FLT3 Internal Tandem Duplication Acute Myeloid Leukemia Research Articles

Ningetinib, a novel FLT3 inhibitor, overcomes secondary drug resistance in acute myeloid leukemia.

FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is a common mutation type in acute myeloid leukemia (AML) and is usually associated with poor patient prognosis. With advancements in molecular diagnostics and the development of tyrosine kinase inhibitors (TKI), the overall survival (OS) of AML patients with FLT3-ITD mutations has been prolonged to some extent, but relapse and drug resistance are still substantial challenges. Ningetinib is a novel TKI against various kinases in relation to tumour pathogenesis and is undergoing clinical trials of lung cancer. In this study, we explored the antitumor activity of ningetinib against AML with FLT3 mutations both in vivo and in vitro. Cell proliferation assays were performed in AML cell lines and Ba/F3 cells expressing various FLT3 mutations to validate the antileukemic activity of ningetinib in vitro. Immunoblot assays were used to verify the effect of ningetinib on the FLT3 protein and downstream pathways. Molecular docking and CETSA were used to validate the interaction of ningetinib with target proteins. The survival benefit of ningetinib in vivo was assessed in Ba/F3-FLT3-ITD-, MOLM13, Ba/F3-FLT3-ITD-F691L-, MOLM13-FLT3-ITD-F691L-induced leukemia mouse models. We also used patient-derived primary cells to determine the efficacy of ningetinib. Ningetinib inhibited cell proliferation, blocked the cell cycle, induced apoptosis and bound FLT3 to inhibit its downstream signaling pathways, including the STAT5, AKT and ERK pathways, in FLT3-ITD AML cell lines. In the mouse models with FLT3-ITD and FLT3-ITD-F691L mutation, ningetinib showed superior anti-leukemia activity to existing clinical drugs gilteritinib and quizartinib, significantly prolongating the survival of mice. In addition, ningetinib exhibited activity against patient-derived primary cells harboring FLT3-ITD mutations. Overall, our study confirmed the therapeutic role of ningetinib in AML with FLT3-ITD mutations, providing a potential new option for clinically resistant patients.

Combining the novel FLT3 and MERTK dual inhibitor MRX-2843 with venetoclax results in promising antileukemic activity against FLT3-ITD AML

FMS-like tyrosine kinase 3 (FLT3) mutations occur in approximately one third of acute myeloid leukemia (AML) patients. FLT3-Internal tandem duplication (FLT3-ITD) mutations are the most common FLT3 mutations and are associated with a poor prognosis. Gilteritinib is a FLT3 inhibitor that is US FDA approved for treating adult patients with relapsed/refractory AML and a FLT3 mutation. While gilteritinib monotherapy has improved patient outcome, few patients achieve durable responses. Combining gilteritinib with venetoclax (VEN) appears to make further improvements, though early results suggest that patients with prior exposure to VEN fair much worse than those without prior exposure. MRX-2843 is a promising inhibitor of FLT3 and MERTK. We recently demonstrated that MRX-2843 is equally potent as gilteritinib in FLT3-ITD AML cell lines in vitro and primary patient samples ex vivo. In this study, we investigated the combination of VEN and MRX-2843 against FLT3-ITD AML cells. We found that VEN synergistically enhances cell death induced by MRX-2843 in FLT3-mutated AML cell lines and primary patient samples. Importantly, we found that VEN synergistically enhances cell death induced by MRX-2843 in FLT3-ITD AML cells with acquired resistance to cytarabine (AraC) or VEN+AraC. VEN and MRX-2843 significantly reduce colony-forming capacity of FLT3-ITD primary AML cells. Mechanistic studies show that MRX-2843 decreases Mcl-1 and c-Myc protein levels via transcriptional regulation and combined MRX-2843 and VEN significantly decreases oxidative phosphorylation in FLT3-ITD AML cells. Our findings highlight a promising combination therapy against FLT3-ITD AML, supporting further in vitro and in vivo testing.

Red blood cell membrane-coated FLT3 inhibitor nanoparticles to enhance FLT3-ITD acute myeloid leukemia treatment

FMS-like tyrosine kinase 3 (FLT3) is a viable and important therapeutic target for acute myeloid leukemia (AML). FLT3 internal tandem duplication (FLT3-ITD) mutations have been identified in approximately 30% of AML patients, and are associated with unfavorable prognosis, higher risk of relapse, drug resistance, and poor clinical outcome. Even FLT3 inhibitors have demonstrated promising efficacy, they cannot cure AML or even significantly extend the lives of patients with FLT3-ITD mutations. This is partly because of poor water solubility, insufficient membrane penetration and short half-life of small molecule inhibitors. Besides, the presence of enzymes like CYP3A4 in bone marrow accelerate the elimination and metabolism of FLT3 inhibitors, resulting in low plasma concentrations and side effects. Here we report the erythrocyte membrane-camouflaged FLT3 inhibitor nanoparticles to enhance FLT3-ITD AML treatment. Briefly, we physically coextruded red blood cell (RBC) membrane vesicles with nanoparticles derived from FLT3 inhibitor F30 to obtain F30@RBC-M, which exhibited comparable potent FLT3-ITD inhibitory effects compared to free F30in vitro, while displaying a higher potent antitumor efficacy in xenograft models due to the prolonged circulation properties. Furthermore, administration of F30@RBC-M significantly extended the survival of mice in a transplanted mouse model than F30 free drug. These findings suggest that RBC membrane-coated nanoparticles derived from FLT3 inhibitors hold promise as a tool to enhance the therapeutic efficacy to treat FLT3-ITD AML.

Pim Kinase Inhibitors Increase Gilteritinib Cytotoxicity in FLT3-ITD Acute Myeloid Leukemia Through GSK-3β Activation and c-Myc and Mcl-1 Proteasomal Degradation.

FLT3-ITD is present in 25% of in AML, with continued poor outcomes. Combining Pim kinase inhibitors with the FDA-approved FLT3 inhibitor gilteritinib increases cytotoxicity in vitro and in vivo through activation of GSK-3β, which phosphorylates and posttranslationally downregulates c-Myc and Mcl-1. The data support efficacy of GSK-3βactivation in FLT3-ITD AML, and also support development of a clinical trial combining the Pim inhibitor TP-3654 with gilteritinib.

Sorafenib plus intensive chemotherapy in newly diagnosed FLT3-ITD AML: a randomized, placebo-controlled study by the ALLG

AbstractSorafenib maintenance improves outcomes after hematopoietic cell transplant (HCT) for patients with FMS-like tyrosine kinase 3–internal tandem duplication (FLT3-ITD) acute myeloid leukemia (AML). Although promising outcomes have been reported for sorafenib plus intensive chemotherapy, randomized data are limited. This placebo-controlled, phase 2 study (ACTRN12611001112954) randomized 102 patients (aged 18-65 years) 2:1 to sorafenib vs placebo (days 4-10) combined with intensive induction: idarubicin 12 mg/m2 on days 1 to 3 plus either cytarabine 1.5 g/m2 twice daily on days 1, 3, 5, and 7 (18-55 years) or 100 mg/m2 on days 1 to 7 (56-65 years), followed by consolidation and maintenance therapy for 12 months (post-HCT excluded) in newly diagnosed patients with FLT3-ITD AML. Four patients were excluded in a modified intention-to-treat final analysis (3 not commencing therapy and 1 was FLT3-ITD negative). Rates of complete remission (CR)/CR with incomplete hematologic recovery were high in both arms (sorafenib, 78%/9%; placebo, 70%/24%). With 49.1-months median follow-up, the primary end point of event-free survival (EFS) was not improved by sorafenib (2-year EFS 47.9% vs 45.4%; hazard ratio [HR], 0.87; 95% confidence interval [CI], 0.51-1.51; P = .61). Two-year overall survival (OS) was 67% in the sorafenib arm and 58% in the placebo arm (HR, 0.76; 95% CI, 0.42-1.39). For patients who received HCT in first remission, the 2-year OS rates were 84% and 67% in the sorafenib and placebo arms, respectively (HR, 0.45; 95% CI, 0.18-1.12; P = .08). In exploratory analyses, FLT3-ITD measurable residual disease (MRD) negative status (<0.001%) after induction was associated with improved 2-year OS (83% vs 60%; HR, 0.4; 95% CI, 0.17-0.93; P = .028). In conclusion, routine use of pretransplant sorafenib plus chemotherapy in unselected patients with FLT3-ITD AML is not supported by this study.

Children's Oncology Group's 2023 blueprint for research: Myeloid neoplasms.

During the past decade, the outcomes of pediatric patients with acute myeloid leukemia (AML) have plateaued with 5-year event-free survival (EFS) and overall survival (OS) of approximately 46 and 64%, respectively. Outcomes are particularly poor for those children with high-risk disease, who have 5-year OS of 46%. Substantial survival improvements have been observed for a subset of patients treated with targeted therapies. Specifically, children with KMT2A-rearranged AML and/or FLT3 internal tandem duplication (FLT3-ITD) mutations benefitted from the addition of gemtuzumab ozogamicin, an anti-CD33 antibody-drug conjugate, in the AAML0531 clinical trial (NCT00372593). Sorafenib also improved response and survival in children with FLT3-ITD AML in the AAML1031 clinical trial (NCT01371981). Advances in characterization of prognostic cytomolecular events have helped to identify patients at highest risk of relapse and facilitated allocation to consolidative hematopoietic stem cell transplant (HSCT) in first remission. Some patients clearly have improved survival with HSCT, although the benefit is largely unknown for most patients. Finally, data-driven refinements in supportive care recommendations continue to evolve with meaningful and measurable reductions in toxicity and improvements in EFS and OS. As advances in application of targeted therapies, risk stratification, and improved supportive care measures are incorporated into current trials and become standard-of-care, there is every expectation that we will see improved survival with a reduction in toxic morbidity and mortality. The research agenda of the Children's Oncology Group's Myeloid Diseases Committee continues to build upon experience and outcomes with an overarching goal of curing more children with AML.

Sitravatinib as a potent FLT3 inhibitor can overcome gilteritinib resistance in acute myeloid leukemia

BackgroundGilteritinib is the only drug approved as monotherapy for acute myeloid leukemia (AML) patients harboring FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation throughout the world. However, drug resistance inevitably develops in clinical. Sitravatinib is a multi-kinase inhibitor under evaluation in clinical trials of various solid tumors. In this study, we explored the antitumor activity of sitravatinib against FLT3-ITD and clinically-relevant drug resistance in FLT3 mutant AML.MethodsGrowth inhibitory assays were performed in AML cell lines and BaF3 cells expressing various FLT3 mutants to evaluate the antitumor activity of sitravatinib in vitro. Immunoblotting was used to examine the activity of FLT3 and its downstream pathways. Molecular docking was performed to predict the binding sites of FLT3 to sitravatinib. The survival benefit of sitravatinib in vivo was assessed in MOLM13 xenograft mouse models and mouse models of transformed BaF3 cells harboring different FLT3 mutants. Primary patient samples and a patient-derived xenograft (PDX) model were also used to determine the efficacy of sitravatinib.ResultsSitravatinib inhibited cell proliferation, induced cell cycle arrest and apoptosis in FLT3-ITD AML cell lines. In vivo studies showed that sitravatinib exhibited a better therapeutic effect than gilteritinib in MOLM13 xenograft model and BaF3-FLT3-ITD model. Unlike gilteritinib, the predicted binding sites of sitravatinib to FLT3 did not include F691 residue. Sitravatinib displayed a potent inhibitory effect on FLT3-ITD-F691L mutation which conferred resistance to gilteritinib and all other FLT3 inhibitors available, both in vitro and in vivo. Compared with gilteritinib, sitravatinib retained effective activity against FLT3 mutation in the presence of cytokines through the more potent and steady inhibition of p-ERK and p-AKT. Furthermore, patient blasts harboring FLT3-ITD were more sensitive to sitravatinib than to gilteritinib in vitro and in the PDX model.ConclusionsOur study reveals the potential therapeutic role of sitravatinib in FLT3 mutant AML and provides an alternative inhibitor for the treatment of AML patients who are resistant to current FLT3 inhibitors.

Microenvironmental CXCL12 deletion enhances Flt3-ITD acute myeloid leukemia stem cell response to therapy by reducing p38 MAPK signaling.

Fms-like tyrosine kinase 3 (Flt3) tyrosine kinase inhibitors (Flt3-TKI) have improved outcomes for patients with Flt3-mutated acute myeloid leukemia (AML) but are limited by resistance and relapse, indicating persistence of leukemia stem cells (LSC). Here utilizing a Flt3-internal tandem duplication (Flt3-ITD) and Tet2-deleted AML genetic mouse model we determined that FLT3-ITD AML LSC were enriched within the primitive ST-HSC population. FLT3-ITD LSC showed increased expression of the CXCL12 receptor CXCR4. CXCL12-abundant reticular (CAR) cells were increased in Flt3-ITD AML marrow. CXCL12 deletion from the microenvironment enhanced targeting of AML cells by Flt3-TKI plus chemotherapy treatment, including enhanced LSC targeting. Both treatment and CXCL12 deletion partially reduced p38 mitogen-activated protein kinase (p38) signaling in AML cells and further reduction was seen after treatment in CXCL12 deleted mice. p38 inhibition reduced CXCL12-dependent and -independent maintenance of both murine and human Flt3-ITD AML LSC by MSC and enhanced their sensitivity to treatment. p38 inhibition in combination with chemotherapy plus TKI treatment leads to greater depletion of Flt3-ITD AML LSC compared with CXCL12 deletion. Our studies support roles for CXCL12 and p38 signaling in microenvironmental protection of AML LSC and provide a rationale for inhibiting p38 signaling to enhance Flt3-ITD AML targeting.

PB2183: RELAPSE PREDICTS POOR OUTCOMES IN FLT3-MUTATED ACUTE MYELOID LEUKEMIA POST ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION

Background: Allogeneic hematopoietic cell transplantation (alloHCT) is the only curative option for patients with FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplication) acute myeloid leukemia (AML). Although sorafenib has been recommended as maintenance treatment, data on risk factors and outcomes in these patients are limited. Aims: We investigated the risk factors and outcomes in patients with FLT3-mutated AML that underwent alloHCT. Methods: We enrolled consecutive adult patients with FLT3-mutated AML that underwent alloHCT at our JACIE-accredited center over the last two decades based on EBMT standards. The following factors were recorded and analyzed: age, disease, phase at transplant, type of donor and transplant, grade II-IV acute and moderate/severe chronic graft-versus-host disease (GVHD), relapse, treatment-related mortality (TRM), disease-free (DFS) and overall survival (OS). Results: We studied 43 patients with FLT3-mutated AML who received alloHCT at a median age of 45 (18-62) years from sibling (21), matched (16) or mis-matched (3) unrelated, and haploidentical donors (3). Phase at transplant was first complete remission/CR1 (31 patients), CR2 (9) or relapsed/refractory disease (3). With a median follow-up of 26.5 (1.2-239) months, relapse was observed in 16 patients, 4 of whom presented only with isolated extramedullary disease. Five-year cumulative incidence (CI) of relapse was 41.7% and was independently associated with previous lines of treatment (p=0.015), disease phase at transplant (25.7% for CR1, 50% for CR2, and 100% for relapsed/refractory disease, p=0.007), and chronic GVHD (p=0.004). Relapse was treated with chemotherapy (15 patients), donor lymphocyte infusions (7), azacytidine (6), or gilteritinib (3). No patient received sorafenib either as maintenance or treatment. Only one relapsed patient survived after treatment of isolated extramedullary disease. Five-year CI of TRM was 17.4% and was associated only with disease phase at transplant (p<0.001). Five-year DFS and OS were 46.5% and 52.5%, respectively. The only significant predictor of OS was relapse (beta=0.303, p<0.001). Summary/Conclusion: r study confirms that relapse post alloHCT is an imminent feature of FLT3-mutated AML, associated with poor outcomes. Disease phase at transplant is an important risk factor for alloHCT outcomes. Nevertheless, relapse incidence remains high, even in patients transplanted in CR1, highlighting the necessity of maintenance therapy with FLT3 inhibitors.

Abstract 3338: Concurrent treatment with Pim kinase inhibitor enhances efficacy of FLT3 inhibitors in AML with FLT3-ITD through GSK-3βactivation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1

Abstract BACKGROUND: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) cells in 30% of patients, with poor patient outcomes. FLT3 inhibitors are in clinical use, but with incomplete responses and onset of resistance. New targeting strategies are needed. The oncogenic serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD, directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop. We previously showed that concurrent treatment of FLT3-ITD cells with Pim kinase and FLT3 inhibitors enhanced apoptosis induction through post-translational downregulation of the master transcription factor c-Myc and the anti-apoptotic protein Mcl-1. The serine/threonine kinase GSK-3β regulates c-Myc and Mcl-1 protein via phosphorylation at T58 and S159, respectively; we hypothesized downregulation of both proteins by combination treatment through phosphorylation by GSK-3β. METHODS: FLT3-ITD cells lines (Ba/F3-ITD, MV4-11, and MOLM-14) and primary FLT3-ITD AML patient blasts were cultured with the pan-Pim kinase inhibitor AZD1208, the FLT3 inhibitors gilteritinib or quizartinib and the GSK-3β inhibitor TCG-24. Protein expression was measured by immunoblotting. To measure protein half-lives, cells were pretreated with cycloheximide with and without the proteasome inhibitor MG-132. Ba/F3-ITD cells were infected with Myc-T58A plasmid, preventing c-Myc T58 phosphorylation, or Mcl-1 S159A plasmid, preventing Mcl-1 S159 phosphorylation, or empty vector controls. Apoptosis was measured by Annexin V labeling, measured by flow cytometry. RESULTS: Combined treatment of FLT3-ITD-expressing cell lines and AML patient blasts with Pim and FLT3 inhibitors caused rapid downregulation of c-Myc, and then Mcl-1, protein expression, relative to single drugs. Combined Pim and FLT3 inhibitor treatment caused marked decrease in half-lives of both proteins, which was rescued by proteasome inhibition, as the mechanism of protein downregulation. Combined Pim and FLT3 inhibitor treatment rapidly activated (dephosphorylated) GSK-3β and culture with GSK-3β inhibitor prevented induction of c-Myc and Mcl-1 downregulation, increased c-Myc and Mcl-1 protein turnover by concurrent Pim and FLT3 inhibitor treatment. Finally, combination treatment of Ba/F3-ITD cells infected with T58A c-Myc or S159A Mcl-1 plasmids did not downregulate these proteins or increase their turnover, with reduced apoptosis induction. CONCLUSION: Pim inhibitors enhance efficacy of FLT3 inhibitors via GSK-3β activation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1, highlighting GSK-3β as a key target in cells with FLT3-ITD. Citation Format: Jonelle Lee, Mario Scarpa, Aditi Chatterjee, Mooath Mustafa Ali, Prerna Singh, Shivani Kapoor, Rossana Trotta, Maria Baer. Concurrent treatment with Pim kinase inhibitor enhances efficacy of FLT3 inhibitors in AML with FLT3-ITD through GSK-3βactivation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3338.

Cytogenetic risk classification maintains its prognostic significance in transplanted FLT3-ITD mutated acute myeloid leukemia patients: On behalf of the acute leukemia working party/European society of blood and marrow transplantation.

FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutational status is a pivotal prognosticator in acute myeloid leukemia (AML) patients and significantly increases the risk of disease relapse. However, it remains unclear whether in FLT3-ITD patients referred for allogeneic stem cell transplantation (allo-SCT), baseline cytogenetics significantly impacts clinical outcome. Using the European Society of Blood and Marrow Transplantation registry, we performed a retrospective analysis of 1631 FLT3-ITD AML patients who underwent allo-SCT with the aim of determining the influence of cytogenetic risk category on patient outcomes. Median patient age was 49 years and median follow-up duration was 36 months. Two-year leukemia-free survival (LFS) and incidence of relapse were 54% and 31.6%, respectively. Non-relapse mortality was experienced by 14.4% with a 2-year overall survival (OS) of 60.1%. On multivariate analysis, LFS was significantly lower in patients with intermediate and adverse risk cytogenetics compared with those with favorable risk cytogenetics, (hazard ratio [HR]=1.48, 95% confidence interval [CI], 1.06-2.06; p= .02), and (HR=01.65, 95% CI, 1.13-2.40; p= .009), respectively. OS was significantly lower in patients with adverse risk cytogenetics compared with patients with favorable risk cytogenetics (HR=1.74, 95% CI, 1.16-2.61; p= .008) with a trend toward lower OS in patients with intermediate risk cytogenetics compared to those with favorable risk cytogenetics (HR=1.43, 95% CI, 1.00-2.05; p= .052). In addition, adverse risk patients and intermediate risk patients experienced higher relapse rates compared with favorable risk patients (HR=1.83, 95% CI, 1.13-2.94; p= .013 and HR=1.82, 95% CI, 1.19-2.77; p= .005). Overall, cytogenetic studies aid in refinement of risk stratification in transplanted FLT3-ITD AML patients.

Mannose Metabolism Is a Metabolic Vulnerability Unveiled By Standard and Novel Therapies in Acute Myeloid Leukemia

IntroductionThe development of resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukemia (AML). Metabolic rewiring has emerged as a therapeutically actionable vulnerability in AML, where specific metabolic adaptations arising as a result of driver mutations or in response to therapy have been reported. Mannose phosphoisomerase (MPI) is the first enzyme in the mannose metabolism (MM) pathway, that leads to the production of GDP-Mannose, a key sugar donor for N-glycosylation reactions. MPI was amongst the top drop-out genes in our published CRISPR-Cas9 screen aiming to identify sensitizers to FLT3 tyrosine kinase inhibitors (TKI) in AML carrying activating FLT3 internal tandem duplication (ITD) mutations (Gallipoli et al., Blood 2018). Moreover, analysis of published genomic datasets indicates that MPI expression levels are higher in AML compared to normal samples, correlate with patient outcome (A) and further increase in paired relapsed to diagnosis samples. We therefore hypothesized that MM and MPI inhibition sensitize AML cells to both FLT3-TKI and standard chemotherapy and tested this in preclinical models.MethodsExperiments were performed in human AML cell lines, primary AML mononuclear cells and normal CD34 + stem/progenitor cells. Liquid chromatography coupled to mass spectrometry and oxygen consumption/extracellular acidification rate as measured by a Seahorse analyser were employed to assess metabolic changes. Gene-expression was measured by RNA-sequencing and confirmed by RT-qPCR. Viability, surface protein expression and lipid peroxidation were assessed by flow-cytometry. Protein expression and localisation was measured by western blot and immunofluorescence. Gene silencing was performed using CRISPR-Cas9 gene editing and inducible short hairpin RNA interference.ResultsWe show that genetic and chemical MPI inhibition sensitizes FLT3 WT and FLT3 ITD AML cell lines in vitro to standard cytarabine chemotherapy and FLT3-TKI respectively and these effects are rescued by the addition of MPI downstream product mannose. We validate these findings in vivo using MPI knock-out (KO) cell line xenografts and in vitro using primary AML samples following MPI knock-down (B). We also demonstrate a lack of toxicity to normal CD34 + cells.Surprisingly, global metabolomic analysis show that MPI KO cells accumulate fatty acids and particularly polyunsaturated fatty acids (PUFA) (C), due to both increased uptake and reduced fatty acid oxidation (FAO). MPI KO cells downregulate several FAO metabolism genes and this is corroborated by the strong positive correlation of MPI expression levels with multiple genes involved in FAO across multiple primary AML datasets. Metabolic profiling demonstrates that MPI KO cells have reduced oxidative phosphorylation metabolism and in particular are unable to oxidise palmitate, an effect rescued by mannose or the FAO activator fenofibrate (D).We link this metabolic defect to the specific activation of the ATF6 arm of the unfolded protein response (UPR) due to a reduction in protein glycosylation in MPI KO cells. We validate this by demonstrating that activating or inhibiting ATF6 respectively phenocopies or rescues the effects of MPI KO (E) and show this to be secondary to ATF6 driving FAO inhibition, via transcriptional downregulation of PPARα, a master regulator of lipid catabolism.Finally, we show that accumulation of PUFA in MPI KO cells is accompanied by reduced cysteine levels and increased markers of oxidative stress and lipid peroxidation, such as 4-hydroxynonenal (4-HNE), all features associated with ferroptosis (F). We validate this by showing that combining FLT3-TKI with MPI KO leads to ferroptotic cell death in AML cells, which can be rescued by the radical-trapping antioxidant ferrostatin.ConclusionsWe provide further evidence to support the role of metabolic rewiring in driving therapy resistance in AML and show for the first time that targeting MPI and MM sensitizes AML cells to cytarabine and FLT3-TKI. Mechanistically we unveil a novel connection between MM and fatty acid metabolism, via activation of the ATF6 arm of the UPR, leading to cellular PUFA accumulation, lipid peroxidation and ferroptotic cell death. Finally, our findings also suggest that triggering ferroptosis can be leveraged as a therapeutically actionable mechanism driving cell death in therapy-resistant AML cells. [Display omitted] DisclosuresVassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy.

Impact of Concomitant Genetic Abnormalities on Sorafenib Therapy in FLT3-ITD Positive Acute Myeloid Leukemia Undergoing Allo-HSCT

AimIn this study, we investigated the co-occurring mutation landscape in acute myeloid leukemia (AML) patients with FLT3 internal tandem duplication (FLT3-ITD), and explored whether post-allogenic hematopoietic stem cell transplantation (allo-HSCT) sorafenib maintenance therapy could improve the outcomes of FLT3-ITD AML patients combined with other co-occurring genetic abnormalities.MethodsA total of 456 FLT3-ITD AML patients receiving first allo-HSCT were included in this study. Gene mutations were detected using direct sequencing or next generation sequencing. Fusion genes were detected using a 53-gene polymerase chain reaction panel. The frequency of each genetic abnormality was investigated, and the mutation landscape was investigated. The primary outcome of this study was 3-year cumulative incidence of relapse. The secondary outcomes were 3-year overall survival (OS), disease-free survival (DFS) and non-relapse mortality (NRM). The outcomes were compared between patients who received post-HSCT sorafenib maintenance and those who did not in the whole population and in subgroups referring to co-occurring mutations.ResultsA total of 190 patients received post transplantation sorafenib maintenance therapy (sorafenib group) and 266 patients did not (control group). Of the patients receiving sorafenib pre-transplantation, the median duration of sorafenib therapy was 106 days (IQR 68 - 132) in the sorafenib group, and 99 days (IQR 71 - 142) in the control group (p = 0.883). Of the patients receiving post-transplantation sorafenib maintenance therapy, sorafenib was initiated at a median of 30 days (IQR, 30 - 52 days) after allo-HSCT, and continued for a median of 148 days (IQR, 112 - 150).Median follow-up time was 38.7 months (IQR, 28.1 - 47.9). Thirty-four patients in the sorafenib group and 93 patients in the control group relapsed. The 3-year cumulative incidence of relapse was 18.0% (95% CI 13.1% - 24.3%) in the sorafenib group and 36.1% (95% CI 30.5% - 42.3%) in the control group (HR 0.43, 95% CI 0.29 - 0.64; p < 0.001). A total of 126 patients died, including 39 in the sorafenib group and 87 in the control group. Three-year DFS was 75.8% in the sorafenib group and 57.5% in the control group (HR 0.47, 95% CI 0.34 - 0.66; p < 0.001). Three-year OS was 79.5% for patients in the sorafenib group and 68.4% for patients in the control group (HR 0.57, 95% CI 0.39 - 0.83, p = 0.004).Gene mutations were detected using a 12-mutation panel direct sequencing including FLT3-ITD, FLT3-TKD, NPM1, KIT, DNMT3A, CEBPA, ASXL1, TP53, TET2, IDH1, IDH2 and RUNX1 in all patients, and a 127-gene panel new generation sequencing in 188 patients.Except FLT3, a total of 920 co-occurring gene mutations and 147 cytogenetic abnormalties were detected. The co-occurring mutations that presented in at least 10% the cases were NPM1 (31.6%), DNMT3A (15.4%), TET2 (11.4%), and CEBPA (10.5%). There were 10.5% patients presented with both NPM1 and DNMT3A mutations (Triple-mutated AML patients), and 13.8% patients combined with at least one additional genetic abnormality classified as adverse according to the 2017 ELN risk stratification other than FLT3-ITD. Cytogenetic abnormalities presented in more than 1% the patients were RUNX1-RUNX1T1, which occurred in 23 patients (5.0%), followed by +8 (2.9%), complex karyotype (2.6%), CBFB-MYH11 (2.4%), DEK-NUP214 (1.3%) and -y (1.3%).Patients combined with NPM1(p = 0.009 and 0.009), DNMT3A (p = 0.036 and 0.086), triple-mutated AML patients (p = 0.030 and 0.027), and patients with at least one additional adverse abnormality (p = 0.014 and 0.005) benefit significantly in DFS and OS from post-transplantation sorafenib maintenance, but not those with CEBPA (p = 0.669 and 0.576) or TET2 (p = 0.375 and 0.178) mutations.ConclusionPost transplantation sorafenib maintenance therapy can improve the prognosis of FLT3-ITD AML patients combined with DNMT3A or NPM1, patients with triple-mutated AML, and patients combined with at least one additional adverse abnormalities, but not of those combined with CEBPA or TET2 mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cigarette Smoke or Cigarette Condensate Exposure Accelerates Growth of FLT3-ITD AML Models, Induces Oxidative Stress, and Alters DNA Methylation

Cigarette Smoke or Cigarette Condensate Exposure Accelerates Growth of FLT3-ITD AML Models, Induces Oxidative Stress, and Alters DNA Methylation

Combination Therapy of FLT3 Tyrosine Kinase Inhibitors and BH3 Mimetics Targeting Antiapoptotic MCL-1 Synergistically Eliminates FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and In Vivo

Combination Therapy of FLT3 Tyrosine Kinase Inhibitors and BH3 Mimetics Targeting Antiapoptotic MCL-1 Synergistically Eliminates FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and In Vivo

Pim kinase inhibitor co-treatment decreases alternative non-homologous end-joining DNA repair and genomic instability induced by topoisomerase 2 inhibitors in cells with FLT3 internal tandem duplication.

Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) relapses with new chromosome abnormalities following chemotherapy, implicating genomic instability. Error-prone alternative non-homologous end-joining (Alt-NHEJ) DNA double-strand break (DSB) repair is upregulated in FLT3-ITD-expresssing cells, driven by c-Myc. The serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD, and inhibiting Pim increases topoisomerase 2 (TOP2) inhibitor chemotherapy drug induction of DNA DSBs and apoptosis. We hypothesized that Pim inhibition increases DNA DSBs by downregulating Alt-NHEJ, also decreasing genomic instability. Alt-NHEJ activity, measured with a green fluorescent reporter construct, increased in FLT3-ITD-transfected Ba/F3-ITD cells treated with TOP2 inhibitors, and this increase was abrogated by Pim kinase inhibitor AZD1208 co-treatment. TOP2 inhibitor and AZD1208 co-treatment downregulated cellular and nuclear expression of c-Myc and Alt-NHEJ repair pathway proteins DNA polymerase θ, DNA ligase 3 and XRCC1 in FLT3-ITD cell lines and AML patient blasts. ALT-NHEJ protein downregulation was preceded by c-Myc downregulation, inhibited by c-Myc overexpression and induced by c-Myc knockdown or inhibition. TOP2 inhibitor treatment increased chromosome breaks in metaphase spreads in FLT3-ITD-expressing cells, and AZD1208 co-treatment abrogated these increases. Thus Pim kinase inhibitor co-treatment both enhances TOP2 inhibitor cytotoxicity and decreases TOP2 inhibitor-induced genomic instability in cells with FLT3-ITD.

Combination of Omacetaxine Mepesuccinate (homoharringtonine) and Sorafenib As an Effective Regimen for Acute Myeloid Leukemia (AML) Carrying FLT3-ITD

Objectives: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (FLT3-ITD) is associated with poor response to chemotherapy and prognosis. In vitro drug screening and laboratory mechanistic studies have identified omacetaxine mepesuccinate (OME, also known as homoharringtonine) as an effective agent that demonstrated synergism with FLT3 inhibitors in this AML subtype. We report herein the clinical outcome of the first 40 FLT3-ITD AML patients treated with sorafenib and OME combination (SOME).Methods: Patients with relapsed or refractory (R/R) FLT3-ITD AML were recruited and treated with sorafenib 200-400 mg b.d. continuously and OME [2 mg daily for 7 (first course) or 5 days (second course onwards) every 21 days] until disease progression or allogeneic hematopoietic stem cell transplantation (HSCT). Complete remission (CR) or CR with incomplete hematologic recovery (CRi), leukemia-free survival (LFS) and overall survival (OS) were evaluated. Partial remission (PR) was defined as marrow or circulating blasts ≤ 50% of the pre-treatment state. LFS and OS were evaluated by Kaplan-Meier analysis and compared by log-rank test. Fisher's exact test was used to compare nominal variables. Cox proportional hazard model was used in the univariate and multivariate analyses.Results: Forty patients (16 men, 24 women) were recruited between June 2013 to July 2017. Their clinicopathologic characteristics were shown in Table 1. Thirty patients (75%) achieved CR/CRi, 2 patients showed PR and 7 patients (of whom 5 have had exposure to FLT3 inhibitors) showed no response. One patient was on day 5 of treatment at the time of writing this abstract. The median LFS of the 30 patients who achieved CR/CRi was 4.0 months. Prior exposure to FLT3 inhibitors (p =0.003) and more than one induction chemotherapy before SOME (p <0.001) were associated with a lower rate of CR/CRi. Non-hematological toxicities were minimal and limited to hand-foot-skin reaction and rash associated with sorafenib. Univariate analysis showed that early treatment with SOME (defined as ≤10 months from diagnosis) and HSCT after SOME induced remission were associated with better LFS with a hazard ratio (HR) of 4.1 (1.2-14.1, p =0.026) and 6.1 (1.7-22.0, p =0.005). Achievement of CR/CRi after SOME (HR 4.1, 1.5-11.5 p =0.007), <2 prior induction chemotherapy regimens (HR 3.6, 1.3-9.8, p =0.013), early treatment with SOME (HR 2.8, 1.1-7.2, p =0.031) and HSCT (HR 6.5, 1.5-29.0, p =0.014) were associated with superior OS. In multivariate analysis, HSCT was the only parameter associated with a better LFS (HR 5.4, 1.5-20.0, p =0.012) and OS (HR 6.0, 1.3-27.4, p =0.022).Conclusion: Combination of sorafenib with OME is an effective and safe regimen for R/R FLT3-ITD AML. The molecular features predictive of treatment response as well as the emergence of tyrosine kinase domain mutations at relapse are being evaluated. Further studies testing combination of OME with other FLT3 inhibitors will soon begin. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Marrow Stromal Cells Secrete FGF2 in Exosomes, Providing Paracrine Protection for Leukemia Cells from Kinase Inhibitors

Marrow Stromal Cells Secrete FGF2 in Exosomes, Providing Paracrine Protection for Leukemia Cells from Kinase Inhibitors

Homoharringtonine synergizes with quizartinib in FLT3-ITD acute myeloid leukemia by targeting FLT3-AKT-c-Myc pathway

Acute myeloid leukemia (AML) with FLT3 internal tandem duplication (FLT3-ITD) has a dismal prognosis. FLT3 inhibitors have been developed to treat patients with FLT3-ITD AML; however, when used alone, their efficacy is insufficient. FLT3 inhibitors combined with chemotherapy may be a promising treatment for FLT3-ITD AML. Homoharringtonine (HHT) is a classical anti-leukaemia drug with high sensitivity to FLT3-ITD AML cells. Here, we showed that HHT synergizes with a selective next-generation FLT3 inhibitor, quizartinib, to inhibit cell growth/viability and induce cell-cycle arrest and apoptosis in FLT3-ITD AML cells in vitro, significantly inhibit acute myeloid leukemia progression in vivo, and substantially prolong survival of mice-bearing human FLT3-ITD AML. Mechanistically, HHT and quizartinib cooperatively inhibit FLT3-AKT and its downstream targets GSK3β, c-Myc, and cyclin D1, cooperatively up-regulate the pro-apoptosis proteins Bim and Bax, and down-regulate the anti-apoptosis protein Mcl1. Most strikingly, HHT and quizartinib cooperatively reduce the numbers of side-population (SP) and aldehyde dehydrogenase (ALDH)-positive cells, which reportedly are rich in LSCs. In conclusion, HHT combined with quizartinib may be a promising treatment strategy for patients with FLT3-ITD AML.

Cytoplasmic DROSHA and non-canonical mechanisms of MiR-155 biogenesis in FLT3-ITD acute myeloid leukemia.

We report here on a novel pro-leukemogenic role of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) that interferes with microRNAs (miRNAs) biogenesis in acute myeloid leukemia (AML) blasts. We showed that FLT3-ITD interferes with the canonical biogenesis of intron-hosted miRNAs like miR-126, by phosphorylating SPRED1 protein and inhibiting the “gatekeeper” Exportin 5 (XPO5)/RAN-GTP complex that regulates the nucleus-to-cytoplasm transport of pre-miRNAs for completion of maturation into mature miRNAs. Of note, despite the blockage of “canonical” miRNA biogenesis, miR-155 remains upregulated in FLT3-ITD+ AML blasts, suggesting activation of alternative mechanisms of miRNA biogenesis that circumvent the XPO5/RAN-GTP blockage. MiR-155, a BIC-155 long noncoding (lnc) RNA-hosted oncogenic miRNA, has previously been implicated in FLT3-ITD+ AML blast hyperproliferation. We showed that FLT3-ITD upregulates miR-155 by inhibiting DDX3X, a protein implicated in the splicing of lnc-RNAs, via p-AKT. Inhibition of DDX3X increases unspliced BIC-155 which is then shuttled by NXF1 from the nucleus to the cytoplasm, where it is processed into mature miR-155 by cytoplasmic DROSHA, thereby bypassing the XPO5/RAN-GTP blockage via “non-canonical” mechanisms of miRNA biogenesis.