ALBERT

Description: Background: L-asparaginase, an important component of ALL therapy, hydrolyzes the nonessential amino acid asparagine, depleting plasma levels and selectively killing leukemic lymphoblasts that are asparagine autotrophs. L-asparaginases are immunogenic and can induce hypersensitivity reactions; high neutralizing antibody titers may limit their therapeutic effect. The inability to receive asparaginase secondary to hypersensitivity has prognostic implications for patients with ALL and has been associated with significantly worse outcomes (Silverman LB, et al. Blood 2001;97:1211-1218; Gupta S, et al. J Clin Oncol. 2019;37[suppl]: Abstract 10005). Alternative preparations are needed to ensure that all patients unable to receive E. coli-derived asparaginase due to hypersensitivity are able to receive adequate treatment. RC-P is a recombinant crisantaspase. Due to the use of a novel Pseudomonas fluorescens technology expression platform, RC-P has no immunologic cross-reactivity to E. coli-derived asparaginases. In a study of RC-P administration in healthy adults (JZP458-101), the enzyme was well tolerated and maintained adequate (≥0.1 IU/mL) serum asparaginase activity (SAA), a surrogate marker for asparagine depletion, for up to 72 hours. Study Design and Methods: This is an open-label, multicenter, dose confirmation and pharmacokinetic (PK) study (JZP458-201) of RC-P in patients with ALL or LBL who develop allergic reactions to an E. coli-derived asparaginase and have ≥1 dose of E. coli-derived asparaginase remaining in their treatment plan (Table). For these patients, 6 doses of RC-P will be substituted for each dose of long-acting E. coli-derived asparaginase. Individual patient treatment duration will vary depending on the number of E. coli-derived asparaginase doses that remain in the patient's original treatment plan. The study will consist of 2 sequential parts: Part A will determine the dose of RC-P for intramuscular (IM) administration and confirm safety and efficacy; Part B will define the optimal dose and schedule of intravenous (IV) RC-P. Blood samples will be collected at prespecified time points to determine SAA levels, and patients will be monitored for adverse events. Immunogenicity of RC-P treatment will also be assessed. The primary objectives are to (1) determine the efficacy of IM RC-P administration measured by the last 72-hour nadir SAA (NSAA) level being ≥0.1 IU/mL during the first course of treatment, and (2) assess the safety and tolerability of IM RC-P in patients with ALL/LBL who are hypersensitive to E. coli-derived asparaginases. Secondary objectives include determination of the efficacy of IM RC-P measured by the last 48-hour and last 72-hour NSAA levels being ≥0.4 IU/mL during the first course, characterization of PK of IM RC-P using a population PK approach, and assessment of immunogenicity following repeat administration of RC-P. Exploratory objectives include determination of the efficacy, safety, PK, and immunogenicity of IV RC-P. Disclosures Raetz: Pfizer: Research Funding. Lin:Jazz Pharmaceuticals: Employment, Equity Ownership. Zhu:Jazz Pharmaceuticals: Employment, Equity Ownership. Kim:Jazz Pharmaceuticals: Employment, Equity Ownership. Chandula:Jazz Pharmaceuticals: Employment, Equity Ownership. McClung:Jazz Pharmaceuticals: Employment, Equity Ownership. Gray:Jazz Pharmaceuticals: Employment, Equity Ownership. Choi:Jazz Pharmaceuticals: Employment, Equity Ownership. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Adamson:Pfizer: Research Funding; Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Adaptive Biotechnologies: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amneal Pharmaceuticals: Equity Ownership; Allergan: Equity Ownership; Gilad Sciences: Equity Ownership; Medtronic: Equity Ownership; Merck: Equity Ownership, Research Funding; Genentech/Roche: Research Funding; Novartis: Research Funding; Eisa: Research Funding; Celator: Research Funding; Seattle Genetics: Research Funding; United Therapeutics: Research Funding; Sanofi/Aventis: Research Funding; Jubilant Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Bayer: Research Funding; Amgen: Research Funding; AstraZeneca: Research Funding; Cancer Prevention Pharmaceuticals: Research Funding; Astellas Pharma: Research Funding; Lilly: Research Funding; Springworks: Research Funding; Millennium: Research Funding. OffLabel Disclosure: The abstract presents data from an investigational agent.

Description: Background: WT1 is a zinc finger transcriptional regulator and acts as a tumor suppressor gene in various cell types. WT1 mutations are reported in approximately 10% of both adult and pediatric patients with acute myeloid leukemia (AML), and at a lower frequency in patients with myelodysplastic syndome (MDS). Reported mutations consist of insertions, deletions or point mutations, and are thought to alter WT1 DNA-binding ability and result in a loss of function. WT1 mutations are associated with FLT3/ITD mutations in AML, suggesting possible leukemogenic cooperativity, and yet WT1 mutations have been independently associated with treatment failure and a poor prognosis. Recently, a physical interaction demonstrated between WT1 and TET2 suggests a common functional pathway, and explains the mutual exclusivity of these mutations in AML. Despite these observations, the functional contribution of WT1 mutations in hematologic malignancies is not entirely understood. To our knowledge, we are the first to describe here a hematologic phenotype in a WT1 mutant mouse model and in a novel WT1 mutant x FLT3/ITD crossbred mouse model. Methods: Knock-in WT1 mutant mice are heterozygous for missense mutation R394W in the DNA-binding domain, which has been described in cases of human AML. Mice with a heterozygous 18-bp ITD knocked into the FLT3 gene were crossbred with the WT1 mutant mice, and Kaplan-Meier survival analysis was performed across genotypes. CBCs and BM cytospin morphology from moribund mutant mice were compared to wild type controls. To create a transplant model, 2e6 whole BM cells from each genotype were injected into lethally irradiated congenic mice. Competitive transplants were performed by injecting a 1:1 ratio of CD45.1 wild type (control) cells with CD45.2 WT1 mutant or wild type (test) cells into lethally irradiated C45.1 recipients. Results: We noted an expansion of lineage negative cells and various progenitor cell compartments in WT1 mutant (WT1mut) BM relative to wild type (wt); including the megakaryocyte-erythroid progenitor (MEP) compartment. WT1mut BM cells from two-month old mice showed an increased ability to serially replate in methylcellulose culture compared to wt BM cells, demonstrating aberrantly enhanced self-renewal capacity. WT1mut mice demonstrated a trend towards an inferior late survival compared to wt in survival analysis, and several moribund WT1mut mice were found to have anemia and erythrodysplasia. Most ITD mice developed a fatal myeloproliferative neoplasm (MPN), as previously described. Interestingly, double mutant mice (WT1mut+ITD) had an inferior survival compared to ITD (p

Description: Background The WT1 gene encodes for a zinc finger-containing transcription factor involved in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with expression maintained in the kidney and in CD34+ hematopoietic progenitor cells. Inactivating mutations of this tumor suppressor gene are well-described in sporadic Wilms tumor and as germline mutations in Wilms tumor predisposition syndromes. WT1 mutations have been reported in approximately 10% of both adult and pediatric patients with cytogenetically-normal acute myeloid leukemia (CN-AML), and have been associated with treatment failure and a poor prognosis. These reported mutations consist of insertions, deletions or point mutations. Many are frameshift mutations in exon 7, can occur as biallelic double mutations, and result in truncated proteins which may alter DNA-binding ability. Missense mutations in exon 9 have also been identified, and reports suggest that these may act in a dominant-negative manner, resulting in a loss of function. Despite these observations, the functional contribution of WT1 mutations to leukemogenesis is still largely undetermined. Methods/Results We obtained a novel knock-in WT1 mutant mouse model, which is heterozygous for the missense mutation R394W in exon 9, and homologous to exon 9 mutations seen in human AML. We hypothesized that WT1 mutations may have an aberrant effect on hematopoiesis, and specifically, could alter progenitor cell differentiation or proliferation. To investigate this, we collected lineage-negative bone marrow (lin- BM) cells from two-month old WT1 mutant (WT1mut) and wild-type (wt) mice. We performed methylcellulose colony-forming assays, serially replating cells every 10-12 days. Strikingly, WT1mut progenitor cells showed higher in vitro colony-forming capacity and an increased ability to serially replate, suggesting aberrantly enhanced self-renewal capability. Furthermore, WT1mut colonies from secondary and tertiary passages were larger and more cohesive than wild-type colonies, demonstrating increased proliferation and morphology consistent with blast colony-forming units (CFU-blast). Flow cytometric analysis of these WT1mut cells at tertiary replating revealed an immature, largely c-Kit+ population. Next, in order to study the effects of WT1mut on HSCs in vivo, we performed serial competitive transplantation of HSC-enriched, lineage-depleted BM into lethally irradiated mice. At 14 weeks post-transplant, the donor bone marrow cells were harvested and analyzed by flow cytometry. We observed a significant expansion of the LT-HSC compartment in the WT1mut mice compared to wild-type mice. These data provide new insight into the biology and functional role of WT1 mutations in the aberrant regulation of hematopoietic stem and progenitor cell expansion. Conclusion Oncogenic WT1 mutations confer enhanced proliferation and renewal of myeloid progenitor cells in vitro and expansion of LT-HSCs in vivo. Our findings suggest that WT1 mutations enhance stem cell self-renewal, potentially priming these cells for leukemic transformation upon acquisition of cooperative events. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Patients who harbor the Philadelphia (Ph+) chromosome t(9;22) translocation account for approximately 20-30% of adult ALL and 2-5% of pediatric ALL. Prior to approval and use of imatinib, a small molecule TKI which targets the Ph+ chromosome BCR-ABL1, these patients had poor survival & EFS - with long term survival rates in the 20% range. With the addition of imatinib and later generation TKIs to chemotherapy backbones and bone marrow transplant, EFS & survival rates have substantially improved - surpassing 50% in studies in adults and even higher in children. However, resistance to imatinib and other TKIs has become a significant problem in Ph+ ALL, especially in adults. ABL1 kinase domain mutations are the dominant form of TKI resistance, however other resistance mechanisms include upregulation of parallel pathways such as SRC family kinases, MAPK and BCL6 pathways. BCL6 is an oncogene that suppresses transcription of tumor suppressor genes such as p53 and CDNK1A. Interestingly, BCL6 has been shown to be upregulated and activated through deacetylation following imatinib treatment in Ph+ ALL, likely leading to its role in resistance. Histone deacetylase inhibitors (HDACi) have been shown to act synergistically with TKIs in imatinib sensitive and resistant Ph+ leukemia though multiple mechanisms including attenuation of BCR-ABL1 levels and other downstream proliferation promoting pathways. We have shown that HDACi treatment acetylates (and thus inactivates) BCL6 in Ph+ ALL, and that the combination of HDACis and TKIs leads to synergistic effects in vitro and in vivo (using xenograft models). Methods: In vitro WST-1 cell viability assays were carried out on TOM1 cells (non-ABL1 mutant, imatinib sensitive Ph+ ALL) and NALM1 cells (non-ABL1 mutant, imatinib resistant CML lymphoid blast crisis) with imatinib and entinostat (a HDACi). Synergy was assessed using Calcusyn software. Western blots were performed assessing BCL6 expression and acetylation, and expression of downstream effectors of apoptosis. Two separate in vivo xenograft mouse experiments were performed transplanting TOM1 and NALM1 cells into Nod SCID Gamma (NSG) mice. Cohorts of TOM1 mice were treated with imatinib 50mg/kg BID, entinostat 15mg/kg QD, imatinib plus entinostat combination, or vehicle control. In the NALM1 mice we added a higher dose imatinib cohort (100 mg/kg BID) due to known imatinib resistance. Results: In vitro, there was substantially more synergy of the imatinib/entinostat combination in imatinib-resistant NALM1 cells vs. the imatinib-sensitive TOM1 cells. Average Combination Index (CI) values in TOM1 cells across multiple entinostat and imatinib doses was 1.2 (CI: =1 suggest additive effect, 1 = antagonism), while the CI in NALM1 cells at the same dose combinations was 0.53. We noted BCL6 upregulation and decreased BCL6 acetylation - signs correlating with resistance - in Western blots of NALM1 and TOM1 cells treated with imatinib, while exposure to entinostat caused increased acetylation of BCL6 and increased expression of downstream tumor suppressors. In the imatinib-sensitive TOM1 xenograft trial, the combination displayed a significant reduction in bone marrow leukemic blast involvement versus control following 6 weeks of dosing as measured by flow cytometry (36.9% mean decrease, p=0.001). There was a trend toward decreased bone marrow involvement between the combination treatment and other active treatment arms. There was no difference in peripheral blood blast percentage between arms. In the imatinib-resistant NALM1 xenograft trial, the combination showed a significant decrease in peripheral blood blast percentage in the combination arms versus all other arms after only two weeks of therapy (p=0.0008). Conclusions: Upregulation of activated BCL6 is a known mechanism of resistance in Ph+ ALL that may be abrogated by acetylation of BCL6 with HDACi, as our in-vitro data suggests. Further, we have shown in xenograft models of Ph+ acute lymphoblastic leukemia that combination therapy with HDACi + imatinib, even in imatinib-resistant leukemia, has significant activity. Interestingly, the combination appears more active in resistant disease than in imatinib-sensitive disease. This combination could prove a viable strategy to attenuate imatinib- (and perhaps other TKI-) resistance in Ph+ ALL relapse, particularly in cases not driven by ABL1 kinase domain mutations. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Acute myeloid leukemia (AML) comprises about 18% of childhood leukemias, with an incidence of 7.7 cases per million in the United States. The evidence for variation in disease distribution by race and ethnicity is limited, although there is a slight increased risk for the promyelocytic subtype in Hispanics (Puumala et al, Pediatr Blood Cancer, 2014). In earlier treatment eras (pre-2002), the Children's Cancer Group reported Hispanics with AML to have inferior overall survival (OS) when compared with non-Hispanics (Lange et al, Blood, 2008; Aplenc et al, Blood, 2006), but their event-free survival did not differ significantly. According to recent SEER data, both Hispanic children and adults with AML demonstrated similar OS disparities (Hossain et al, Cancer Epidemiol, 2015; Patel et al, Am J of Clin Oncol, 2015), despite the fact that age at presentation and cytogenetic features were more favorable in Hispanics compared with non-Hispanics. In order to better understand the impact of Hispanic ethnicity upon AML outcomes, we examined relapse-free survival (RFS) and OS in children diagnosed with AML at Texas Children's Hospital (TCH), which has a large Hispanic population, and compared host, disease, and treatment factors that may have affected outcomes. Methods: We retrospectively reviewed medical records from children (age 0-21 years) with newly diagnosed AML treated at TCH between 1998 and 2015. Subjects with acute promyelocytic leukemia or therapy-related AML were excluded. Self-reported race and ethnicity were used to categorize the study population into Hispanics (of any race) and non-Hispanics. Differences in proportions of host, disease, and treatment characteristics between the two groups were compared using Pearson's X2 test. The Kaplan-Meier method was applied to estimate RFS and OS. We then used the Wilcoxon-Breslow-Gehan test to determine if survival functions (RFS and OS) were statistically different by ethnicity, adjusting for treatment era (pre-vs, post 2002). RFS was defined as time from the date of diagnosis until date of relapse. Patients without an event were censored at the date of last known contact. Research was performed under a local Institutional Review Board-approved protocol and in accord with the Declaration of Helsinki. Results: Of the 99 AML cases with available clinical information, 37 (37%) self-identified as Hispanic. Host, disease, and treatment factors in Hispanic and non-Hispanic subjects with AML did not differ according to prognostic factors such as age at diagnosis or favorable cytogenetic features (Table 1). Additionally, Hispanics and non-Hispanics did not differ significantly in cause of death (disease-related or other). The groups did not differ significantly in OS, but Hispanics had significantly poorer RFS (p=0.03) (Figure 1). Conclusions: Despite no significant differences in frequency of known AML risk factors, the TCH Hispanic population was both significantly more likely to relapse and had an earlier time to relapse than did non-Hispanics. This effect was even more surprising given that this population was twice as likely to have AML characterized by favorable cytogenetic features, although this enrichment did not reach significance. Of note, the RFS difference we observed is unlikely to be related to treatment compliance or socioeconomic factors, as all AML patients were hospitalized throughout treatment. Further study is needed to confirm this finding in a larger pediatric AML cohort, and to identify host factors related to Hispanic ancestry that may be responsible for the differences observed in RFS. Figure 1. Patient characteristics *Number of subjects in each category is shown in parentheses unless otherwise specified. SD=Standard deviation, CBF = core binding factor, MRD = minimal residual disease, BMT = bone marrow transplant Figure 1. Patient characteristics. / *Number of subjects in each category is shown in parentheses unless otherwise specified. / SD=Standard deviation, CBF = core binding factor, MRD = minimal residual disease, BMT = bone marrow transplant Figure 2. Comparison of AML relapse-free survival in Hispanics vs. non-Hispanics, adjusted for treatment era Figure 2. Comparison of AML relapse-free survival in Hispanics vs. non-Hispanics, adjusted for treatment era Disclosures No relevant conflicts of interest to declare.

Description: Mutations of the DNA methyltransferase, DNMT3A, occur in approximately 20% of adult patients with acute myeloid leukemia (AML), and portend a poor prognosis. The most common of these mutations results in a dominant negative loss of function. Our lab observed that upon conditional inactivation of Dnmt3a in the murine hematopoietic system, Dnmt3a–/– hematopoietic stem cells (HSCs) expanded dramatically while their differentiation was inhibited, consistent with a pre-leukemic state. The likely mechanism by which Dnmt3a loss contributes to leukemogenesis is altered DNA methylation and the attendant gene expression changes, however our current understanding is incomplete. In analyses of gene expression data, we observed that murine Dnmt3a–/– HSCs markedly overexpress the histone 3, lysine 79 (H3K79) methyltransferase, Dot1l. This is of interest given the known functional interplay between DNA methylation and histone modifications. Additionally, DOT1L plays a critical role in leukemia with MLL-rearrangements, lesions that essentially never occur concomitantly with DNMT3A mutations in AML. The mutual exclusion of these lesions combined with the observed overexpression of Dot1l in our murine model, led us to postulate that MLL-rearrangements and DNMT3A mutations are distinct epigenetic aberrations that converge on a common mechanism resulting in dysregulated gene expression, specifically mediated by H3K79 methylation (H3K79me). Therefore, in the pathogenesis of DNMT3A-mutant AML, like in MLL-rearranged leukemia, DOT1L-induced H3K79me may play a central role, and may represent a viable therapeutic target. Throughout the genome of normal HSCs, expansive regions with low DNA methylation (canyons) exist. These canyons span conserved domains frequently containing transcription factors. In our Dnmt3a-/- model, canyon borders, particularly flanking genes frequently dysregulated in human leukemia such as HOX genes, are highly prone to DNA methylation loss when Dnmt3a is deleted, resulting in canyon expansion. However, not all canyons expand with Dnmt3a loss. We found a close association between canyon behavior and the associated histone marks, with expanding canyons characterized by a lack of the repressive histone mark, H3K27me. To determine if in Dnmt3a-mediated malignant hematopoiesis, H3K79me also correlates with altered DNA methylation, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) for H3K79 di-methylation (H3K79me2) and aligned these data with whole genome DNA methylation data. This revealed that H3K79me2 specifically coats canyons that lose methylation with Dnmt3a loss, including the HoxA and HoxB clusters, but is not present at canyons without methylation loss. This strong correlation between H3K79me and DNA hypomethylation with Dnmt3a loss suggests a functional interaction. To examine whether this also occurred in human samples with DNMT3A mutations, we analyzed TCGA data, which confirmed many canyon borders as regions with marked DNA methylation loss. Further, many canyon-associated genes, including HOX genes are significantly changed in human DNMT3A-mutant AML. To explore the role of H3K79me, and specifically of DOT1L in human DNMT3A-mutant AML, we utilized the DNMT3A-mutant human AML cell lines OCIAML2 and OCIAML3. These cell lines were found to have increased total H3K79me compared to DNMT3A-wild type controls, consistent with the increased Dot1l expression in Dnmt3a–/– HSCs. We then tested the in vitro efficacy of two selective DOT1L inhibitors, SYC-522 (Anglin. J Med Chem. 2011) and the Epizyme compound, EPZ004777 (Daigle. Cancer Cell. 2011), against DNMT3A-mutant cells. Both compounds led to a dose- and time-dependent inhibition of proliferation and induction of apoptosis in the DNMT3A-mutant cell lines at concentrations comparable to those used for MLL-rearranged cell lines. With treatment, DNMT3A-mutant cells also had evidence of induction of differentiation with increased expression of the mature monocyte marker, CD14. Importantly, oncogenic HOX genes overexpressed in DNMT3A-mutant AML were repressed in a time-dependent fashion with DOT1L inhibitor treatment. In conclusion, our data suggest that DOT1L may be a novel, immediately actionable therapeutic target for the treatment of DNMT3A-mutant AML. Disclosures Rau: Epizyme: Honoraria.

Description: There is a critical need for new agents with novel therapeutic targets and improved safety profiles in high-risk acute lymphoblastic leukemia (ALL), which is a significant cause of morbidity and mortality in pediatric and adult populations. Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of their mechanistically relevant targets remains a major experimental challenge. We applied a chemical genetics approach involving sequential unbiased high-throughput chemical and ultra-complex, genome-scale shRNA screens to address this challenge and identify novel agents in ALL. A cell-based phenotypic high-throughput chemical screen of 115,000 compounds identified 640 compounds that inhibited growth of one or both ALL cell lines with high-risk Mixed Lineage Leukemia (MLL) genetic abnormalities, but did not inhibit the growth of a cell line lacking MLL rearrangement. The most potent and selective 64 were tested on an expanded panel of eight human B-ALL cell lines to identify lead compound STF-118804. STF-118804 inhibited the growth of most B-ALL cell lines with high potency demonstrating IC50 values in the low nanomolar range. Leukemic samples from five pediatric ALL patients were also sensitive to STF-118804 in the low nanomolar range. STF-118804 displayed 5–10 fold more potency against most leukemias in comparison to cycling human (lineage-negative cord blood) and murine (c-kit+ bone marrow) progenitor cells, demonstrating a therapeutic index. STF-118804 displays distinctive cytotoxicity by inducing apoptosis without causing a phase-specific cell cycle arrest. To discover the molecular target of STF-118804, a functional genomic screen was performed to identify shRNAs that conferred sensitivity or resistance to STF-118804, utilizing an ultra-complex (∼25 shRNAs per gene) library targeting in total ∼9300 human genes and 1000s of negative control shRNAs. NAMPT was the most statistically significant gene to confer sensitivity to STF-118804, suggesting that STF-118804 functioned as a NAMPT inhibitor. NAMPT encodes nicotinamide phosphoribosyl transferase, a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), a crucial cofactor in many biochemical processes. STF-118804 was confirmed as a novel class of NAMPT inhibitor through metabolic rescue, enzymatic, and genetic studies. STF-118804 displayed strong inhibitory activity in in vitro NAMPT enzymatic assays. Over-expression of wild-type or mutant NAMPT in cells indicated that STF-118804 cytotoxicity is a result of its ability to inhibit NAMPT, and that STF-118804 does not have significant off-target effects on cell viability. The potential efficacy of STF-118804 in vivo was assessed in an orthotopic xenograft model of ALL. Sublethally irradiated immunodeficient mice were transplanted with human ALL cells engineered to constitutively express firefly luciferase. Dosing of STF-118804 was initiated two weeks post-transplant when ALL cells had engrafted and bioluminescent signal was detectable. Mice treated with STF-118804 showed regression of leukemia by bioimaging and significantly extended survival. The leukemia initiating cell (LIC) frequency in STF-118804 treated mice was significantly lower (∼8 fold) than vehicle treated mice, showing that STF-118804 was effective in reducing LICs. In summary, tandem high-throughput screening identified a highly-specific, potent, and structurally novel small molecule inhibitor of NAMPT that is active in ALL. Tandem high throughput screening using chemical and ultra-complex shRNA libraries provides a rapid chemical genetics approach for seamless progression from small molecule lead identification to target discovery and validation. Disclosures: No relevant conflicts of interest to declare.

Description: The de novo DNA methyltransferase (DNMT) 3A is mutated in 50% of patients with mixed phenotype acute leukemia, 20% with acute myeloid leukemia (AML) and 18% with T-cell acute lymphoblastic leukemia (T-ALL). The mechanisms through which mutant DNMT3A contributes to hematologic malignancy are poorly understood. In mice, deletion of Dnmt3a in hematopoietic stem cells (HSCs) leads to abnormal DNA methylation and inhibition of differentiation, but is insufficient for leukemic transformation. To study the role of Dnmt3a in leukemia, we combined Dnmt3a-deletion with the activated FLT3 proto-oncogene (FLT3-ITD), a frequent co-mutation with DNMT3A in AML patients, to establish a murine model of Dnmt3a-associated malignancy. In mice transplanted with Dnmt3a-knockout (KO) or wild-type (WT) bone marrow cells transduced with a FLT3-ITD retrovirus, Dnmt3a-loss dramatically impacted the disease phenotype. Dnmt3aKO/ITD transplanted mice had significantly shortened survival (79 days vs. 116 days) and increased rate of acute leukemia compared to mice with ITD alone. The mice developed CD4+CD8+ Notch activation-associated T-ALL or myeloproliferative disease (MPD), or concurrently both, consistent with previous studies of FLT3-ITD in mice. To determine the leukemia-initiating population, we transplanted sorted HSC, myeloid, and lymphoid progenitors transduced with FLT3-ITD. All mice transplanted with HSC and myeloid progenitors succumbed to both malignancies. To uncover the mechanisms by which Dnmt3a-deletion accelerated acute leukemia, we analyzed changes in DNA methylation in T-ALL blasts by whole genome bisulfite sequencing. Compared to Dnmt3aWT/ITD, Dnmt3aKO/ITD blasts exhibited global hypomethylation, particularly at distal enhancer sites. These hypomethylated enhancer sites were associated with genes in signaling pathways, transcription regulators, and metabolic pathways in cancer (KEGG and GO Analysis). Transcriptome analysis showed that relative to Dnmt3aWT/ITD, the Dnmt3aKO/ITD blasts had 1577 significantly differentially expressed genes positively related to cancer, cellular growth, and proliferation, and negatively to apoptosis by Ingenuity Pathway Analysis (IPA). Surprisingly, we observed increased expression of genes related to HSCs and myeloid function and decreased expression of genes related to lymphocyte function. Human AML signature genes (Oncomine) were also upregulated in our mouse model. Predicted activated pathways include Myc, Nfe2l2, Eif4e, E2f1, Csf2, Cebpb, Vegf, Rxra, Ezh2, and Brd4 and inhibited pathways include tumor suppressors Rb, let7, Cdkn2a, and Tob1 (IPA). We did not observe changes in genomic copy number variation by chromosomal comparative hybridization (cCGH). To test whether Dnmt3a-deletion could functionally bestow stem cell properties on pre-leukemic cells, we examined self-renewal capabilities of malignant cells of Flt3+/ITD knock-in mouse (an ITD mutation knocked in to the endogenous murine Flt3 allele causing MPD). Remarkably, when Dnmt3aKO; Flt3+/ITD bone marrow cells were serially transplanted, MPD was seen in all recipients, compared to none in Dnmt3aWT; Flt3+/ITD transplanted mice (n=7). Further, we transplanted sorted CLP, CMP, GMP, MPP, ST-HSC, LT-HSC populations and observed myeloproliferation in transplanted non-stem (CMP, GMP, ST-HSC) and stem cell (LT-HSC) populations. This strongly suggests that Dnmt3aKO synergized with Flt3-ITD to confer stem cell self-renewal abilities to transformed progenitor and stem cells. Increasingly, decitabine is being used to treat patients with AML and MDS, but whether patients with DNMT3A mutations could benefit is unclear, so we examined the impact of decitabine treatment on the retroviral transduced Dnmt3aKO/ITD mice. Monthly treatment led to significantly increased survival of Dnmt3aKO/ITD mice from T-ALL and MPD and reduced presence of ITD-transduced KO cells. Together, we demonstrate that Dnmt3aKO accelerated malignancies induced by FLT3-ITD in mouse and may shed light on how DNMT3A mutations contribute to lymphoid and myeloid disease in patients. Dnmt3a deletion ignited multilineage and stem cell programs at the expense of lymphoid programs to accelerate disease, but was extinguishable by decitabine therapy. The findings from our mouse model can be used for the development and testing of targeted epigenetic therapy for DNMT3A-associated malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Juvenile myelomonocytic leukemia (JMML) is a rare mixed myelodysplastic/myeloproliferative neoplasm seen in early childhood. JMML in general is a poor prognosis disease, including risk of transformation to acute leukemic blast crisis in approximately 15% of patients. Like adult chronic myeloid leukemia (CML) the blast phase of JMML is typically myeloid, however B cell and T cell transformations have been reported. Interestingly, in CML, a minority of patients will have a small aberrant B-lymphoblast population that does not inevitably herald progression to B-lymphoblastic blast crisis, however this has not been reported in JMML. Here, we report a case of a child with JMML found to have an aberrant population of precursor B lymphoid blasts. An 11-month-old boy presented to our clinic with splenomegaly and complete blood count (CBC) findings of a low platelet count of 69 x103/UL, low absolute neutrophil count of 0.61 X103/UL and elevated monocytes (36.3%), with an absolute monocyte count of 3.2 x 103/UL. Bone marrow aspirate and biopsy were consistent with JMML with no abnormal blast population reported at the time. Karyotyping confirmed the presence of monosomy 7, with concurrent fluorescent in-situ hybridization (FISH) evaluation showing monosomy 7 in 74% of cells examined. Next generation sequencing on the aspirate revealed a KRAS p.G12A activating mutation at an allele frequency of 35%, further validating the diagnosis of JMML. The patient did not have a matched related or unrelated donor available, thus he was followed closely with serial physical exams, blood counts and bone marrow evaluations. His splenomegaly resolved, blood counts remained stable to improved without intervention, but repeat bone marrow 6 months after the diagnosis showed similar frequencies of monosomy 7 and mutant KRAS. With still no suitable donor available, we opted to pursue a trial of hypomethylating agent, 5-azacitidine that has shown promising results in recent reports. A bone marrow evaluation was done prior to starting azacitidine to establish a pre-therapy baseline. Surprisingly, we were halted by detecting a B lymphoid blast population (6%), with characteristic flow findings, including positive CD45 (dim), CD19, CD10, CD20 (partial), CD22 (dim), CD34, HLA-DR, CD52, CD99, CD58 (heterogeneous) and CD38. Additionally, a new partial CDKN2A deletion was detected by FISH in 10% of cells. These findings were concerning for an emerging precursor B-acute lymphoblastic leukemia, possibly driven by the new CDKN2A deletion. However, we flow sorted the aberrant B cell population from the marrow aspirate, which showed the partial CDKN2A deletion in only 1.25% of these cells, unlike monosomy 7, which was seen in 100% of the abnormal B cell population. A retrospective review of the flow data from the marrow 4 months prior revealed the same aberrant B cell population in a similar percentage (5%) that was originally attributed to hematogones with low CD45 expression. The stability of this population over the course of months was inconsistent with an evolving lymphoid blast crisis. We observed the patient closely, and his blood counts and clinical exam remained stable over the following weeks. A repeat bone marrow showed that the abnormal B-cell population had decreased slightly to 2.6% and the partial CDKN2A deletion was now seen in only 0.4% of the examined cells. Thereafter, treatment with azacitidine (5 daily doses-100mg/m2/dose) was initiated with an excellent response after one cycle, with monosomy 7 detected in only 12% of cells and KRAS allelic burden down to 4.3%. Additionally, the aberrant B cell population declined to 0.15% and partial CDKN2A deletion was not detected by FISH. While longer follow up and additional patients will be necessary to reach definitive conclusions, our case suggests that the immunophenotypic detection of a small, stable abnormal B-lymphoblast population in patients with JMML does not necessarily herald impending acute leukemia. Disclosures No relevant conflicts of interest to declare.

Description: In B lymphoblastic leukemia (B-ALL), genome-wide association studies have revealed that deletions and mutations of the gene IKAROS family zinc finger 1 (IKZF1) are present in nearly 30% of patients. These lesions are most prevalent in high-risk subsets, including greater than 60% of patients with Philadelphia chromosome positive (Ph+) and Ph-like ALL. IKZF1 deletions are associated with an increased risk of relapse, therapy resistance, and inferior survival. It is therefore imperative to devise new treatment strategies for this poor-prognosis subset of patients. To this regard, using novel CRISPR-Cas9 genome editing strategies, we developed a series of human B-ALL cell lines with IKZF1 deletions. These robust model systems have allowed us to investigate the underlying biology of IKZF1-deleted B-ALL. Our studies have thus far shown that IKZF1 deletion results in a stem cell-like gene expression profile, enhanced bone marrow homing and engraftment, and cell-intrinsic chemoresistance, consistent with the relapsing disease phenotype observed in affected patients. We are using these model systems to explore possible mechanisms of chemoresistance and delineate new strategies to improve response to therapy. Global gene expression analysis of the engineered Nalm-6 IKZF1-deleted cells by RNA-seq revealed potential therapeutic vulnerabilities. IKZF1-deleted cells are characterized by increased activation of the JAK/STAT pathway with overexpression of JAK1, JAK3, STAT3, and STAT5. Aberrant activation of this pro-survival, anti-apoptosis pathway is associated with poor-prognosis leukemia; thus, we postulated this is a contributor to the chemoresistance inherent to IKZF1-deleted B-ALL. We explored the therapeutic potential of targeting the JAK/STAT pathway by treating IKZF1-deleted cells with selective inhibitors of JAK1/3 (tofacitinib) and STAT3 (MM-206) and calculated the IC50 by Annexin V/7-AAD double-negative population after 48 to 72 hours of treatment. The IKZF1 wild-type cells were sensitive to both compounds, suggesting activated JAK/STAT signaling is critical to cell survival. In comparison, the IKZF1-deleted cells were relatively resistant to both compounds (MM-206 IC50 : 5.6 µM vs. 8.2 µM, p 〈 0.001; tofacitinib IC50 : 43 nM vs. 55 nM, p = 0.05) similar to the relative resistance to ABL1-tyrosine kinase inhibition observed in Ph+ B-ALL cells with loss of function IKZF1 mutations. However, we postulated that inhibition of the JAK/STAT pathway could still augment the effects of standard chemotherapy. Indeed, whereas IKZF1-deleted Nalm-6 cells are highly resistant to glucocorticoid chemotherapy alone, when the cells were also treated with sub-IC50 levels of MM-206, we observed a significant re-sensitization to dexamethasone-induced apoptosis. A similar pattern of re-sensitization was seen with the combination of sub-IC50 MM-206 and vincristine treatment. Additionally, our gene expression analysis of the IKZF1-deleted Nalm-6 cells revealed significantly increased expression of the receptor tyrosine kinase, FLT3. Overexpression was confirmed at the protein level by flow cytometry for cell-surface FLT3. We treated our engineered cell lines with the potent and selective FLT3 inhibitor quizartinib and again found that the IKZF1-deleted cells were relatively resistant compared to the wild type cells (IC50 : 240 nM vs. 282 nM, p 〈 0.01). Postulating that parallel activation of the JAK/STAT pathway may contribute to this resistance, we treated our cells with pacritinib, a combined JAK/FLT3 inhibitor. The IKZF1-deleted cells were as sensitive to this compound as the wild type cells, suggesting dual targeting of FLT3 and JAK may be efficacious for the treatment of IKZF1-deleted B-ALL. Our data support that IKZF1-deleted B-ALL is an aggressive disease characterized by cell-intrinsic chemoresistance. We found that loss of IKAROS amplifies pro-survival, anti-apoptotic signaling pathways, a likely contributing mechanism to chemoresistance. IKZF1 deletion confers relative resistance to targeted inhibitors of these pathways. However, the combined JAK/FLT3 inhibition exhibits therapeutic efficacy. Additionally, the combination of a JAK/STAT pathway inhibitor with conventional chemotherapy including dexamethasone and vincristine may be a promising strategy to overcome the chemoresistance inherent to this poor-prognosis subset of B-ALL. Disclosures No relevant conflicts of interest to declare.