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

Inhibition of MCL1 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) or 10 (n = 5), 25 (n = 5), or 75 (n = 5) mg/kg of VU661013. Peripheral blood, bone marrow (BM), and spleen (SPL) were harvested for tricompartmental chimerism analysis. A nonparametric, unpaired, two-tailed t test was used to calculate significance. B, IHC of femurs and spleen (20×) 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 = 0.001; n = 5 per arm).

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 the 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 the x-axis. For B–E, data shown as mean ± SEM (n = 3). F, The combination of VEN and VU661013 in vivo resulted in a survival benefit in an MV-4-11 AML model mice via Kaplan–Meier analysis. Statistical significance was calculated using log-rank (Mantel–Cox) test (P < 0.001; n = 5 per arm). G, The combination of VEN and VU661013 in vivo significantly decreased tumor burden in an MOLM-13 AML xenograft. Per arm vehicle (n = 7), VEN (n = 9), VU661013 (n = 6), and VU661013/VEN (n = 8). A nonparametric, unpaired, two-tailed t test was used to calculate significance. Data are combined from two independent experiments. H, IHC of bone marrow (femur) and spleen (20×), stained with monoclonal antibody for hCD45 in experimental mice. Scale bars, 50 μm.

A, Human UCB-derived CD34⁺ cells were transplanted in NSGS mice. After confirmation of chimerism with notation of hCD45⁺ cells in the peripheral blood at 2 weeks, mice were treated with vehicle, 30 mg/kg VEN, 75 mg/kg VU661013, or VEN 15 mg/kg and 75 mg/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–enriched cells are noted by hCD34⁺CD38⁻. Per arm: vehicle (n = 3), VEN (n = 2), VU661013 (n = 3), and VU661013/VEN (n = 3). Data, 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).

BH3 profiling supports in vitro findings of specific BCL2 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 profiling 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).

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 and C) treated with dose titrations of VEN and VU661013 (mean ± SEM). D, Coimmunoprecipitation (IP) experiment of patient samples AML 001 and AML 002 illustrate that AML 001 predominantly expressed high levels of BCL2, and AML 002 expressed high levels of MCL1 [input samples; immunoblot (IB)]. The BCL2 from the AML 001 patient sample and the MCL1 from AML 002 were dimerized with BIM. In patient sample AML 002, BCL2 and BCL-xL are also associated with BIM to a lesser degree. E, Samples from patients with AML who later failed VEN + LDAC treatment. Overall viability after VU661013/VEN treatment showed significant decreases in viability. F, After early during treatment (24 hours), blast cells from these samples began to undergo apoptosis with decreases in viability shown by Annexin V/PI staining. G, Comparison of pretreatment and posttreatment sensitivity to VU661013 + VEN combination therapy in samples from patients with AML 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 concomitantly 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 nonparametric, unpaired, two-tailed t test was used to calculate significance. I, Posttreatment, reduction of human leukemia was noted through IHC staining of bone marrow for hCD45 in MCL1-dependent AML 002 (20×).