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FLT3 internal tandem duplication (FLT3ITD) are common mutations in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide CRISPR/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations, and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI, and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation driven leukemias.

Functionally critical decreases in levels or activity of the ETS family transcription factor PU.1 are present in approximately 2/3 of patients with acute myeloid leukemia (AML), across different AML subtypes (Sive, Leukemia 2016) including at the stem cell level (Steidl, Nat Genet 2006; Will, Nat Med 2015). Thus, targeting PU.1 could be an appealing option for treatment. As complete loss of PU.1 leads to stem cell failure (Iwasaki, Blood 2005), we hypothesized that PU.1 inhibition could eradicate leukemic cells harboring already low levels of PU.1, with modest effects on normal cells. We initially tested this hypothesis using 3 different shRNAs, and found that PU.1 inhibition led to a significant decrease in proliferation and clonogenicity, and increased apoptosis of mouse and human leukemic cell lines with low PU.1 levels, as well as the majority of primary human AML cells tested. We demonstrated that these effects were indeed due to decreased PU.1 levels by retroviral add-back experiments. The direct pharmacologic targeting of transcription factors has proven challenging in the past. Besides the core ETS binding motif (GGAA) in the DNA major groove, PU.1 binding to chromatin depends on additional minor groove contacts enriched for AT nucleotides upstream of the ETS motif, which determine selectivity for PU.1. Using an integrated screening strategy utilizing biosensor surface plasmon resonance, DNA footprinting, and cell-based dual-color PU.1 reporter assays, we developed novel small molecules of the heterocyclic diamidine family acting as first-in-class PU.1 inhibitors. Targeted occupancy by our compounds in the minor groove induces perturbations in DNA conformation that are transmitted to the PU.1 site in the major groove and thus inhibits PU.1 binding via an allosteric mechanism. Consistent with this, the inhibitory effects were selective for PU.1 versus other ETS transcription factors. Treatment with 3 different compounds led to cell growth inhibitory effect with respect to PU.1 level and preferentially affects PU.1low AML cells. Similarly to what we observed with shRNAs, treatment with our novel inhibitors led to decreased proliferation and colony forming capacity, increased apoptosis, and disrupted serial replating capacity of PU.1low AML cells and a majority of primary AML cell samples. Targeted ChIP and expression analysis showed that the compounds disrupt PU.1-promoter interaction and lead to downregulation of canonical PU.1 transcriptional targets in AML cells, confirming on-target activity in AML cells. Genome-wide analysis showed highly significant enrichment of known transcriptional targets of PU.1, and selectivity over genes regulated by other ETS family members. Comparison with published transcriptomic and PU.1 ChIP-seq data sets, as well as ARACNe analysis of the PU.1 regulon in primary AML cells, demonstrated that the inhibitors antagonize PU.1-regulated pathways at a genome-wide level. ChIP-seq performed in PU.1low AML cells confirmed a genome-wide decrease of PU.1 peaks after treatment and provides novel insight into the molecular mechanisms mediating the anti-leukemic effects of pharmacological PU.1 inhibition. To test the effects of PU.1 inhibition on normal hematopoiesis, we treated normal hematopoietic stem/progenitors cells (HSPC) in colony forming assays and saw decreased production of mature granulo-monocytic cells, consistent with PU.1's known role in this lineage. However, this effect was reversible upon drug removal, and serial replating capacity was not affected suggesting no significant effects on more immature HSPC. Congenic transplantation assays of treated normal bone marrow cells led to no change in myeloid and T-cells and only a modest decrease in B-cell numbers. Lastly, in vivo treatment with PU.1 inhibitors in mouse and human AML (xeno)transplantation models significantly decreased tumor burden and increased survival. To conclude, our study provides proof-of-principle for PU.1 inhibition as a novel therapeutic strategy in AML. Furthermore, we present the development of first-in-class PU.1 inhibitors acting via an allosteric minor groove-mediated mechanism. Our work shows that the specific pharmacological targeting of the DNA interaction of transcription factors such as PU.1 is feasible in principle, and may open the way for targeting of other transcription factors through minor groove-directed approaches. Disclosures Will: Novartis Pharmaceuticals: Consultancy, Research Funding. Steidl: Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding; Bayer Healthcare: Consultancy.

Introduction Chemotherapy-inducedmyelosuppressionis a major complication for cancer patients causing high rates of morbidity and mortality. Furthermore,myelosuppressionis frequently managed by delaying and/or reducing the scheduled dose, and as a consequence the efficacy of the treatment can be compromised.Since chemotherapy remains the cornerstone for treating many cancers, the development of compounds that inhibitmyelosuppressionwould provide considerable health benefits. By repurposing quizartinib we have identified a novel approach to managemyelosuppression, which led to the development of an effective treatment for a mouse model of acute myeloidleukemia(AML). Methods Quizartinib was administered to C57BL/6 mice by oral gavage with a single priming dose of 10 or 30 mg/kg. Fluorouracil (5-FU) was administered byintraperitonealinjection at 150 mg/kg 6-12 hours later. This schedule was repeated every 10 days to examine the protective effects of quizartinib from multiple cycles of 5-FU. The approach of quizartinib priming plus 5-FU every 10 days was examined as a treatment for a mouse model of AML. Results This work is based on studies from mice showing that a single dose of the FLT3 inhibitor quizartinib induces a rapid but transient quiescence of multipotentprogenitors (MPPs). MPPs are highly proliferative FLT3-expressing cells that are responsible for maintaining life-long blood production. They are markedly depleted by chemotherapy so their loss is a significant component of myelosuppression. We found that a single dose of quizartinib 6-12 hours before 5-FU resulted in a 10-fold increase in the survival of MPPs two days after administration. Quizartinib priming also provided significant protection to hematopoietic stem cells and committed progenitors within the lin-, Sca-1-, c-Kit+ population.Analysis of bone marrow at 2-10 days after a single 5-FU injection revealed the rapid recovery conferred to quizartinib-primed mice compared to mice primed with vehicle (Fig. 1). To test whether quizartinib provided protection from multiple cycles of 5-FU we injected mice once every 10 days with 5-FU following a priming dose of quizartinib or vehicle 12 hrsearlier. This 10-day cycle of 5-FU is a potent regimen that causes bone marrow failure and lethality. We found that quizartinib priming promoted a marked level of protection, with only 1 of 5 mice dying over a period of 15 cycles, whereas all vehicle-primed mice succumbed between the 4th and 6th cycles. In addition, we found that quizartinib-primed mice did not lose weight and showed a rapid recovery of white and red blood cells and platelets. From these findings we hypothesized that quizartinib priming before chemotherapy may provide a safe and effective approach for treating cancers that are independent of FLT3signaling. We tested this with an AML model developed from Npm1c x FLT3-ITD(F692L) mutant mice. The F692L mutation confers resistance to quizartinib. The suitability of this model was confirmed from experiments showing that quizartinib dosing did not induce quiescence of Npm1c x FLT3-ITD(F692L) AML cells, nor did it protect them from 5-FU. A cohort of non-irradiated B6.CD45.1 mice transplanted with Npm1c x FLT3-ITD(F692L) AML cells was therefore established. At 16 days after transplantation, when AML cells were evident in the peripheral blood, the mice were divided into three groups: (i) untreated, (ii) induction therapy treated, and (iii) quizartinib primed + 5-FU treated.The induction therapy involved two rounds ofcytarabineat 50 mg/kg for 5 days and doxorubicin at 1.5 mg/kg for 3 days (i.e. 5+3 therapy). All mice in the untreated and induction chemotherapy-treated groups succumbed within 30 and 56 days respectively (Fig. 2). In contrast, only 1 of 5 mice treated by 9 cycles of quizartinib priming + 5-FU succumbed (at day 121). The remaining 4 mice were healthy at 176 days after transplantation, and no AML cells were detectable in their blood or BM. Furthermore, surviving mice had WBC counts within the normal range. Conclusions This work has shownthat priming mice with quizartinib provides significant protection to the hematopoietic system from 5-FU cytotoxicity. This has resulted in the development of a novel quizartinib + 5-FU treatment protocol for a preclinical model of AML whereby it markedly enhanced the survival of mice compared to mice receiving conventional therapy. Disclosures No relevant conflicts of interest to declare.

High levels of expression of wild-type Flt3 characterize many hematopoietic proliferative diseases and neoplasms, providing a potential therapeutic target. Using the c-Cbl RING finger mutant mouse as a model of a myeloproliferative disease (MPD) driven by wild-type Flt3, in the present study, we show that treatment with the Flt3 kinase inhibitor AC220 blocks MPD development by targeting Flt3+ multipotent progenitors (MPPs). We found that daily administration of AC220 caused a marked reduction in Flt3 expression, induction of quiescence, and a significant loss of MPPs within 4 days. Unexpectedly, a robust Flt3 ligand–associated proliferative recovery response soon followed, preventing further loss of MPPs. However, continued AC220 treatment limited MPP recovery and maintained reduced, steady-state levels of cycling MPPs that express low levels of Flt3. Therefore, a finely tuned balance between the opposing forces of AC220 and Flt3 ligand production was established; whereas the Flt3 ligand blunted the inhibitory effects of AC220, the disease was held in remission for as long as therapy was continued. The net effect is a potent therapy indicating that patients with c-Cbl mutations, or those with similarly enhanced Flt3 signaling, may respond well to AC220 even after the induction of high levels of Flt3 ligand.

Abstract 642 Introduction: c-Cbl is a RING finger based E3 ubiquitin ligase that is highly expressed in hematopoietic cells where it directs the polyubiquitylation and degradation of protein tyrosine kinases. c-Cbl also functions as a multi-domain adaptor that recruits and activates proteins such as PI 3-kinase. Oncogenic forms c-Cbl that have lost E3 ligase show enhanced activity of these adaptor functions in addition to the inability to downregulate activated tyrosine kinases. c-Cbl mutations are found in patients with a range of myeloid malignancies that include myelodysplastic syndromes, chronic myelomonocytic leukaemia, juvenile myelomonocytic leukaemia, atypical chronic myeloid leukaemia and acute myeloid leukaemia. Sequencing has shown that the mutations are located in either the linker or RING finger domains and that these mutations abolish E3 ligase activity. Thus the development of leukemia is caused, at least in part, through the loss of this activity. To investigate c-Cbl associated malignancies we generated a mouse with a knockin mutation in the RING finger domain that develops a myeloproliferative disease (MPD) progressing to lethal leukemia. The mutation is a substitution of a cysteine for alanine in the RING finger at 379 (i.e. C379A) that disrupts c-Cbl's ability to function as an E3 ubiquitin ligase. The majority of the c-Cbl C379A mutant mice succumb within a year with markedly elevated white blood cell (WBC) counts, splenomegaly and extensive infiltrations of myeloid cells into peripheral organs. These mice therefore provide a pre-clinical model for studying myeloid malignancies associated with oncogenic forms of c-Cbl. We have found that hematopoietic progenitors from c-Cbl(C379A) mice have enhanced FLT3 signaling and an expanded population of FLT3high cells compared to WT and c-Cbl KO mice. When c-Cbl(C379A) mice are mated to FLT3 ligand KO mice the doubly mutant mice do not develop a severe MPD or leukemia and the FLT3high population is markedly suppressed. Thus targeting FLT3 may offer an effective therapy. AC220 is a FLT3 inhibitor that shows excellent potency, selectivity and pharmacokinetic properties and is currently being investigated in clinical trials treating patients with activating FLT3 mutations. In this study we examined the effects of treating c-Cbl(C379A) mice with AC220 to determine whether AC220 can prevent the development of myeloid malignancies associated with aberrant wild-type FLT3 signaling. Results: To determine the effectiveness of AC220 for treating c-Cbl-associated MPD we established two cohorts, one comprising 16 C57BL/6.CD45.1 mice repopulated with bone marrow (BM) from an 8 week-old c-Cbl(C379A) mouse and one comprising 18 c-Cbl(C379A) mice aged 8–9 months. Mice were bled 5 days before dosing to determine pre-treatment WBC counts and both cohorts were then divided into two groups for daily dosing by oral gavage with either AC220 (10mg/kg) or vehicle (5% cyclodextrin) and bled at 3 and 6 weeks. Dosing with AC220 resulted in a marked decrease in WBC counts in all lineages. The mean WBC counts for the AC220-treated mice transplanted with c-Cbl(C379A) marrow at 3 and 6 weeks were 14.0 and 14.5×109/L respectively, compared to a mean WBC count of 33.5×109/L before treatment commenced. For the 8–9 month old c-Cbl(C379A) mice the 3 and 6 wk counts were 28.5 and 20.0 ×109/L respectively, compared to pre-treatment levels of 64.5×109/L. In contrast the WBC counts remained constantly high in the vehicle treated groups. After 6 weeks of dosing the mice were sacrificed and the BM analyzed by flow cytometry to determine the effect on FLT3high progenitors. The AC220 treatment reduced the FLT3high population of LSK cells by more than two fold in both cohorts. Similarly AC220 treatment had a significant impact on spleen weights with a greater than two-fold reduction in the AC220-treated groups. In addition AC220 dosing resulted in a marked decrease in the myeloid invasiveness into peripheral organs, an outcome that suggests AC220 treatment would enhance survival. Conclusions: These studies are the first to demonstrate in vivo that AC220 is a very effective treatment for diseases or conditions that are driven by WT FLT3. Future studies investigating aberrant FLT3 expression on progenitors from a range of MDS/MPN patients may therefore reveal therapeutic options that would not have otherwise been appreciated without the preclinical studies of the c-Cbl RING finger mutant mouse. Disclosures: No relevant conflicts of interest to declare.