Anti-Leukemic Activity of Phosphoinositide 3-Kinase Inhibitor, Copanlisib in ABL Tyrosine Kinase Resistant Leukemia Cells
Authors: Okabe Seiichi , Tauchi Tetsuzo , Tanaka Yuko , Katagiri Seiichiro , Ohyashiki Kazuma 
Published: October 2019

Abstract
Introduction: Chronic myelogenous leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric oncoprotein BCR-ABL. ABL tyrosine kinase inhibitor (TKI) therapy such as imatinib, nilotinib and dasatinib has improved the survival of Ph-positive leukemia patients. Although impressive clinical responses are obtained in the majority of CML patients, increasing evidence of acquired resistance to TKIs have been documented in clinically. Moreover, ABL TKIs cannot eradicate leukemia stem cells, thus, TKIs do not appear to lead to a cure the diseases. Therefore, new approach against BCR-ABL mutant cells and leukemia stem cells may improve the outcome of Ph-positive leukemia patients. Phosphoinositide 3-kinase (PI3K) pathway regulates cell metabolism, proliferation and survival. Furthermore, aberrant activation of PI3K signaling pathway has been shown to be important in initiation maintenance of human cancers. Copanlisib, also known as BAY80-6946 is a PI3K inhibitor with potential antineoplastic activity. Copanlisib is being investigated in a pivotal phase 2 clinical trial against hematological malignancies such as malignant lymphoma. We hypothesized that targeting PI3K, in combination with ABL TKI, would result in enhanced therapeutic activity in Ph-positive leukemia cells including T315I mutation and ABL TKI resistant.
Materials and methods: We investigated the combination therapy with a copanlisib and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, nilotinib resistant K562, ponatinib resistant Ba/F3 cells and primary sample.
Results: 72 h treatment of copanlisib exhibits cell growth inhibition against K562 cells in a dose dependent manner. The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells co-cultured with feeder cell line, HS-5. We found that the treatment of copanlisib abrogated the protective effects of HS-5 in K562 cells. We examined the intracellular signaling after treatment of copanlisib. High concentration of copanlisib reduced the phosphorylation of BCR-ABL and Crk-L. Activity of caspase 3 and poly (ADP-ribose) polymerase (PARP) was increased. We next investigated the efficacy between ABL TKI and copanlisib by using these cell line. Phosphorylation of BCR-ABL, Crk-L and Akt was reduced after imatinib and copanlisib treatment. We investigated the copanlisib activity against T315I positive cells. Copanlisib potently induced cell growth inhibition of Ba/F3 T315I cells. Combined treatment of Ba/F3 T315I cells with ponatinib and copanlisib caused significantly more cytotoxicity than each drug alone. Phosphorylation of BCR-ABL and Crk-L was reduced and cleaved PARP was increased after ponatinib and copanlisib treatment. To assess the activity of copanlisib and ponatinib, we performed to test on CML tumor formation in mice. We injected nude mice subcutaneously with Ba/F3 T315I mutant cells. A dose of 20 mg/kg/day p.o of ponatinib and 6 mg/kg/three times per week i.p of copanlisib inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of copanlisib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells, K562 nilotinib resistant cells and primary sample.
Conclusion: These results indicated that administration of the PI3K inhibitor, copanlisib may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells.

Disclosures
Ohyashiki: Bristol: Honoraria, Research Funding.