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: 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: Abstract 145 NPMc+ mutations occur in up to 60% of adult and 20% of childhood acute myeloid leukemia (AML) with normal karyotype. Flt3 ITD mutations occur in 25% of adult and 15% of childhood AML. Flt3 ITD mutations occur twice as frequently in patients with NPMc+ mutations compared to those who lack a NPMc+ mutation. The presence of Flt3 ITD portends a poor prognosis. NPMc+ is associated with improved outcome, but only in the absence of a concomitant Flt3 ITD mutations. Given the high frequency with which these mutations occur together it is plausible that they cooperate to cause leukemia. However, this has yet to be demonstrated experimentally. To examine this, we crossed mice expressing a knock-in of an 18-bp ITD mutation in the juxtamembrane domain of the murine Flt3 gene (Flt3 wt/ITD) with transgenic mice expressing Flag-tagged type A NPMc+ mutation driven by the myeloid-specific human MRP8 promoter. Flt3wt/ITD mice develop a fatal myeloproliferative disorder (Li L, et al. Blood. 2008;111:3849-58) and NPMc+ transgenic mice develop a non-fatal myeloproliferation (Cheng K, et al. Blood. 2010;115-18), but neither develop leukemia, suggesting that cooperating events are required. Progeny were characterized by: 1) H&E staining of peripheral blood smears, bone marrow (BM) cytospins, and spleen sections; 2) FACS analysis of BM cells, splenocytes and thymocytes to determine the disease phenotype; 3) RT PCR to examine the expression of Flt3 ITD and NPMc+; and 4) immunofluorescence (IF) using an anti-Flag antibody to determine localization of the NPMc+ protein. Mice harboring both mutations develop acute leukemia with a median onset of 285 days (Figure 1). All the leukemic mice exhibit a moribund appearance, leukocytosis (mean WBC 69.3±32.7 vs 11.7±8.3K/μ L in wt/wt mice), splenomegaly (mean weight 0.63±0.3 vs 0.12±0.02g in wt/wt mice), anemia and thrombocytopenia. Four disease phenotypes based on FACS and histologic analysis have been observed (Figure 2). Thirty-three percent develop AML with infiltration of the BM and spleen with Mac1+/Gr1+ myeloblasts. Thiry-three percent develop T cell ALL with infiltration of BM, spleen, thymuses and other organs with abnormal CD3+/CD4+/CD8+ lymphoblasts. An additional 33% develop a mixed phenotype acute T/myeloid leukemia with both Mac1+/Gr1+ myeloblasts and CD3+/CD4+/CD8+ lymphoblasts. One mouse developed an undifferentiated acute leukemia with primitive blasts expressing no markers specific for either lymphoid or myeloid lineage. RT-PCR of bulk leukemia cells demonstrates expression of NPMc+ and Flt3 ITD. IF of BM cells from leukemic mice demonstrates cytoplasmic localization of the NPMc+ protein. In summary, we have utilized a mouse model to demonstrate that NPMc+ and Flt3 ITD mutations cooperate to cause leukemia. Many of the mice with both mutations develop myeloid leukemia with features similar to the human disease. There is also a striking incidence of T cell leukemia, which is particularly interesting as the NPMc+ mutation is driven by the myeloid-specific hMRP8 promotor. It is possible that there is aberrant expression of NPMc+ in lymphoid cells due to position effects of the transgene or the activation of an endogenous leukemogenic retrovirus. Other disease models using this promoter have documented similar phenomenon (Jaiswal, et al. PNAS. 2003;100:10002-7). There is a long latency of disease onset which may be due to a relatively low level of expression of NPMc+ or because additional genetic or epigenetic events are required. Perhaps the Flt3 ITD mutation causes proliferation and NPMc+ impairs DNA repair resulting in the accumulation of additional mutations that contribute to leukemogenesis. This mouse model provides the first in vivo model of NPMc+/Flt3 ITD+ leukemia. The model will allow for the further study of this disease entity, including the examination of involved pathways and the exploration for potential therapeutic targets. Kaplan-Meier Survival Curve. Mice with both NPMc+ and Flt3 ITD mutations have a median survival of 413 days. FACS analysis of leukemic mice BM cells and thymocytes. Mice with AML have cKit+/Mac1+/Gr1+ myelobasts. Mice with T cell ALL have infiltration of the BM, spleen (not shown) and thymus with CD3+/CD4+/CD8+ lymphoblasts. Mice with T/myeloid leukemia have both Mac1+/Gr1+ myeloblasts and CD3+/CD4+/CD8+ lymphoblasts. Leukemic mice have depletion of maturing Ter119+ erythroid cells. Figure 1 Kaplan-Meier Survival Curve. Mice with both NPMc+ and Flt3 ITD mutations have a median survival of 413 days. Figure 1. Kaplan-Meier Survival Curve. Mice with both NPMc+ and Flt3 ITD mutations have a median survival of 413 days. Figure 2 FACS analysis of leukemic mice BM cells and thymocytes. Mice with AML have cKit+/Mac1+/Gr1+ myelobasts. Mice with T cell ALL have infiltration of the BM, spleen (not shown) and thymus with CD3+/CD4+/CD8+ lymphoblasts. Mice with T/myeloid leukemia have both Mac1+/Gr1+ myeloblasts and CD3+/CD4+/CD8+ lymphoblasts. Leukemic mice have depletion of maturing Ter119+ erythroid cells. Figure 2. FACS analysis of leukemic mice BM cells and thymocytes. Mice with AML have cKit+/Mac1+/Gr1+ myelobasts. Mice with T cell ALL have infiltration of the BM, spleen (not shown) and thymus with CD3+/CD4+/CD8+ lymphoblasts. Mice with T/myeloid leukemia have both Mac1+/Gr1+ myeloblasts and CD3+/CD4+/CD8+ lymphoblasts. Leukemic mice have depletion of maturing Ter119+ erythroid cells. Disclosures: No relevant conflicts of interest to declare.

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. WT1 mutations are reported in approximately 10-15% of both adult and pediatric patients with acute myeloid leukemia (AML), and have been associated with treatment failure and a poor prognosis. Reported mutations consist of insertions, deletions or point mutations; and occur primarily in exon 7 or exon 9 of the WT1 gene. These mutations are thought to alter WT1 DNA-binding ability or result in a loss of function. Despite these observations, the functional contribution of WT1 mutations in leukemogenesis is still largely undetermined. Results and Methods: We have shown that transduction and expression of wild type WT1 in murine 32D cells enhances granulocytic differentiation upon treatment with G-CSF, and that expression of mutant WT1 inhibits this effect. To investigate this in a human AML cell model, we transduced U937 cells with the same WT1 vectors. Strikingly, shortly after transduction, U937 cells expressing wild type WT1 spontaneously differentiate towards a mature monocytic phenotype, but U937 cells expressing mutant WT1 do not differentiate and maintain an immature phenotype (Fig A). This relative block in U937 differentiation with mutant WT1 expression was overcome with differentiation-inducing treatment with all-trans retinoic acid (ATRA). These results suggest that mutant WT1 alters the ability of myeloid cells to terminally differentiate. We obtained a novel knock-in WT1 mutant (WT1mut) mouse model that is heterozygous for the missense mutation R394W in exon 9, and homologous to exon 9 mutations observed in human AML. We evaluated cohorts of two-month old mice and noted an expansion of lineage negative cells and various progenitor cell compartments; particularly, the megakaryocyte-erythroid progenitor (MEP) compartment; in WT1mut bone marrow (BM) relative to wild type. We also found that lineage negative WT1mut BM cells from two-month old mice show higher in vitro colony-forming capacity and an increased ability to serially replate in methylcellulose culture compared to wild type BM cells. Flow cytometry of WT1mut cells at tertiary replating revealed an immature, largely c-kit+ population, suggesting an aberrantly enhanced self-renewal capability of myeloid progenitors in WT1mut mice. Furthermore, survival analysis of the WT1mut mice demonstrates inferior survival compared to wild type, and several WT1mut mice were found to have anemia and myelodysplasia. To address the possibility of germ line WT1mut syndromes causing renal failure and anemia, and thereby influencing survival, we transplanted BM from each genotype into lethally irradiated congenic mice. Upon engraftment with donor marrow, the expression of WT1mut is confined to the hematopoietic system in this model. The Kaplan-Meier survival curve, based on absolute age of the BM, shows statistically significant decreased survival of WT1mut BM transplant recipients compared to wild type BM recipients (Fig B). Anemia and dysplasia were also seen in these WT1mut BM recipients; findings that are suggestive of dysfunctional hematopoiesis, and may be secondary to the changes in progenitor cell self-renewal and differentiation we have observed. Conclusions: Leukemogenic WT1 mutations confer enhanced self-renewal of hematopoietic progenitor cells and a block in terminal myeloid differentiation in vitro, which could potentially prime cells for leukemic transformation upon acquisition of cooperative events. Mice with WT1 mutant bone marrow develop anemia and evidence of myelodysplasia, which may contribute to their decreased survival. These data provide new and important insights into the aberrant functional effects of WT1 mutations on hematopoiesis, and are the first to characterize the hematopoietic phenotype of a WT1 mutation in vivo. Figure: (A) U937 cells expressing wild type WT1 spontaneously differentiate, demonstrated here by gain of monocytic markers CD11a and CD11b as measured by flow cytometry, whereas cells expressing mutant WT1 vectors 101 and 126 remain undifferentiated. (B) Mice transplanted with WT1mut bone marrow have inferior survival compared to mice transplanted with wild type bone marrow. Figure:. (A) U937 cells expressing wild type WT1 spontaneously differentiate, demonstrated here by gain of monocytic markers CD11a and CD11b as measured by flow cytometry, whereas cells expressing mutant WT1 vectors 101 and 126 remain undifferentiated. (B) Mice transplanted with WT1mut bone marrow have inferior survival compared to mice transplanted with wild type bone marrow. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2437 Introduction/Background: Wilms tumor 1 (WT1) gene encodes for a zinc finger-containing transcription factor thought to play a role in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with adult expression maintained in kidney cells and early CD34+ hematopoietic progenitor cells. Inactivating mutations of this tumor suppressor gene are well described in sporadic Wilms tumor and as germline mutations in WAGR and Denys-Drash syndromes. Approximately 10% of adult and pediatric patients with cytogenetically normal acute myeloid leukemia (CN-AML) harbor WT1 mutations. Some studies suggest that patients with WT1 mutations may have a worse overall prognosis, particularly in combination with other poor prognostic indicators, such as FLT3ITD mutations. Interestingly, WT1 and FLT3ITD mutations are commonly found together, suggesting they may cooperate to cause AML. Despite these clinical observations, the functional contribution of WT1 mutations to leukemogenesis, both alone and in cooperation with FLT3ITD, is still largely undetermined. Methods/Results: Ba/F3 cells were stably transfected with the WT1 gene, including 3 different engineered mutated vectors: two missense point mutations (WT1mut101 and WT1mut146) and one nonsense insertional mutation (WT1mut126), which produces a truncated protein. These mutations have been described in pediatric patients with acute leukemia. The expression of each WT1 vector was confirmed by PCR and cDNA sequencing. We compared the proliferative rate of WT1 wildtype (WT1wt) to WT1mutated (WT1mut) transfected cells using trypan blue cell counting, and saw an early increased proliferative rate for high-expressing WT1mut compared to WT1wt and WT1 empty vector (EV). To further investigate these differences, we performed cell cycle analysis with propidium iodide (PI) staining. After synchronizing all cell lines by arresting cells in G0-G1 phase, the cells were seeded at equal concentrations and assayed for cell cycle changes at various time points. Interestingly, all WT1mut cell lines consistently showed earlier entry into S phase and therefore a decreased G1/S ratio at 24 hrs after synchronization, compared to EV and WT1wt (Fig A). This suggests that the “check-point” controlling entry into S phase is altered in cells expressing WT1 mutations, which manifests as a proliferative advantage in these cells. Next, we stably transfected Ba/F3 cells with a FLT3ITD construct, and co-transfected additional cells with both WT1mut and FLT3ITD constructs. All cells were synchronized by arresting in G0-G1 phase, achieved with 24 hrs of serum and cytokine starvation and treatment with 20nM of CEP-701 (ensuring G0-G1 arrest of cells with FLT3 vectors). The cells were then washed and re-suspended in serum and cytokine-containing media, and seeded at equal concentrations. We observed that FLT3ITD cells and WT1mut126 cells had decreased and nearly equivalent G1/S ratios at early time points compared to EV and WT1wt cells, conferring similar early proliferative advantages for cells with these mutations. Interestingly, we found that cells co-transfected with both mutations enhanced this effect: the absolute number of WT1mut126+FLT3ITD cells undergoing transition into S phase was increased and this effect was seen at an earlier time point compared to WT1mut126, FLT3ITD, WT1wt or EV cells (Fig B). Conclusions: The functional and contributory role of WT1 mutations in leukemogenesis has yet to be characterized. Our preliminary in vitro data with a cell line transfected with WT1 vectors suggests that cell cycle regulation, and therefore proliferation, is aberrant in cells expressing mutated WT1. Consistent with previous reports, our data reaffirms a proliferative advantage in cells transfected with FLT3ITD mutations. We showed that these cells transition to S phase at an early time point, conferring an early proliferative advantage, and do so at an equivalent rate and time to WT1mut cells. In addition, we found that cells co-transfected with both WT1mut126+FLT3ITD demonstrated an earlier and more pronounced entry into S phases compared to either individual mutation alone. These observations deserve further investigation, as they may help explain how mutated WT1 contributes to the initiation and progression of leukemia, and how WT1 and FLT3ITD mutations may cooperate in leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1410 Background: Activation of CXCR4 by the chemokine SDF-1 (CXCL12) results in the migration of leukemia cells to marrow niches that may contribute to chemoresistance and relapse. We previously showed that in vitro chemotherapy (chemo) treatment modulates CXCR4 expression in leukemia cell lines and primary pediatric AML samples, and that chemo-induced increases in surface CXCR4 (s-CXCR4) results in increased chemotaxis toward an SDF-1 gradient and decreased chemo-induced apoptosis when co-cultured with human marrow stroma feeder layers. We hypothesized that 1) CXCR4 inhibition by plerixafor (P) would sensitize leukemias to chemo through the interruption of leukemia-stromal cell signaling and 2) the degree of chemo-induced s-CXCR4 upregulation would be a predictive biomarker of the efficacy of P as a chemosensitizer. Because B-precursor ALL are known to highly express CXCR4, we tested these hypotheses in vitro using ALL cell lines and in vivo using a xenograft model of a high-risk pediatric leukemia, infant ALL. In Vitro Methods/Results: ALL cell lines were pretreated for 72 hours with araC (A), dauno, vcr, and vehicle control (C). Chemo pretreatment induced upregulation of s-CXCR4 compared to C. Viable cells were then isolated using Ficoll and plated off stroma (O), on stroma (S), or pretreated with P for 30 minutes prior to plating on stroma (P+S). Cells were then treated for an additional 72 hours with full dose ranges of chemo. Apoptosis was measured with Annexin V/7-AAD, and IC50 was calculated. Overall, IC50 values were highest in S, followed by P+S, then O, demonstrating that upregulation of s-CXCR4 leads to stromal protection, and that stromal protection is diminished by treatment with P. Cells with higher levels of s-CXCR4 upregulation had greater differences between S IC50 and O IC50, compared to cells with lower s-CXCR4 upregulation, suggesting that the degree of s-CXCR4 upregulation is predictive of the degree of stromal protection. Cells with higher s-CXCR4 upregulation also had greater differences between S IC50 and P+S IC50, suggesting that P diminishes stromal protection more effectively in leukemias that highly upregulate s-CXCR4 in response to chemo. Xenograft Methods/Results: Infant ALL patient samples were transplanted into sublethally irradiated NOG mice. After 3 weeks, we treated cohorts (n=5) with single doses of P, A, P followed by A 4 hours later (P+A), or C. We dosed A below the maximal tolerated dose (MTD) to facilitate assessment of P+A synergy. Mice were sacrificed 4 weeks post treatment and cells were isolated from bone marrow (BM), spleen, liver, and peripheral blood (PB) and analyzed by FACS. Leukemic blasts were defined as human CD19+ and CD45+. S-CXCR4 MFI was measured in the blast population. Overall, leukemic burden was similar in C, A, and P, consistent with conservative dosing of A and minimal direct anti-leukemic effect of P. A resulted in increased blasts in spleen and liver compared to C, possibly due to higher levels of s-CXCR4, while P resulted in increased blasts in liver, possibly due to mobilization of blasts. The key finding was that P+A resulted in decreased blasts in BM, spleen, liver, and PB, demonstrating a synergistic effect between P and A. Interestingly, P+A led to a higher reduction in blasts in a sample with A-induced s-CXCR4 upregulation, compared to a sample that did not upregulate s-CXCR4 in response to A. In all treatment cohorts, s-CXCR4 expression was highest in PB blasts, followed by liver, and BM/spleen. Conclusions: Chemo-induced s-CXCR4 upregulation confers stromal-mediated chemoprotection in vitro that can be reversed by P. In vivo, P is an effective chemosensitizer. S-CXCR4 expression is increased in blasts located outside the BM, suggesting that blasts migrating from BM into PB upregulate s-CXCR4 as they home to new niches. Extramedullary disease may develop as a result of chemo-induced upregulation of s-CXCR4 or through mobilization of blasts by P alone. Importantly, P+A resulted in decreased leukemic burden in our infant ALL xenografts, suggesting that chemo-induced increases in s-CXCR4 and P-induced blast mobilization can be overcome. Finally, the efficacy of P as a chemosensitizer was predicted by the degree of chemo-induced s-CXCR4 upregulation, identifying a biomarker with the potential to identify optimal patients for CXCR4 inhibition. P in combination with chemo may thus prove useful in the treatment of high-risk pediatric ALL. Disclosures: No relevant conflicts of interest to declare.