Superior preclinical efficacy of co-treatment with BRG1/BRM and FLT3 inhibitor against AML cells with FLT3 mutations

Abstract

Although treatment with standard frontline therapies, including a FLT3 inhibitor (FLT3i) reduces AML burden and achieves clinical remissions, most patients with AML with FLT3 mutation relapse due to therapy-resistant stem/progenitor cells. The core ATPases, BRG1 (SMARCA4) and BRM (SMARCA2) of the canonical (c) BAF (BRG1/BRM-associated factor) complex is a dependency in AML cells, including those harboring FLT3 mutations. We have previously reported that treatment with FHD-286, a BRG1/BRM ATPases inhibitor, induces differentiation and loss of viability of AML stem/progenitor cells. Findings of present studies demonstrate that treatment with FHD-286 induces lethality in AML cells, regardless of sensitivity or resistance to FLT3i. This efficacy is associated with the induction of gene-expression perturbations responsible for growth inhibition, differentiation, as well as a reduced AML-initiating potential of the AML cells. Additionally, co-treatment with FHD-286 and FLT3i exerts superior pre-clinical efficacy against AML cells and patient-derived (PD) xenograft (PDX) models of AML with FLT3 mutations.

Fig. 1. Treatment with FHD-286 depleted mRNA and protein expression of FLT3 and induced lethality in FLT3-ITD expressing AML cells.

A MOLM13, MV4-11 and quizartinib-resistant MOLM13-QR cells were treated with the indicated concentrations of FHD-286 for 96 h. Then, cells were stained with TO-PRO-3 iodide and the % non-viable cells were determined by flow cytometry. Columns, mean of three experiments, Bars, S.E.M. B Our publicly available RNA-Seq data of MOLM13 cells treated with 100 nM of FHD-286 for 16 h (as biologic replicates) were used to generate a volcano plot (log2 fold-change versus –log10 p-value) of mRNA expression changes in FLT3 and FLT3 target genes at greater than 1.25-fold up or down and p < 0.05. C. MOLM13 cells were treated with 100 nM of FHD-286 for 48 h. Cells were harvested and pellets were processed for total proteome profiling by tandem mass tag mass spectrometry. Volcano plot (log2 fold-change versus -log10 p-value) of mass spectrometry-determined protein expression changes (>1.20-fold up or down and p < 0.05) in FLT3 and FLT3 target genes in FHD-286-treated compared to control cells. D, E MOLM13, MV4-11 and quizartinib-resistant MOLM13-QR cells were treated with the indicated concentrations of quizartinib or gilteritinib for 96 h. Following this, the % non-viable cells were determined by TO-PRO-3 iodide staining and flow cytometry. Columns, mean of three experiments, Bars, S.E.M. F. MOLM13 and MOLM13-QR cells were treated with the indicated concentration of quizartinib for 24 h or FHD-286 for 48 h. Following this, cells were harvested and total cell lysates were prepared. Immunoblot analyses were conducted on the total cell lysates. The expression levels of GAPDH in the cell lysates served as the loading control. Representative immunoblots from two experiments are shown.

Fig. 2. Co-treatment with FHD-286 and quizartinib or gilteritinib induced synergistic in vitro lethality in AML cells expressing MLL-r and FLT3-ITD while FHD-286 pretreatment reversed resistance to FLT3-TKI in MOLM13-QR cells.

A MV4-11 and MOLM13 cells were treated with FHD-286 (dose range: 10 nM–50 nM) and quizartinib (dose range: 0.5-10 nM), or gilteritinib (dose range: 2 nM–20 nM) for 96 h. At the end of treatment, the % non-viable cells were determined by staining with TO-PRO-3 iodide and flow cytometry analysis. Delta synergy scores were determined by the ZIP method within the SynergyFinder web application. Synergy scores >1.0 indicate a synergistic interaction of the two agents in the combination. Mean Delta Synergy scores for each combination are shown. B MOLM13 cells were treated with the indicated concentrations of FHD-286 and/or quizartinib for 24–48 h. At the end of treatment, total cell lysates were harvested and immunoblot analyses were conducted. The expression levels of β-Tubulin in the cell lysates served as the loading control. Representative immunoblots from two experiments are shown. C MOLM13-QR (FLT3-D835Y), MOLM13 TP53-R175H, and MOLM13 TP53-R248Q cells were treated with FHD-286 (dose range: 10 nM to 50 nM) and quizartinib (dose range: 0.5–50 nM), or gilteritinib (dose range: 2 nM–20 nM) for 96 h. At the end of treatment, the % non-viable cells were determined by staining with TO-PRO-3 iodide and flow cytometry analysis. Delta synergy scores were determined by the ZIP method within the SynergyFinder web application. Synergy scores >1.0 indicate a synergistic interaction of the two agents in the combination. Mean Delta Synergy scores for each combination are shown. D MOLM13-QR cells were treated with the indicated concentrations of FHD-286 for 96 h. Following this, total cell lysates were prepared and immunoblot analyses were conducted. The expression levels of β-Tubulin in the cell lysates served as the loading control. Representative immunoblots from two experiments are shown. E, F MOLM13-QR cells were treated with or without 30 nM of FHD-286 for 96 h, followed by treatment with the indicated concentrations of quizartinib or gilteritinib for 96 h. Cells were then stained with TO-PRO-3 iodide and the % non-viable cells were determined by flow cytometry. Columns, mean of three experiments, Bars, S.E.M. *=p < 0.05; **=p < 0.01; ***=p < 0.005 compared to cells with no FHD-286 pretreatment.

Fig. 3. Treatment with FHD-286 depleted MYC target gene sets and induced loss of viability of OCI-AML3-FLT3-ITD cells.

A–D OCI-AML3-FLT3-ITD cells were treated with or without 100 nM of FHD-286 for 16 h in biologic duplicates. Total RNA was isolated and utilized for RNA-Seq analysis. A Gene set enrichment analysis of OCI-AML3-FLT3-ITD-expressing cells over OCI-AML3-vector cells compared to HALLMARK and REACTOME pathways. Normalized enrichment scores are shown. All q-values are <0.1. B Volcano plot (log2 fold-change versus –log10 p value) of RNA-Seq-determined mRNA expression changes (greater than 1.25-fold up or down and p < 0.05) in OCI-AML3-FLT3-ITD cells over OCI-AML3-vector cells. C Gene set enrichment analysis of FHD-286-treated OCI-AML3-FLT3-ITD cells compared to HALLMARK and REACTOME pathways. Normalized enrichment scores are shown. All q-values are <0.1. D Volcano plot (log2 fold-change versus –log10 p-value) of RNA-Seq-determined mRNA expression changes (greater than 1.25-fold up or down and p < 0.05) in FHD-286-treated OCI-AML3-FLT3-ITD cells compared to untreated OCI-AML3-FLT3-ITD cells. E OCI-AML3-vector and OCI-AML3-FLT3-ITD cells were treated with the indicated concentrations of FHD-286 for 48 h. At the end of treatment, total cell lysates were prepared and immunoblot analyses were conducted. The expression levels of GAPDH in the cell lysates served as the loading control. F OCI-AML3-FLT3-ITD cells were treated with the indicated concentrations of FHD-286 for 96 h. Following this, the % non-viable cells were determined by TO-PRO-3 iodide staining and flow cytometry. Columns, mean of three experiments, Bars, S.E.M. G OCI-AML3-FLT3-ITD cells were treated with FHD-286 (dose range: 10 nM–50 nM) and quizartinib (dose range: 10–100 nM), or gilteritinib (dose range: 10 nM–50 nM) for 96 h. At the end of treatment, the % non-viable cells were determined by staining with TO-PRO-3 iodide and flow cytometry analysis. Delta synergy scores were determined by the ZIP method within the SynergyFinder web application. Synergy scores >1.0 indicate a synergistic interaction of the two agents in the combination. Mean Delta Synergy scores for each combination are shown.

Fig. 4. Treatment with FHD-286 significantly depleted FLT3 expression in bulk AML cells and phenotypically defined AML stem cells with mtNPM1 and FLT3-ITD and FHD-286-based combinations exerted synergistic in vitro lethality in patient-derived AML cells expressing mtNPM1 with FLT3-ITD and/or additional FLT3 mutation.

A Immunoblot analysis of FLT3 expression in three patient-derived AML samples with FLT3-ITD and one non-FLT3-ITD AML sample showing variation in FLT3 expression. The expression levels of GAPDH in the cell lysates served as the loading control. B PD mtNPM1 + FLT3-ITD AML cells were treated with 100 nM of FHD-286 for 48 h. Cells were harvested and pellets were processed for total proteome profiling by tandem mass tag mass spectrometry. Volcano plot (log2 fold-change versus -log10 p-value) of mass spectrometry-determined protein expression changes in FLT3 and FLT3 target genes in FHD-286-treated compared to control cells (fold change >0 or <0 and p < 0.05). C Patient-derived mtNPM1 + FLT3-ITD AML cells were treated with 100 nM of FHD-286 for 48 h. CyTOF analysis was conducted utilizing cocktails of rare metal element-tagged antibodies. The heat map shows the log2 fold-change (FHD-286-treated over Control) of depleted and induced proteins in phenotypically defined AML stem/progenitor cells (CLEC12A hi, CD123 hi, CD99 hi, CD33 hi and CD11b low). D Four patient-derived AML samples with mtNPM1 + FLT3-ITD and/or additional FLT3 mutation were treated with the indicated concentrations of gilteritinib or FHD-286 (in duplicate) for 72 h. At the end of treatment, cells were stained with TO-PRO-3 iodide and the % non-viable cells were determined by flow cytometry. E Patient-derived AML cells with mtNPM1 + FLT3-ITD with or without FLT3-TKD or gatekeeper (F691L) mutation were treated with FHD-286 (dose range: 20 nM–50 nM) and quizartinib (dose range: 0.5–2.0 nM), or gilteritinib (dose range: 5 nM–20 nM) for 72 h. At the end of treatment, the % non-viable cells were determined by staining with TO-PRO-3 iodide and flow cytometry analysis. Delta synergy scores were determined by the ZIP method within the SynergyFinder web application. Synergy scores >1.0 indicate a synergistic interaction of the two agents in the combination. Delta Synergy scores for each combination are shown.

Fig. 5. Compared to treatment with either agent alone, co-treatment with FHD-286 and gilteritinib significantly reduced leukemia burden and improved survival of NSG mice bearing luciferized MOLM13-QR AML xenografts.

A Total photon counts [flux] (determined by bioluminescent imaging) in NSG mice engrafted with luciferized MOLM13-QR cells and treated for 1 week with FHD-286 and/or quizartinib or gilteritinib at the indicated doses. *=p < 0.05; **=p < 0.01; ****=p < 0.001. B Representative bioluminescent images of mice from panel (A). C Kaplan-Meier survival plot of NSG mice engrafted with luciferized MOLM13-QR cells and treated with 1.5 mg/kg of FHD-286 (daily x 5 days, P.O.) and/or 5 mg/kg of quizartinib (daily x 5 days, P.O.) or 20 mg/kg of gilteritinib (daily x 5 days, P.O.) for 4 weeks. Significance between cohorts was determined by a Mantel-Cox log-rank test. *=p < 0.05; **=p < 0.01; ***=p < 0.005; ****=p < 0.001.

Fig. 6. Co-treatment with FHD-286 and gilteritinib significantly reduced leukemia burden and markedly improved survival of NSG mice bearing luciferized mtNPM1 + FLT3-ITD + FLT3-D835Y-expressing AML PDX cells.

A Total photon counts [flux] (determined by bioluminescent imaging) in NSG mice engrafted with luciferized mtNPM1 + FLT3-ITD + FLT3-D835Y AML PDX cells and treated for 5 weeks with FHD-286 and/or quizartinib or gilteritinib at the indicated doses. ***=p < 0.005; ****=p < 0.001. B Representative bioluminescent images of mice from panel (A). C Kaplan-Meier survival plot of NSG mice engrafted with luciferized mtNPM1 + FLT3-ITD + FLT3-D835Y AML PDX cells and treated with 1.5 mg/kg of FHD-286 (daily x 5 days, P.O.) and/or 5 mg/kg of quizartinib (daily x 5 days, P.O.) or 20 mg/kg of gilteritinib (daily x 5 days, P.O.) for 8 weeks. Significance between cohorts was determined by a Mantel-Cox log-rank test. ****=p < 0.001.