Abstract A189: Expression of fusion proteins in acute myeloid leukemia cells increases sensitivity to histone deacetylase inhibitors

Abstract Acute Myeloid Leukemias (AMLs) are often characterized by chromosomal rearrangements that result in fusion proteins with aberrant transcriptional regulatory activities. These fusion proteins bind to gene promoters and recruit corepressors such as histone deacetylases (HDACs), which remodel chromatin into a closed conformation thereby silencing genes and contributing to a malignant phenotype. Aberrantly silenced genes include tumor suppressor and pro-differentiation genes. In addition, multiple groups have reported a DNA repair deficient phenotype concurrent with the expression of different fusion proteins in leukemia. Small molecule HDAC inhibitors (HDACi) were devised as a strategy to reverse transcriptional repression. Indeed, many studies have demonstrated the ability of HDACi to re-sensitize leukemic cells to differentiating stimuli. However, other studies have revealed alternate methods by which HDACi exert anti-tumor activity, including induction of apoptosis that may be dependent on induction of ROS, MAPK signaling etc. We find that low doses of HDACi, including Vorinostat and LBH589, induce cell death in the AML cell line U937 in a dose and time-dependant manner. Further, Vorinostat induced cell death in U937 cells is preceded by DNA damage and a G2/M arrest. This correlates with reports that HDACi repress DNA repair, by down-regulating DNA repair genes and by acetylating repair proteins thereby impairing their repair function. Due to the inhibitory effect of leukemic fusion proteins on DNA repair, we predicted that DNA damage induced by HDACi, and thus cell death, would be amplified in AML cells expressing PML-RARα and PLZF-RARα fusion proteins. Sensitivity to Vorinostat and LBH589 was tested in three U937 derived cell lines: PR9 (PML-RARα inducible), B412 (PLZF-RARα inducible) and SN4 (mock transfected control). Indeed, induction of PLZF-RARα increased sensitivity of B412 cells to both Vorinostat- and LBH-mediated cell death. Assaying for DNA damage using alkaline comet assay, PR9 and B412 cells displayed an increase in DNA damage when their respective fusion protein is expressed. Nonetheless, the contribution of DNA damage to HDACi-mediated cell death remains unclear and further study is necessary. These findings are significant as they point to fusion protein expressing AMLs as a target group that may respond better to HDACi-based therapies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A189.</jats:p

Loss of KDM6A/UTX Accelerate the Development of Multiple Myeloma

Abstract In multiple myeloma (MM), inactivating mutations and deletions affecting the histone demethylase KDM6A locus are found in up to 10% of newly diagnosed patients and associated with poor prognosis. KDM6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) belongs to a family of Jumonji-C (Jmj-C)-containing demethylases that work as a scaffold for a multiprotein complex containing H3K4 specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300 and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex appears to add activation marks on histones and remove methylation of lysine 27 on histone H3 (H3K27me) associated with gene repression. Importantly, all these coregulators are found significantly mutated in MM and their function may converge into a tumor suppressive pathway. Our goal is to understand how KDM6A loss contributes to the development of MM. We modeled the loss of KDM6A in MM cell lines using CRISPR-Cas9 ribonucleotide protein (RNP) technology. Mutant allele frequency over time post electroporation of RNP revealed a growth advantage of KDM6A mutant alleles. By 2 weeks of growth most of the cells in culture harbored KDM6A gene disruption and exhibit elimination of KDM6A protein confirming the tumor suppressive role of KDM6A in MM. We used these isogenic polyclonal edited cell lines with KDM6A wild type or mutated to identify KDM6A binding sites and enhancers affected by the loss of KDM6A. As well, we knock-in an HA tag on endogenous KDM6A to identify DNA regions occupied by KDM6A. To understand the importance of KDM6A demethylase activity in the tumor suppressive effect of KDM6A, we developed stable cell lines with a doxycycline-inducible form of KDM6A wild-type (WT) or lacking demethylase activity (JmjC-dead). We found that about 20% of the genes deregulated by re-expression of WT and jmjC-dead KDM6A overlap suggesting that demethylase activity is not essential for all KDM6A functions in MM. Importantly, we confirmed the tumor suppressive role of KDM6A in a novel mouse model of MM in which KDM6A is deleted specifically in the B cell compartment. Briefly, we isolated CD19cre-/+ (control) or CD19cre-/+ Kdm6afl/fl fetal liver cells and transduced these cells with a constitutively activated form of the IL-6 coreceptor (L-GP130) that activates the JAK/STAT pathway. Mice transplanted with CD19cre-/+ Kdm6afl/fl fetal liver cells developed MM by 7 weeks post transplantation while mice transplanted with CD19cre+/- fetal liver cells did not developed MM by20 weeks. Necroscopy and flow cytometry analysis demonstrated infiltration of CD138+ cells in bone marrow, spleen, liver and kidney of mice that developed MM. In the future we will use this model to explore how loss of KDM6A affects chromatin structure in vivo and how it changes the characteristics of MM. These studies are expected to provide new insights that lead to the development of more effective MM therapies which directly target mechanisms of chromatin structure regulation. Disclosures Licht: Celgene: Research Funding. </jats:sec

KDM6A Controls Genes Modulating Immune Surveillance in Multiple Myeloma

Background: In multiple myeloma (MM), inactivating mutations and loss of the histone demethylase KDM6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) locus are found in up to 50% of patients and are associated with poorer prognosis. KDM6A belongs to a family of Jumonji-C (Jmj-C)-containing demethylases that work as a scaffold for a multiprotein complex containing H3K4 specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300 and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex activates enhancers by adding activation marks on histones and removing methylation of lysine 27 on histone H3 (H3K27me) associated with gene repression. The H3K27me3 methyltransferase EZH2 that catalyzes the reverse reaction of KDM6A, is often overexpressed in MM and this also correlates with poor prognosis. As well, KMT2C, KMT2D and SWI/SNF components of its complex are all found significantly mutated in MM which emphasize the importance of enhancer deregulation in myeloma. Aim: Our goal is to understand how loss of KDM6A affects gene transcription networks and enhancer function to affect MM pathogenesis or progression. Method: We explored the transcriptional consequences of KDM6A deficiency in patient tumors using the gene expression dataset included within the Multiple Myeloma Research Foundation (MMRF) Researcher Gateway and in MM cell lines using data from the Cancer Cell Line Encyclopedia (CCLE). We further modeled the loss of KDM6A in female MM cell lines using CRISPR-Cas9 ribonucleotide protein (RNP) technology and by re-expressing KDM6A in cells null for KDM6A. Using ChIP-sequencing analysis in isogenic KDM6A knockout or replete cell lines, we identified KDM6A binding site in MM and explored how KDM6A deficiency affects chromatin structure to regulate a transcriptional network. Results: Epithelial-to-mesenchymal-transition (EMT)-related pathways were downregulated in low KDM6A expressing tumor from the MMRF patient cohort. In both male and female patients, low expression of KDM6A was associated with decreased expression of genes involved in immune recognition. In MM cell lines, we observed a significant positive correlation between KDM6A expression level and expression of the transcriptional regulators of Major Histocompatibility complex I and II (MHCII and MCHII), NLRC5 and CIITA, respectively. With ChIP-sequencing analysis we find that KDM6A directly binds NLRC5 and CIITA genes, among many other genes involved in immune surveillance. KDM6A null MM cells displayed upregulation of pathways promoting EMT and downregulation of genes regulating immune function, such as HLA-A/B/C, CCL3/5, HHLA2 and ITGAL. Moreover, re-expression of KDM6A in KDM6A null cells upregulated the expression of these gene along with surface expression of MHCI proteins. Conclusion: Together, our data demonstrates that KDM6A modulates EMT and directly regulates expression of major regulators of immune surveillance. KDM6A deficiency may drive MM towards an EMT program to promote systemic spread and facilitate escape from the immune surveillance. Disclosures No relevant conflicts of interest to declare. </jats:sec

Cells with DNMT3A Mutations Are More Sensitive to Cytarabine-Induced DNA Damage

Abstract Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) (20-35%) and are associated with poor prognosis. Although studies demonstrated survival benefit after daunorubicin dose intensification for DNMT3A mutant AML, these patients tend to be older (median age at diagnosis 67 y.o. in DNMT3A-mutated cases compared to 60 y.o. in DNMT3A-WT) and more likely to be unfit for high-dose chemotherapy. In older patients, cytarabine monotherapy, a nucleoside analog chain terminator that stalls DNA replication, may be preferred due to lower toxicity. However, its efficacy in DNMT3A mutant AML has not been evaluated. Previous gene expression studies in large AML cohorts and in a mouse model of Dnmt3a-mutant hematopoiesis demonstrated negative enrichment of the cell cycle related signatures, including G2/M checkpoint and E2F targets. Hence, we hypothesized cell cycle-specific drugs cytarabine and fludarabine may be effective in DNMT3A-mutant context. DNMT3A mutant AML cell lines (MUT) SET-2 and KO-52 were more sensitive to cytarabine and fludarabine than DNMT3A wild-type (WT) cells KU-812 and K-562 in vitro (cytarabine IC50 46.1 and 165.8 μM in MUT SET-2 and KO-52 compared to 465.2 μM and not reached in WT KU-812 and K-562, respectively; fludarabine IC50 1.1 and 1.3 μg/ml in MUT vs 16 μg/ml and not reached in WT, Figure 1A). Higher proportion of cells with mutant DNMT3A were undergoing apoptosis 24 hours after cytarabine exposure (26.5±2.3% and 13.1±2.3% Annexin V+ cells in SET-2 and KO-52 vs 6.9±2.6% and 4.6±2.5% in KU-812 and K-562, respectively, p&lt;0.05). Analysis of the DNA damage signaling revealed increased levels of phospho-CHK1, γH2A.X, and cleaved PARP (stalled replication forks, DNA damage, and apoptosis markers). To investigate the mechanism of differential sensitivity to cytarabine-induced DNA damage, we overexpressed WT or R882 mutant forms of DNMT3A in U2OS cells, a well-established model for DNA damage studies. MUT cells were more sensitive to cytarabine compared to WT (IC50 38 μM vs 213 μM after 48 hours of exposure, Figure 1B), which was accompanied by increased apoptosis (22.3±7.6% in MUT vs 7.2±0.3% in WT Annexin V+ cells, p&lt;0.03). DNMT3A-MUT cells showed persistence of the phospho-CHK1 levels over 12 hours of continuous cytarabine exposure, whereas WT showed resolution of the CHEK1 signaling after initial activation. Consistently, MUT cells accumulated significantly more DNA damage over 36 hours of continuous exposure compared to WT, as evidenced by γH2A.X immunofluorescent staining, and by Comet assay (Figure 1C). Fewer MUT cells were able to complete replication and progress to the G2 phase of the cell cycle (5.3±0.2% in MUT vs 11.2±0.6% in WT, flow-cytometric analysis by DNA content, p&lt;0.01). Notably, both WT and MUT cells were proficient in DNA repair by homologous recombination (HR, tested by RAD51 foci formation visualized by immunofluorescence) and by non-homologous end joining (NHEJ, 53BP1 foci). These data demonstrate that enhanced sensitivity to cytarabine in cells expressing mutant DNMT3A is due to increased susceptibility to DNA damage during replication, and not to defects in DNA repair. Consistently, even though MUT cells showed more DNA damage by Comet assay after 12 hours of cytarabine treatment, it was largely repaired 60 minutes after drug wash-out, similar to WT, Figure 1D). These observations support continuous infusion of cytarabine as a delivery method of choice in the clinic, rather than bolus administration. Finally, bone marrow cells derived from Dnmt3a-mut conditional knock-in mice showed impaired clonogenic survival in MethoCult when exposed to 25 nM cytarabine ex vivo, compared to Dnmt3a-WT (p&lt;0.003). We are currently investigating gene expression, DNA methylation, and chromatin accessibility profiles in WT and MUT cells; and cytarabine efficacy in a pre-clinical mouse model of Dnmt3a(mut):Flt3(ITD):Npm1(c) leukemia. In conclusion, our studies show cells with DNMT3A mutations may be sensitive to DNA damage induced by clinically relevant nucleoside analogs such as cytarabine. Our data support continuous infusion of cytarabine rather than bolus administration, and establish its mechanistic basis. These results demonstrate, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication. Figure 1. Figure 1. Disclosures Licht: Celgene: Research Funding. </jats:sec

A Gain of Function Mutation in the NSD2 Histone Methyltransferase Drives Glucocorticoid Resistance of Acute Lymphoblastic Leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common diagnosed pediatric cancer. Despite improvements in chemotherapy that have increased the 5-year survival rate to close to 90%, 15-20% of these patients may relapse with the majority of such children succumbing to this disease. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biological processes mediated by mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in two B-ALL cell lines (RCH-ACV and SEM) and one T-ALL cell line (RPMI-8402) and inserted the E1099K mutation into three ALL cell lines (697, CEM, MOLT4). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells compared to cells in which the mutation is removed demonstrate enhanced cell growth, colony formation and migration. NSD2 mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system (CNS) in xenografts. The NSD2 mutation is found prominently in children who relapse early from therapy for ALL, and NSD2E1099K cells are particularly resistant to glucocorticoids (GC). Reversion of NSD2E1099K mutation to wild type NSD2 conferred glucocorticoid sensitivity to both B and T cell lines. GC response upon disruption of mutant NSD2 was accompanied by cell cycle arrest and apoptosis. Mice xenografted with NSD2E1099K cells were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2 wild-type cells led to significant tumor reduction and survival extension. RNA-Seq analysis showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2 wild-type cells. Furthermore, in NSD2 mutant cells, there was lower basal expression level of glucocorticoid receptor (GR) and GR levels were not significantly induced by GC. Accordingly, after treatment with GC, there was significantly less DNA-binding activity of the GR in NSD2E1099K cells than that of NSD2 wild-type cells. The key pro-apoptotic regulators Bim and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. In conclusion, these studies demonstrate that the NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by causing GC resistance. Future studies will determine how NSD2 which generally activates genes paradoxically blocks the ability of GC and the GR to induce critical pro-death genes. Disclosures Licht: Celgene: Research Funding. </jats:sec

NSD2-E1099K Mutation Leads to Glucocorticoid-Resistant B Cell Lymphocytic Leukemia in Mice

Background: NSD2 (nuclear receptor binding SET domain protein 2) is a histone methyltransferase specific for dimethylation of histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. In pediatric acute lymphoblastic leukemia (ALL), particularly at relapse, a gain of function mutation (E1099K) of NSD2 is found in 10-15% of cases. The NSD2 mutation is found in addition to fusion proteins such as E2A-PBX and ETV6-RUNX1. The mutation can be subclonal at diagnosis and dominant at relapse, suggesting a link to therapeutic resistance. The NSD2-E1099K mutation affects gene expression through an increase in H3K36me2 and a decrease in H3K27me3. Using CRISPR/Cas9-edited isogenic ALL cell lines, we found that NSD2-E1099K mutation drove oncogenic programming by altering chromatin architecture, gene expression and enhancing cell growth, migration and infiltration to the central neural system (CNS). NSD2 mutation caused resistance of ALL cells to glucocorticoids (GC) by blocking genome wide binding of the glucocorticoid receptor (GR, encoded by NR3C1 gene) preventing GC-mediated induction of pro-apoptotic genes. NR3C1 levels were depressed in NSD2-E1099K cells and GC failed to induce autoactivation of NR3C1. While H3K27me3 was globally decreased by NSD2-E1099K, increased H3K27me3 was noted at the promoter of NR3C1, suggesting a novel role of polycomb repressive complex 2 as a therapeutic target for relapsed ALL with NSD2 mutation. While NSD2 is highly expressed in B cells and NSD2 knockout causes defects in B cell development, how the NSD2 mutation affects B cell development and leukemia occurrence in vivo is uncertain. Aims: To determine the role of NSD2 mutation in the pathogenesis of lymphocytic malignancies and GC resistance in a mouse model. Methods: We generated a conditional NSD2-E1099K knock-in mouse model in which the NSD2-E1099K allele was placed in the Rosa26 locus and expressed in B cells under the control of Cd19-Cre (Cd19+/-NSD2E1099K/WT). The resulting phenotype was characterized through peripheral blood counts, cellular morphology and histology of blood smears, bone marrow (BM), spleen and liver, flow cytometric analysis, germinal center B cells (GCB) immunization, BM transplantation, and hematopoiesis analysis in a CD3-/- background. We further established mouse leukemia cell lines with NSD2 mutation for functional analysis. RNA-Seq, real time PCR, immunoblotting, and apoptosis analysis (Annexin V/PI staining) following GC treatment were performed to demonstrate the effects of NSD2 mutation on histone modifications, transcriptome and GC resistance. Results: The NSD2-E1099K mutation increased H3K36me2 and decreased H3K27me3 in isolated B cells from mouse BM and spleen. Mice were aged and did not develop signs of malignancy and RNA-sequencing showed few differences between B cells with or without the NSD2 mutation. However, after immunizing the mice with sheep red blood cells (SRBC), more GCBs were seen in the spleen of NSD2 mutant mice, suggesting mutant NSD2 stimulated germinal center hyperplasia. Transplantation of BM cells from mice expressing NSD2-E1099K into lethally irradiated recipients lead to an expansion of B cells while myeloid and T cells and life span of the recipients impaired. The NSD2 knock-in mouse model was crossed with Cd3-/- mice to create Cd19+/-Cd3-/-NSD2E1099K/WT mice, which within 2 months of birth developed a disease resembling an immature B lymphocytic leukemia (B220+CD19+IgM+IgD-CD5-) with infiltration of the spleen, liver and CNS and a median survival of 4.8 months. These tumors could be transplanted into immunodeficient mice but not immunocompetent mice. RNA seq analysis of these cells revealed 6,815 genes (3,295 upregulated and 3,520 downregulated) differentially expressed in NSD2 mutant B cells compared to normal B cells. The upregulated genes were related to abnormal immunoglobulin level , B cell activation, T-helper 1 physiology, and decreased B cell apoptosis. Importantly, the NSD2 mutant leukemic cells displayed depressed level of NR3C1 gene expression and GC resistance. Conclusions: The NSD2 mutation alters B cell development, particularly in an immunodeficient background and causes B cells to become resistant to glucocorticoids. The inability of the mutation to generate disease on its own except in an immunodeficient background suggests genes that collaborate with NSD2 in ALL may play a role in immune escape. Disclosures No relevant conflicts of interest to declare. </jats:sec

Dysregulation of Epigenetic Landscape Uncovered the Mechanisms Underlying the Relapse of Pediatric Acute Lymphoblastic Leukemia with NSD2 Mutation

Abstract Background: Relapse from acute lymphoblastic leukemia (ALL) is one of the most common causes of pediatric cancer-related death. Early relapse of ALL is associated with recurrent mutations of histone methyltransferase NSD2 (nuclear receptor binding SET domain protein 2), which is specific for H3K36me2, suggesting a link to therapy resistance or other mechanisms underlying relapse such as central neural system (CNS) infiltration. NSD2 p.E1099K affects gene expression through disturbing the balance of H3K36me2/H3K27me3. Using CRISPR/Cas9-edited isogenic ALL cell lines and PDX cells, we found that NSD2 p.E1099K drives oncogenic programming, CNS infiltration and glucocorticoid (GC) resistance. However, the molecular mechanisms underlying the relapse of this subtype of ALL are still under investigation. Aim: To elucidate the epigenetic landscape underlying the mechanism of the relapse of pediatric ALL with NSD2 mutation. Methods: We performed in vivo experiments to observe tumor burden, leukemia cell infiltration and survival of the NOD/SCID mice injected with a NSD2 p.E1099K mutation knock-out SEM cell line and knock-in CEM cell line. We determined transcriptome (RNA-Seq), chromatin accessibility (ATAC-Seq) in isogenic RCH-ACV, SEM, RPMI-8402 and CEM cell lines, transcription factor binding and histone modification (ChIP-Seq) and 3D organization (Hi-C) in RCH-ACV cells. Finally, we integrated analysis of RNA-Seq, ATAC-Seq, ChIP-Seq and Hi-C to comprehensively disclose the epigenetic landscape in ALL with NSD2 mutation. Results: NOD/SCID mice xenografted with NSD2 mutant cells developed high tumor burden and infiltration to spleen, liver and brain while the mice injected with WT cells accumulated significant less tumor cells and had extended survival. RNA-Seq analysis showed that reversion of NSD2 mutation to WT caused more genes downregulated while insertion of NSD2 mutation to WT cells led to more genes upregulated (Mutant vs WT: RCH-ACV 838 vs 494, SEM 1567 vs 1158, RPMI-8402 1922 vs 1745, CEM 1809 vs 1031). 50 upregulated genes and 3 downregulated genes were in common in B-ALL and T-ALL with NSD2 mutation. Most of the upregulated genes correlated with neural development and adhesion which might contribute to CNS infiltration (e.g., NCAM1 and NEO1). A few genes were associated with GC resistance such as decreased NR3C1 and increased NR3C2. Accordingly, ATAC-Seq showed that NSD2 mutant cells had more open chromatin peaks than those of WT (RCH-ACV 4853 vs 3212, SEM 10052 vs 7595, RPMI-8402 20392 vs 12133, CEM 10155 vs 6437). ChIP-Seq revealed general large gains of H3K36me2 in intergenic regions in NSD2 mutant cells. Importantly, genes upregulated with NSD2 mutation (e.g., NCAM1 and NEO1) lost H3K27me3 at promoters but gained H3K36me2 at promoters and whole gene bodies, accompanied with increased H3K27ac at enhancers. Conversely, a small subset of genes gained H3K27me3 and lost H3K36me2 in their promoters. Concentrated H3K36me2 in gene bodies diffused and broadened was less prominent and H3K27me3 accumulation became dominant. This for example was associated with repression of NR3C1 to drive GC resistance of NSD2 mutant cells. Genes upregulated in NSD2 mutant cells were enriched for binding sites for lymphoid transcriptional activators such as EBF1 and IRF2. The promoters of the downregulated genes had motifs for transcription factors poorly expressed in lymphoid cells and were enriched for binding sites for the BCL6 transcriptional repressor. Hi-C analysis revealed 430 topologically associated domains (TADs) with increased loop interactions while 136 TADs with decreased interactions were in NSD2 mutant cells compared to WT cells. Overall, 491 regions switched from compartment A to B and 444 regions switched from B to A in NSD2 mutant cells compared to WT cells. Compartment switching from inactive B to active A correlated with upregulated gene expression levels while the reverse was true for switching from A to B. Increased intra-TAD interactions were linked to upregulated genes while decreased intra-TAD interactions were linked to downregulated genes. Conclusions: The NSD2 mutation led to increased tumor burden, CNS infiltration and glucocorticoid resistance due to dysregulation of epigenetic patterns and 3D chromatin architecture, indicating mechanisms underlying the relapse of pediatric ALL and potential therapeutic targets in ALL with NSD2 mutation. Disclosures Licht: Epizyme: Research Funding. </jats:sec

DNMT3A with Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novoacute myeloid leukemia (AML) and are associated with poor prognosis. Although studies demonstrated survival benefit of induction chemotherapy dose intensification, outcomes remain unsatisfactory in most patients due to advanced age, comorbidities, and hence inability to tolerate treatment. Clinical trials of low-intensity regimens combining cytarabine and cladribine, nucleoside analog chain terminators that stall DNA replication, appear to be safe and effective, and tend to particularly benefit patients with DNMT3Amutations. Consistently, we observe increased sensitivity to cytarabine, fludarabine, and cladribine in multiple cellular systems harboring mutant DNMT3Ain vitro (Figure 1A, B). Differential sensitivity to cytarabine was confirmed in normal and leukemic primary bone marrow cells derived from mice with and without Dnmt3a mutations ex vivo (Figure 1C). Dynamic chromatin organization plays a pivotal role in DNA-associated cellular processes including DNA replication and damage repair. We previously found altered chromatin remodeling in cells expressing mutant DNMT3A after genotoxic stress. Gene expression studies by us and others demonstrated negative enrichment of cell cycle related signatures including G2/M checkpoint adaptation, in cells with DNMT3A mutations. These signatures are implicated in DNA damage response and replication fork integrity and suggest sensitivity to replication stress. To investigate the mechanism of differential sensitivity to cytarabine-induced DNA damage, we overexpressed wildtype (WT) or R882 mutant (MUT) forms of DNMT3A in U2OS cells, a well-established model for DNA damage studies. Analysis of the DNA damage signaling proficiency in response to cytarabine revealed persistent intra-S phase checkpoint activation (phospho-CHK1), accompanied by accumulation of DNA damage visualized by γH2A.X foci and by Comet assay in the DNMT3A(mut) overexpression cells (Figure 1D). This damage was only partially resolved after drug had been removed and cells were allowed to repair the DNA (Figure 1E), and was carried through mitosis, resulting in increased rate of micronucleation.At the same time, DNMT3A mutant cells remained proficient in initiating homology-directed repair (HDR) and non-homologous end joining (NHEJ) pathways, evidenced by RAD51 and 53BP-1 foci formation, respectively. These data demonstrate enhanced sensitivity to cytarabine in cells expressing mutant DNMT3A is due to increased susceptibility to DNA damage during replication, rather than defects in double-strand DNA break repair. In support of this, cells with mutant DNMT3Awere characterized by accentuated replication stress as evidenced by high levels of phospho-RPA, which persisted after drug wash-out (Figure 1F). Consistently, DNMT3A-mutant cells treated with cytarabine were characterized by a higher number and a larger area of PCNA foci. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that while the overall kinetics of replication restart remained unchanged, cells with DNMT3A(mut) showed higher rate of fork collapse and increased reliance on latent replication origins (Figure 1G). Gene expression profiling by RNA-seq identified dysregulation of pathways associated with cell cycle progression, specifically G1/S phase transition, DNA replication, DNA integrity checkpoint, and chromatin. Our studies show cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication and suggest that cytarabine-induced replication fork stalling may further synergize with other agents aimed at DNA damage and replication. Figure 1 Disclosures No relevant conflicts of interest to declare. </jats:sec

PRC2 Inhibitors Overcome Glucocorticoid Resistance Driven by NSD2 Mutation in Pediatric Acute Lymphoblastic Leukemia.

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. While H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1/(GR) promoter. Pre-treatment of NSD2 p.E1099K cell lines and PDX 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 pro-apoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation