Evaluation of Repotrectinib's Anti-MM Effects in Combination with Bortezomib, Carfilzomib, Cyclophosphamide, and Dexamethasone in Vitro and Ex Vivo

Abstract

Introduction: The proteasome inhibitors bortezomib and carfilzomib are anticancer drugs which are indicated for the treatment of multiple myeloma (MM). The alkylating agent cyclophosphamide is also an effective drug against MM. Glucocorticoids including dexamethasone induce MM cell apoptosis and are effective in MM. However, the disease remains incurable with currently available therapies. Repotrectinib (Turning Point Therapeutics, San Diego, CA) is a novel next-generation ROS1/TRK/ALK-tyrosine kinase inhibitor (TKI). Repotrectinib also inhibits the Janus kinase (JAK) 2 and indirectly down-regulates STAT5 phosphorylation in the SET2 cancer cell line harboring a JAK2 V617F tyrosine kinase mutation (Drilon et al 2018). We have previously shown the preclinical anti-MM effects of JAK1/2 inhibitors in combination with corticosteroids and other anti-MM drugs in vitro and in vivo. Although activation of JAK2 and PI3K signaling is often detected in hematological malignancies, there is no data regarding the potential anti-MM effects of repotrectinib alone or in combination with other anti-MM drugs. Thus, we evaluated the anti-myeloma effects of repotrectinib alone and in combination with bortezomib, carfilzomib, cyclophosphamide, and dexamethasone in vitro using MM cell lines and ex vivo using the human MM xenograft model LAG- λ1. Methods: The human MM cell lines U266, RPMI8226 and MM1S were obtained from ATCC. MCs were isolated from MM patients' BM aspirates. Cell viability was quantified using the MTS assay. For the ex vivo studies, cells were isolated from the MM xenograft LAG-λ1 after it was established in SCID mice. We determined the IC curves using the MM cell lines and fresh BMMCs from 6 MM patients. Next, we combined repotrectinib with other active anti-MM drugs (carfilzomib, bortezomib, cyclophosphamide, and dexamethasone) using these same three MM cell lines and BMMCs from 6 MM patients to evaluate the anti-proliferative effects of these combinations. Results: We first evaluated the anti-MM effects of repotrectinib in combination with bortezomib or carfilzomib by using the MM1S, RPMI-8226, and U266 MM cell lines. Specifically, the results with repotrectinib in combination with carfilzomib showed enhanced anti-MM effects when compared to repotrectinib alone or carfilzomib alone. We also determined the anti-MM effects of repotrectinib alone and in combination with carfilzomib by using isolated LAG- λ1 MM xenograft cells. Repotrectinib alone inhibited tumor cell proliferation in a concentration dependent manner (IC50 4.5 µM) and in combination with carfilzomib synergistically inhibited LAG- λ1 MM tumor cell proliferation. These results were not seen when it was combined with bortezomib. We studied BMMCs from 3 PD and 3 CR patients using a cell proliferation assay. The results showed that repotrectinib alone minimally inhibited MM tumor cell proliferation among patients with PD. Repotrectinib combined with carfilzomib showed enhanced anti-proliferative effects compared with either drug alone among BMMCs from PD patients but not those in CR. In contrast, bortezomib did not show this enhanced anti-proliferative effect. We also determined the anti-MM effects of repotrectinib in combination with cyclophosphamide using ex vivo and in vivo studies. The results showed that repotrectinib in combination with cyclophosphamide enhanced the inhibition of LAG- λ1 MM tumor cell and fresh MM BMMC proliferation compared with repotrectinib or cyclophosphamide alone. We further determined the anti-MM effects of repotrectinib in combination with dexamethasone. The results showed that repotrectinib in combination with dexamethasone reduced LAG- λ1 MM cell proliferation in a concentration dependent manner and enhanced inhibition in 2 PD patients' samples but not the other PD patient or 3 CR patients' samples. Summary Single agent repotrectinib decreased MM tumor cell proliferation in all 3 MM cell lines and reduced LAG- λ1 tumor cell proliferation ex vivo. Repotrectinib also reduced proliferation of BMMCs from MM patients with PD but not those in CR. Repotrectinib in combination with carfilzomib, cyclophosphamide or dexamethasone showed enhanced anti-proliferative effects compared with the drugs alone. The results suggest that this kinase inhibitor may have activity in treating MM patients in combination with other active anti-MM agents. Further evaluation is warranted.