FLT3-mutated Acute Myeloid Leukemia Cells Research Articles

Concomitant targeting of FLT3 and SPHK1 exerts synergistic cytotoxicity in FLT3-ITD+ acute myeloid leukemia by inhibiting β-catenin activity via the PP2A-GSK3β axis

BackgroundApproximately 25–30% of patients with acute myeloid leukemia (AML) have FMS-like receptor tyrosine kinase-3 (FLT3) mutations that contribute to disease progression and poor prognosis. Prolonged exposure to FLT3 tyrosine kinase inhibitors (TKIs) often results in limited clinical responses due to diverse compensatory survival signals. Therefore, there is an urgent need to elucidate the mechanisms underlying FLT3 TKI resistance. Dysregulated sphingolipid metabolism frequently contributes to cancer progression and a poor therapeutic response. However, its relationship with TKI sensitivity in FLT3-mutated AML remains unknown. Thus, we aimed to assess mechanisms of FLT3 TKI resistance in AML.MethodsWe performed lipidomics profiling, RNA-seq, qRT-PCR, and enzyme-linked immunosorbent assays to determine potential drivers of sorafenib resistance. FLT3 signaling was inhibited by sorafenib or quizartinib, and SPHK1 was inhibited by using an antagonist or via knockdown. Cell growth and apoptosis were assessed in FLT3-mutated and wild-type AML cell lines via Cell counting kit-8, PI staining, and Annexin-V/7AAD assays. Western blotting and immunofluorescence assays were employed to explore the underlying molecular mechanisms through rescue experiments using SPHK1 overexpression and exogenous S1P, as well as inhibitors of S1P2, β-catenin, PP2A, and GSK3β. Xenograft murine model, patient samples, and publicly available data were analyzed to corroborate our in vitro results.ResultsWe demonstrate that long-term sorafenib treatment upregulates SPHK1/sphingosine-1-phosphate (S1P) signaling, which in turn positively modulates β-catenin signaling to counteract TKI-mediated suppression of FLT3-mutated AML cells via the S1P2 receptor. Genetic or pharmacological inhibition of SPHK1 potently enhanced the TKI-mediated inhibition of proliferation and apoptosis induction in FLT3-mutated AML cells in vitro. SPHK1 knockdown enhanced sorafenib efficacy and improved survival of AML-xenografted mice. Mechanistically, targeting the SPHK1/S1P/S1P2 signaling synergizes with FLT3 TKIs to inhibit β-catenin activity by activating the protein phosphatase 2 A (PP2A)-glycogen synthase kinase 3β (GSK3β) pathway.ConclusionsThese findings establish the sphingolipid metabolic enzyme SPHK1 as a regulator of TKI sensitivity and suggest that combining SPHK1 inhibition with TKIs could be an effective approach for treating FLT3-mutated AML.

Abstract 1746 The Effects of Vitamin A Intake and FLT3/JAK2 Inhibition on FLT3-Mutated Acute Myeloid Leukemia Cells as Potential Novel Treatment Options

Abstract 1746 The Effects of Vitamin A Intake and FLT3/JAK2 Inhibition on FLT3-Mutated Acute Myeloid Leukemia Cells as Potential Novel Treatment Options

Synergistic CRISPR/Cas9 Gene Editing and CAR T-cell Therapy for FLT3-Mutated in AML

Acute Myeloid Leukemia (AML) with FLT3 (FMS-like Tyrosine Kinase 3) mutations poses a significant challenge in oncology. This paper explores the potential of a combined treatment approach involving CRISPR/Cas9 gene editing and CAR T-cell therapy. CRISPR/Cas9 technology can precisely target and correct the FLT3 gene mutation responsible for AML proliferation. Simultaneously, CAR T-cell therapy harnesses the immune system to target FLT3-mutated AML cells using engineered T cells. While promising, these approaches come with challenges, such as off-target effects for CRISPR/Cas9 and complications like cytokine release syndrome for CAR T-cell therapy. Nevertheless, they offer a groundbreaking paradigm shift in AML treatment, potentially providing more effective and personalized therapies for patients with FLT3 mutations. Further research and clinical trials are essential to fully realize their potential and address associated hurdles. This combination therapy offers hope for improving outcomes in AML patients with FLT3 mutations, representing a significant advancement in precision oncology.

Autophagy Induction Constitutes a Targetable Vulnerability in FLT3-Mutant Acute Myeloid Leukemia Cells Treated with FLT3-Inhibitors

Recently, we and others identified autophagy induction as a protective mechanism in FLT3-ITD mutant acute myeloid leukemia (AML) cells upon treatment with FLT3-inhibitors (FLT3i), constituting an increasing issue in clinical practice. Here, we investigated the molecular consequences of autophagy induction upon FLT3i treatment, exploring resistance mechanisms and interrogated whether autophagy inhibition can improve FLT3i antileukemic therapy. Drug response of primary FLT3-ITD AML samples ex vivo treated with FLT3i, quizartinib (AC220, n= 59) and midostaurin (PKC412, n=64), was associated with their transcriptional profile to unravel which molecular programs were enriched in poor responders. Both gene set enrichment analysis (GSEA) and single sample GSEA (ssGSEA) revealed enriched molecular signatures compatible with autophagy activation in poor responders to FLT3i, while sensitive samples enriched molecular signatures consonant with mitochondrial function. Enrichment scores for programs associated with “regulation of autophagy” and “autophagosome organization” were positively correlated to ex vivo response area under the curve values for AC220 and PKC412. To functionally address the molecular pathways associated with autophagy activation upon FLT3i treatment, MOLM-13 and MV4-11 AML cells ( FLT3-ITD) and primary AML samples, were treated with AC220 and PKC412 alone or in combination with the autophagy inhibitor chloroquine (CQ, 5-10μM). In FLT3-ITD AML models, treatment with FLT3i significantly induced the formation of acidic vesicular organelles, suggesting autophagy induction. We also observed a reduction of autophagy regulators in a dose- and time-dependent manner, evidenced by the conversion of LC3B-I to LC3B-II, with degradation of LC3B-II and p62. Furthermore, FLT3i treatment resulted in phosphorylation inhibition of AKT Ser473, mTOR Ser2448 and its downstream protein p70S6K Thr421/Ser424, and phosphorylation reduction of ULK1 Ser757. Combination of FLT3i with CQ (10 μM) significantly reduced cell viability and survival for both PKC412 and AC220 inhibitors. To rule out the off target effects associated with CQ treatment, we performed a knockdown (KD) on the key autophagy mediator ATG5 using shRNA-mediated inhibition in FLT3-ITD cells. In MOLM13 cells, FLT3i treatment in ATG5-KD cells significantly reduced the IC50 (control: 31nM vs. ATG5-KD: 17nM) and increased apoptosis induction (control: 41% vs. ATG5-KD: 58%). We validated these observations in ex vivo treated primary AML samples, where we observed that in FLT3-ITD but not in FLT3 wild-type samples, the combination of FLT3i (PKC412 and AC220) plus autophagy inhibitors (CQ) reduced cell proliferation and survival. In summary, we demonstrated that autophagy induction is a biological process intrinsically associated with drug resistance in FLT3-ITD mutant AMLs when treated with FLT3i. Hence, autophagy inhibition can be leveraged to increase the antileukemic effect of FLT3i and highlights the autophagy as a major acquired biological process associated with FLT3i therapy.

Translational Control of FLT3-Mutated AML Cell Survival

Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults with a five-year survival rate of ~30%. Approximately 30% of AML patients harbor an internal tandem duplication (ITD) mutation in the FMS related receptor tyrosine kinase 3 (FLT3) gene, which is an indicator of poor overall survival and a high relapse rate. These mutations cause constitutive activation of the FLT3 receptor, which amplify proliferation and survival signaling that supports leukemia progression and chemotherapy resistance. Although FLT3 inhibitors (FLT3i) improve median survival by a couple of months, and they have reduced toxicity relative to conventional chemotherapy in clinical trials, patients eventually acquire resistance and relapse. Clinical trials have revealed that AML blasts in the bone marrow (BM) were not effectively eliminated compared to AML blasts in peripheral blood, suggesting a protective role of the BM microenvironment that counteracts FLT3i therapy. A clear understanding of FLT3-mutated AML biology and how it is influenced by the microenvironment is imperative for developing new molecular approaches for improving efficacy of FLT3i-based therapies. We have discovered that BM stromal cell mediated regulation of Ataxia Telangiectasia Mutated (ATM) promotes Mammalian Target of Rapamycin Complex 1 (mTORC1) activity despite FLT3 inhibition, which maintains translation of oxidative phosphorylation genes that are critical for AML cell survival upon treatment with FLT3i [PMID: 36259537]. Key findings show that FLT3i inhibits mTOR and mTOR-dependent translation leading to cell death. However, in the presence of BM stromal factors, survival is restored upon FLT3i by regulating ATM/mTOR-dependent translation of essential phosphorylation genes to overcome FLT3i. Moreover, we demonstrated that the combination of FLT3i and mTORC1 inhibition (mTORC1i) synergistically kills human FLT3-mutated AML cells in vitro and substantially reduces tumor burden and prevents relapse in mouse models of FLT3-mutated AML. Further studies measuring translation in FLT3-mutated AML cells revealed that there are at least two different translation pathways dictated by FLT3. One of these pathways is mTOR-dependent, as expected, and the other is mTOR-independent. Additional studies have sought to characterize FLT3-controlled translation, independent of mTOR, and its importance for FLT3-mutated AML cell survival. Although highly sensitized to FLT3i, we observed that ATM/mTOR-deficient cells maintain near-normal translation and survival. Additionally, mounting evidence suggests that there are other kinases, such as CDK1, GSK3β, and p38, that drive mTOR-independent translation by phosphorylating 4E-BP1. With this knowledge, we took an unbiased approach and used O-Propargyl Puromycin (OPP)-translation assays to assess the impact of inhibitors against these kinases on translation activity in FLT3-mutated AML cell lines. This approach revealed CDK1 as a critical regulator of translation downstream of FLT3. Interestingly, myeloid progenitors reprogram to an mTOR-independent translational program requiring phosphorylation of 4E-BP1 by CDK1 [PMID: 32386556], which could be relevant for myeloid leukemias. Further corroborating my findings, RNA sequencing data suggest that FLT3 positively regulates CDK1 expression/activity, suggesting a downstream position in parallel with mTOR. In conclusion, we have uncovered a FLT3-controlled translation pathway that promotes FLT3-mutated AML cell survival independent of mTOR. Further characterization of this pathway could help predict mechanisms of resistance to FLT3 and mTOR inhibitors in AML and could identify new targets to promote more durable remissions in patients.

Blockade of ROS production inhibits oncogenic signaling in acute myeloid leukemia and amplifies response to precision therapies.

Mutations in the type III receptor tyrosine kinase FLT3 are frequent in patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. AML is characterized by the overproduction of reactive oxygen species (ROS), which can induce cysteine oxidation in redox-sensitive signaling proteins. Here, we sought to characterize the specific pathways affected by ROS in AML by assessing oncogenic signaling in primary AML samples. The oxidation or phosphorylation of signaling proteins that mediate growth and proliferation was increased in samples from patient subtypes with FLT3 mutations. These samples also showed increases in the oxidation of proteins in the ROS-producing Rac/NADPH oxidase-2 (NOX2) complex. Inhibition of NOX2 increased the apoptosis of FLT3-mutant AML cells in response to FLT3 inhibitors. NOX2 inhibition also reduced the phosphorylation and cysteine oxidation of FLT3 in patient-derived xenograft mouse models, suggesting that decreased oxidative stress reduces the oncogenic signaling of FLT3. In mice grafted with FLT3 mutant AML cells, treatment with a NOX2 inhibitor reduced the number of circulating cancer cells, and combining FLT3 and NOX2 inhibitors increased survival to a greater extent than either treatment alone. Together, these data raise the possibility that combining NOX2 and FLT3 inhibitors could improve the treatment of FLT3 mutant AML.

Synergistic Targeting of DNA-PK and KIT Signaling Pathways in KIT Mutant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common and aggressive form of acute leukemia, with a 5-year survival rate of just 24%. Over a third of all AML patients harbor activating mutations in kinases, such as the receptor tyrosine kinases FLT3 (receptor-type tyrosine-protein kinase FLT3) and KIT (mast/stem cell growth factor receptor kit). FLT3 and KIT mutations are associated with poor clinical outcomes and lower remission rates in response to standard-of-care chemotherapy. We have recently identified that the core kinase of the non-homologous end joining DNA repair pathway, DNA-PK (DNA-dependent protein kinase), is activated downstream of FLT3; and targeting DNA-PK sensitized FLT3-mutant AML cells to standard-of-care therapies. Herein, we investigated DNA-PK as a possible therapeutic vulnerability in KIT mutant AML, using isogenic FDC-P1 mouse myeloid progenitor cell lines transduced with oncogenic mutant KIT (V560G and D816V) or vector control. Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK in the T2599/T2605/S2608/S2610 cluster in KIT mutant cells, indicative of DNA-PK activation. Accordingly, proliferation assays revealed that KIT mutant FDC-P1 cells were more sensitive to the DNA-PK inhibitors M3814 or NU7441, compared with empty vector controls. DNA-PK inhibition combined with inhibition of KIT signaling using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Global phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib and M3814 single-agent treatments inhibited extracellular signal–regulated kinase and AKT (RAC-alpha serine/threonine-protein kinase)/MTOR (serine/threonine-protein kinase mTOR) activity, with greater inhibition of both pathways when used in combination. Combined dasatinib and M3814 treatment also synergistically inhibited phosphorylation of the transcriptional regulators MYC and MYB. This study provides insight into the oncogenic pathways regulated by DNA-PK beyond its canonical role in DNA repair and demonstrates that DNA-PK is a promising therapeutic target for KIT mutant cancers.

Dual inhibition of CHK1/FLT3 enhances cytotoxicity and overcomes adaptive and acquired resistance in FLT3-ITD acute myeloid leukemia.

FLT3 inhibitors (FLT3i) are widely used for the treatment of acute myeloid leukemia (AML), but adaptive and acquired resistance remains a primary challenge. Inhibitors simultaneously blocking adaptive and acquired resistance are highly demanded. Here, we observed the potential of CHK1 inhibitors to synergistically improve the therapeutic effect of FLT3i in FLT3-mutated AML cells. Notably, the combination overcame adaptive resistance. The simultaneous targeting of FLT3 and CHK1 kinases may overcome acquired and adaptive resistance. A dual FLT3/CHK1 inhibitor 30 with a good oral PK profile was identified. Mechanistic studies indicated that 30 inhibited FLT3 and CHK1, downregulated the c-Myc pathway and further activated the p53 pathway. Functional studies showed that 30 was more selective against cells with various FLT3 mutants, overcame adaptive resistance in vitro, and effectively inhibited resistant FLT3-ITD AML in vivo. Moreover, 30 showed favorable druggability without significant blood toxicity or myelosuppression and exhibited a good oral PK profile with a T1/2 over 12 h in beagles. These findings support the targeting of FLT3 and CHK1 as a novel strategy for overcoming adaptive and acquired resistance to FLT3i therapy in AML and suggest 30 as a potential clinical candidate.

C/EBPα Confers Dependence to Fatty Acid Biosynthetic Pathways and Vulnerability to Lipid Oxidative Stress in FLT3-Mutant Leukemia

Despite therapeutic breakthroughs, drug resistance and relapse remain the major barrier for the treatment of patients with acute myeloid leukemia (AML). Therefore, understanding and targeting therapy-resistant AML cells responsible for relapse ("persisters") represent an urgent need in the development of new treatments. In this context, we demonstrated that the hyperactivation of mitochondrial oxidative metabolism plays a crucial role in drug resistance of AML. This high oxidative phosphorylation (OxPHOS) phenotype associated with enhanced mitochondrial BCL2 dependency and ROS detoxification is the consequence of a mitochondrial adaptation driven by inflammatory stress responses in a cAMP-PKA-ATF4-dependent manner. Targeting elevated oxidative phosphorylation (OxPHOS) activity with direct and indirect mitochondrial inhibitors sensitizes resistant cells to chemotherapy cytarabine (AraC) in AML. Furthermore, we have recently shown that the mitochondrial OxPHOS state supports IDH mutant cell proliferation and chemoresistance in a C/EBPa-dependent manner. Importantly, multi-omics analyses uncovered a coordinated activation of CEBPA and Fms-like tyrosine kinase 3 (FLT3) to control lipid metabolism in FLT3-mutant AML cells from primary AML patient specimens, cell lines, and PDX models. Mechanistically, C/EBPα regulated FASN-SCD axis to promote uptake, biosynthesis and desaturation of fatty acids (FA). We further demonstrated that FLT3 or C/EBPα inactivation decreased mono-unsaturated FAs incorporation to membrane phospholipids through SCD downregulation in leukemic cells. Consequently, SCD inhibition enhanced susceptibility to lipid redox stress and accumulation of poly-unsaturated FA increased lipid peroxidation and vulnerability to redox stress. Accordingly, this C/EBPα-dependent adaptation of FA homeostasis was exploited by combining FLT3 and glutathione peroxidase 4 (GPX4) inhibition to trigger lipid oxidative stress, enhancing ferroptotic death of FLT3-mutant AML cells ex vivo and in vivo. Altogether, this study reveals a non-canonical C/EBPα function in lipid homeostasis and adaptation to redox stress, and a previously unreported vulnerability of FLT3-mutant AML with promising therapeutic application. Altogether, our works highlight the role of metabolic adaptations to support the plasticity and drug resistance in AML.

Combination of midostaurin and ATRA exerts dose-dependent dual effects on acute myeloid leukemia cells with wild type FLT3

BackgroundMidostaurin combined with chemotherapy is currently used to treat newly diagnosed acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3 (FLT3)-mutations. However, midostaurin acts as an antagonist to some chemotherapeutic agents in leukemia cell lines without FLT3 mutations. All-trans retinoic acid (ATRA) induces apoptosis when used in combination with midostaurin in FLT3-mutated AML cells. This combination has been shown to be safe in AML patients. However, the effect of this combination has not been investigated in AML without FLT3 mutations.MethodsCell proliferation was assessed by a cell counting assay. Cell death was evaluated by cell viability and Annexin-V assays. Cell differentiation was assessed by CD11b expression profiling and morphological analysis. To explore the underlying mechanisms, we studied the role of caspase3/7, Lyn, Fgr, Hck, RAF, MEK, ERK, AKT, PU.1, CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPε by Western blot analysis and immunoprecipitation assays. Antitumor activity was also confirmed in mouse xenograft models established with AML cells.ResultsIn this study, 0.1 − 0.25 μM midostaurin (mido(L)) combined with ATRA induced differentiation while 0.25 − 0.5 μM midostaurin (mido(H)) combined with ATRA triggered apoptosis in some AML cell lines without FLT3-mutations. Midostaurin combined with ATRA (mido-ATRA) also exhibited antitumor activity in mouse xenograft models established with AML cells. Mechanistically, mido(H)-ATRA-induced apoptosis was dependent on caspase-3/7. Mido(L)-ATRA inhibited Akt activation which was associated with decreased activity of Lyn/Fgr/Hck, resulted in dephosphorylation of RAF S259, activated RAF/MEK/ERK, along with upregulating the protein levels of C/EBPβ, C/EBPε and PU.1. A MEK specific inhibitor was observed to suppress mido(L)-ATRA-induced increases in the protein levels of C/EBPs and PU.1 and mido(L)-ATRA-induced differentiation. Furthermore, inhibition of Akt activity promoted mido(L)-ATRA-induced downregulation of RAF S259 phosphorylation and mido(L)-ATRA-induced differentiation. Therefore, Lyn/Fgr/Hck-associated Akt inhibition activated RAF/MEK/ERK and controlled mido(L)-ATRA-induced differentiation by upregulation of C/EBPs and PU.1. Mido(L)-ATRA also promoted assembly of the signalosome, which may facilitate RAF activation.ConclusionsMidostaurin combined with ATRA exerts antitumor activity against AML with wild-type FLT3 mutations in vitro and in vivo. These findings may provide novel therapeutic strategies for some AML patients without FLT3 mutations and imply a new target of midostaurin.

Inhibition of BCL2A1 by STAT5 inactivation overcomes resistance to targeted therapies of FLT3-ITD/D835 mutant AML

Tyrosine kinase inhibitors (TKIs) are established drugs in the therapy of FLT3-ITD mutated acute myeloid leukemia (AML). However, acquired mutations, such as D835 in the tyrosine kinase domain (FLT3-ITD/D835), can induce resistance to TKIs. A cap analysis gene expression (CAGE) technology revealed that the gene expression of BCL2A1 transcription start sites was increased in primary AML cells bearing FLT3-ITD/D835 compared to FLT3-ITD. Overexpression of BCL2A1 attenuated the sensitivity to quizartinib, a type II TKI, and venetoclax, a selective BCL2 inhibitor, in AML cell lines. However, a type I TKI, gilteritinib, inhibited the expression of BCL2A1 through inactivation of STAT5 and alleviated TKI resistance of FLT3-ITD/D835. The combination of gilteritinib and venetoclax showed synergistic effects in the FLT3-ITD/D835 positive AML cells. The promoter region of BCL2A1 contains a BRD4 binding site. Thus, the blockade of BRD4 with a BET inhibitor (CPI-0610) downregulated BCL2A1 in FLT3-mutated AML cells and extended profound suppression of FLT3-ITD/D835 mutant cells. Therefore, we propose that BCL2A1 has the potential to be a novel therapeutic target in treating FLT3-ITD/D835 mutated AML.

AXL/MERTK inhibitor ONO-7475 potently synergizes with venetoclax and overcomes venetoclax resistance to kill FLT3-ITD acute myeloid leukemia.

FMS-like Tyrosine Kinase 3 (FLT3) mutation is associated with poor survival in acute myeloid leukemia (AML). The specific Anexelekto/MER Tyrosine Kinase (AXL) inhibitor, ONO-7475, kills FLT3-mutant AML cells with targets including Extracellular-signal Regulated Kinase (ERK) and Myeloid Cell Leukemia 1 (MCL1). ERK and MCL1 are known resistance factors for Venetoclax (ABT-199), a popular drug for AML therapy, prompting the investigation of the efficacy of ONO-7475 in combination with ABT-199 in vitro and in vivo. ONO-7475 synergizes with ABT-199 to potently kill FLT3-mutant acute myeloid leukemia cell lines and primary cells. ONO-7475 is effective against ABT-199-resistant cells including cells that overexpress MCL1. Proteomic analyses revealed that ABT-199-resistant cells expressed elevated levels of pro-growth and anti-apoptotic proteins compared to parental cells, and that ONO-7475 reduced the expression of these proteins in both the parental and ABT-199-resistant cells. ONO-7475 treatment significantly extended survival as a single in vivo agent using acute myeloid leukemia cell lines and PDX models. Compared to ONO-7474 monotherapy, the combination of ONO-7475/ABT-199 was even more potent in reducing leukemic burden and prolonging the survival of mice in both model systems. These results suggest that the ONO-7475/ABT-199 combination may be effective for AML therapy.

The Combined Treatment With the FLT3-Inhibitor AC220 and the Complex I Inhibitor IACS-010759 Synergistically Depletes Wt- and FLT3-Mutated Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a high mortality rate and relapse risk. Although progress on the genetic and molecular understanding of this disease has been made, the standard of care has changed minimally for the past 40 years and the five-year survival rate remains poor, warranting new treatment strategies. Here, we applied a two-step screening platform consisting of a primary cell viability screening and a secondary metabolomics-based phenotypic screening to find synergistic drug combinations to treat AML. A novel synergy between the oxidative phosphorylation inhibitor IACS-010759 and the FMS-like tyrosine kinase 3 (FLT3) inhibitor AC220 (quizartinib) was discovered in AML and then validated by ATP bioluminescence and apoptosis assays. In-depth stable isotope tracer metabolic flux analysis revealed that IACS-010759 and AC220 synergistically reduced glucose and glutamine enrichment in glycolysis and the TCA cycle, leading to impaired energy production and de novo nucleotide biosynthesis. In summary, we identified a novel drug combination, AC220 and IACS-010759, which synergistically inhibits cell growth in AML cells due to a major disruption of cell metabolism, regardless of FLT3 mutation status.

CG '806, a Novel Pan-FLT3/BTK Multi-Kinase Inhibitor, Induces Cell Cycle Arrest, Apoptosis or Autophagy in AML Cells Depending on FLT3 Mutation Status

Gain-of-function mutations of the fms-like tyrosine kinase 3 (FLT3) play a pivotal role in hematopoietic malignancies (Gilliland et al., 2002). Therefore, FLT3 kinase inhibitors are important component in the treatment of acute myeloid leukemia (AML). However, relapse or resistance to these inhibitors are frequent because of secondary acquired mutations in FLT3. Recently, we reported on a novel small molecular multi-kinase inhibitor, CG'806, which showed promising effects in AML harboring FLT3-internal-tandem-deletion (ITD) mutations, tyrosine kinase domain (TKD) mutations, or both by inhibiting FLT3/Bruton's tyrosine kinase (BTK)/aurora kinase (AuroK) activation. We observed an impressive inhibition of leukemic cell proliferation (i.e., IC50s at sub-nanomolar or low nanomolar levels, Zhang, et al., AACR Hematological Malignancies, 2017).To further characterize the mechanisms underlying this anti-leukemia effect, we investigated the impact of CG'806 on cell cycle progression. Measuring BrdU incorporation by flow cytometry, CG'806 triggered profound G1 cell cycle arrest in FLT3-ITD-mutated cells. Interestingly, CG'806 triggered G2/M phase arrest in FLT3 wild type (WT) cells. Immunoblotting demonstrated that the G1 arrest was mediated by downregulation of signaling associated with p-FLT3-ITD, the p-AKT/p-mTOR/cyclin D1/p-Rb axis, and that of cyclin B1/A2, cdk1/cdc2, and cdk4/6 in FLT3-ITD-mutated cells. This was not observed in FLT3 WT cells. In addition, c-Myc, a primary regulator of the G1/S transition, was downregulated in FLT3-mutated but not in WT cells treated with CG'806. In support of this finding, knockdown of c-Myc with siRNA increased the G1-arrested population in FLT3-mutated cells, suggesting a critical role of c-Myc in the CG'806-induced G1 arrest. However, only suppression of p-BTK and p-AuroK but not p-FLT3, and no modulation of G1 arrest-related proteins was observed in FLT3 WT cells.Interestingly, we observed autophagy induction in FLT3 WT cells but induction of apoptosis in FLT3 mutated cells after exposure to low nanomolar CG'806 for 24 hours as evidenced by modulated LC3-II and cleaved-caspase 3 levels. Inhibiting autophagy with 3-methyladenine (3-MA) partially reversed CG'806-induced G2/M arrest in FLT3 WT cells, suggesting that autophagy induction may also be involved in G2/M arrest besides suppression of AuroK and BTK in FLT3 WT cells.Next, we investigated if nucleoside analogues and intercalating agents enhance activity of CG'806, and observed that combination of CG'806 with cytarabine or idarubicin profoundly enhanced pro-apoptotic effects.Conclusions: CG'806 exerts profound suppression of cell proliferation by arresting cell cycle progression at G1 phase in FLT3-mutant AML cells which is associated with inhibition of mutant FLT3 and downstream p-AKT/p-mTOR/cyclin D1/p-Rb axis. CG'806 exerts a G2/M arrest in FLT3 WT cells associated with inhibition of aurora and BTK kinases and induction of autophagy. CG'806 sensitized AML to standard chemotherapeutic agents-mediated cytotoxicity. Taken together, these data support the development of CG'806 for AML patients with FLT3-ITD, FLT3-ITD with additional TKD mutations, as well as FLT3-WT. DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

A Phase I/II Study of Selinexor (SEL) with Sorafenib in Patients (pts) with Relapsed and/or Refractory (R/R) FLT3 Mutated Acute Myeloid Leukemia (AML)

Background: Responses to FLT3-inhibitors are usually transient due to emergence of resistance through the acquisition of kinase domain point mutations and other non-mutational mechanisms. SEL is a potent first-in-class Selective Inhibitor of Nuclear Export/SINE™ that exerted marked cell killing of human and murine FLT3 -mutant AML cells, including those with ITD, D835Y, ITD+Y842C or ITD+F691L mutations by modulating the cdk inhibitor p27 and anti-apoptotic Mcl-1 (Zhang W et al, Blood 2015 126). The combination of SEL+sorafenib had synergistic pro-apoptotic effects in FLT3-mutated AML cells by suppressing phosphorylation levels of FLT3 and its downstream signaling mediators ERK/AKT, and by inducing myeloid differentiation in ITD and D835 mutated cell lines.Methods: We designed a phase I/II trial of SEL with sorafenib for FLT3-ITD and/or -D835 R/R AML pts. In phase I, the primary objectives were to determine the maximum tolerated dose (MTD), the recommended phase 2 dose (RP2D), and dose-limiting toxicity (DLT) of the combination. In phase II, primary objectives included the rate of complete remission (CR) + CR with incomplete count recovery (CRi) + partial response + >50% blast reduction within 3 months of therapy initiation. Secondary objectives were safety and overall survival (OS). SEL was given orally twice a week for 3-weeks on and 1-week off in 28-day cycles and sorafenib was given continuously at a dose of 400mg twice daily from cycle 1 day 1 of SEL. In phase I, SEL dose was de-escalated in “3+3” fashion starting at dose level 0 and going down if DLTs were exceeded (dose levels 0, -1, -2 = 80, 60, 40 mg, respectively). Once MTD was established, the phase II enrolled pts in 2 cohorts: prior FLT3-inhibitor failure (cohort 1) and FLT3-inhibitor naïve (cohort 2). PB or BM samples were collected at pre-dose (C1D1), 24 h (C1D2) and day 28 (C1D28) of cycle 1, and apoptosis induction was determined by measuring annexin V positivity with FACS.Results: Fourteen pts with a median age of 71 years (range, 24-81) were enrolled. All pts had baseline next generation sequencing for AML specific mutations (Figure 1A). The median number of prior therapies was 3 (range, 1-5) as follows: salvage (S) 1: n=1, S2: n=5, S3+: n=8. 11 (79%) pts had prior FLT3-inhibitors: 1 prior FLT3-inhibitor (n=9); 2 prior FLT3-inhibitors: n= (2). Three pts had prior SCT. Four pts were treated at dose level 0 (SEL 80mg) with DLTs in 2 (Grade (G) 3 sepsis, n=1; G3 mucositis, syncope, adrenal insufficiency, n=1). Three pts were subsequently treated at dose level -1 (SEL 60mg) with no DLTs and this was established as the MTD/RP2D. Seven pts were treated in expansion: 6 had prior FLT3-inhibitor therapy (cohort 1) and 1 had no prior FLT3-inhibitor (cohort 2).The median duration of treatment for all 14 pts was 28 days (range, 14-122) and therapy is ongoing in 1 pt. All pts are evaluable for response. CRp/CRi and >50% blast reduction as best responses were seen in 4/14 (29%) and 2/14 (14%) pts, respectively. All 6 responders had a prior FLT3 inhibitor yielding a response rate of 55% (6/11) pts with prior FLT3 inhibitor therapy. 3 pts with CRp/CRi (2 with FLT3-ITD and 1 with ITD+D835) achieved negative RT-PCR for FLT3. (Figure 1B). One pt in CRi was bridged to SCT and remains alive at 1 year. The most common G 3/4 adverse events irrespective of causality were bleeding (gastrointestinal, n=4; intracranial, n=1), febrile neutropenia (n=4), pneumonia (n=3) and syncope (n=2). The reasons for discontinuing study therapy were disease progression (n=9; 64%), toxicity (n=3; 21%) and SCT (n=1; 7%). The median OS for pts on study was 3.5 months (range, 0.9-15). Translational analysis showed that SEL+sorafenib induced apoptosis in 4 of 5 tested patients, especially in the BM samples (Figure 1C)Conclusions: The combination of SEL and sorafenib is safe with clinical activity and apoptosis induction in R/R FLT3+ AML. The benefit was especially encouraging in pts exposed to prior FLT3 inhibitors, with a response rate of 55% (6/11; including 2 CRi and 2 CRp). The RP2D is 60 mg twice weekly of SEL and the study is accruing (NCT01607892). [Display omitted] DisclosuresDaver:Kiromic: Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Otsuka America Pharmaceutical, Inc.: Consultancy; Incyte Corporation: Honoraria, Research Funding; Pfizer Inc.: Consultancy, Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Immunogen: Research Funding. Jabbour:Bristol-Myers Squibb: Consultancy. DiNardo:Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Cortes:BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; ARIAD: Consultancy, Research Funding; Teva: Research Funding; Pfizer: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding. Kantarjian:Novartis: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding; Delta-Fly Pharma: Research Funding.

Inhibition of translation initiation factor eIF4a inactivates heat shock factor 1 (HSF1) and exerts anti-leukemia activity in AML.

Eukaryotic initiation factor 4A (eIF4A), the enzymatic core of the eIF4F complex essential for translation initiation, plays a key role in the oncogenic reprogramming of protein synthesis, and thus is a putative therapeutic target in cancer. As important component of its anticancer activity, inhibition of translation initiation can alleviate oncogenic activation of HSF1, a stress-inducible transcription factor that enables cancer cell growth and survival. Here, we show that primary acute myeloid leukemia (AML) cells exhibit the highest transcript levels of eIF4A1 compared to other cancer types. eIF4A inhibition by the potent and specific compound rohinitib (RHT) inactivated HSF1 in these cells, and exerted pronounced in vitro and in vivo anti-leukemia effects against progenitor and leukemia-initiating cells, especially those with FLT3-internal tandem duplication (ITD). In addition to its own anti-leukemic activity, genetic knockdown of HSF1 also sensitized FLT3-mutant AML cells to clinical FLT3 inhibitors, and this synergy was conserved in FLT3 double-mutant cells carrying both ITD and tyrosine kinase domain mutations. Consistently, the combination of RHT and FLT3 inhibitors was highly synergistic in primary FLT3-mutated AML cells. Our results provide a novel therapeutic rationale for co-targeting eIF4A and FLT3 to address the clinical challenge of treating FLT3-mutant AML.

The Combined Treatment with the FLT3-Inhibitor AC220 and the Complex I Inhibitor Iacs-010759 Synergistically Depletes Wt- and FLT3-Mutated Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) cells are highly dependent on mitochondrial function for survival 1. We have recently reported a novel oxidative phosphorylation (OXPHOS) inhibitor IACS-010759 that potently inhibits mitochondrial complex I, suppresses OXPHOS and selectively inhibits the growth of AML cells in vitro and in vivo2. In this study, we aimed to identify chemotherapeutic agents that synergistically deplete AML cells when administered in combination with IACS-010759. We performed a high-throughput screening of a drug library (289 anti-cancer compounds) administered either individually or in combination with IACS-010759 on two leukemia cell lines (OCI-AML3, MOLM-13) and three bone marrow stromal cell lines (HS-5, HS-27A, MSC) in both hypoxia (1% O2) and normoxia conditions. Based on the cell viability datasets, we selected top candidates for combinations based on the following criteria: either bliss index > 0.1 (synergy of the combination treatment; red in Fig. 1A&B), or high cytotoxicity to leukemia cells (relative cell viability < 0.5, blue in Fig. 1A&B), as well as low toxicity against normal cells (relative cell viability in normal cells > 0.8, yellow in Fig. 1A&B). Twenty-four compounds satisfied the selection criteria above, either in normoxia or hypoxia, or both. Out of the 24 compounds, 5 agents (Fig. 1C) are known FLT3 (FMS-like tyrosine kinase 3) inhibitors, including AC220 (quizartinib), dovitinib, nintedanib, SGI-1776, and rebastinib, pointing to a molecular target of great potential interest in the design of synergistic drug combinations with IACS-010759. Thus, we investigated more in-depth the synergism between IACS-010759 (10nM) and 13 FLT3 inhibitors, all currently in clinical trials (AC220, sorafenib, gilteritinib, sunitinib, ponatinib, midostaurin, ibrutinib, TP-0903, crenolanib, tandutinib, FF-10101, lestaurtinib, and KW-2449; 0.0128:5x:5000nM), in AML cell lines (FLT3-wt KG-1, U937, OCI-AML2, OCI-AML3; and FLT3-mutant MOLM-13 and MOLM-14). Among the 13 FLT3 inhibitors, only AC220 combined with IACS-010759 showed concentration windows with bliss index higher than 0.1 across different lines. Next, we further characterized the synergism between AC220 and IACS-010759 in AML cell lines (U937 and OCI-AML3) under hypoxic conditions using metabolic flux analysis (MFA) to trace the incorporation of 13C5,15N2-glutamine and 1,2-13C2-glucose and study the metabolic modulation associated with the synergy. Leukemia cells were incubated with unlabeled/labeled medium for 24h and concurrently treated with 5nM IACS-010759 and/or 500nM AC220. While both individual agents modulate glutamine consumption and TCA cycle dynamics, by far the most dramatic metabolic effects on TCA cycle intermediates are observed following administration of the combined treatment. Severe drops in the levels of TCA cycle metabolites, (Fig. 1D) point to a reduced mitochondrial activity following the combined treatment, which is also validated by the increased ratio of oxidized/reduced forms of nicotinamide adenine dinucleotide (NAD/NADH). Interestingly, the total pool of the oncometabolite 2-hydroxyglutarate, while increasing following the individual treatments, significantly dropped to very low levels in response to the combined treatment. The significantly reduced metabolite levels as well as the glucose-derived enrichment fractions of glucose 6-phosphate, fructose bisphosphate, phosphoenolpyruvate and ribose 5-phosphate in the AC220-containing treatment groups (significantly more pronounced in the combined treatment) point to impaired glycolysis /pentose phosphate pathway (Fig. 1E). In turn this results in lower de novo nucleotide biosynthesis (based on the decreased glutamine and glucose incorporation). Similar results were observed in OCI-AML3 cells. Overall, the combinatorial treatment with IACS-010759 and AC220 impaired AML cell metabolism tremendously and to a much greater extent than any of the individual treatments alone. Influx inhibition of both the two main carbon sources, glucose and glutamine, was observed leading to impairment of the TCA cycle and glycolysis for energy production, as well as pentose phosphate pathway and de novo nucleotide biosynthesis. In conclusion, we identified a novel drug combination AC220 and IACS-010759 which synergistically inhibits AML cell growth regardless of FLT3 mutation at least by metabolism disruption. Disclosures Konopleva: Kisoji: Consultancy; Agios: Research Funding; Amgen: Consultancy; Cellectis: Research Funding; Eli Lilly: Research Funding; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Sanofi: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; AbbVie: Consultancy, Research Funding; AstraZeneca: Research Funding; Calithera: Research Funding; Forty-Seven: Consultancy, Research Funding; Ascentage: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding.

Targeting AML-associated FLT3 mutations with a type I kinase inhibitor.

Tyrosine kinase domain (TKD) mutations contribute to acquired resistance to FMS-like tyrosine kinase 3 (FLT3) inhibitors used to treat FLT3-mutant acute myeloid leukemia (AML). We report a cocrystal structure of FLT3 with a type I inhibitor, NCGC1481, that retained potent binding and activity against FLT3 TKD and gatekeeper mutations. Relative to the current generation of advanced FLT3 inhibitors, NCGC1481 exhibited superior antileukemic activity against the common, clinically relevant FLT3-mutant AML cells in vitro and in vivo.

Overcoming Adaptive Therapy Resistance in AML By Targeting Immune Response Pathways

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course, however, most patients will relapse due to target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. One example involves the FMS-like receptor tyrosine kinase (FLT3). Activating mutations of FLT3 result in its autophosphorylation and initiation of intracellular signaling pathways, which induce abnormal survival and proliferation of leukemic cells.One of the most common mutations in acute myeloid leukemia (AML) involves the internal tandem duplication (ITD) of FLT3, which occurs in ~25% of all cases of newly diagnosed AML and confers a particularly poor prognosis. FLT3 inhibitors (FLT3i) evaluated in clinical studies as monotherapy and combination therapies have shown good initial response rates; however, patients eventually relapse with FLT3i-resistant disease. The absence of durable remission in patients treated with potent and selective FLT3i highlights the need to identify resistance mechanisms and develop additional treatment strategies. Several mechanisms contribute to resistance to selective FLT3i, including mutations in the tyrosine kinase domain of FLT3 (20-50%) or activation of parallel signaling mechanisms that bypass FLT3 signaling, referred to as adaptive resistance (30-50%). Here we describe mechanisms of adaptive resistance in FLT3-mutant AML by examining in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). Through this integrative approach, we identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i. Utilizing genetic approaches, we demonstrated that innate immune pathway activation via IRAK1 and IRAK4 contributes to adaptive resistance in FLT3-mutant AML cells. The immediate nature of IRAK1/4 activation in adaptively resistant FLT3-ITD AML cells requires concomitant inhibition of these targets to avoid compensatory signaling and cell survival. Achieving optimal multi-drug combination regimens that yield extended overlapping exposure while avoiding unwanted toxicities is challenging. Therefore, we desired a small molecule inhibitor that simultaneously targeted the FLT3 and IRAK1/4 kinases to eradicate adaptively resistant FLT3-ITD AML. To overcome this adaptive resistance mechanism, we developed and optimized a novel small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The FLT3-IRAK1/4 inhibitor exhibited potent binding affinity for IRAK1 (KD= 2.9 nM), IRAK4 (KD= 0.3 nM), and FLT3 (KD= 0.3 nM), as well as acceptable pharmacokinetic properties in mice. Moreover, a high-resolution crystal structure demonstrates that the FLT3-IRAK1/4 inhibitor binds as a type I inhibitor (ATP-competitive binding to the active state). The FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cell lines and patient-derived samples in vitro and in vivo, and displayed superior efficacy as compared to current targeted FLT3 therapies. Our study demonstrates that therapies that simultaneously inhibit FLT3 signaling and compensatory IRAK1/4 activation have the potential to improve the therapeutic efficacy in patients with FLT3-mutant AML. In conclusion, these findings reveal that inflammatory stress response pathways contribute to adaptive resistance in FLT3-mutant AML and suggests that this mechanism may extend to other malignant cells undergoing a stress-induced response to therapy. Disclosures Hoyt: Kurome Therapeutics: Consultancy. Berman:Astellas: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees. Rosenbaum:Kurome Therapeutics: Consultancy, Employment. Perentesis:Kurome Therapeutics: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy.

Overcoming adaptive therapy resistance in AML by targeting immune response pathways.

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course; however, most patients will relapse because of target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. Here, we describe mechanisms of adaptive resistance in FMS-like receptor tyrosine kinase (FLT3)-mutant acute myeloid leukemia (AML) by examining integrative in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). We identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i and showed that innate immune pathway activation via the interleukin-1 receptor-associated kinase 1 and 4 (IRAK1/4) complex contributes to adaptive resistance in FLT3-mutant AML cells. To overcome this adaptive resistance mechanism, we developed a small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The multikinase FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cells in vitro and in vivo and displayed superior efficacy as compared to current targeted FLT3 therapies. These findings uncover a polypharmacologic strategy for overcoming adaptive resistance to therapy in AML by targeting immune stress response pathways.