Delineation of a KIT-Independent Oncogenic Pathway in Neoplastic Mast Cells That Involves Lyn and Btk, and Can Be Disrupted by the KIT/Lyn/Btk-Targeting Drug Dasatinib

Abstract

Systemic mastocytosis (SM) is a myeloid neoplasm characterized by increased growth and survival of neoplastic mast cells (MC). Aggressive SM (ASM) and MC leukemia (MCL) are advanced disease variants that usually are drug-resistant and have an unfavorable prognosis. In most patients, the D816V-mutated ′oncogenic′ variant of KIT is detectable. However, the mutant is also detectable in patients with indolent SM exhibiting a normal life-expectancy, and therefore is not considered to represent a fully transforming oncoprotein. This assumption is also supported by studies in Ba/F3 cells, and whether KIT D816V-targeting drugs are able to induce long-term remission in ASM or MCL, remains to be seen. Therefore, it has been hypothesized that in addition to KIT, other pro-oncogenic molecules and signaling pathways play a role in malignant transformation/progression in SM. We here describe a novel KIT D816V-independent oncogenic pathway in neoplastic MC that involves Lyn and Bruton's tyrosine kinase (Btk). Western blotting and immunostaining revealed that neoplastic MC display the Btk- and Lyn protein. Both molecules were found to be constitutively phosphorylated in primary neoplastic MC and in the MC leukemia cell line HMC-1. Lyn/Btk-activation was not only detectable in KIT D816V-positive HMC-1.2 cells, but also in the KIT D816V-negative HMC-1.1 subclone. In studies employing Ba/F3 cells with doxycycline-inducible expression of KIT, we were able to show that KIT D816V induces activation of STAT5 and Akt, but does not induce activation of Btk. Correspondingly, pharmacologic deactivation/dephosphorylation of KIT in HMC-1 cells by midostaurin (PKC412) (Novartis, Basel, Switzerland) was not accompanied by a decrease in phosphorylation of Lyn or Btk. The functional significance of Btk expression/activation in neoplastic MC could be demonstrated by a Btk-specific siRNA that reduced the proliferation and survival in HMC-1 cells, and was found to cooperate with midostaurin in producing growth inhibition. In consecutive experiments, we identified the Src/Abl kinase-targeting drug dasatinib (BMS, Princeton, NJ) as a potent inhibitor of Lyn/Btk activation in neoplastic MC. In particular, dasatinib (1 μM) was found to block Lyn and Btk activity in HMC-1.1 cells as well as in HMC-1.2 cells, and corresponding results were obtained with primary neoplastic MC. Finally, as assessed by a chemical proteomics approach, we were able to show that dasatinib directly binds to Btk and Lyn in neoplastic MC. In summary, our data show that a KIT-independent Lyn/Btk-driven signaling pathway contributes to growth and survival of neoplastic MC, and possibly to disease progression in SM. Our study also identifies dasatinib as a potent inhibitor of the Lyn/Btk pathway, which may have clinical implications and may explain some of the synergistic effects obtained with combinations of dasatinib and other KIT-targeting TK inhibitors in neoplastic MC.