Concomitant targeting of BCL2 with venetoclax and MAPK signaling with cobimetinib in acute myeloid leukemia models

Abstract

The pathogenesis of acute myeloid leukemia (AML) involves serial acquisition of mutations controlling several cellular processes, requiring combination therapies affecting key downstream survival nodes in order to treat the disease effectively. The BCL2 selective inhibitor venetoclax has potent anti-leukemia efficacy; however, resistance can occur due to its inability to inhibit MCL1, which is stabilized by the MAPK pathway. In this study, we aimed to determine the anti-leukemia efficacy of concomitant targeting of the BCL2 and MAPK pathways by venetoclax and the MEK1/2 inhibitor cobimetinib, respectively. The combination demonstrated synergy in seven of 11 AML cell lines, including those resistant to single agents, and showed growth-inhibitory activity in over 60% of primary samples from patients with diverse genetic alterations. The combination markedly impaired leukemia progenitor functions, while maintaining normal progenitors. Mass cytometry data revealed that BCL2 protein is enriched in leukemia stem/progenitor cells, primarily in venetoclax-sensitive samples, and that cobimetinib suppressed cytokine-induced pERK and pS6 signaling pathways. Through proteomic profiling studies, we identified several pathways inhibited downstream of MAPK that contribute to the synergy of the combination. In OCI-AML3 cells, the combination downregulated MCL1 protein levels and disrupted both BCL2:BIM and MCL1:BIM complexes, releasing BIM to induce cell death. RNA sequencing identified several enriched pathways, including MYC, mTORC1, and p53 in cells sensitive to the drug combination. In vivo, the venetoclax-cobimetinib combination reduced leukemia burden in xenograft models using genetically engineered OCI-AML3 and MOLM13 cells. Our data thus provide a rationale for combinatorial blockade of MEK and BCL2 pathways in AML.

Figure 1

Anti-leukemia efficacy of cobimetinib and venetoclax in acute myeloid leukemia cell lines. Eleven acute myeloid leukemia cell lines were left untreated or treated with cobimetinib or venetoclax as single agents at 0.001, 0.01, 0.1, or 1.0 μM for 72 h. Calcusyn software was used to calculate the median inhibitory concentration (IC₅₀) values. Combinations of the two drugs were then tested on the same cell lines at dose ranges of 0.25, 0.5, 1, 2, and 4 times the IC₅₀ value of each compound. The combination index of each combination in each cell line was calculated on the basis on the luminescent intensity that correlated with number of viable cells determined by the CellTiter-Glo assay. Responses to treatment were categorized into four patterns: (i) sensitive to both drugs; (ii) sensitive only to cobimetinib and showing synergy for the combination; (iii) sensitive only to venetoclax and showing synergy for the combination; (iv) resistant to both drugs. AML: acute myeloid leukemia; Cobi: cobimetinib; Ven: venetoclax; CI: combination index

Figure 2

Treatment with cobimetinib and venetoclax causes on-target suppression of cell proliferation and impairs leukemia progenitor function in a subset of primary acute myeloid leukemia cases. (A) Primary acute myeloid leukemia (AML) peripheral blood mononuclear or bone marrow cells from AML cases were cultured in serum-free expansion medium supplemented with BIT 9500 Serum Substitute and cytokines, including stem cell factor (SCF; 100 ng/mL), Flt3 ligand (50 ng/mL), interleukin 3 (IL3; 20 ng/mL), and granulocyte colony-stimulating factor (G-CSF; 20 ng/mL) as well as StemRegenin 1 (SR1; 1 μM). Cells were left untreated or treated with cobimetinib (Cobi) or venetoclax (Van), both at 0.1 μM, as single agents or in combination (Combo). After culture for 5 days, cells were stained with CD45-PE, Annexin-V-APC, and DAPI. Apoptotic leukemia blasts (CD45dimAnnexin-V⁺) were isolated by flow cytometry. Results are expressed as percentage of specific apoptosis calculated by the formula: 100 × (% apoptosis of treated cells – % apoptosis of control cells)/(100 – % apoptosis of control cells). Percentage of growth inhibition was calculated on the basis of the number of control viable cells (Annexin-V⁻/DAPI⁻). **P<0.01; ***P<0.001. (B) Mononuclear cells collected from AML patients (100,000 cells) or healthy donors (50,000 cells; NBM) were plated in methylcellulose, then treated with venetoclax or cobimetinib (both at 0.1 μM) as single agents or in combination. Colonies were scored on day 14. Data are presented as percentage inhibition compared to control groups. (C) The absolute cell counts of AML13 and AML14 samples as determined in (A) are shown in comparison with those of untreated controls (Contr), with median inhibitory concentration (IC₅₀) values indicated for each sample. (D) AML13 and AML14 samples were treated with cobimetinib 1.0 μM overnight followed by 10 min with or without (Unstim) stimulation with SCF or G-CSF (100 ng/mL). Cells were fixed, permeabilized, and processed for time-of-lfight mass spectrometry. Spanning-tree progression analysis of density-nor-malized events (SPADE) trees were generated by using markers shown in Online Supplementary Figure S2. The leukemia stem/progenitor populations were manually annotated and highlighted by analysis of all surface markers. The median intensities of pERK and pS6 in gated populations are shown. BCL2 expression in CD34⁺ and CD34⁻ fractions in both samples is shown. DMSO: dimethylsulfoxide.

Figure 3

Pharmacodynamic markers of drug response identified through reverse-phase protein arrays and RNA sequencing. Acute myeloid leukemia (AML) cell lines were left untreated or treated with cobimetinib or venetoclax as single agents or in combination at 0.5, 1, or 2 times the median inhibitory concentration (IC₅₀) value of each compound in each cell line for 24 h. Cell pellets were harvested after treatment and subjected to reverse-phase protein array (RPPA) analysis as previously reported. (A) Plots depict proteins differentially expressed between cobimetinib-sensitive and cobimetinib-resistant cells. (B) The mean values of corresponding proteins in cell lines showing synergy to the combination treatment (CI<0.8 as presented in Table 1) are shown in the heatmap. Only the proteins showing significant differences (P<0.05) between control and treated groups are shown. C: untreated control; T: treated; S: sensitive; R: resistant; Syn: synergy. (C) Cells were treated with cobimetinib (Cobi), venetoclax (Ven), or a combination (Combo) at 10, 100 and 1000 nM for 4 h and subjected to lysis; proteins were separated and probed with the antibodies indicated. (D) AML cell lines were left untreated (control) or treated with cobimetinib or venetoclax as single agents or in combination at 10 times the median inhibitory concentration (IC₅₀) value of each compound in each cell line for 4 h. Cell pellets were harvested after treatment and subjected to electrochemiluminescent enzyme-linked immunosorbent assay. The levels of BCL2, MCL1, BCL2:BIM and MCL1:BIM complexes were plotted based on percentages of the levels in the control group. (E) AML cells were treated and processed as described above for the RPPA assay. RNA was isolated using the RNeasy kit and sent for mRNA sequencing. The enriched pathways in cell types showing synergy in response to the combination are shown.

Figure 4

In vivo administration of cobimetinib in combination with venetoclax demonstrated anti-leukemia efficacy in acute myeloid leukemia xenograft mouse models. (A) NSGS mice were injected intravenously with OCI-AML3-Luci-GFP cells (1.0×10⁶). Leukemia engraftment was confirmed 1 week later through a noninvasive in vivo bioluminescence imaging (BLI) system following injection with a D-luciferin (4 mg/mouse) substrate. Mice were dosed daily with oral vehicle or an orally active form of cobimetinib (Cobi; 10 mg/kg) or venetoclax (Ven; 100 mg/kg) or their combination (Combo) for 4 weeks. BLI data over time are shown. (B) Luciferase intensity [mean ± standard deviation(SD)] at week 5. Human CD45 engraftment in bone marrow and spleen was determined by time-of-flight mass spectrometry (C) BLI data over time from the leukemia model established with MOLM13-Luc-GFP cells (1×10⁶ per animal) in NSGS mice. Mice received treatment as for the OCI-AML3/Luc/GFP model for 14 days. (D) Quantification of BLI signals (mean ± SD) on day 17 in the MOLM13 model. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.