Bosutinib Blocks Lyn and Btk Activation and Synergizes with the KIT D816V-Targeting Drug Midostaurin in Inducing Apoptosis in Neoplastic Human Mast Cells.
Authors: Gleixner Karoline Veronika , Mayerhofer Matthias , Hörmann Gregor , Schuch Karina , Cerny-Reiterer Sabine , Peter Barbara , Herrmann Harald , Pickl Winfried F. , Sillaber Christian , Valent Peter 
Published: October 2019

Abstract
Abstract 1717
Poster Board I-743
Advanced systemic mastocytosis (SM) is a malignant hematopoietic neoplasm characterized by destructive growth of neoplastic mast cells (MC) in various organ systems. In these patients, the response to conventional cytoreductive therapy is poor and the prognosis is grave. The D816V-mutated variant of c-KIT is found in most patients and is considered to be a major transforming oncoprotein in SM that leads to abnormal survival and growth of neoplastic MC. Therefore, agents interfering with the kinase activity of KIT D816V have been developed. One promising agent is midostaurin (PKC412). However, in most patients with advanced SM, therapy with midostaurin is not sufficient to induce long term remissions. In addition, midostaurin is unable to block all pro-oncogenic signaling molecules, such as Lyn and Btk, in neoplastic MC, suggesting that additional oncoproteins and survival factors may play a role in malignant transformation in SM, and that novel therapeutic strategies are required to block such KIT-independent oncogenic pathways. Especially Lyn and Btk have attracted attention as potential new targets in neoplastic MC. Bosutinib (SKI-606) is a novel multikinase inhibitor that targets a broad spectrum of kinases including Lyn and Btk. The aim of the current study was to evaluate the effect of bosutinib on neoplastic MC, and potential cooperative drug interactions between bosutinib and midostaurin. As assessed by 3H-thymidine uptake, bosutinib was found to inhibit the growth of the MC leukemia cell line HMC-1, including the HMC-1.1 subclone that lacks KIT D816V and HMC-1.2 cells expressing KIT D816V, with similar IC50 values (1-5 μM). Furthermore, bosutinib was found to induce apoptosis in both HMC-1 subclones. Growth-inhibitory and apoptosis-inducing effects of bosutinib were also seen in primary neoplastic MC obtained from the bone marrow of patients with SM (n=3). As assessed by phosphoblotting, bosutinib did not inhibit the autophosphorylation of mutant KIT in HMC-1 cells, but was found to completely inhibit the phosphorylation of Lyn and Btk. To confirm the target-function of Lyn and Btk in neoplastic MC, siRNA experiments were performed. Knockdown of Lyn or Btk resulted in induction of apoptosis and growth-inhibition in HMC-1 cells. We next attempted to exploit target-specific and complementing effects of midostaurin and bosutinib by combining both substances. As expected, combined application of bosutinib and midostaurin resulted in a complete inhibition of phosphorylation of KIT, Lyn, and Btk in HMC-1.1 and HMC-1.2 cells. We were also able to show that bosutinib synergizes with midostaurin in inducing apoptosis in both HMC-1 subclones. Synergistic effects were also observed when combining midostaurin with Lyn- or Btk-siRNA. Together, we have identified Lyn and Btk as novel KIT-independent survival molecules in neoplastic MC. Inhibition of these kinases by siRNA-knockdown or by bosutinib leads to growth-inhibition and apoptosis. Synergistic pro-apoptotic effects were observed with the combination “bosutinib + midostaurin”, suggesting that simultaneous targeting of KIT and Lyn/Btk may be a powerful strategy to counteract the survival of neoplastic MC. This drug combination may therefore be an interesting approach to overcome drug-resistance in advanced forms of SM.

Disclosures
Valent: Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.