Effective Menin inhibitor-based combinations against AML with MLL rearrangement or NPM1 mutation (NPM1c)

Abstract

Treatment with Menin inhibitor (MI) disrupts the interaction between Menin and MLL1 or MLL1-fusion protein (FP), inhibits HOXA9/MEIS1, induces differentiation and loss of survival of AML harboring MLL1 re-arrangement (r) and FP, or expressing mutant (mt)-NPM1. Following MI treatment, although clinical responses are common, the majority of patients with AML with MLL1-r or mt-NPM1 succumb to their disease. Pre-clinical studies presented here demonstrate that genetic knockout or degradation of Menin or treatment with the MI SNDX-50469 reduces MLL1/MLL1-FP targets, associated with MI-induced differentiation and loss of viability. MI treatment also attenuates BCL2 and CDK6 levels. Co-treatment with SNDX-50469 and BCL2 inhibitor (venetoclax), or CDK6 inhibitor (abemaciclib) induces synergistic lethality in cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1. Combined therapy with SNDX-5613 and venetoclax exerts superior in vivo efficacy in a cell line or PD AML cell xenografts harboring MLL1-r or mt-NPM1. Synergy with the MI-based combinations is preserved against MLL1-r AML cells expressing FLT3 mutation, also CRISPR-edited to introduce mtTP53. These findings highlight the promise of clinically testing these MI-based combinations against AML harboring MLL1-r or mtNPM1.

Fig. 1. Depletion or degradation of Menin increases sensitivity to treatment with venetoclax or abemaciclib in AML cells.

A MOLM13 was transfected with sgRNA against Exon 2 or Exon 6 of Menin or a negative control sgRNA (sgNeg) and incubated for 5 days. Then, total cell lysates were prepared and immunoblot analyses were conducted. The expression levels of GAPDH served as the loading control. B MOLM13 cells were transfected with sgNeg or sg Menin Ex 2 or Ex 6 and incubated for 72 h. Then, cells were treated with the indicated concentrations of venetoclax for 48 h. The % of annexin V-positive, apoptotic cells were determined by flow cytometry. Mean of two independent experiments performed in duplicate ±S.D. **p < 0.01 compared to sgNeg-transfected MOLM13 cells (determined by a two-tailed, unpaired t test). C MOLM13 cells were transfected with sgNeg or sg Menin Ex 2 or Ex 6 and incubated for 72 h. Following this, cells were treated with the indicated concentrations of abemaciclib for 96 h. The % of TO-PRO-3 iodide-positive, non-viable cells were determined by flow cytometry. Mean of two independent experiments performed in duplicate ±S.D. **p < 0.01 compared to sgNeg-transfected MOLM13 cells (determined by a two-tailed, unpaired t test). D MOLM13-Menin-FKBP12^F36V cells were treated with the indicated concentrations of dTAG-13 for 72 h. At the end of treatment, cell lysates were prepared and immunoblot analyses were conducted for HA-tagged Menin, endogenous Menin, MEIS1, FLT3, CDK6, PBX3, BCL2, and p27. The expression levels of GAPDH served as the loading control. E MOLM13-Menin-FKBP12^F36V cells were treated with the indicated concentrations of venetoclax for 48 h alone or co-treated with 500 nM of dTAG-13. The % of TO-PRO-3 iodide-positive, non-viable cells were determined by flow cytometry. Mean of two independent experiments performed in duplicate ±S.D. **p < 0.01 compared to MOLM13-Menin-FKBP12^F36V cells not treated with dTAG-13 (determined by a two-tailed, unpaired t test in GraphPad V8). F MOLM13-Menin-FKBP12^F36V cells were treated with the indicated concentrations of abemaciclib for 96 h without or with 500 nM of dTAG-13. The % of TO-PRO-3 iodide-positive cells was determined by flow cytometry. Mean of two independent experiments performed in duplicate ±S.D. **p < 0.01 compared to MOLM13-Menin-FKBP12^F36V cells not treated with dTAG-13 (determined by a two-tailed, unpaired t test in GraphPad V8).

Fig. 2. Treatment with Menin inhibitor SNDX-50469 depletes MEIS1, FLT3, CDK6, and BCL2 with concomitant induction of MCL1 and CD11b expression and features of morphologic differentiation in AML cells.

A, B MOLM13 and OCI-AML3 cells were treated with the indicated concentrations of SNDX-50469 for 48 h. Following this, total cell lysates were prepared and immunoblot analyses were conducted. The expression levels of β-Actin in the lysates served as the loading control. C, D MOLM13 and OCI-AML3 cells were treated with the indicated concentrations of SNDX-50469 for 96 h or 7 days. Morphologic differentiation (% myelocytes, meta-myelocytes, or bands) was determined by light microscopy. Mean of three experiments ±S.E.M. ***p < 0.005; ****p < 0.001 (determined by two-tailed, unpaired t test in GraphPad V8). E MV4-11, OCI-AML3, and MOLM13 cells were treated with the indicated concentrations of SNDX-50469 or SNDX-5613 for 96 h. At the end of treatment, the % of TO-PRO-3 iodide-positive, non-viable cells were determined by flow cytometry. Columns, mean of three experiments ±S.E.M. F Kaplan–Meier survival curve of mice infused with MLL-AF9 + FLT3-TKD expressing PDX (AML#4) cells and treated with SNDX-5613 as indicated for 4 weeks.

Fig. 3. Menin inhibitor treatment depletes H3K27 acetyl mark on chromatin, depletes mRNA expression of MYC, MEIS1, and FLT3 in cultured and primary AML blasts and depletes Menin, BCL2, and MLL target gene expression levels in phenotypically defined leukemia stem cells.

A IGV plots showing signal tag density of H3K27Ac ChIP-Seq at the MEIS1 and FLT3 gene in MOLM13 cells treated with SNDX-50469 for 16 h. Black arrows mark the direction of the coding sequence of each gene. Blue bars beneath the gene indicate significant, log2 fold-changes in signal tag density of H3K27Ac in SNDX-50469-treated cells compared to control cells. B–D MOLM13, OCI-AML3, and PD MLL-AF9, FLT3-TKD AML cells were treated with the indicated concentrations of SNDX-50469 for 16 h. Total RNA was isolated, and reverse transcribed. The resulting cDNAs were used for qPCR as shown. The expression of GAPDH served as the normalization control. E Patient-derived AML cells were treated with 1000 nM of SNDX-50469 for 16 h. Cells were harvested and analyzed by CyTOF analysis utilizing a cocktail of rare metal element-tagged antibodies. Leukemia stem cells were defined by high expression of CLEC12A, CD123, CD244, CD99, and CD33 but low expression of CD11b.

Fig. 4. Co-treatment with Menin inhibitor and venetoclax exerts synergistic in vitro lethality in cultured AML cells expressing MLL1-r or mtNPM1.

A MV4-11, MOLM13, and OCI-AML3 cells were treated with the indicated concentrations of venetoclax for 96 h. At the end of treatment, the % of TO-PRO-3 iodide-positive, non-viable cells were determined by flow cytometry. Columns, mean of three experiments ±S.E.M. B–D. MV4-11, MOLM13, and OCI-AML3 cells were treated with the indicated concentrations of SNDX-50469 and/or venetoclax 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. Synergy scores > 1.0 indicate a synergistic interaction of the two agents in the combination.

Fig. 5. Combined treatment with Menin inhibitor and venetoclax induces synergistic in vitro lethality in patient-derived AML cells expressing MLL1-r or mtNPM1 with or without FLT3 alterations.

A–F Patient-derived AML cells with MLL1 rearrangement or mtNPM1 with or without FLT3 alterations were treated with the indicated concentrations of SNDX-50469 and/or venetoclax for 48–72 h. 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. Synergy scores > 1.0 indicate a synergistic interaction of the two agents in the combination.

Fig. 6. Co-treatment with Menin inhibitor and venetoclax or FLT3 inhibitor gilteritinib induces synergistic in vitro lethality in MOLM13 cells with isogenic TP53 mutations or TP53 knockout.

A–C MOLM13 TP53-R175H, MOLM13 TP53-R248Q, and MOLM13 TP53^−/− cells were treated with the indicated concentrations of SNDX-50469 and/or venetoclax 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 calculated by the ZIP method. Synergy scores > 1.0 indicate a synergistic interaction of the two agents in the combination. D–F MOLM13 TP53-R175H, MOLM13 TP53-R248Q, and MOLM13 TP53^−/− cells were treated with the indicated concentrations of SNDX-50469 and/or gilteritinib 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. Synergy scores > 1.0 indicate a synergistic interaction of the two agents in the combination.

Fig. 7. Treatment with SNDX-5613 and venetoclax reduced leukemia burden and significantly improved survival of NSG mice bearing MLL1-r or mtNPM1 with mtFLT3 AML xenografts.

A Total photon count [flux] (determined by bioluminescent imaging) in NSG mice engrafted with MOLM13 GFP/Luc cells and treated for 2 weeks with SNDX-5613 and/or venetoclax at the indicated doses. B Kaplan–Meier survival plot of NSG mice engrafted with MOLM13 GFP/Luc cells and treated with 50 mg/kg of SNDX-5613 (B.I.D. ×5 days, P.O.) and/or 30 mg/kg of venetoclax (daily ×5 days, P.O.) for 3 weeks. Significance was calculated by a Mantel–Cox log-rank test. C Total photon counts [flux] (determined by bioluminescent imaging) in NSG mice engrafted with PD, AML blasts expressing mtNPM1, and mtFLT3 and treated with vehicle or SNDX-5613 and/or venetoclax at the indicated doses for 2 weeks. D Kaplan–Meier survival plot of NSG mice bearing a mtNPM1 and mtFLT3-expressing AML PDX and treated with 75 mg/kg of SNDX-5613 (B.I.D. ×5 days, P.O.) and/or 30 mg/kg of venetoclax (daily ×5 days, P.O.) for 4 weeks. Significance was calculated by a Mantel–Cox log-rank test.