Targeting 1q21 Amplification with APG2575 and Lenalidomide to Sensitize BCL-2 Inhibition with the Decrease of MCL-1 Protein in High Risk MM Models

Background Multiple myeloma (MM) is a heterogenous plasma cell malignancy. About 30-40% of newly diagnosed and 20-60% of relapsed/refractory MM patients carry 1q21 amplification worldwide, a high-risk indicator for poor prognosis in RRMM. Different BCL-2 family anti-death proteins play important roles in MM survival and drug resistance. High expression of BCL-2 due to t (11;14) renders cell vulnerability to BCL-2 antagonist, however, patients carrying other genetic abnormality including 1q21 amplification have limited response to the newly emerged treatment choice. With MCL-1 upregulation accompanied with 1q21 amplification, we tested whether BCL-2 antagonist combined with IMiDs (immunomodulatory imide drugs), improves cell killing in high-risk MM patient models, including those resistant to bortezomib and lenalidomide. For that aim, we studied APG-2575, a novel and potent BCL-2 inhibitor developed by Ascentage Pharma Group and it is currently in clinical trials for hematologic malignances, including MM. Methods 1. Cells were treated with APG-2575 single agent or in combination with lenalidomide; 2. Cell line viability was assessed by CellTiter-Glo (CTG) assay; 3. Flow cytometry analysis was used to detect CD138+ cell surface marker in primary cells derived from MM patients; 4. Western blot analysis for BCL-2 family and IMiD signaling proteins. Results We first determined the cell sensitivity of APG-2575 as a single agent in a panel of MM cell lines, and as expected, those carrying t (11;14) were very sensitive to APG-2575, with low IC50 values ranging 7-23 nM. The IC50 values for cells carrying other genetic markers were greater than 5-10 μM. We then evaluated cell-death inducing activity of APG-2575 in MM patient-derived primary cells ex vivo, which were freshly prepared from patients’ bone marrow aspirates. Primary cells were treated with APG-2575 or ABT-199 (venetoclax) for 18-24 hours, and the loss of CD138 surface marker was used to quantify cell death. As shown in Figure 1a, APG-2575 induced cell death (CD138+ loss) in a dose-dependent manner, and significant cell death was observed at 0.37-3.3 μM of APG-2575, indicating primary cells more sensitive than MM cell lines. Interestingly, the sensitivity to APG-2575 is similar in primary MM cells with or without 1q21 amplification. Since moderate cell death inducing activity was observed in MM cells when APG-2575 used as a single agent, we combined APG-2575 with lenalidomide. Cell death was increased in