Ponatinib Induced Thrombocytopenia Might Inhibit Proplatelet Formation of Megakaryocytes Via the Pathways Including Rho/Rock and Rac.
Authors: Murai Kazunori , Kowata Shugo , Oyake Tatsuo , Shimoyama Tadashi , Asahi Maki , Norifumi Sugawara , Sasaki Ryosei , Yuzo Suzuki , Yukiteru Fujishima , Tsukushi Yasuhiko , Ito Shigeki , Ishida Yoji 
Published: October 2019

Abstract
Abstract 2186

Background:
Ponatinib (AP24534) is identified as a pan-BCR-ABL inhibitor that potently inhibits the T315I gatekeeper mutant, and has advanced into clinical development for the treatment of refractory or resistant CML (Chronic Myeloid Leukemia). Ponatinib potently inhibited in vitro proliferation of Ba/F3 cells expressing BCR-ABL T315I mutation (IC 50; 11 nM). In PACE (Ponatinib Ph+ ALL and CML Evaluation) clinical trial indicated that 57% had CCyR (complete cytogenetical response), and 47% had MMR (major molecular response) in CML-chronic phase with T315I mutation. Ponatinib has substantial activity in heavily pretreated patients and those with refractory T315I mutation. However, approximately one third of ponatinib-treated patients had moderate to severe thrombocytopenia (J.E. Cortes et al. 2011 ASH Annual Meeting). The mechanism of ponatinib-induced thrombocytopenia remains unknown. In this study, we evaluated the effects of ponatinib on megakaryocytic progenitor cells, megakaryocytopoiesis, megakaryocyte and platelet production in mice.

Method:
All animal procedures were approved by the Institutional Animal Care and Use Committee of Iwate Medical University. Male ddY mice at 8 weeks of age were used in all experiments. We studied in vitro culture of megakaryocytic colonies (CFU-Meg), megakaryocyte ploidy analyses in vitro culture and proplatelet formation (PPF) assay in vitro. Murine bone marrow cells were cultured in methylcellulose with mIL-3, mIL-6 and TPO at 37°C in 5% CO2 and 20% O2 for 7 days in the presence of ponatinib (0.01, 0.1, 1, 10, 100 nM). PPF: Murine megakarocytes were partially purified from bone marrow cells using BSA gradient. They were plated in 96 micro-well culture plates (300 megakaryocyte/well) and cultured in IMDM, supplemented with 1% ITS-G (serum-free medium) in the presence of ponatinib (0.01, 0.1, 1, 10, 100 nM), at 37°C in 5% CO2and 20% O2. After 24 hr incubation, the megakaryocytes with proplatelets in each well were counted. Activated Rho and activated Rac in murine platelets were measured by the Western blot using Rhotekin-binding domain (RBD) beads and PAK-PBD Affinity beads respectively. The phosphorylation of Lyn (Src family kinase) in murine platelets was also evaluated by the Western blots.

Results:
CFU-Megs did not decrease significantly at 0.01, 0.1, 1, 10 and 100 nM (31.8 +/− 1.4 to 42.3 +/− 2.4 cells) and decreased significantly (17.0 +/− 1.6 cells p<0.01) at 1000 nM of ponatinib. PPF were decreased significantly at 0.1, 1, 10, 100 nM ponatinib (0 nM: 26.4 ± 0.8 %, 0.1 nM: 19.2 ± 1.7% p<0.05, 1 nM: 19.4 ± 2.1 % p<0.05, 10 nM: 17.9 ± 1.1% p<0.01, 100 nM: 12.5 ± 1.1 % p<0.001, 1000 nM: 11.6 ± 0.9 % p<0.001). The decreases in PPF were cancelled significantly by the addition of Y27632, Rho-associate kinase ROCK inhibitor (ponatinib 10 nM; 17.9 ± 1.1%, ponatinib 10 nM + Y27632 10μM; 29.8 ± 1.7% p<0.001, ponatinib 100 nM; 12.5 ± 1.1%, ponatinib 100 nM + Y27632 10μM; 19.3 ± 1.4% p<0.001). There was no difference in DNA ploidy of cultured megakaryocytes in the presence of ponatinib (0.01 to 100 nM). Next we have tried to understand the precise role of Rho/Rock and Rac pathways in platelets. Our data showed that Rho was upregulated and that activated Rac was downregulated at 50 nM of ponatinib. Ponatinib reduced the levels of phosphorylated Lyn (p-Lyn).

Discussion and Conclusion:
The Rho/ROCK pathway was reported to be negative regulators (Blood 2007 109; 4229) and positive regulators (Blood 2007 110; 3637) in PPF. Ponatinib induced thrombocytopenia might not be due to the inhibition of megakaryocyte colony formations but the inhibition of PPF of megakaryocytes via pathways including Rho/Rock and Rac.

Disclosures:
No relevant conflicts of interest to declare.