Comparative TKI Profiling Analyses to Explore Potential Mechanisms of Ponatinib-Associated Arterial Thrombotic Events

Background: Ponatinib is a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) indicated for patients with T315I positive or treatment-refractory CML and Ph+ ALL. To develop hypotheses regarding molecular and cellular targets of ponatinib that could contribute to arterial thrombotic events observed in some patients we are undertaking a broad comparative profiling analysis of ponatinib and other TKIs.

Methods: Cellular activities of ponatinib and additional BCR-ABL (imatinib, nilotinib, dasatinib, bosutinib) and VEGFR2/multi-targeted (sunitinib, regorafenib) TKIs were examined in panels of Ba/F3 cell lines expressing activated kinase variants (N=61), tumor cell lines (N=246), and primary lines derived from human vasculature (aortic smooth muscle cells [ASMC] and umbilical vein [HUVEC], aortic [HAEC] and pulmonary artery [HPAEC] endothelial cells). Human steady-state Cave concentrations of 45 mg ponatinib (101 nM) were corrected for the functional effects of protein binding (3.6-fold) to derive the clinically-effective concentration.

Results: Ponatinib inhibits the in vitro activity of multiple kinases with IC50s within 10-fold of ABL, including members of the VEGFR, PDGFR, FGFR, EPH receptor and SRC families of kinases, KIT, RET, TIE2, and FLT3. This profile was largely recapitulated in cellular assays using engineered Ba/F3 cells, with ponatinib demonstrating substantially greater potency against VEGFRs, FGFRs, TIE2, RET and FLT3 than other ABL TKIs. Across a broad panel of tumor cell lines, ponatinib inhibited viability with a median IC50 of 598 nM. Ponatinib only inhibited 16 cell lines (6.5%) with IC50s below its clinically effective concentration (28 nM) with the 5 most sensitive lines (IC50 <1 nM) all being BCR-ABL positive. Within the vasculature-derived cell panel, ponatinib inhibited viability of HUVECs grown in full serum with an IC50 of 261 nM, with all of the other ABL and non-ABL TKIs tested having IC50s >2000 nM. Effects of ponatinib on HAECs, HPAECs and ASMCs were more modest (IC50s 1533, 490 and 750 nM, respectively). Finally, ponatinib (IC50 20 nM) and other VEGFR2 inhibitors potently inhibited survival of HUVECs grown in VEGF-dependent conditions, while other BCR-ABL inhibitors, except dasatinib (IC50 14 nM), did not.

Conclusions: Ponatinib is a potent BCR-ABL inhibitor that also inhibits VEGFR2 and other kinases at clinically achievable concentrations in vitro. Modest effects of ponatinib on endothelial cells have been observed that warrant further exploration in vivo. Developing a precise understanding of the mechanism by which ponatinib contributes to arterial thrombotic events should facilitate development of strategies to optimize its benefit/risk in patients.