A Novel MCL1 Inhibitor Combined with Venetoclax Rescues Venetoclax-Resistant Acute Myelogenous Leukemia

Abstract

Suppression of apoptosis by expression of antiapoptotic BCL2 family members is a hallmark of acute myeloblastic leukemia (AML). Induced myeloid leukemia cell differentiation protein (MCL1), an antiapoptotic BCL2 family member, is commonly upregulated in AML cells and is often a primary mode of resistance to treatment with the BCL2 inhibitor venetoclax. Here, we describe VU661013, a novel, potent, selective MCL1 inhibitor that destabilizes BIM/MCL1 association, leads to apoptosis in AML, and is active in venetoclax-resistant cells and patient-derived xenografts. In addition, VU661013 was safely combined with venetoclax for synergy in murine models of AML. Importantly, BH3 profiling of patient samples and drug-sensitivity testing ex vivo accurately predicted cellular responses to selective inhibitors of MCL1 or BCL2 and showed benefit of the combination. Taken together, these data suggest a strategy of rationally using BCL2 and MCL1 inhibitors in sequence or in combination in AML clinical trials. SIGNIFICANCE: Targeting antiapoptotic proteins in AML is a key therapeutic strategy, and MCL1 is a critical antiapoptotic oncoprotein. Armed with novel MCL1 inhibitors and the potent BCL2 inhibitor venetoclax, it may be possible to selectively induce apoptosis by combining or thoughtfully sequencing these inhibitors based on a rational evaluation of AML.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494.

Figure 1.

VU661013 blocks expansion of AML cell lines in vitro. A, The chemical structure of VU661013. B, Measurement of growth sensitivity to inhibition of MCL-1 with VU661013 in several cell lines, C, with resistance in some lines (mean ± SEM). D, AML cell lines were subjected to inhibition of MCL-1 (VU661013), BCL-2/BCL-xL/BCL-w [navitoclax (NAV)], BCL-2 (venetoclax) or BCL-xL (A1155463), and GI₅₀ at 48 hours was calculated. Cell lines with GI₅₀ values above 10uM are listed as >10uM as a specific value is unknown above our maximum concentration tested. E, Western blot analysis of AML cell lines reveal a wide variation in the protein content of BCL-2 family members. F,G Correlation of MCL-1 protein content to growth inhibiton in response to VU661013 treatment.

Figure 2.

Inhibition of MCL-1 reduces AML in an in vivo murine model. A, NSGS mice were engrafted with MV-4–11 human leukemia cells and were then treated with either vehicle (n=6), 10 (n=5), 25 (n=5) or 75 (n=5) mg/kg of VU661013. Peripheral blood (PB), bone marrow (BM) and spleen (SPL) were harvested for tri-compartmental chimerism analysis. A non-parametric, unpaired, two-tailed t-test was used to calculate significance. B,Immunohistochemistry of femurs and spleen (20x) stained with monoclonal antibody for hCD45 reveal AML cells left within the bone marrow and spleen of experimental mice at each dose level. C, Ratio of spleen to total body weight measurements from above mentioned experiments. D, Kaplan-Meier analysis. Statistical significance was calculated using Log-rank (Mantel-Cox) test (p=.001)(n= 5 per arm).

Figure 3.

BH3-targeted inhibitors drive specific resistance in human cell lines which can be overcome with alternating or combining inhibitors. A, Human MV-4–11 cells were isolated from the bone marrow of premorbid vehicle treated mice at D28, and VU661013-treated mice at D42 and were tested ex vivo with VU661013 (mean ± SEM; n= 3). B, Naïve MV-4–11 cells (parent) and cells made resistant to VU661013 (VU661013-resistant) or venetoclax (VEN-resistant) were tested in growth inhibition assays with VEN and C, VU661013 treatment. D, VU661013-resistant MV-4–11 cells treated with VU661013, VEN or a combination of VU661013 and VEN, concentrations of each compound (Cmpd) are noted on x-axis; E, VEN-resistant MV-4–11 cells treated with VU661013, VEN or a combination of VU661013 and VEN, concentrations of each compound (Cmpd) are noted on x-axis. For Figures B-E data shown as mean ± SEM (n= 3). F, The combination of VEN and VU661013 in vivo resulted in a survival benefit in a MV-4–11 AML model mice via Kaplan-Meier analysis. Statistical significance was calculated using Log-rank (Mantel-Cox) test (p<.001) (n= 5 per arm). G, The combination of VEN and VU661013 in vivo significantly decreased tumor burden in a MOLM-13 AML xenograft. Per arm Vehicle (n= 7), VEN (n=9), VU661013 (n= 6) and VU661013/VEN (n= 8). A non-parametric, unpaired, two-tailed t-test was used to calculate significance. Data are combined from 2 independent experiments. H, Immunohistochemistry of bone marrow (femur) and spleen (20x), stained with monoclonal antibody for hCD45 in experimental mice.

Figure 4.

A, Human UCB-derived CD34⁺ cells were transplanted in NSGS mice. After confirmation of chimerism with notation of hCD45⁺ cells in the PB at 2 weeks, mice were treated with vehicle, 30 mg/kg VEN, 75mg/kg VU661013, or VEN 15mg/kg and 75mg/kg VU661013 in combination. Mice were sacrificed at D42, and chimerism in bone marrow was assessed. Human chimerism is measured by hCD45⁺. Progenitor cells are noted by hCD34⁺, and human hematopoietic stem cell (HSC)-enriched cells are noted by hCD34⁺CD38⁻. Per arm: Vehicle (n= 3), VEN (n=2), VU661013 (n= 3) and VU661013/ VEN (n= 3). Data is represented as mean ± SEM. B, hCD34⁺ cells from three normal bone marrow samples were treated for 48 hours in vitro with VEN or VU661013 (mean ± SEM; n= 3).

Figure 5.

BH3 profiling supports in vitro findings of specific BCL-2 family inhibitor sensitivities. A, BH3 profiling was used with BIM, MS1 and HRK peptides, as well as navitoclax (NAV) and VEN to define the apoptotic priming of AML cell lines (mean ± SEM). B, BH3 proiling using MS1 correlated with GI₅₀ sensitivity to VU661013 lines (mean ± SEM). C, BH3 profiling of MV-4–11 cells (parent) and engineered MV-4–11 cell lines resistant to VU661013 (VU0661013^Res) or VEN (VEN^Res).

Figure 6.

BH3 profiling of patient samples, and improvement in disease control with combination therapy in a PDX model. A, AML patient samples analyzed using BH3 profiling were then B,C treated with dose titrations of VEN and VU661013 (mean ± SEM) D, Co-immunoprecipitation (IP) experiment of patient samples AML001 and AML002 illustrate that AML001 predominantly expressed high levels of BCL-2, and AML-002 expressed high levels of Mcl-1 [input samples; immunoblot (IB)]. The BCL-2 from the AML001 patient sample and the MCL-1 from AML002 were dimerized with Bim. In patient sample AML002, BCL-2 and BCL-xL are also associated with BIM to a lesser degree. E, Samples from AML patients who later failed VEN+LDAC treatment. Overall viability after VU661013/VEN treatment showed significant decreases in viability. F, After early during treatment (24hrs), blast cells from these samples began to undergo apoptosis with decreases in viability shown by Annexin V/PI staining. G, Comparison of pre-treatment and post-treatment sensitivity to VU661013+VEN combination therapy in samples from AML patients who were treated with VEN+LDAC and relapsed; For E-G, Individual patients are represented by shapes; in G, relative viability after ex vivo exposure to VU661013+VEN in samples taken from patients prior to therapy and after therapy with VEN+LDAC in the clinic is noted; p = n.s. H, In patient-derived xenografts, VEN and VU661013 were given concominantly at low doses with bone marrow harvested at Day 42. For AML 001 [vehicle (n= 4), VEN (n=4), VUO661013 (n= 5) and VU661013/VEN (n= 3)], there was no significant difference between treatments. For AML 002 vehicle (n= 6), VEN 15 (n=6), VUO661013 (n= 5) and VU661013/VEN (n= 4), VU661013/VEN combination treatments led to reduction in engrafted human leukemia (mean ± SEM). A non-parametric, unpaired, two-tailed t-test was used to calculate significance. I, Post-treatment, reduction of human leukemia was noted through immunohistochemical staining of bone marrow for hCD45 in MCL-1 dependent AML 002 (20x).

Figure 7.

MCL-1 and BCL-2 inhibitors in the treatment of AML. While anti-apoptotic dependence is heterogeneous across patients and intrapatient with AML, individual patients may have greater MCL-1 or BCL-2 anti-apoptotic dependence at diagnosis, and this may be interrogated to guide initial treatment. Resistance to BH3 mimetics may arise from upregulation of another anti-apoptotic protein family member, and a patient may switch selective BH3 mimetic at that time. A, In some patients, this sequential targeting of anti-apoptotic family members (with or without chemotherapy priming agent) may continue to provide disease remissions and clinical benefit. This may occur in a tumor that is initially MCL-1 dependent. B, Or initially, BCL-2 dependent AML. C, Combination therapy with MCL-1 inhibition and BCL-2 inhibition at diagnosis has not been tested in patients, but may be tolerable and lead to tumor involution by targeting two important anti-apoptotic proteins heterogeneously upregulated in AML.