Inhibition of PI3K Alpha and PI3K Delta with Copanlisib Shows Preclinical Activity As a Single Agent and in Combination in Multiple Myeloma

Abstract Background: The reversible, pan-class I PI3K inhibitor, copanlisib (BAY 80-6946), has preferred activity for the alpha and delta isoforms. This is in contrast to the delta and delta/gamma isoform-selective PI3K inhibitors developed primarily for Non-Hodgkin Lymphoma. We hypothesize that a copanlisib-based combination strategy could provide an effective, novel therapy for multiple myeloma with a pharmacodynamic biomarker to identify patients most likely to benefit from the therapy. Here we demonstrate the preclinical efficacy of copanlisib as a single agent and in combination with carfilzomib, a BET bromodomain inhibitor (BETi) and venetoclax. Methods: A panel of 20 human multiple myeloma (MM) cell lines were evaluated for the effects of copanlisib on cell proliferation after a 5-day exposure to the drug. Sensitive NCI-H929, MM.1S, L-363, and resistant AMO-1, JJN3, COLO-677 MM cell lines were selected for further analysis. All mechanism of action (MOA) studies were performed by treating the MM cells at a dose of 100 nM copanlisib. Cell cycle analysis and induction of apoptosis was performed by FACS after propidium iodide or Annexin V FITC staining respectively on cells treated for 72 hours. Reverse phase protein array (RPPA) was performed at baseline and post treatment for proteomic analysis with confirmatory western blots. Phospho-flow was performed to measure pre- and post-treatment levels of the ribosomal protein phospho-S6 (p-S6). The combination studies utilized serial dilutions of copanlisib with carfilzomib, a BETi, and venetoclax. Excess over highest single agent (EOHSA) was used to evaluate for potentiation. These findings were confirmed in vivo using a SCID mouse model. Results: Following copanlisib exposure, we observed apoptosis in the sensitive cell lines (50-80% AN-V+ cells) but not in resistant cell lines (1-5% AN-V+ cells). We saw an increased cell cycle arrest in G1 in the sensitive cell lines, but not in the resistant cell lines. RPPA performed at baseline revealed a pattern of low p-S6 at the serine positions 235/236 and 240/244 in sensitive compared to resistant cell lines. Western blot analyses validated the RPPA results. We next performed RPPA following exposure to copanlisib, which resulted in a greater decrease in p-S6 in the sensitive cell lines NCI-H929 and L363 (53-83%, 73-93% respectively) compared to resistant cell lines COLO-677 and JJN3 (5-27%, and 38-67%, respectively). The pharmacodynamic p-S6 response was maintained following 24hr and 48hr of drug exposure. These findings were further supported with western blot analysis and phospho-flow. Treatment with copanlisib in combination with carfilzomib or BETi showed potentiation by EOHSA statistics, but this was not seen in with venetoclax. In xenograft models, treatment with copanlisib 10mg/kg by tail vein injection twice daily every 7 days resulted in regression of tumor growth compared to vehicle control. In the L-363 xenograft model, single agent copanlisib resulted in greater tumor regression than single agent carfilzomib. Discussion: Copanlisib demonstrated pre-clinical anti-tumor activity as a single agent and in combination with conventional and novel agents in vitro and in vivo . The low nanomolar range IC50 values correlated with low baseline and post-treatment p-S6, which may allow for the development of a pharmacodynamic FACS based biomarker assay to select patients most likely to benefit from a copanlisib based combination therapy. Disclosures Slamon: Pfizer: Equity Ownership, Honoraria, Research Funding; Eli Lilly: Consultancy; Bayer: Consultancy; Biomarin: Consultancy, Equity Ownership; Novartis: Honoraria, Research Funding.