Gab2 deficiency prevents Flt3-ITD driven acute myeloid leukemia in vivo

Internal tandem duplications (ITD) of the FMS-like tyrosine kinase 3 (FLT3) predict poor prognosis in acute myeloid leukemia (AML) and often co-exist with inactivating DNMT3A mutations. In vitro studies implicated Grb2-associated binder 2 (GAB2) as FLT3-ITD effector. Utilizing a Flt3-ITD knock-in, Dnmt3a haploinsufficient mouse model, we demonstrate that Gab2 is essential for the development of Flt3-ITD driven AML in vivo, as Gab2 deficient mice displayed prolonged survival, presented with attenuated liver and spleen pathology and reduced blast counts. Furthermore, leukemic bone marrow from Gab2 deficient mice exhibited reduced colony-forming unit capacity and increased FLT3 inhibitor sensitivity. Using transcriptomics, we identify the genes encoding for Axl and the Ret co-receptor Gfra2 as targets of the Flt3-ITD/Gab2/Stat5 axis. We propose a pathomechanism in which Gab2 increases signaling of these receptors by inducing their expression and by serving as downstream effector. Thereby, Gab2 promotes AML aggressiveness and drug resistance as it incorporates these receptor tyrosine kinases into the Flt3-ITD signaling network. Consequently, our data identify GAB2 as a promising biomarker and therapeutic target in human AML.

FLT3-ITD allelic ratio and HLF expression predict FLT3 inhibitor efficacy in adult AML

FLT3 internal tandem duplication (FLT3-ITD) is a frequent mutation in acute myeloid leukemia (AML) and remains a strong prognostic factor due to high rate of disease recurrence. Several FLT3-targeted agents have been developed, but determinants of variable responses to these agents remain understudied. Here, we investigated the role FLT3-ITD allelic ratio (ITD-AR), ITD length, and associated gene expression signatures on FLT3 inhibitor response in adult AML. We performed fragment analysis, ex vivo drug testing, and next generation sequencing (RNA, exome) to 119 samples from 87 AML patients and 13 healthy bone marrow controls. We found that ex vivo response to FLT3 inhibitors is significantly associated with ITD-AR, but not with ITD length. Interestingly, we found that the HLF gene is overexpressed in FLT3-ITD+ AML and associated with ITD-AR. The retrospective analysis of AML patients treated with FLT3 inhibitor sorafenib showed that patients with high HLF expression and ITD-AR had better clinical response to therapy compared to those with low ITD-AR and HLF expression. Thus, our findings suggest that FLT3 ITD-AR together with increased HLF expression play a role in variable FLT3 inhibitor responses observed in FLT3-ITD+ AML patients.

Momelotinib is a highly potent inhibitor of FLT3-mutant AML

Kinase activating mutation in FLT3 is the most frequent genetic lesion associated with poor prognosis in acute myeloid leukemia (AML). Therapeutic response to FLT3 tyrosine kinase inhibitor (TKI) therapy is dismal, and many patients relapse even after allogenic stem cell transplantation. Despite the introduction of more selective FLT3 inhibitors, remissions are short-lived, and patients show progressive disease after an initial response. Acquisition of resistance-conferring genetic mutations and growth factor signaling are two principal mechanisms that drive relapse. FLT3 inhibitors targeting both escape mechanisms could lead to a more profound and lasting clinical responses. Here we show that the JAK2 inhibitor, momelotinib, is an equipotent type-1 FLT3 inhibitor. Momelotinib showed potent inhibitory activity on both mouse and human cells expressing FLT3-ITD, including clinically relevant resistant mutations within the activation loop at residues, D835, D839, and Y842. Additionally, momelotinib efficiently suppressed the resistance mediated by FLT3 ligand (FL) and hematopoietic cytokine activated JAK2 signaling. Interestingly, unlike gilteritinib, momelotinib inhibits the expression of MYC in leukemic cells. Consequently, concomitant inhibition of FLT3 and downregulation of MYC by momelotinib treatment showed better efficacy in suppressing the leukemia in a preclinical murine model of AML. Altogether, these data provide evidence that momelotinib is an effective type-1 dual JAK2/FLT3 inhibitor and may offer an alternative to gilteritinib. Its ability to impede the resistance conferred by growth factor signaling and activation loop mutants suggests that momelotinib treatment could provide a deeper and durable response; thus, warrants its clinical evaluation.

A Phase-Ib/II Clinical Evaluation of Ponatinib in Combination with Azacitidine in FLT3-ITD and CBL-Mutant Acute Myeloid Leukemia (PON-AZA study)

Introduction Despite the advent of targeted therapy for FLT3-mutated AML, unmet need still exists for patients unfit for intensive chemotherapy, with no evidence that overall survival (OS) can be improved by combining either venetoclax (Konopleva et al., ASH 2020) or gilteritinib (Astellas press release, December 2020) with azacitidine. Although gilteritinib has been shown to improve median OS from 5.5 to 9.8 months, the majority will relapse (Perl et al., 2019). Adaptive on-target gilteritinib resistance may be due to the FLT3-F691L gatekeeper mutation, whereas off-target resistance may be due to loss-of-function variants in CBL, which encodes an E3 ubiquitin-protein ligase that negatively regulates FLT3 (McMahon et al, 2019). Ponatinib is a type-1 FLT3 inhibitor that is active in vitro against FLT3 F691L (Smith et al., 2013) and had an overall response rate (ORR) of 43% in a small pilot phase-I study (Talpaz et al., 2011). Combination of a FLT3 inhibitor with azacitidine may antagonize the synergistic hypermethylation reported for FLT3-ITD in association with epigenetic mutations (Shih et al., 2015). CBL loss-of-function mutations may also enhance responsiveness to FLT3 inhibitors (Taylor et al, 2015). We thus hypothesize that the combination of ponatinib and azacitidine could mitigate the rapid evolution of drug resistance typical of more selective FLT3 inhibitors used as single agents. Methods A phase-Ib study was conducted with the primary objective safety and key secondary objective preliminary efficacy of azacitidine in combination with ponatinib in patients with FLT3-ITD AML failing prior therapy or unfit for intensive chemotherapy. Exploratory objectives included mechanisms of ponatinib resistance and responsiveness of CBL-mutant AML to FLT3 inhibition. At dose level 1 (DL1), patients received azacitidine 60 mg/m 2 on days 1-5 and 8-9 and ponatinib 30 mg daily on days 5-25 of each cycle. In patients not achieving CR or CRi after cycle 1, the ponatinib dose was increased to 45 mg during cycle 2. For dose level 2 (DL2), the dose of azacitidine was increased to 75 mg/m 2. Results Thirty-one patients were evaluable for response. Median age was 67 years (range, 26-87). Frequency of prior lines of therapy was 0 (15%), 1 (46%), 2 (23%) or 3 (8%). Four patients had a history of prior allogeneic hematopoietic cell transplant and one had previously received a FLT3 inhibitor. FLT3-ITD was present in 28 patients (median VAF 0.33; range, 0.009-17.95) and 3 had inactivating CBL mutations. A total of 20 patients were treated at DL1 and 12 patients at DL2. There were two grade-4 DLTs (raised AST/ALT [DL1] and tubulointerstitial nephritis [DL2]). Three grade-2 thromboembolic events were observed (two cannula-related DVTs and a distal lower-limb DVT). There were two grade-5 AEs (infection and cardiac failure), which were not considered drug related. The most common grade-3-4 AEs were febrile neutropenia (57%), neutropenia (47%), infections (47%), thrombocytopenia (40%) and anaemia (27%). Cardiac arrhythmias (atrial fibrillation/flutter, bradycardia, sinus tachycardia and ventricular tachycardia [1 patient]) were observed in 30% of patients. Of these, 80% were grade 1 or 2 and only one was considered by the investigator to be related to study treatment. Response was evaluable in 23 of 31 patients. Nine patients (39%) achieved CR or CRi, 3 (13%) achieved a PR and 8 (35%) achieved SD (ORR 52%). ORR at DL1 and DL2 was 43% and 66%, respectively. Median time to best response was 1.4 months (range 1.0-11.9). Median duration of best response was 12.9 months at both dose levels. Median OS for DL1 was 6.5 months and not reached for DL2. Despite shorter follow-up, DL2 patients experienced better OS than DL1 patients (p = 0.015). Responses were seen in 2 of 4 patients with post-allograft relapse. Two of three patients with a CBL mutation responded (1 CR and 1 CRi). Eradication of the CBL mutation was seen in one patient, who remains on therapy after 15 cycles. Molecular studies to investigate dynamic changes in molecular architecture are ongoing. Conclusions The recommended phase-II dose of ponatinib is 30 mg on days 5-25 and that of azacitidine is 75 mg/m 2 for seven doses each cycle. The ORR was 52% and durable disease control was observed, especially in patients receiving DL2. Preliminary efficacy was observed in CBL-mutated patients. Further clinical investigation of this regimen is warranted in patients with FLT3- or CBL-mutant AML. Figure 1 Figure 1. Disclosures Kipp: Novartis: Honoraria. Perkins: Celgene: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau. Lane: Novartis: Consultancy; Geron: Consultancy; BMS: Consultancy, Research Funding; Abbvie: Honoraria; Astellas: Membership on an entity's Board of Directors or advisory committees. Enjeti: Sanofi: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Honoraria; Roche: Speakers Bureau; Astra Zeneca: Honoraria. Bajel: Abbvie, Amgen, Novartis, Pfizer: Honoraria; Amgen: Speakers Bureau. Reynolds: Novartis AG: Current equity holder in publicly-traded company; Abbvie: Research Funding; Alcon: Current equity holder in publicly-traded company. Wei: Abbvie, Amgen, Astellas, AstraZeneca, Celgene/BMS, Genentech, Janssen, MacroGenics, Novartis, Pfizer, and Servier: Membership on an entity's Board of Directors or advisory committees; Novartis, Abbvie, Celgene/BMS: Speakers Bureau; Former employee of Walter and Eliza Hall Institute: Patents & Royalties: Prof. Andrew Wei is a former employee of the Walter and Eliza Hall Institute and is eligible for a fraction of the royalty stream related to Venetoclax; Abbvie, Amgen, Astellas, AstraZeneca, Celgene/BMS, Genentech, Janssen, MacroGenics, Novartis, Pfizer, and Servier: Honoraria; Servier: Consultancy; Abbvie, Amgen, AstraZeneca, Celgene/BMS, Novartis, Servier and F. Hoffmann-La Roche: Research Funding. OffLabel Disclosure: Ponatinib - used as an experimental therapy for AML in combination with azacitidine

FLT3 Inhibitor Quizartinib Facilitates Proliferation and CXCL12-Induced Chemotaxis in Quizartinib Resistant FLT3/ITD + Hematopoietic Cells By Increasing RUNX1

RUNX1 generally functions as a tumor suppressor in the hematopoietic system. However, RUNX1 expression is significantly elevated in human AML cells with FLT3/ITD mutations, promotes leukemogenesis induced by FLT3/ITD (Behrens et al. JEM 2017) and enhances the resistance of FLT3/ITD + cells to type-II FLT3 inhibitor quizartinib (Hirade et al IJH 2016). We previously reported that RUNX1 expression is higher in CXCR4-low FLT3/ITD + cells compared to Cxcr4-high FLT3/ITD + cells, even though Cxcr4 expression is trans-activated by RUNX1. This difference in RUNX1 expression level was associated with divergent response to CXCL12 in FLT3/ITD + cells harboring different CXCR4 expression levels that were exposed to quizartinib (Fukuda S. et al. ASH 2019). Our data also demonstrated that RUNX1 expression is down-regulated following withdrawal of quizartinib in FLT3/ITD + cells that became refractory to quizartinib (Hirade et al. IJH 2016), suggesting that RUNX1 expression may be up-regulated by quizartinib in FLT3/ITD + cells. Since RUNX1 regulates proliferation of FLT3/ITD + AML cells, the present study investigated association between RUNX1 expression levels and proliferation of quizartinib resistant FLT3/ITD + cells that are exposed to quizartinib. In the sensitive FLT3/ITD + Ba/F3 cells, RUNX1 protein expression was transiently up-regulated but eventually down-regulated by 5 nM quizartinib, coincident with decline in the viable cells. In contrast, RUNX1 expression was up-regulated by quizartinib and remained elevated in the resistant FLT3/ITD + Ba/F3 cells. Since RUNX1 enhances proliferation of FLT3/ITD + cells, we next examined whether proliferation FLT3/ITD + cells that acquired resistance to quizartinib is facilitated by quizaritinib as a result from quizartinib-mediated up-regulation of RUNX1, using the Cxcr4-low and Cxcr4-high FLT3/ITD + cells that acquired resistance to quizartinib. Although CXCL12 barely enhanced the proliferation of refractory FLT3/ITD + Ba/F3 cells, 5 nM quizartinib significantly increased the proliferation of both Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells that acquired resistance to quizartinib compared to those without quizartinib. This increase in the proliferation of Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells coincided with the elevation in RUNX1 and CXCR4 protein expression. Moreover, the resistant Cxcr4-low FLT3/ITD + Ba/F3 cells proliferated significantly faster than Cxcr4-high FLT3/ITD + cells, with concomitant higher expression of RUNX1 in Cxcr4-low FLT3/ITD + cells than in Cxcr4-high FLT3/ITD + cells. Likewise, type-I FLT3 inhibitor gilteritinib significantly enhanced proliferation of Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells that acquired resistance to gilteritinib. Knocking down Runx1 using shRNAs significantly decreased the enhanced proliferation induced by quizartinib in refractory FLT3/ITD + Ba/F3 cells, coincident with reduction in CXCR4 expression. Since CXCR4 expression level was elevated by quizartinib in the FLT3/ITD + cells refractory to quizartinib, we next examined CXCL12-induced migration in quizartinib-resistant FLT3/ITD + cells following exposure to quzartinib. Pre-incubating the quizartinib resistant Cxcr4-low or Cxcr4-high FLT3/ITD + Ba/F3 cells with 5 nM quizartinib for 72 hours significantly enhanced their migration to 100 ng/ml of Cxcl12 compared to those without quizartinib, coincident with elevation in RUNX1 levels. Surprisingly, migration of CXCR4-low FLT3/ITD + cells to CXCL12 was significantly elevated compared to CXCR4-high cells, with concomitant higher expression of RUNX1 in Cxcr4-low FLT3/ITD + cells than in Cxcr4-high FLT3/ITD + cells. Silencing Runx1 using shRNAs significantly decreased migration to CXCL12 in refractory Cxcr4-low FLT3/ITD + Ba/F3 cells. These data indicate that the FLT3 inhibitor itself can facilitate the proliferation and migration to CXCL12 in FLT3/ITD + cells that are refractory to FLT3 inhibitors by up-regulating RUNX1. The results implicate that FLT3 inhibitors may worsen the disease progression in the patients that became refractory to FLT3 inhibitors by facilitating proliferation and migration to CXCL12 of the resistant FLT3/ITD + AML cells. In this regard, targeting RUNX1 may represent additional strategy to eradicate resistant FLT3/ITD + AML cells, in which their proliferation and migration are supported by FLT3 inhibitors. Disclosures No relevant conflicts of interest to declare.

Synergistic Combination Activity of the Novel GSPT1 Degrader CC-90009 in Acute Myeloid Leukemia Models

Introduction: CC-90009 is a novel cereblon E3 ligase modulator (CELMoD ®) agent that is a first-in-class degrader of translation termination factor G1 to S phase transition 1 (GSPT1). CC-90009 has demonstrated antileukemic activity as a single agent and is currently under investigation in patients with acute myeloid leukemia (AML; NCT02848001). Treatment with CC-90009 led to rapid reductions in peripheral and bone marrow blasts, and demonstrated preliminary promising efficacy, including several complete remissions, in patients with relapsed or refractory AML. Here, we describe the identification and preclinical activity of select anti-AML agents as potential combination partners of CC-90009 to further improve its efficacy and therapeutic index. Based on these results, the combination activity of CC-90009 with venetoclax (VEN)/azacitidine (AZA) is being evaluated in a phase 1/2 trial in patients with AML (NCT04336982). Methods: A high-throughput cell viability screen was performed to identify synergistic partners of CC-90009. AML cell lines (HL-60, HNT-34, KG-1, ML-2, NOMO-1, MOLM-13, MV4-11, F-36P, OCI-AML2, OCI-AML3) were treated with CC-90009 in combination with > 70 compounds, including standard anti-AML agents, tyrosine kinase inhibitors (TKIs), unfolded protein response inducers, transcription inhibitors, and epigenetic agents. MOLM-13 and MV4-11 are fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) cell lines. Hits were validated in a colony formation (CF) assay using primary AML cells and bone marrow mononuclear cells (BMMC) from healthy donors. Synergy of the combination partners with CC-90009 was further assessed in AML patient-derived xenograft (PDX) models. Synergy between the isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib and CC-90009 was evaluated in a TF-1 erythroleukemia cell line overexpressing IDH2 R140Q mutant, and an IDH2 R140Q PDX model, AM7577. Results: Our high-throughput combination screen revealed multiple TKIs, epigenetic agents, and pro-apoptotic agents as potential combination partners of CC-90009 in AML cell lines. FLT3 inhibitors, including sunitinib, pexidartinib, midostaurin, lestaurtinib, crenolanib, and gilteritinib, synergized with CC-90009 to reduce viability in FLT3-ITD AML cell lines MV4-11 and MOLM-13. Similarly, the B-cell lymphoma 2 (BCL2) inhibitor VEN potentiated CC-90009-induced apoptosis and accelerated cell-autonomous killing. Reduction in levels of MCL-1, an anti-apoptotic factor, by CC-90009 most likely contributed to the synergy with VEN. We prioritized the evaluation of FLT3, BCL2, and IDH2 inhibitors as partners of CC-90009. In CF assays, midostaurin enhanced the inhibitory effect of CC-90009 in primary AML cells, without augmenting the effect of CC-90009 in CD34+ BMMC from healthy donors. Similarly, VEN enhanced the reduction in CF by CC-90009 in AML patient-derived BMMC without exacerbating the decrease in CF by CC-90009 in BMMC from healthy donors. We characterized FLT3 inhibitor/CC-90009 and BCL2 inhibitor/AZA/CC-90009 combinations in a FLT3-ITD PDX murine model, PDX1. The FLT3 inhibitor quizartinib significantly prolonged survival when combined with CC-90009 compared with either agent alone (P < 0.001). Similarly, VEN/AZA/CC-90009 combination markedly extended survival compared with single agents or VEN/AZA doublets (P < 0.001). The synergy between CC-90009 and epigenetic modulators was validated and further characterized in customized cell differentiation assays. Addition of CC-90009 to enasidenib, a mutant IDH2 inhibitor, enhanced differentiation and killing of CD34+ stem and progenitor cells, and increased differentiated CD235a+ erythroblasts, compared with enasidenib alone in a TF-1 cell line overexpressing IDH2 R140Q. Enasidenib/CC-90009 combination treatment reduced CD45+ malignant populations and increased differentiated CD14+ cells, leading to significantly prolonged animal survival in an IDH2 R140Q PDX model, AM7577, compared with either agent alone (P < 0.0001). Conclusion: Using a high-throughput combination screen, we identified rational combination partners that synergize with CC-90009 in in vitro and in vivo AML models. Collectively, these results support the clinical evaluation of CC-90009 in combination with FLT3, BCL2, and IDH2 inhibitors to further improve treatment outcomes for patients with AML. Disclosures Pierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Yao: Bristol Myers Squibb: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months, Research Funding. Pace: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Wang: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Flandin-Blety: Bristol Myers Squibb: Current Employment. Benitez: Bristol Myers Squibb: Current Employment. Guarinos: Bristol Myers Squibb: Current Employment. Hoffmann: Bristol Myers Squibb: Current Employment. Carrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Pim Kinase Inhibitor Enhances FLT3 Inhibitor Efficacy through GSK-3β Activation and GSK-3β-Mediated Proteasomal Degradation of c-Myc

BACKGROUND Internal tandem duplication in the fms-like tyrosine kinase 3 receptor tyrosine kinase (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients, associated with poor treatment outcomes due to rapid relapse. FLT3 inhibitors are used in the clinic, but with incomplete efficacy and development of resistance. Further treatment options are needed. The serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD; it directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop in cells with FLT3-ITD. Dual targeting of Pim-1 and FLT3 is a promising treatment strategy. The c-Myc transcription factor contributes to dysregulation of cell growth and apoptosis in cancers, including AML. In addition to transcriptional regulation, c-Myc is regulated post-translationally by T58 phosphorylation by the serine/threonine kinase glycogen synthase kinase-3- β (GSK-3β). Here we show that concurrent treatment of cells with FLT3-ITD with Pim and FLT3 inhibitors activates GSK-3β, which phosphorylates and post-translationally downregulates c-Myc. METHODS Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and FLT3-ITD AML patient blasts were cultured with the pan-Pim inhibitor AZD1208 (1 μM) and/or the FLT3 inhibitors gilteritinib or quizartinib (15 nM, 1 nM), with and without the GSK-3β inhibitor TCG-24 (20 μM). c-Myc, p-GSK3-α/β (S21/9) and GSK3-α/β protein expression was measured by immunoblotting. c-Myc mRNA was measured by qPCR. Cells were also cultured with cycloheximide (100 μg/mL) with and without the proteasome inhibitor MG-132 (20 μM) to measure protein half-life and proteasomal degradation. To study the role of c-Myc overexpression and activation, Ba/F3-ITD cells were infected with retroviral estrogen receptor (ER)-Myc plasmid, causing c-Myc nuclear translocation when activated by 4-hydroxytamoxifen (4-OHT; 300 nM). To study the role of c-Myc phosphorylation at T58, Ba/F3-ITD cells were infected with MycT58A plasmid, preventing c-Myc phosphorylation at T58. Apoptosis was detected by Annexin V and propidium iodide staining, measured by flow cytometry. RESULTS Treatment with Pim inhibitor AZD1208 and FLT3 inhibitor gilteritinib or quizartinib combination rapidly downregulated c-Myc protein expression in Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and in primary FLT3-ITD AML patient blasts, compared to quizartinib or gilteritinib alone. Pim inhibitor and FLT3 inhibitor combination treatment did not decrease c-Myc mRNA levels, but markedly decreased c-Myc protein half-life, from 36 mins without drugs and 24 mins with gilteritinib to 18 mins with combination. Half-life did not decrease when cells were pre-treated with the proteasome inhibitor MG-132, consistent with post-translational downregulation through proteasomal degradation. Apoptosis induction by Pim inhibitor and FLT3 inhibitor combination decreased by more than 50% in Ba/F3-ITD cells infected with ER-Myc plasmid and treated with 4-OHT, demonstrating the major role of c-Myc downregulation in apoptosis induction by combination treatment. GSK-3b is inactivated by phosphorylation, and combination treatment rapidly decreased p-GSK-3b levels, while total GSK-3b levels were unchanged, indicating activation of GSK-3b. Treatment of cells with FLT3-ITD with the GSK-3b inhibitor TCG-24 in addition to Pim and FLT3 inhibitors abrogated c-Myc protein downregulation, demonstrating that Pim and FLT3 inhibitor combination downregulates c-Myc through activation of GSK-3b. Finally, Pim and FLT3 inhibitor combination treatment did not downregulate c-Myc in Ba/F3-ITD cells transfected with c-Myc T58A, preventing c-Myc phosphorylation at T58, showing that c-Myc phosphorylation at T58 is necessary for its downregulation by combination treatment. CONCLUSIONS Concurrent treatment of cells with FLT3-ITD with Pim kinase inhibitor enhances the efficacy of FLT3 inhibitors through activation of GSK-3β and GSK-3β-mediated phosphorylation of c-Myc at T58, with resulting c-Myc downregulation through increased proteasomal degradation. This work and previous work in our laboratory on PP2A activating drugs and FLT3 inhibitor combination (Mol Cancer Ther 20:676, 2021) support GSK-3β activation as a mechanism for enhancing efficacy of FLT3 inhibitors in AML with FLT3-ITD. Disclosures No relevant conflicts of interest to declare.

Gilteritinib for Relapsed Acute Myeloid Leukaemia with FLT3 Mutation during the COVID-19 Pandemic: Real World Experience from the UK National Health Service

Background Early data suggest that patients undergoing salvage chemotherapy for relapsed or refractory (R/R) acute myeloid leukaemia (AML) have poor outcomes if infected with SARS-CoV-2, and nosocomial transmission has been a major problem worldwide. Gilteritinib is effective in R/R FLT3 mutated AML, is significantly less immunosuppressive and does not require hospital admission, however at the start of the pandemic this was not yet approved for routine use in all countries. In the United Kingdom, the National Health Service (NHS) made gilteritinib available as an emergency measure from late April 2020 to patients aged >16y with R/R FLT3 mutated AML, with the aim of reducing both mortality and healthcare resource use. We report a health-system-wide real world data collection for toxicity and patient outcomes across 27 NHS Hospitals. Methods Each patient was registered on a central NHS database, with clinicians certifying that their patient met the above criteria. Anonymised data were retrospectively collected by treating physicians. Gilteritinib dose, duration and toxicity information was requested for the first 4 cycles of therapy. Response definitions were as per European Leukaemia Network (ELN) guidelines. A total of 81 patients have been registered on the scheme, with outcomes reported here for those with follow-up information at a data cut on 1st August 2021. Results Fifty patients were included with a median age of 59y (range 19 - 77) and 50% male. The majority (83%) had an ECOG performance status of 0-1. AML was secondary to a previous haematological disorder in 12%, therapy-related in 4% and de novo in the remaining 84%. The disease was refractory to the last therapy in 38%. Most patients had previously received 1 (65%) or 2 (33%) lines of therapy, including intensive chemotherapy in a majority (86%). A FLT3 inhibitor had previously been administered to 45% and 35% were post allogeneic transplant. The FLT3 mutation was an internal tandem duplication (ITD) in 80% and tyrosine kinase domain (TKD) mutation in 22%. NPM1 mutations were detected in 34%. Next-generation sequencing results were available for 94% of patients, with mutations in IDH1 or IDH2 in 12.5%, ASXL1 in 2%, RUNX1 in 21% and no TP53 mutations. Patients spent a median 3.5 days in hospital in cycle 1, 0 days in cycles 2 and 3 and 1 day in cycle 4. In cycles 1, 2, 3 and 4, the median number of days of grade 4 neutropenia was 18, 7, 7.5, and 6.5 respectively, and the grade 4 thrombocytopenia was 2, 7, 0.5 and 0.5. The composite complete remission (CR) / CR with incomplete haematological recovery (CRi) rate was 27%. MRD data is being collected. The best response was morphological leukaemia free state (MLFS) in 4%, partial remission (PR) in 25% and refractory disease in 38%. The rate of combined CR/CRi did not differ in those with previous exposure to FLT3 inhibitors (23% vs 32%, p=0.6) or with past allogeneic transplant (29% vs 27%, p=0.3). There were no CR/CRi in patients with adverse cytogenetic risk. Median follow-up was 10.5 months (95%CI 7.3 - 12.3) with median overall survival (OS) 6.7 months (95%CI 4.5 - not reached). Mortality at day 30 was 0% and day 60 was 14%. 12-month overall survival was 38%. Patients who achieved a CR/CRi had a 12-month OS of 83%, and for PR this was 35%. Survival did not differ in those with previous FLT3 inhibitor exposure (HR 1.0, p>0.9) or allogeneic transplant (HR 0.63, p=0.3). Seven patients (14%) so far have been bridged with gilteritinib to allogeneic transplant. Conclusion Our data demonstrate that gilteritinib is well tolerated and clinically active in adults with relapsed FLT3 mutated AML. Importantly, during the COVID-19 pandemic, its availability has permitted the great majority of treatment to be delivered as an outpatient with significant resource saving at a time of critically constrained inpatient resources. Patients who achieve CR/CRi have good short-term outcomes and are able to proceed to a potentially curative allogeneic stem cell transplant. Figure 1 Figure 1. Disclosures Belsham: Celgene: Other: meeting attendance; Abbvie: Other: meeting attendance. Byrne: Incyte: Honoraria. Khan: Abbvie: Honoraria; Astellas: Honoraria; Takeda: Honoraria; Jazz: Honoraria; Gilead: Honoraria; Novartis: Honoraria. Khwaja: Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Latif: Kite: Consultancy, Honoraria, Speakers Bureau; Jazz: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Speakers Bureau; Takeda UK: Speakers Bureau. Loke: Amgen: Honoraria; Daichi Sankyo: Other: Travel Support; Janssen: Honoraria; Novartis: Other: Travel Support; Pfizer: Honoraria. Munisamy: Jazz Pharmaceuticals: Speakers Bureau; Roche: Speakers Bureau. Murthy: Abbvie: Other: support to attend educational conferences.. Smith: Daiichi Sankyo: Speakers Bureau; Pfizer: Speakers Bureau; ARIAD: Honoraria. Craddock: Novartis Pharmaceuticals: Other: Advisory Board ; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dillon: Amgen: Other: Research support (paid to institution); Astellas: Consultancy, Other: Educational Events , Speakers Bureau; Menarini: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Session chair (paid to institution), Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: educational events; Jazz: Other: Education events; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research Support, Educational Events.

RNA Polymerase II Is a Therapeutic Target to Overcome FLT3 Inhibitor Resistance Mediated By the Bone Marrow Microenvironment

Background: Gilteritinib is a clinically active FLT3 tyrosine kinase inhibitor (TKI) approved for relapsed/refractory FLT3-mutant AML, but nearly all patients treated with gilteritinib and other FLT3 TKIs eventually develop clinical resistance. Activating RAS/MAPK pathway mutations are a predominant non-FLT3 dependent resistance mechanism in patients treated with gilteritinib. AML blasts can also develop FLT3 TKI resistance secondary to paracrine MAPK activation stimulated by FLT3 Ligand, FGF2, or other protective cytokines within the bone marrow microenvironment (BME). To identify potential targets that sensitize AML cells to gilteritinib-induced apoptosis in a model of the BME, we performed a genome-wide CRISPR/Cas9 death screen in MOLM-14 FLT3-ITD+ human AML cells cultured in bone marrow stromal cell conditioned media. We hypothesize that identified genes represent promising combinatorial therapeutic targets that can enhance clinical efficacy of FLT3 TKIs in AML. Methods: To model stroma-mediated TKI resistance, we used the HS5 human bone marrow stromal cell line that secretes multiple cytokines (G-CSF, GM-CSF, FGF2) and supports myeloid progenitor proliferation in co-culture. MOLM-14 CRISPRi cells transduced with CRISPRi-v2 genome-wide sgRNA library were cultured in HS5 conditioned media for 24 hours and then treated with gilteritinib 250 nM. Cells were stained with a fluorogenic caspase 3/7 reagent and then fixed after 24 hours of drug treatment. Caspase-3 positive cells were sorted from the entire drug-treated cell population by FACS and guide RNAs enriched or depleted in this sample as compared to an untreated T0 sample were determined by NGS. Results: We identified several gene-level hits that were enriched in the apoptotic population (FDR <0.2). Among these, we identified multiple transcription factors or regulators of transcriptional activation. The latter included multiple components of RNA pol II machinery (POLR2G, RTF1) and multiple subunits of Mediator (MED12, MED30, MED21, MED11), a complex that regulates RNA pol II activity and has been shown to modulate super-enhancer-associated genes in AML cells. To validate select hits, we transduced MOLM-14 and MV411 CRISPRi cells with a tetracycline-inducible sgRNA expression vector. Using this system, we found that conditional knockdown of MED12, the top scoring Mediator subunit in our screen, significantly sensitized MOLM-14 (FgH1) cells to gilteritinib while modestly augmenting the cytotoxicity of gilteritinib in MV411 (FgH1) cells when compared to a non-targeting sgRNA. Functionally, MED12 associates with MED13, Cyclin C, and CDK8 to form the CDK8 kinase module of Mediator. MED12 knockdown, as expected, led to suppression of STAT1 S7272 and STAT5 S726 phosphorylation, known targets of CDK8. Based on these results, we hypothesized that CDK8 inhibition would augment apoptosis induced by gilteritinib in HS5 conditioned media. Using SEL120, a novel CDK8 inhibitor already in early phase AML clinical trials, we performed an 8 x 8 dose matrix drug synergy analysis of gilteritinib and SEL120 in multiple FLT3-mutant AML cell lines using Bliss independence modeling. We found that SEL120 was synergistic with gilteritinib in inducing apoptosis in MOLM-14 and MV411 cells in HS5 conditioned media (Bliss synergy scores of 2.44 and 11.45 with most synergistic area scores of 10.91 and 26.85, respectively). We also found combinatorial activity against MOLM-14 cells harboring secondary NRAS activating mutations (G12C and Q61K), suggesting the therapeutic combination could potentially overcome cell intrinsic and extrinsic MAPK-activating resistance mechanisms. Lastly, we found that gilteritinib and SEL120 combined to impart greater cytotoxicity than either drug alone in a primary sample (AML #1) from a patient with newly diagnosed AML possessing a FLT3-ITD mutation at high mutant allele ratio. Conclusions: The results and validation of our CRISPRi screen suggest that combined CDK8 and FLT3 inhibition is a novel strategy for augmenting gilteritinib cytotoxicity. Assessment of the activity of the combination in additional primary AML samples and in vivo murine models of AML is planned. Additional candidate targets already described and other Mediator and RNA pol II subunits from our screen are also being further evaluated to precisely define the transcriptional programs that influence FLT3 inhibitor resistance. Disclosures Logan: Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding; Amgen, Pfizer, AbbVie: Consultancy. Gilbert: Denali Therapeutics: Ended employment in the past 24 months, Other: Spouse/Significant Other's Employment; GSK: Consultancy, Research Funding; AstraZeneca: Research Funding; Chroma Medicine: Consultancy, Other: Co-founder. Smith: Revolutions Medicine: Research Funding; AbbVie: Research Funding; Daiichi Sankyo: Consultancy; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Biology and Clinical Implications of Complex Landscape of Cooperating Events in FLT3-ITD Acute Myeloid Leukemia

FLT3-ITD mutations are among the most common somatic mutations in acute myeloid leukemia (AML) and are important in prognostic determination as well as therapeutic allocation. Recent studies have demonstrated improved outcomes with the addition of FLT3 inhibitors and for some patients hematopoietic stem cell transplant (HSCT) in first remission (CR1). We have previously demonstrated that the outcome of FLT3-ITD patients can be quite heterogeneous based on the co-occurrence of a few specific risk stratifying mutations, including NPM1 and NUP98-NSD1. We sought to interrogate the complex landscape of cooperating events with FLT3-ITD AML and potential impacts on outcome in the context of contemporary therapies, including the FLT3 inhibitor sorafenib. Of the 1296 children and young adult patients with de novo AML enrolled on COG AAML1031, 229 had FLT3-ITD mutations and were included in this study. Patients with high allelic ratio (HAR; >0.4) FLT3-ITD were allocated to Arm C, received sorafenib in combination with chemotherapy and received HSCT in CR1. Those with low allelic ratio (LAR; £ 0.4) FLT3-ITD were treated on Arm A/B and received chemotherapy, no sorafenib, and did not receive HSCT in CR1 unless they had evidence of residual disease following induction I (MRD³ 0.1%) or a high-risk cytogenetic feature. FLT3-ITD status and allelic ratio were determined by PCR and all samples also underwent karyotyping, FISH, and next generation sequencing in 195 (85%) of cases for determination of comprehensive co-occurring mutational profile. Among the 229 FLT3-ITD positive patients, allelic ratio ranged from <0.1-20.8, with 96 (42%) patients classified as LAR and 133 (58%) patients as HAR. Among the cohort overall, the significant majority of 85% (n=195) harbored a cooperating genomic aberration. The most common co-occurring single gene mutations were: WT1 (31%, n=71), NPM1 (20%, n=46), NRAS (9.2%, n=21), FLT3-TKD (7%, n=16), CEBPA (6.5%, n=15), KMT2A-PTD (5.7%, n=13) (Figure 1A). KMT2A-PTD lesions were significantly more prevalent among FLT3-ITD vs non ITD patients, 5.7% vs. 0.65% (p<0.001). Normal karyotype was detected in 50% of patients. The most common recurring cytogenetic abnormalities were NUP98-NSD1/t(5;11) fusions (19.2%, n=44), trisomy 8 (10%, n=23), DEK-NUP214/t(6;9) fusions (7%, n=16), KMT2A rearrangements (3.9%, n=9)(Figure 1A). In contrast, the other high risk abnormalities (monosomy 5/del5q, monosomy 7) were absent or exceedingly rare, while the low risk lesions t(8;21) and inv(16) were also rare (3%, n=7 each). We have previously reported outcome of the more common and risk stratifying mutations with co-occurring NUP98-NSD1 resulting in dismal prognosis regardless of treatment arm, while outcome for those with WT1 was improved with Arm C treatment and approached that of other FLT3-ITD patients(Figure 1B). Evaluation of the FLT3-ITD/trisomy 8 patients demonstrated those treated on Arm C experienced poor outcomes with an EFS of 30% and was equivalent to 29% for those on Arm A/B (p=0.96, Figure 1C), with a corresponding OS of 40% vs. 34% (p=0.66) respectively. In contrast, evaluation of outcome of the KMT2A-PTD patients demonstrated those treated on Arm C had a favorable 5-year event-free survival (EFS) of 71% vs. 23% (p=0.05) for those on Arm A/B (Figure 1D), with a corresponding 5-year overall survival (OS) of 86% vs. 46% (p=0.15) respectively. Comprehensive sequencing demonstrated the FLT3-ITD samples identified co-occurring genetic mutations or cytogenetic abnormalities in the majority of cases. Although KMT2A-PTD is rarely reported in pediatric compared to adult AML, we found it was enriched in FLT3-ITD patients and this cohort experienced favorable outcomes when treated with transplant and sorafenib. Patients with dual FLT3-ITD/trisomy 8 had suboptimal outcomes similar to other poor risk co-occurring lesions and comparable regardless of AR or treatment arm. While there was some overlap with WT1 mutations in this cohort, further investigation into prognostic impact of this cooperating event is warranted. The prognostic implications FLT3-ITD mutations vary and we provide further data that the comprehensive cooperating mutational profile is critical to understanding the prognostic implications in specific patients, and may also impact response to FLT3 inhibitor therapy. Figure 1 Figure 1. Disclosures Hylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pollard: Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees.