostic boundaries. This article is highlighted in the In This Issue feature, p. 2659

Notch activation is highly prevalent among cancers, in particular T-cell acute lymphoblastic leukemia (T-ALL). However, the use of pan-Notch inhibitors to treat cancers has been hampered by adverse effects, particularly intestinal toxicities. To circumvent this barrier in T-ALL, we aimed to inhibit ETS1, a developmentally important T-cell transcription factor previously shown to cobind Notch response elements. Using complementary genetic approaches in mouse models, we show that ablation of Ets1 leads to strong Notch-mediated suppressive effects on T-cell development and leukemogenesis but milder intestinal effects than pan-Notch inhibitors. Mechanistically, genome-wide chromatin profiling studies demonstrate that Ets1 inactivation impairs recruitment of multiple Notch-associated factors and Notch-dependent activation of transcriptional elements controlling major Notch-driven oncogenic effector pathways. These results uncover previously unrecognized hierarchical heterogeneity of Notch-controlled genes and point to Ets1-mediated enucleation of Notch–Rbpj transcriptional complexes as a target for developing specific anti-Notch therapies in T-ALL that circumvent the barriers of pan-Notch inhibition. Significance: Notch signaling controls developmentally important and tissue-specific activities, raising barriers for developing anti-Notch therapies. Pivoting away from pan-Notch inhibitors, we show antileukemic but less toxic effects of targeting ETS1, a T-cell NOTCH1 cofactor. These results demonstrate the feasibility of context-dependent suppression of NOTCH1 programs for the treatment of T-ALL. This article is highlighted in the In This Issue feature, p. 127

Long-range enhancers govern the temporal and spatial control of gene expression; however, the mechanisms that regulate enhancer activity during normal and malignant development remain poorly understood. Here, we demonstrate a role for aberrant chromatin accessibility in the regulation of MYC expression in T-cell lymphoblastic leukemia (T-ALL). Central to this process, the NOTCH1-MYC enhancer (N-Me), a long-range T cell–specific MYC enhancer, shows dynamic changes in chromatin accessibility during T-cell specification and maturation and an aberrant high degree of chromatin accessibility in mouse and human T-ALL cells. Mechanistically, we demonstrate that GATA3-driven nucleosome eviction dynamically modulates N-Me enhancer activity and is strictly required for NOTCH1-induced T-ALL initiation and maintenance. These results directly implicate aberrant regulation of chromatin accessibility at oncogenic enhancers as a mechanism of leukemic transformation. Significance: MYC is a major effector of NOTCH1 oncogenic programs in T-ALL. Here, we show a major role for GATA3-mediated enhancer nucleosome eviction as a driver of MYC expression and leukemic transformation. These results support the role of aberrant chromatin accessibility and consequent oncogenic MYC enhancer activation in NOTCH1-induced T-ALL. This article is highlighted in the In This Issue feature, p. 1631

Despite recent advances, overall survival for children with acute myeloid leukemia (AML) remains relatively low at 70 to 80%. Further refinement of risk stratification is paramount to guiding optimal therapy selection and improving outcomes. In this study, we leverage machine learning and network-based systems biology approaches that infer tumor protein activity and define key disease drivers - master regulator (MR) proteins - in pediatric AML to further refine existing patient risk stratification and identify potential therapeutic targets. Using the ARACNe and VIPER algorithms1, we reverse-engineered an AML gene regulatory network and inferred protein activity from enrichment of the proteins' transcriptional targets in gene expression signatures from a novel 1080-patient cohort treated on COG AAML1031. We integrated ensemble clustering with feed-forward feature selection on VIPER-inferred protein activity to identify groups of patients with unique protein activity signatures and correlated them to survival by Cox proportional-hazards regression analysis. We identified features predictive of each group by Monte Carlo cross-validation and binary classification. We found that the activity of 2-4 MR proteins was sufficient to predict patient stratification for each group with areas under the receiver-operator characteristic curve above 0.9. We validated our findings in three external test datasets: St-Jude Children's Research Hospital (n=249), TARGET (n=682) and Erasmus MC Sophia's Children's Hospital (n=237). We identified 10 groups with significant survival differences (P < 0.0001, overall survival range 42-85%). This includes a new high-risk group that is not associated with any known cytogenomic features, but shows high activity of NOTCH signaling pathway regulators HES1 and HEY2. Other groups show aberrant activity for known transcription regulators; for example RUNX1, KDM5B and POU4F1. In conclusion, we propose novel patient risk stratification based on VIPER-inferred MR protein activity in pediatric AML. We define 10 groups with differential survival, including refinement of a classically-characterized intermediate-risk cytogenomically normal group as high-risk. We classify patients into therapeutically-relevant risk groups that can guide treatment decision-making. We define the MR proteins driving these groups to offer mechanistic insights into AML tumorigenesis as well as suggest potential molecular targets for therapies. Reference: 1. Califano A, Alvarez M, The recurrent architecture of tumour initiation, progression and drug sensitivity, Nat Rev Cancer (2017), 17(2): 116-130 Citation Format: Alexandre P. Alloy, Jovana Pavisic, Rhonda E. Ries, Tanja Gruber, C Michel Zwaan, Monique L. den Boer, James R. Downing, Adolfo Ferrando, Soheil Meshinchi, Andrea Califano. Novel pediatric AML patient risk stratification by inferred protein activity through integrative network analysis and machine learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1.

Peripheral T-cell lymphomas (PTCL) are highly aggressive, malignant hematologic tumors that arise from clonal proliferation of mature T cells. Among these, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T-cell lymphomas not otherwise specified (PTCL, NOS) account for >45% diagnoses, show limited response to intensified chemotherapy treatment, and are associated with dismal survival. Genomic studies from our group have uncovered recurrent mutations and novel cancer-associated gene fusions involving the guanine nucleotide exchange factor VAV1 in AITL and PTCL, NOS. Interestingly, mutation co-occurrence analysis in AITL and PTCL, NOS showed significant mutual exclusivity of VAV1 genomic alterations and the highly prevalent RHOA mutations (p-value 0.0142), supporting a common mechanism of action. Gene fusions involving VAV1 are characterized by the substitution of their auto-inhibitory C-terminal SH3 domains by different domains from their fusion partners, leading to increased activation of VAV1-dependent signaling pathways. Among them, the VAV1-MYO1F fusion shows the strongest increase in VAV1 activity and activation of the mitogen-activated protein kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and nuclear factor of activated T-cells (NFAT) pathways. To study the role of VAV1-MYO1F, we engineered a conditional knock-in mouse that expresses the Vav1-myo1f fusion in CD4+ T-cells. Expression of Vav1-myo1f in CD4 T cells induces cell activation and alterations in T-cell specification, associated with upregulation of master transcription factors involved in helper T-cell cell differentiation. Moreover, Vav1-Myo1f increased CD4+ T-cell survival upon cytokine withdrawal and enhanced Vav1 phosphorylation and activation of the MAPK pathway, resulting in increased cell activation and proliferation both in vivo and in vitro in response to TCR engagement. Notably, expression of Vav1-Myo1f fusion in CD4+ T cells is sufficient to induce development of fatal malignant lymphomas with a latency of 6-14 months. Histologic examination showed disrupted splenic architecture associated with clonal expansion of CD4+ cells indicative of T-cell lymphoma with PTCL, NOS phenotype. Similar results were obtained in a genetic model that combined the expression of Vav1-myo1f with the deletion of the Tet2 epigenetic regulator. In both genetic models, tumor cells specifically present a memory cell-associated immunophenotype (CD44+ CD62L-) and characteristic Th2-like features including increased expression of the transcription factors Gata3 and c-Maf and the IL4 and Il10 cytokines. Overall, these results demonstrate a direct oncogenic role for Vav1-Myo1f in the pathogenesis of PTCL, associated with deregulation of T-cell specification and of signaling programs critical for the control of T-cell proliferation. Citation Format: Jose Rodriguez Cortes, Robert Albero, Ioan Filip, Juan Angel Patino, Anisha R. Cooke, Wen-Hsuan Lin, Anouchka P. Laurent, Bobby B. Shih, Aidan S. Quinn, Raul Rabadan, Adolfo Ferrando, Teresa Palomero. Expression of Vav1-Myo1F fusion affects T-cell differentiation and induces T-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-41.

Activating mutations in the cytosolic 5’-nucleotidase II (NT5C2) gene drive resistance to chemotherapy with 6-mercaptopurine (6-MP) in acute lymphoblastic leukemia (ALL) highlighting NT5C2 as a potential therapeutic target. In efforts to reverse 6-MP therapy resistance, we performed high throughput screens to identify small molecule inhibitors of NT5C2. Using a high throughput malachite-green based NT5C2 nucleotidase assay, we identified HTP_2 as a lead compound with low micromolar inhibitory activity against NT5C2 R367Q mutant recombinant protein. HTP_2 reverses 6-MP resistance in Nt5c2+/R367Qmutant mouse lymphoblasts and NT5C2 R29Q mutant expressing human cell lines. In addition, HTP_2 treatment results in increased sensitivity to 6-MP therapy in NT5C2 wild-type cells, highlighting the role of wild-type NT5C2 activity in the clearance of 6-MP. These results support a potential therapeutic use for NT5C2 inhibitors in potentiating the effects of 6-MP based chemotherapy in NT5C2 wild-type cells as well as NT5C2 mutant cells. Overall, this study presents a framework for the development of a NT5C2 inhibitor for the reversal of 6-MP resistance in relapsed ALL patients. Citation Format: Chelsea Dieck, Clara Reglero-Gomez, Arie Zask, Brent Stockwell, Adolfo Ferrando. NT5C2 small molecule inhibitor for the reversal of 6-MP resistance in acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C005. doi:10.1158/1535-7163.TARG-19-C005

Acute lymphoblastic leukemia (ALL) tumors are dependent on purine biosynthesis for proliferation and survival and relapsed ALL is particularly associated with transcriptional up-regulation of this pathway. Increased dependency on purine biosynthesis highlights IMPDH (inosine monophosphate dehydrogenase), the rate-limiting enzyme in guanosine production, as a potential therapeutic target for the treatment of ALL. To assess the role of IMPDH1 and IMPDH2 in ALL development and progression, we generated NOTCH1-induced leukemias from Impdh1 knockout and Impdh2 conditional knockout mice. In addition, we have completed whole exome sequencing and RNAseq profiling of 12 pairs of ALL xenografts corresponding to matched diagnostic and relapsed ALL samples and have performed dose response assays with the IMPDH inhibitor mizoribine. Additionally and towards the identification of synthetic lethal genetic vulnerabilities with IMPDH inhibition in ALL we have performed a genome wide CRISPR screen in REH ALL cells with the IMPDH inhibitor mizoribine. Overall, these studies highlight the therapeutic potential for IMPDH inhibitors for the treatment of ALL. Citation Format: Chelsea Dieck, Koichi Oshima, Alberto Ambesi-Impiombato, Adolfo Ferrando. IMPDH1 and IMPDH2 as therapeutic targets in acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B134. doi:10.1158/1535-7163.TARG-19-B134

The identification of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) has led to the clinical development of JAK kinase inhibitors, including ruxolitinib. Ruxolitinib reduces splenomegaly and systemic symptoms in myelofibrosis and improves overall survival; however, the mechanism by which JAK inhibitors achieve efficacy has not been delineated. Patients with MPN present with increased levels of circulating proinflammatory cytokines, which are mitigated by JAK inhibitor therapy. We sought to elucidate mechanisms by which JAK inhibitors attenuate cytokine-mediated pathophysiology. Single-cell profiling demonstrated that hematopoietic cells from myelofibrosis models and patient samples aberrantly secrete inflammatory cytokines. Pan-hematopoietic Stat3 deletion reduced disease severity and attenuated cytokine secretion, with similar efficacy as observed with ruxolitinib therapy. In contrast, Stat3 deletion restricted to MPN cells did not reduce disease severity or cytokine production. Consistent with these observations, we found that malignant and nonmalignant cells aberrantly secrete cytokines and JAK inhibition reduces cytokine production from both populations.Our results demonstrate that JAK-STAT3-mediated cytokine production from malignant and nonmalignant cells contributes to MPN pathogenesis and that JAK inhibition in both populations is required for therapeutic efficacy. These findings provide novel insight into the mechanisms by which JAK kinase inhibition achieves therapeutic efficacy in MPNs.

The TLX1 transcription factor oncogene is frequently activated by chromosomal translocations in T-cell acute lymphoblastic leukemia (T-ALL) and defines a distinct molecular group of tumors characterized by differentiation arrest at the early cortical stage of thymocyte differentiation and excellent response to therapy. Recent developments from the analysis of genomic data on TLX1-specific transcriptional targets and analysis of the molecular mechanisms of TLX1 transformation in human- and mouse-induced leukemias have shown novel insight into the activity of this transcription factor oncogene. Aberrant expression of TLX1 in T-cell progenitors disrupts normal T-cell development and triggers the development of aneuploidy during T-cell transformation. Importantly, the disruption of the mitotic checkpoint in TLX1-induced tumors may be linked not only to the acquisition of secondary genetic alterations in T-ALL but also to increased sensitivity of these tumors to chemotherapy with drugs targeting the formation of the mitotic spindle. Clin Cancer Res; 17(20); 6381–6. ©2011 AACR.

The identification of activating mutations in NOTCH1 in the majority of T-cell acute lymphoblastic leukemias and lymphomas (T-ALL) has brought much interest in inhibiting NOTCH1 signaling as therapeutic target in this disease. Small-molecule inhibitors of the γ-secretase complex, which mediates a critical proteolytic cleavage required for NOTCH1 activation, hold the promise of becoming an effective molecular therapy against relapsed and refractory T-ALL. Recent progress in the elucidation of the transcriptional regulatory networks downstream of oncogenic NOTCH1 has uncovered a central role of NOTCH1 signaling in promoting leukemic cell growth and revealed an intricate circuitry that connects NOTCH1 signaling with MYC and the PI3K-AKT signaling pathway. The identification of the downstream effector pathways controlled by NOTCH1 should pave the way for the rational design of anti-NOTCH1 therapies for the treatment of T-ALL.

Over 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL) harbor activating mutations in Notch1 gene, making this receptor a promising therapeutic target for T-ALL. Υ-secretase inhibitors (GSIs), which block a proteolytic cleavage required for NOTCH1 activation, have been clinically explored for the treatment of T-ALL. However, lack of primary responses and relapses have prevented further development of GSIs in the clinic, highlighting the need to identify novel therapies synergistic with NOTCH1 inhibition for the treatment of highly aggressive NOTCH1-mutant T-ALL. To identify new antileukemic drugs for T-ALL we designed an expression-based drug screen utilizing drug profiles contained in the Connectivity Map (cMAP) to search for positive connections with gene signatures driven by NOTCH1 inhibition in this disease. This analyses uncovered 17 compound candidates for synergistic activity with NOTCH1 inhibition, including drugs with known mechanism of action: inhibitors of the mTOR/PI3K/AKT pathway and histone deacetylase inhibitors; as well as compounds with unrelated mechanisms, pyrvinium pamoate (antihelmintic) and withaferin A (antiangiogenic). Pair-wise gene set enrichment analyses on the expression profile driven by each of these drugs in T-ALL cells revealed compounds with transcriptional programs partially overlapping those driven by PI3K/AKT inhibitors, and drugs with distinctive expression profiles, such as withaferin A, suggestive of a unique mode of action. We next characterized the activity of each of the cMAP hits alone and in combination with the GSI DBZ in vitro using a panel of 5 NOTCH1-mutant T-ALL cell lines (CUTLL1, DND41, HPBALL, RPMI and CCRF-CEM). In this analysis, 5 drugs scored as highly active (IC50 Functional annotation and DeMAND algorithm analyses on the expression profile of withaferin A revealed downregulation of genes and pathways involved in protein translation and identified 21 components of the ribosome and the translation machinery as potential effectors of the antitumoral activity of the drug in T-ALL, including two subunits of the eIF2A translation complex (eIF2S1 and eIF2S2) which controls translation initiation via phosphorylation of the eIF2S1 subunit under conditions of cellular stress. Polysome profiling, nascent protein, and Western blot analyses of cells treated with withaferin A confirmed an inhibitory effect of the drug in protein translation mediated by eIF2S1 phosphorylation. Consistently, expression of a non-phosphorylable mutant form of eIF2S1 in leukemia cell lines rescued the effects of withaferin A in vitro. Our results illustrate the power of our expression-based and in vivo validation approach toward the identification of antitumor drug combinations for the treatment of human cancer. Citation Format: Marta Sanchez-Martin, Alberto Ambesi-Impiombato, Yue Qin, Daniel Herranz, Mukesh Bansal, Tiziana Girardi, Elisabeth Paietta, Martin S. Tallman, Jacob M. Rowe, Kim De Keersmaecker, Andrea Califano, Adolfo Ferrando. Synergistic antileukemic therapies in NOTCH1-induced T-ALL [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 49.

Activating mutations in cytosolic 5'-nucleotidase II (NT5C2) drive resistance to 6-mercaptopurine (6-MP) in relapsed acute lymphoblastic leukemia (ALL). These gain-of function alleles encode proteins with increased nucleotidase activity, yet their specific molecular mechanisms are poorly understood. Allosteric activation of NT5C2 is primarily mediated by a marked conformational change in the helix A region, which reconfigures the catalytic center for substrate access and catalysis. Here we demonstrate that constitutively active NT5C2 mutant proteins (K359Q and L375F) adopt active helix A region structures in the absence of allosteric activator. Moreover, crystallographic, structural modeling, genetic, and functional antibody analyses identify a switch-off mechanism mediated by the dynamic mobilization of Asp407 in the tip region of the arm segment which, guided by positively charged residues located in the surface of a basic inter-monomeric cavity exposed upon allosteric activation, turns off NT5C2 by interacting with and disrupting the helical conformation of helix A. Loss of this regulatory mechanism in NT5C2 mutants involving the tip region of the arm segment and the inter-monomeric cavity drives increased nucleotidase activity and allosteric response. Finally, increased allosteric activation by the Q523X mutation results from loss of the NT5C2 C-terminal acidic tail segment, which promotes a closed inactive state in basal conditions. These results uncover dynamic mechanisms responsible for NT5C2 regulation targeted by chemotherapy resistance-driving NT5C2 mutations, with important implications for the development of NT5C2 inhibitor therapies. This abstract is also being presented as Poster 05. Citation Format: Chelsea L. Dieck, Gannie Tzoneva, Farhad Forouhar, Zachary Carpenter, Marta Sanchez-Martin, Alberto Ambesi-Impiombato, Renate Kirschner-Schwabe, Scott Lew, Jayaraman Seetharaman, Liang Tong, Adolfo Ferrando. Mechanisms of NT5C2 activating mutations driving thiopurine resistance in relapsed lymphoblastic leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR02.

Advances in risk-adapted cytotoxic chemotherapy, hematopoietic stem cell transplantation and supportive care have contributed to significant improvements in the survival of patients with acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) over the past few decades. However, despite such progress, a significant percentage of both adult and pediatric leukemia patients become refractory to therapy or relapse and eventually die of disease. Hence, there remains an urgent need for the development of effective and targeted therapies for acute leukemia. Recent genetic profiling of solid and hematologic malignancies has identified epigenetic factors as a critical group of genes recurrently mutated in cancer. Additionally, epigenetic dysregulation has been shown to play an important role in the development, progression and maintenance of leukemia. Therefore, pharmacological inhibition of epigenetic factors represents a potential avenue for the development of novel epigenetic-targeted therapies. In order to identify epigenetic vulnerabilities in leukemia, we developed an epigenetic-focused shRNA screen to search for novel therapeutic targets in human leukemia cell lines both in vitro and in vivo. Specifically, T- and B-ALL cell lines were transduced with a library of shRNAs targeting 449 genes including epigenetic readers, writers and erasers and other chromatin-related factors. Selected cells were subsequently cultured in vitro and concurrently injected into mice. Engraftment of inoculated cells and disease progression were monitored through bioluminescence imaging. Amongst the universe of epigenetic regulatory proteins, the arginine methyl transferase, PRMT5, emerged as the most significantly depleted factor in both in vitro and in vivo screenings. Chemical inhibition of PRMT5 enzymatic activity effectively reduced protein symmetric dimethyl arginine methylation, altered splicing, inhibited cell growth and promoted apoptosis of both ALL and AML cell lines in vitro. In addition, inhibition of PRMT5 in vivo using patient-derived xenograft (PDX) T-ALL mouse models demonstrated diminished tumor growth and prolonged survival. Notably, quantification of peripheral blood cell numbers showed that pharmacologic PRMT5 inhibition was well tolerated and did not affect normal hematopoiesis in mice suggesting that a therapeutic window exists for anticancer drugs targeting PRMT5 in acute leukemia. Overall, our data indicates that pre-mRNA processing and in particular RNA splicing modulation may represent novel therapeutic targets in leukemia. Note: This abstract was not presented at the meeting. Citation Format: Yunyue Wang, Hui Huang, Daniel Diolaiti, Marta Sanchez Martin, Beata Modzelewski, Lianna J. Marks, Allison R. Rainey, Ervin S. Gaviria, Maria L. Sulis, Filemon S. Dela Cruz, Adolfo A. Ferrando, Andrew L. Kung. Identification of arginine methyltransferase PRMT5 as a novel therapeutic target in T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1128. doi:10.1158/1538-7445.AM2017-1128

Aberrant NOTCH1 signaling plays a major role in the pathogenesis of over 60% of T-ALLs harboring activating mutations in the NOTCH1 gene. Most notably, oncogenic NOTCH1 has been proposed as a therapeutic target in NOTCH1-mutant leukemias and small molecule γ-secretase inhibitors (GSIs), which effectively block NOTCH activation via inhibition of a critical intramembrane proteolytic cleavage required for NOTCH signaling. However, the clinical development of anti-NOTCH1 therapies in T-ALL has been hampered by the limited and delayed therapeutic response to these drugs, making the identification of highly effective and synergistic drug combinations capable of delivering strong antileukemic responses a top priority in the field. Here we identify glutaminolysis as a critical pathway for leukemia cell growth downstream of NOTCH1 and a key determinant of clinical response to anti-NOTCH1 therapies. Mechanistically, inhibition of NOTCH1 signaling in T-ALL induces a metabolic shutdown with prominent inhibition of glutaminolysis and triggers autophagy as a salvage pathway supporting leukemia cell metabolism. Consequently, both inhibition of glutaminolysis and inhibition of autophagy strongly and synergistically enhance the antileukemic effects of anti-NOTCH1 therapies. Moreover, Pten loss and consequent activation of the PI3K pathway induces increased glycolysis and consequently rescues leukemic cell metabolism abrogating the antileukemic effects of NOTCH1 inhibition. Overall, these results identify glutaminolysis as a major node in cancer metabolism controlled by NOTCH1 and as therapeutic target for the treatment of T-ALL. These results highlight the fundamental importance of NOTCH1 signaling in the control of leukemia cell metabolism, extend our understanding of the prominent role of PTEN and the PI3K pathway in the control of oncogenic cell growth and provide the basis for the design of new therapeutic strategies targeting cell metabolism for the treatment of T-ALL. Citation Format: Adolfo Ferrando. Oncogenic signaling pathways and resistance to therapy in T-ALL. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA05.

This abstract is being presented as a short talk. A full abstract is printed in the Proffered Abstracts section (PR03) of the Conference Proceedings. Citation Format: Panagiotis Ntziachristos, Grant Welstead, Thomas Trimarchi, Sofia Bakogianni, Luyao Xu, Pieter Van Vlierberghe, Rab Prinjha, Adolfo Ferrando, Rudolf Jaenisch, Charles Mullighan, Aifantis Iannis. Delineating the roles of lysine 27 methylation-associated epigenetic modulators in T cell leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B24.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy arising in immature T cell progenitors. Despite improved survival rates, relapse remains a major therapeutic obstacle and intensive treatment regimens expose T-ALL patients to serious long-term side-effects. Hence more effective, less toxic treatment options are required. While the localization of leukemia cells determines the course of disease, little is known about T-ALL migration. Upon profiling chemokine receptor expression by T-ALL cells, we observed high surface levels of the CXCL12 receptor, CXCR4. Intravital imaging revealed that T-ALL cells are in direct contact with CXCL12-producing bone marrow stromal cells in vivo. Genetic targeting of CXCR4 in T-ALL cells resulted in a marked reduction of leukemia burden and significantly prolonged survival. Moreover, disruption of CXCR4 signaling with a small molecule inhibitor severely impaired T-ALL progression in mouse and human xenograft models of the disease. We found that CXCR4 promotes c-Myc expression, which is required in leukemia initiating cells. Given the relatively subtle role for CXCR4 in normal developing T cells, CXCR4 inhibition to our knowledge has not been proposed as a therapy for T cell malignancies. Our data strongly suggest that targeting CXCR4 could be a promising strategy for combating T-ALL. Citation Format: Lauren A. Pitt, Anastasia N. Tikhonova, Thomas Trimarchi, Bryan King, Hai Hu, Yixiao Gong, Aris Tsirigos, Marta Sanchez-Martin, Dan R. Littman, Adolfo Ferrando, Sean J. Morrison, David R. Fooksman, Iannis Aifantis, Susan Schwab. The chemokine receptor CXCR4 is essential for the maintenance of T cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A06.

The cure rate for childhood acute lymphoblastic leukemia (ALL) has dramatically improved, yet up to 25% of patients experience disease recurrence. T cell ALL (T-ALL) represents 15% of pediatric and 25% of adult ALL and it is classified as a high-risk type of ALL. The aim of the hitherto studies is to specifically block the effects of cancer inducing oncogenes. Recently, we identified inactivating mutations of the polycomb repressive complex 2 (PRC2), the “writer” of Histone 3 lysine 27 methylation, in primary samples from human patients revealing a tumor suppressor role for the complex in T-ALL. Extending our work on the H3K27me3 mark, we now show the oncogenic role for the Jumonji d3 (JMJD3) demethylase. Functionally, genomic ablation of the JMJD3 modulator as well as targeting with a specific inhibitor, GSKJ4, leads to apoptosis and cell cycle arrest of T-ALL lines and primary cells. Genetic ablation of JMJD3 leads to slower initiation of the disease with significantly improved survival rates of the mice. Surprisingly, UTX acts as a tumor suppressor in the context of the same disease, as part of different transcriptional complexes, and we found that it is genetically inactivated in T-ALL patients. Importantly, in light of recent developments on novel epigenetic inhibitors against JMJD3, these findings pave the way to specific pharmacological targeting of T cell leukemia. Citation Format: Panagiotis Ntziachristos, Grant Welstead, Thomas Trimarchi, Sofia Bakogianni, Luyao Xu, Pieter Van Vlierberghe, Rab Prinjha, Adolfo Ferrando, Rudolf Jaenisch, Charles Mullighan, Aifantis Iannis. Delineating the roles of lysine 27 methylation-associated epigenetic modulators in T cell leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr PR03.

Most genetic abnormalities found in cancer are located in intergenic regions and their role in cancer development remains poorly understood. Here we report the identification of a long range enhancer driving NOTCH1-induced transformation in T-ALL. Using genome wide ChIPseq mapping of NOTCH1 binding and functional epigenetic marks in T-ALL we identified a NOTCH1-occupied enhancer located in a gene desert island in 8q24, +1,427 kb 3' from the MYC locus. This regulatory element named N-Me for NOTCH-bound MYC enhancer activates MYC reporter constructs in T-ALL cells and directly interacts with MYC promoter sequences via long range chromatin loops. Notably, high resolution aCGH analysis revealed recurrent focal duplications at chromosome 8q24 encompassing this enhancer in 8/160 (5%) T-ALL cases. Functional characterization of N-Me in T-cell development and transformation using N-Me knockout and conditional knockout mice revealed a major role for this enhancer during thymocyte development and in T-ALL. Thus N-Me null animals showed a marked and selective reduction in thymus size and cellularity and were completely resistant to NOTCH1-induced transformation. Moreover, secondary deletion of N-Me in established NOTCH1 induced leukemias from tamoxifen-inducible N-Me conditional knockout mice induced profound antileukemic effects with extended survival and almost complete suppression of leukemia initiating cell activity. Altogether, these results identify N-Me as a major downstream effector NOTCH1 critically required for MYC expression in T-cell development and leukemia. Citation Format: Adolfo Ferrando. An oncogenic enhancer drives NOTCH1-induced leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA22.