ALBERT

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: 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.