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The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. Significance: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression. This article is highlighted in the In This Issue feature, p. 1307

DNA is compacted into higher order structures that have major implications in gene regulation. These structures allow for long-range interactions of DNA elements, such as the association of promoters with their cognate enhancers. In recent years, mutations in genes that control these structures, including the cohesin-complex and the insulator-binding protein CTCF, have been found in a spectrum of hematologic disorders, and especially in acute leukemias. Cohesin and CTCF are critical for mediating looping and establishing boundaries within chromatin. Cells that harbor mutations in these genes display aberrant chromatin architecture and resulting differences in gene expression that contribute to leukemia initiation and progression. Here, we provide detailed discussion of the nature of 3D interactions and the way that they are disrupted in acute leukemia. Continued research in this area will provide new insights into the mechanisms of leukemogenesis and may shed light on novel treatment strategies.

Deubiquitinases are enzymes that remove ubiquitin moieties from the vast majority of cellular proteins, controlling their stability, interactions, and localization. The expression and activity of deubiquitinases are critical for physiology and can go awry in various diseases, including cancer. Based on recent findings in human blood cancers, we discuss the functions of selected deubiquitinases in acute leukemia and efforts to target these enzymes with the aim of blocking leukemia growth and improving disease outcomes. We focus on the emergence of the newest generation of preclinical inhibitors by discussing their modes of inhibition and their effects on leukemia biology.

Ubiquitination is a versatile and tightly regulated post- translational protein modification with many distinct outcomes affecting protein stability, localization, interactions, and activity. Ubiquitin chain linkages anchored on substrates can be further modified by additional post-translational modifications, including phosphorylation and SUMOylation. Deubiquitinases (DUBs) reverse these ubiquitin marks with matched levels of precision. Over hundred known DUBs regulate a wide variety of cellular events. In this review, we focus on ubiquitin-specific protease 7 (USP7, also known as herpesvirus-associated ubiquitin-specific protease, or HAUSP) as one of the best studied, disease-associated DUBs. By highlighting the functions of USP7, particularly in the nucleus, and the emergence of the newest generation of USP7 inhibitors, we illustrate the importance of individual DUBs in the nucleus, and the therapeutic prospects of DUB targeting in human disease.

FOXA1 is a nonhistone substrate of EZH2, promoting cell growth and sensitizing some prostate cancer to enzymatic EZH2 inhibitors., Forkhead box protein A1 (FOXA1) is essential for androgen-dependent prostate cancer (PCa) growth. However, how FOXA1 levels are regulated remains elusive and its therapeutic targeting proven challenging. Here, we report FOXA1 as a nonhistone substrate of enhancer of zeste homolog 2 (EZH2), which methylates FOXA1 at lysine-295. This methylation is recognized by WD40 repeat protein BUB3, which subsequently recruits ubiquitin-specific protease 7 (USP7) to remove ubiquitination and enhance FOXA1 protein stability. They functionally converge in regulating cell cycle genes and promoting PCa growth. FOXA1 is a major therapeutic target of the inhibitors of EZH2 methyltransferase activities in PCa. FOXA1-driven PCa growth can be effectively mitigated by EZH2 enzymatic inhibitors, either alone or in combination with USP7 inhibitors. Together, our study reports EZH2-catalyzed methylation as a key mechanism to FOXA1 protein stability, which may be leveraged to enhance therapeutic targeting of PCa using enzymatic EZH2 inhibitors.

UBR7 supports nucleotide biosynthesis and NOTCH1-driven T cell acute lymphoblastic leukemia through regulation of PRPS enzymes., Ubiquitin protein ligase E3 component N-recognin 7 (UBR7) is the most divergent member of UBR box–containing E3 ubiquitin ligases/recognins that mediate the proteasomal degradation of its substrates through the N-end rule. Here, we used a proteomic approach and found phosphoribosyl pyrophosphate synthetases (PRPSs), the essential enzymes for nucleotide biosynthesis, as strong interacting partners of UBR7. UBR7 stabilizes PRPS catalytic subunits by mediating the polyubiquitination-directed degradation of PRPS-associated protein (PRPSAP), the negative regulator of PRPS. Loss of UBR7 leads to nucleotide biosynthesis defects. We define UBR7 as a transcriptional target of NOTCH1 and show that UBR7 is overexpressed in NOTCH1-driven T cell acute lymphoblastic leukemia (T-ALL). Impaired nucleotide biosynthesis caused by UBR7 depletion was concomitant with the attenuated cell proliferation and oncogenic potential of T-ALL. Collectively, these results establish UBR7 as a critical regulator of nucleotide metabolism through the regulation of the PRPS enzyme complex and uncover a metabolic vulnerability in NOTCH1-driven T-ALL.

Differences in three-dimensional (3D) chromatin architecture can influence the integrity of topologically associating domains (TADs) and rewire specific enhancer–promoter interactions, impacting gene expression and leading to human disease. Here we investigate the 3D chromatin architecture in T cell acute lymphoblastic leukemia (T-ALL) by using primary human leukemia specimens and examine the dynamic responses of this architecture to pharmacological agents. Systematic integration of matched in situ Hi-C, RNA-seq and CTCF ChIP–seq datasets revealed widespread differences in intra-TAD chromatin interactions and TAD boundary insulation in T-ALL. Our studies identify and focus on a TAD ‘fusion’ event associated with absence of CTCF-mediated insulation, enabling direct interactions between the MYC promoter and a distal super-enhancer. Moreover, our data also demonstrate that small-molecule inhibitors targeting either oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions found in leukemia. Overall, our study highlights the impact, complexity and dynamic nature of 3D chromatin architecture in human acute leukemia. Analysis of 3D chromatin architecture in T-ALL identifies differences in intra-TAD interactions and TAD boundary insulation. Inhibition of oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions.

Purpose: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. Experimental Design: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. Results: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. Conclusions: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.

The lysine methyltransferase KMT2C (also known as MLL3), a subunit of the COMPASS complex, implements monomethylation of Lys4 on histone H3 (H3K4) at gene enhancers. KMT2C (hereafter referred to as MLL3) frequently incurs point mutations across a range of human tumor types, but precisely how these lesions alter MLL3 function and contribute to oncogenesis is unclear. Here we report a cancer mutational hotspot in MLL3 within the region encoding its plant homeodomain (PHD) repeats and demonstrate that this domain mediates association of MLL3 with the histone H2A deubiquitinase and tumor suppressor BAP1. Cancer-associated mutations in the sequence encoding the MLL3 PHD repeats disrupt the interaction between MLL3 and BAP1 and correlate with poor patient survival. Cancer cells that had PHD-associated MLL3 mutations or lacked BAP1 showed reduced recruitment of MLL3 and the H3K27 demethylase KDM6A (also known as UTX) to gene enhancers. As a result, inhibition of the H3K27 methyltransferase activity of the Polycomb repressive complex 2 (PRC2) in tumor cells harboring BAP1 or MLL3 mutations restored normal gene expression patterns and impaired cell proliferation in vivo. This study provides mechanistic insight into the oncogenic effects of PHD-associated mutations in MLL3 and suggests that restoration of a balanced state of Polycomb-COMPASS activity may have therapeutic efficacy in tumors that bear mutations in the genes encoding these epigenetic factors.

Cardiac metabolism affects systemic energetic balance. Previously, we showed that Krüppel-like factor (KLF)-5 regulates cardiomyocyte PPARα and fatty acid oxidation-related gene expression in diabetes. We surprisingly found that cardiomyocyte-specific KLF5 knockout mice (αMHC-KLF5(−/−)) have accelerated diet-induced obesity, associated with increased white adipose tissue (WAT). Alterations in cardiac expression of the mediator complex subunit 13 (Med13) modulates obesity. αMHC-KLF5(−/−) mice had reduced cardiac Med13 expression likely because KLF5 upregulates Med13 expression in cardiomyocytes. We then investigated potential mechanisms that mediate cross-talk between cardiomyocytes and WAT. High fat diet-fed αMHC-KLF5(−/−) mice had increased levels of cardiac and plasma FGF21, while food intake, activity, plasma leptin, and natriuretic peptides expression were unchanged. Consistent with studies reporting that FGF21 signaling in WAT decreases sumoylation-driven PPARγ inactivation, αMHC-KLF5(−/−) mice had less SUMO-PPARγ in WAT. Increased diet-induced obesity found in αMHC-KLF5(−/−) mice was absent in αMHC-[KLF5(−/−);FGF21(−/−)] double knockout mice, as well as in αMHC-FGF21(−/−) mice that we generated. Thus, cardiomyocyte-derived FGF21 is a component of pro-adipogenic crosstalk between heart and WAT.

In the current issue of Blood, Miyagi et al investigated the role of the plant homeodomain-like finger protein 6 (PHF6) in hematopoietic stem cell (HSC) self-renewal in an attempt to further clarify the previously suggested tumor suppressive role of this protein in human leukemia.1 Interestingly, Phf6 ablation rendered HSCs less responsive to inflammatory stress signals, but was able to increase HSCs proliferation rates, which might, at least in part, contribute to oncogenic transformation (see figure).

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Treatments1-6 show high response rates but have debilitating effects and carry risk of relapse5,7,8. Previous work implicated NOTCH1 and other oncogenes1,2,9-20. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Here, we demonstrate that ubiquitin-specific protease 7 (USP7)21-32 controls leukemia growth by stabilizing the levels of the NOTCH1 and JMJD3 demethylase. USP7 is overexpressed T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 through deubiquitination. USP7 is bound to oncogenic targets and controls gene expression through H2B ubiquitination and H3K27me3 changes via stabilization of NOTCH1 and JMJD3. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and USP7 inhibition alters associated gene activity. These results provide a new model for deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. USP7 inhibition33 significantly blocked T-ALL cell growth in vitro and in vivo. Our studies also show that USP7 is upregulated in the aggressive high-risk cases of T-ALL and suggest that USP7 expression might be a prognostic marker in ALL and its inhibition could be a therapeutic tool against aggressive leukemia.

SummarySequencing efforts led to the identification of somatic mutations that could affect the self-renewal and differentiation of cancer-initiating cells. One such recurrent mutation targets the binding pocket of the ubiquitin ligase Fbxw7. Missense FBXW7 mutations are prevalent in various tumors, including T cell acute lymphoblastic leukemia (T-ALL). To study the effects of such lesions, we generated animals carrying regulatable Fbxw7 mutant alleles. Here, we show that these mutations specifically bolster cancer-initiating cell activity in collaboration with Notch1 oncogenes but spare normal hematopoietic stem cell function. We were also able to show that FBXW7 mutations specifically affect the ubiquitylation and half-life of c-Myc protein, a key T-ALL oncogene. Using animals carrying c-Myc fusion alleles, we connected Fbxw7 function to c-Myc abundance and correlated c-Myc expression to leukemia-initiating activity. Finally, we demonstrated that small-molecule-mediated suppression of MYC activity leads to T-ALL remission, suggesting an effective therapeutic strategy.

Notch behaves as a tumor suppressor in AML, and Notch activation induces cell cycle arrest, differentiation, and apoptosis of AML-initiating cells., Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 10-15% of pediatric and 25% of adult ALL cases. Despite improved prognosis driven by the significant advances in the molecular understanding of T-ALL, the outcome of T-ALL patients with primary resistant and relapsed leukemia remains poor. Recent evidences have highlighted a role for changes in 3D chromatin architecture in cancer progression. Herein, for the first time we explored genomewide changes in chromatin organization in primary T-ALL samples relative to T cells from healthy donors. We show how the organization of topologically associated domains (TADs) influences key oncogenic and tumor-suppressive loci by modifying the promoter-enhancer landscape, deregulating the transcriptional program and ultimately causing disease progression. To comprehensively characterize the chromatin landscape of clinically relevant loci in T-ALL, we sought to identify changes in intra-TAD activity and TAD disruptions on a genome-wide scale. We developed and implemented new computational approaches for Hi-C data analysis with a focus on Hi-C data integration with ChIP-Seq and RNA-Seq. We found significant correlations of TAD activity with CTCF occupancy, super-enhancer activity and gene expression of leukemia relevant loci. Furthermore, among the dozens of TAD disruptions we identified, we found a novel TAD “fusion” event around the MYC locus, which allows for chromatin interactions between a recently reported Notch-dependent MYC super-enhancer and promoter to drive overexpression of MYC. This particular TAD fusion is accompanied by a complete CTCF loss in the TAD boundary and increased MYC expression in T-ALL samples. Overall, this study sheds new light into how changes in chromatin architecture influence disease progression by restructuring oncogene and tumor suppressor landscapes. Citation Format: Palaniraja Thandapani, Andreas Kloetgen, Charalampos Lazaris, Xufeng Chen, Panagiotis Ntziachristos, Aristotelis Tsirigos, Iannis Aifantis. Dynamic 3d chromosomal landscapes in acute leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2998.

Rationale: Fatty acid oxidation is transcriptionally regulated by peroxisome proliferator–activated receptor (PPAR)α and under normal conditions accounts for 70% of cardiac ATP content. Reduced Ppara expression during sepsis and heart failure leads to reduced fatty acid oxidation and myocardial energy deficiency. Many of the transcriptional regulators of Ppara are unknown. Objective: To determine the role of Krüppel-like factor 5 (KLF5) in transcriptional regulation of Ppara . Methods and Results: We discovered that KLF5 activates Ppara gene expression via direct promoter binding. This is blocked in hearts of septic mice by c-Jun, which binds an overlapping site on the Ppara promoter and reduces transcription. We generated cardiac myocyte–specific Klf5 knockout mice that showed reduced expression of cardiac Ppara and its downstream fatty acid metabolism–related targets. These changes were associated with reduced cardiac fatty acid oxidation, ATP levels, increased triglyceride accumulation, and cardiac dysfunction. Diabetic mice showed parallel changes in cardiac Klf5 and Ppara expression levels. Conclusions: Cardiac myocyte KLF5 is a transcriptional regulator of Ppara and cardiac energetics.

Krüppel-like factors (KLF) affect metabolism. Lipopolysaccharide-induced sepsis reduced cardiac PPARα and increased KLF5 (8-fold) more than any cardiac KLF isoform detected by whole genome microarray analysis. In silico analysis of ppara gene promoter predicted two KLF5 binding sites that overlap with c-Jun (AP-1) binding sites: -792/-772 bp and -719/-698 bp. Infection of a mouse cardiomyocyte cell line (HL-1) with adenovirus expressing constitutively active c-Jun reduced, while Ad-KLF5 increased PPARα mRNA in a dose-dependent manner. Chromatin immunoprecipitation (ChIP) showed that c-Jun binds both -792/-772 bp and -719/-698 on ppara promoter while KLF5 binds on -792/-772 bp. ChIP on LPS-treated HL-1 cells showed that c-Jun binding on -792/-772 bp prohibits KLF5 binding. We generated a cardiomyocyte-specific KLF5 knockout mouse (αMHC-KLF5-/-), which had 50% normal cardiac function. Cardiomyocyte-specific KLF5 ablation reduced PPARα (50%) and several fatty acid metabolism-associated genes such as CD36 (40%), LpL (20%), PGC1α (45%), AOX (28%) and Cpt1 (45%). As PPARα regulates cardiac fatty acid metabolism, we tested whether cardiac KLF5 is modulated in diabetes, when cardiac PPARα and lipid changes occur. I.p. injection of streptozotocin (STZ) in C57BL/6 mice increased plasma glucose (2.9-fold) and reduced cardiac KLF5 and PPARα gene expression; similar to STZ-treated rats but unlike what had been found in a different mouse strain (C57BL/6 x DBA2) treated with STZ. Treatment of HL-1 cells with increased glucose-containing medium (1 mg/ml) reduced KLF5 (80%) and PPARα (65%), as well as fatty acid metabolism markers, such as AOX (85%), Cpt1β (70%), LCAD (80%) and VLCAD (85%). On the other hand GLUT1 and GLUT4 were increased (30% and 20%) and PDK4 was reduced (65%) indicating increased glucose utilization. A model of non-insulin dependent hyperglycemia (ob/ob mice) had reduced cardiac KLF5 (60%) and PPARα (65%). Correction of hyperglycemia in STZ-treated C57BL/6 mice by pharmacological (dapagliflozin) or antisense oligonucleotide inhibition of kidney’s sodium glucose transporter 2 (SGLT2), restored KLF5 and PPARα gene expression. Thus, KLF5 is a transcriptional regulator of cardiac PPARα that is driven by changes in plasma glucose levels

International audience; Tight control of antigen-receptor gene rearrangement is required to preserve genome integrity and prevent the occurrence of leukaemia and lymphoma. Nonetheless, mistakes can happen, leading to the generation of aberrant rearrangements, such as Tcra/d-Igh inter-locus translocations that are a hallmark of ataxia telangiectasia-mutated (ATM) deficiency. Current evidence indicates that these translocations arise from the persistence of unrepaired breaks converging at different stages of thymocyte differentiation. Here we show that a defect in feedback control of RAG2 activity gives rise to bi-locus breaks and damage on Tcra/d and Igh in the same T cell at the same developmental stage, which provides a direct mechanism for generating these inter-locus rearrangements. Both the RAG2 C-terminus and ATM prevent bi-locus RAG-mediated cleavage through modulation of three-dimensional conformation (higher-order loops) and nuclear organization of the two loci. This limits the number of potential substrates for translocation and provides an important mechanism for protecting genome stability.

Over the past decade, it has become clear that both genetics and epigenetics play pivotal roles in cancer onset and progression. The importance of epigenetic regulation in proper maintenance of cellular state is highlighted by the frequent mutation of chromatin modulating factors across cancer subtypes. Identification of these mutations has created an interest in designing drugs that target enzymes involved in DNA methylation and posttranslational modification of histones. In this review, we discuss recurrent genetic alterations to epigenetic modulators in both myeloid and lymphoid leukemias. Furthermore, we review how these perturbations contribute to leukemogenesis and impact disease outcome and treatment efficacy. Finally, we discuss how the recent advances in our understanding of chromatin biology may impact treatment of leukemia.

Acute lymphoblastic leukemia (ALL) is a highly aggressive blood cancer affecting children and adults. Certain high-risk disease subsets have poor outcomes and often debilitating therapy-related toxicities stemming from direct inhibition of the oncogenes. We hypothesize that the process of oncogenic transformation is driven by aberrant activity of oncogene-associated chromatin modifying partners. These changes create a chromatin environment unique to the malignant state and, therefore, disruption of critical oncogenic chromatin signatures would not likely affect healthy tissues. We have generated strong evidence for the intertwined roles between the NOTCH1 oncogenic pathway and deubiquitinase enzymes in T cell leukemia, members of the ubiquitin-specific proteases (USP) family in particular. Members of the USP family physically interact with NOTCH1 and the lysine 27 on histone H3 (H3K27) demethylase JMJD3 and this methylation-ubiquitination biology-related axis coordinates regulation of transcriptional initiation and elongation, vital for the survival of leukemia cells. Interestingly transcription of USP genes is positively controlled by NOTCH1 creating a feedback loop in leukemia. We have further characterized this oncogenic axis using a combination of small molecule inhibitors and genetic engineering of USPs in ALL cell lines, primary patient samples and primagraft models of disease. We are able to show that a) USP activity is important for certain oncogenic pathways (such as NOTCH1) in leukemia; b) Oncogenes and USP enzymes co-bind certain areas in the leukemia genome; c) Ubiquitination of histone H2B acts in a combinatorial fashion with H3K27me, is a major epigenetic change affected by the USP activity in leukemia and controls d) transcriptional elongation. Finally we demonstrate that f) chemical inhibition or down-regulation of USPs affect leukemia growth in vitro and in vivo. Ongoing and future studies include manipulation of USP levels in mouse models of leukemia as well as combinatorial use of USP inhibitors with chemotherapeutic regiments in vitro using matched diagnosis-relapsed primary samples and in xenograft studies. Information gained from these studies will lend rationale towards the use of small molecule inhibitors against USP proteins in clinical trials for the treatment of aggressive and relapsed ALL. Disclosures Kumar: Progenra Inc.: Employment. Wang:Progenra Inc.: Employment. Wu:Progenra Inc.: Employment.

Introduction. Prior studies have described a subset of B-progenitor ALL cases with a distinct gene expression profile and/or deletions involving ERG (encoding the ETS family member v-ets avian erythroblastosis virus E26 oncogene), however the relationship of these alterations and their role in leukemogenesis are poorly understood. We performed integrated genomic and epigenetic analyses, biochemical studies and leukemogenesis assays to define the genetic basis of this form of ALL. Methods. We studied 1674 childhood, adolescent and young adult B-progenitor ALL cases with microarray gene expression profiling and/or RNA-sequencing data to enable the identification of ERG ALL by unsupervised clustering and predictive analysis of microarrays. Detailed genomic analysis was performed for 144 ERG ALL cases, including whole genome (N=38), exome (n=46) and/or RNA-sequencing (n=57) cases, and single nucleotide polymorphism array analysis. Epigenetic profiling, including whole genome bisulfite sequencing, chromatin immunoprecipitation and sequencing for ERG and histone modifications and ATAC-sequencing were performed for a subset of 8 xenografted ERG tumors and reference cell lines. ERG transcript expression was measured by analysis of RNA-seq analysis and quantitative RT-PCR assays, and by interrogation of TCGA and PCGP RNA-seq data. The function of ERG isoforms was evaluated by EMSA and transcriptional reporter assays, immunofluoresence, colony forming assays and retroviral bone marrow transplant assays. Results. One hundred and forty four cases (8.6%) of B-ALL cases exhibited a distinct gene expression profile and lacked known chromosomal rearrangements (ERG ALL). Such cases had favorable outcome. Eighty cases (55.6%) had focal deletions of ERG with no evidence of oncogenic or chimeric ERG fusions. The deletions were most commonly heterozygous and involving exons 3-7 (n=27) or 3-9 (n=22) of 10 coding exons, and less commonly involving exon 1, or a larger region of the gene. No ERG deletions were identified in non-ERG ALL. Two cases harbored missense mutations in the ETS domain. Analysis of whole genome and exome sequencing data of 71 cases identified a high frequency of alterations of lymphoid transcription factors (46.5%; IKZF1 36.7%, PAX5 11.3%); mutation of transcription factors otherwise uncommon in ALL (21%; MYC, MYCBP2, MGA, ZEB2, GATA3); activation of signaling pathways, most commonly NRAS or KRAS (35.2%); cell cycle regulation (22.5%); and epigenetic modifiers (56.3%), most commonly KMT2D, SETD2, ARID2 and NCOR1. Notably, the five year event-free survival of ERG ALL cases with IKZF1 alterations exceeded 85% in both St Jude and Children's Oncology Group cohorts. We observed striking transcriptional deregulation at the ERG locus. Most (51/56) ERG- deleted cases expressed an ERG isoform encoded by a novel exon in intron 6 that splices in frame to distal exons, resulting in expression of a truncated C-terminal ERG protein that lacks the pointed and central regulatory domains, but retains the ETS and transactivation domain (ERGalt). ERGalt was also present in most (36/44) cases lacking an ERG deletion, and was strongly associated with presence of ERGalt protein in leukemic cells. We also identified expression of an Antisense Long non-coding RNA associated with the ERG locus (ALE) in ERG ALL. ERGalt and ALE were absent, or uncommonly expressed at very low levels in non-ERG ALL. ERGalt was absent, and ALE rarely expressed in non-ALL PCGP and TCGA samples. ERGalt and point mutant ERG were retained in the nucleus, bound DNA targets and acted as competitive inhibitors of wild type (WT) ERG in transcriptional reporter assays. Lineage-negative Arf -null bone marrow cells transduced with ERG WT induced an aggressive erythro-megakaryoblastic leukemia; in contrast ERGalt induced an immature lymphoid progenitor leukemia. Conclusions. Genomic alterations drive aberrant transcription of ERG, resulting on expression of a truncated, C-terminal oncogenic ERG protein. This represents a novel mechanism of transcription factor deregulation in leukemia. As a subset of ERG ALL cases lack ERG deletion, and as IKZF1 alterations are not associated with inferior outcome in this form of ALL, diagnostic approaches must incorporate gene expression profiling in addition to identification of ERG and IKZF1 alterations to accurately identify this form of leukemia. Disclosures Evans: Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Stock:Gilead: Membership on an entity's Board of Directors or advisory committees. Voorhees:Oncopeptides: Consultancy; Onyx Pharmaceuticals: Research Funding; GSK: Consultancy; Oncopeptides: Research Funding; Janssen: Research Funding; A Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy; Millennium Pharmaceuticals: Consultancy, Research Funding; Acetylon Pharmaceuticals, Inc.: Research Funding; Novartis: Consultancy; Array BioPharma: Consultancy; GSK: Research Funding; Celgene: Research Funding. Hunger:Spectrum Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Sigma Tau: Consultancy; Merck: Equity Ownership. Mullighan:Incyte: Consultancy; Amgen: Honoraria.

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.

Recent studies have identified recurrent mutations in SETBP1, the gene that encodes SET-binding protein 1, in several types of myeloid malignancies, including chronic myeloid and acute myeloid leukemias. The identified mutations frequently target the SKI-homologous domain, although the exact pathogenic mechanisms remain unknown.

Although the cure rate for acute lymphoblastic leukemia (ALL), a frequent pediatric leukemia, has improved dramatically, the overall prognosis remains dismal due to frequent disease relapse and the absence of non-cytotoxic targeted therapy options. Up to 25% of children fail frontline therapy and in these cases prognosis is dismal and the cure rate is approximate 20%. Main current therapies are based on intensive induction chemotherapy that is most frequently coupled to intrathecal chemotherapy alone or with cranial irradiation for central nervous system prophylaxis, which has severe short and long-term side effects. Thus, the ultimate and most critical aim for developing new treatments in different types of leukemia is to block the effects of specific cancer-inducing oncogenes. Others and we have previously shown that T cell ALL (T-ALL) is characterized by activating mutations in the NOTCH signaling pathway. It is currently unclear how key transcription factors in T-ALL such as NOTCH1 recruit the epigenetic machinery and bring together different chromosomal domain, in order to carry out the oncogenic transformation program. We generated evidence that NOTCH1 oncogenic action leads to important epigenetic changes through antagonizing the polycomb repressive complex 2 (PRC2) and leads to loss of the repressive mark histone 3 lysine 27 di/tri-methylation (H3K27me2/3). Moreover, 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. Further extending our work on the H3K27me3 mark, we showed the oncogenic role for the Jumonji d3 (JMJD3) demethylase. Functionally, genomic ablation of the JMJD3 modulator as well as targeting with a specific chemical inhibitor, GSKJ4, generated by GlaxoSmithKline, 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. In light of recent developments on novel epigenetic inhibitors against JMJD3, these findings pave the way to specific pharmacological targeting of T cell leukemia. Based on this activity of Notch1 oncogene on epigenetic marks we further hypothesized that the switch from physiological to oncogenic activity might be mediated by changes in enhancer-promoter interaction networks forming chromosomal domains. A substantial percentage of these interactions are likely to be specific for the malignant state, and their disruption with epigenetic pharmacological inhibitors would not potentially affect healthy tissues. Studies in our laboratory show for the first time in leukemia that NOTCH1 chromatin binding sites are associated with enhancer-promoter interactions at oncogenic loci, using up-to-date chromosome conformation capture technology. We hereby show the importance of these interactions for oncogenic gene expression and pharmacological targeting of leukemic cells. These findings lend further rationale to the use of epigenetic drugs for targeted treatment of T cell leukemia. Disclosures Kruidenier: GlaxoSmithKline: Employment. Prinjha:GlaxoSmithKline: Employment.

T cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In a recently published work, we identified and characterized a tumor suppressor role for the Polycomb Repressive Complex 2 (PRC2), that was underlined through the identification of loss-of-function mutations and deletions of members of the PRC2 complex in 25% of the patients with T cell leukemia. Further inhibition of PRC2 action in T cell leukemia comes through competition with master oncogenes, such as Notch1, for chromatin binding. Extending our work on the H3K27me3 mark, we now show that the activity of the corresponding H3K27me3 demethylases is also very important for the initiation, progression and maintenance of the disease. More specifically, transcriptional data analysis and exome sequencing of primary T-ALL samples demonstrates a significant correlation between levels and mutational status of these demethylases and major oncogenic pathways in leukemia. Functionally, genomic ablation of these epigenetic modulators as well as targeting with a specific-recently produced and characterized-inhibitor-GSKJ4, leads to apoptosis and cell cycle arrest of T-ALL lines in vitro and in vivo, accompanied by major epigenetic and transcriptional changes at specific anti-apoptotic, oncogenic and metabolic genes. Furthermore, genetic ablation of these epigenetic modulators leads to slower initiation of the disease in a classic mouse model of leukemia with highly improved survival rates. In light of recent developments of novel epigenetic inhibitors against these molecules, these findings pave the way to specific pharmacological targeting of T cell leukemia. Citation Format: Panagiotis Ntziachristos, Aris Tsirigos, Thomas Trimarchi, Evangelia Loizou, Xu Luyao, Pieter Van Vlierberghe, Bryan King, Grant Welstead, Charles Mullighan, Rudolf Jaenisch, Adolfo Ferrando, Iannis Aifantis. Characterizing the specific oncogenic and tumor suppressor roles of H3K27me3 epigenetic modulators in T cell leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A07.