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. 2023 Feb 1;108(2):522-531.
doi: 10.3324/haematol.2022.280631.

Selective inhibition of MCL1 overcomes venetoclax resistance in a murine model of myelodysplastic syndromes

Melissa A Fischer  1 , Yuanbin Song  2 , Maria P Arrate  3 , Rana Gbyli  4 , Matthew T Villaume  1 , Brianna N Smith  5 , Merrida A Childress  1 , Thomas P Stricker  6 , Stephanie Halene  4 , Michael R Savona  7
Affiliations

Selective inhibition of MCL1 overcomes venetoclax resistance in a murine model of myelodysplastic syndromes

Melissa A Fischer et al. Haematologica. .

Abstract

Treatment for myelodysplastic syndromes (MDS) remains insufficient due to clonal heterogeneity and lack of effective clinical therapies. Dysregulation of apoptosis is observed across MDS subtypes regardless of mutations and represents an attractive therapeutic opportunity. Venetoclax (VEN), a selective inhibitor of anti-apoptotic protein B-cell lymphoma- 2 (BCL2), has yielded impressive responses in older patients with acute myeloid leukemia (AML) and high risk MDS. BCL2 family anti-apoptotic proteins BCL-XL and induced myeloid cell leukemia 1 (MCL1) are implicated in leukemia survival, and upregulation of MCL1 is seen in VEN-resistant AML and MDS. We determined in vitro sensitivity of MDS patient samples to selective inhibitors of BCL2, BCL-XL and MCL1. While VEN response positively correlated with MDS with excess blasts, all MDS subtypes responded to MCL1 inhibition. Treatment with combined VEN + MCL1 inhibtion was synergistic in all MDS subtypes without significant injury to normal hematopoiesis and reduced MDS engraftment in MISTRG6 mice, supporting the pursuit of clinical trials with combined BCL2 + MCL1 inhibition in MDS.

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Figures

Figure 1.
Figure 1.
Dual inhibition of MCL1 and BCL2 is synergistic in myelodysplastic syndrome cells resulting in increased apoptosis. (A) Cell viability of primary myelodysplastic syndrome (MDS) samples was measured by CellTiter-Glo at 48 hours after treatment with threefold dilutions of S63845, venetoclax (VEN) or a combination of both. Contour plots of synergy scores generated from the cell viability dose matrix of S63845 and VEN using the zero interaction potency (ZIP) model. The synergy scores were represented by pseudocoloring 2-D contour plots over the dose matrix, giving rise to the overall synergy landscape. Red color indicates synergy, while green color indicates antagonism for the various concentrations of the S63845/VEN combination (note different pseudocoloring scale for ZIP synergy scores between the samples). (B and C) Apoptosis in CD34+ cells was measured by annexin V/PI staining using flow cytometry after 24 hours of treatment in all samples combined (B) or broken down into MDS subtypes; MDS-RS-SLD is MDS with ring sideroblast with single lineage dysplasia; MLD is multi-lineage dysplasia; EB1 is excess blasts 5-9%; EB2 is 10-19% blasts (C). The percent of live cells relative to DMSO control were calculated. Statistical comparisons between S63845 or VEN monotherapy and the combination are shown (ANOVA followed by two-tailed t-test; n.s.: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
Figure 2.
Figure 2.
Combined inhibition of MCL1 and BCL2 reduces clonogenicity at doses that are tolerable to normal hematopoietic stem and progenitor cells. (A) Colony forming assays of normal (NML) whole bone marrow (WBM), MDS WBM, NML CD34+, and MDS CD34+ patient cells. The percent of CFU-GM colonies relative to dimethyl sulfoxide (DMSO) for 100 nM S63845, 100 nM venetoclax (VEN), or S/V (100 nM S63845 + 100 nM VEN) treatment groups were calculated. Statistical comparisons of S/V combination treatment between NML WBM vs. MDS WBM and NML CD34+ vs. MDS CD34+ cells are shown (two-tailed t-test; *P<0.05). (B) Colony-forming assays of NML CD34+ cells treated with a range of combined concentrations of S63845 + VEN (SV). The percent of CFU-GM colonies relative to DMSO were calculated. (C) Colony-forming assays of normal (NML) whole bone marrow (WBM), MDS WBM, NML CD34+, and MDS CD34+ patient cells. The percent of CFU-GM colonies relative to DMSO for 50 nM S63845, 50 nM VEN, or S/V (50 nM S63845 + 50 nM VEN) treatment groups were calculated. Statistical comparisons of S/V combination treatment between NML WBM vs. MDS WBM and NML CD34+ vs. MDS CD34+ cells are shown (two-tailed t-test; *P<0.05; ***P<0.001). CFU-GM is colony-forming unit – granulocyte, macrophage. Data in (A) and (C) is represented as mean ± standard error of the mean, and individual patient samples in (A to C) are represented by unique symbols.
Figure 3.
Figure 3.
Myeloid engrafment of low and medium-risk myelodysplastic syndromes in MISTRG6 mice. (A to C) Confirmed engraftment of 3 primary myelodysplastic syndrome (MDS) patient cells in MISTRG6 mice. (A) Flow cytometric analysis of mouse vs. human CD45 (mCD45 vs. huCD45; left panels) and huCD34 vs. huCD33 (right panels). (B) Percent of huCD45+ cells in the bone marrow (BM) for each patient sample. (C) Relative distribution of myeloid CD33+ (red), B-lymphoid CD19+ (blue), and T-lymphoid CD3+ (gray) cells as percent of human CD45+ cells for each patient sample.
Figure 4.
Figure 4.
Evidence of reduced myelodysplastic syndrome engrafment in MISTRG6 mice afer co-inhibition of BCL2 and MCL1. (A to D) Patient sample MDS019-EB1 was allowed to engraft in MISTRG6 mice for 16 weeks before beginning treating with vehicle, venetoclax (VEN) (15 mg/kg; 5 days on, 2 days off), or a combination of VEN (15 mg/kg; 5 days on, 2 days off) and S63845 (12.5 mg/kg; 2 days on, 5 days off) for a total of 4 weeks (n=4 per treatment group). The percent (A) or total number (B) of huCD45+ cells in the bone marrow (BM) were compared between pre and post-treatment for each treatment group. (C) After completion of the treatment, the percent of huCD45+/lin-/huCD38-/huCD34+ in the BM was determined. (D) Representative histologic images of BM from mice in (A to C) that were treated with vehicle, VEN, or S63845/VEN and underwent immunohistochemical staining for huCD34 (top panel, original magnification 10x, scale bars: 100 mm; lower panel, original magnification 60x, scale bars:10 µm). The data in (B and C) are represented by mean ± standard error of the mean, and statistical comparisons between vehicle and VEN or combination treatments are shown (two-tailed t-test; n.s.: not significant, *P<0.05, **P<0.01, ***P<0.001).
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