Ponatinib Exerts Growth-Inhibitory Effects on Neoplastic Mast Cells and Synergizes with Midostaurin in Producing Growth Arrest and Apoptosis,
Authors: Gleixner Karoline V. , Blatt Katharina , Peter Barbara , Hadzijusufovic Emir , Valent Peter 
Published: October 2019

Abstract
Abstract 3497
Aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) have a poor prognosis. In these patients, neoplastic mast cells (MC) usually harbor the D816V-mutated variant of KIT and are resistant to conventional cytoreductive drugs and to several tyrosine kinase inhibitors (TKI) such as imatinib. More recently, various KIT kinase blockers including midostaurin (PKC412), have been described to overcome KIT D816V-mediated resistance in neoplastic MC. However, despite encouraging first results observed in clinical trials, these novel kinase blockers are unable to induce long-lasting complete remissions in all patients with ASM and MCL. One reason for the poor response in these patients may be the expression and activation of additional KIT-independent pro-oncogenic signalling molecules and pathways that trigger survival of neoplastic MC. Therefore, current research is seeking novel broadly acting drugs and drug combinations directed against the pro-oncogenic signaling machinery of neoplastic MC. Ponatinib (AP24534) is a broadly acting novel multikinase inhibitor that has been shown to exert major anti-leukemic effects in chronic myeloid leukemia. The aim of our current study was to evaluate the effects of ponatinib on growth and survival of neoplastic MC. Ponatinib was applied as single agent or in combination with midostaurin (PKC412). As assessed by Western blotting, ponatinib was found to inhibit KIT-phosphorylation in both subclones of the human MC leukemia cell line HMC-1, namely HMC-1.1 harboring KIT G560V but not KIT D816V, and HMC-1.2 cells harboring KIT G560V and KIT D816V. Interestingly, the D816V mutation of KIT was found to induce relative resistance against ponatinib. Ponatinib was also found to counteract the phosphorylation of Lyn, a Src-kinase that serves as a major KIT-independent signalling molecule and survival factor in neoplastic MC. Activated STAT5 in MC was also blocked by ponatinib in a dose-dependent manner. In a next step, we examined the effects of ponatinib on proliferation of neoplastic MC by 3H-thymidine uptake experiments. Ponatinib was found to induce dose-dependent growth inhibition in both HMC-1 subclones, with higher IC50-values in HMC-1 cells harbouring KIT D816V (IC50: 100–500 nM) compared to cells lacking KIT D816V (IC50: 1–10 nM). Furthermore, ponatinib was found to inhibit the proliferation of primary neoplastic MC isolated from patients with indolent SM (ISM, n=2) and ASM (n=1), with IC50-values ranging between 50 nM and 500 nM. Growth inhibitory effects of ponatinib on neoplastic MC were accompanied by induction of apoptosis as assessed by light microscopy, flow cytometry, and TUNEL assay. Finally, we were able to demonstrate that ponatinib synergizes with midostaurin in producing growth-inhibition and apoptosis in HMC-1.1 cells and HMC-1.2 cells. Synergistic effects obtained with suboptimal concentrations of single agents were accompanied by a complete blockage of all relevant kinase targets tested including KIT, Lyn, and STAT5. In conclusion, ponatinib exerts major growth-inhibitory effects on neoplastic MC. KIT D816V-expressing MC are less sensitive to ponatinib. This relative resistance of MC against ponatinib can be overcome by combining ponatinib with midostaurin in an in vitro assay. Whether the drug-combination also exerts major anti-neoplastic effects in vivo in patients with ASM and MCL remains to be determined.

Disclosures:
Valent: Novartis: Consultancy, Honoraria, Research Funding.