Inhibitory Effects Of Midostaurin And Blu-285 On Myeloid Progenitor Cells Derived From Patients With Multi-Mutated Kit D816v+Advanced Systemic Mastocytosis

Recent data have highlighted that the molecular pathogenesis of advanced systemic mastocytosis (advSM) is complex. In addition to the phenotypically most important mutations in KIT , e.g. KIT D816V in 80-90% of patients, one or more additional mutations, e.g. in SRSF2 , ASXL1 , RUNX1 , CBL , JAK2 and others, are present in 60-70% of patients (Jawhar et al. , Leukemia 30, 2016). In individual patients, a complex mutational profile is detected not only in mature mast cells (MCs) but also in myeloid progenitors derived from granulocyte-macrophage colony-forming progenitor cells (CFU-GM), indicating multi-lineage involvement of all identified mutations in the vast majority of patients (Jawhar et al. , Leukemia 29, 2015). Midostaurin, a multi-targeted kinase inhibitor, has demonstrated an overall response rate of 60% in advSM patients (Gotlib et al. , NEJM 374, 2016). BLU-285 is a highly selective KIT D816V kinase inhibitor which has demonstrated biochemical activity on the mutated KIT enzyme ( KIT D816V IC 50 = 0.27 nM). In the current study, we sought to a) investigate the inhibitory effects of midostaurin and BLU-285 on single-cell-derived CFU-GM from bone marrow mononuclear cells derived from multi-mutated KIT D816V+ advSM patients and b) correlate the midostaurin CFU-GM data with clinical and various response parameters in midostaurin-treated advSM patients. The mutational status of CFU-GM colonies (median colonies per patient, n=20; range 10-30) was analyzed for KIT D816V and additional mutations by PCR followed by Sanger Sequencing. In 10 multi-mutated advSM patients (aggressive SM [n=8] or mast cell leukemia [n=2] with an associated hematological neoplasm), CFU-GM colonies were screened prior to midostaurin (month 0, n=10) and, if available, at month 6 on midostaurin (n=8). At month 0, a median of 90% (range, 40-100) CFU-GM colonies were KIT D816V+, while at month 6 a median of 70% (range, 5-100) CFU-GM colonies were KIT D816V+. A significant relative reduction (≥50%) in the proportion of KIT D816V+ colonies at month 6 was observed in 4/8 (50%) patients. Midostaurin- naive CFU-GM were incubated with midostaurin at concentrations up to 1000 nM and showed a dose-dependent significant reduction (≥50%) of KIT D816V+ colonies in 1/7 (14%) patients. Overall, the in vitro effects correlated with the in vivo effects of midostaurin on CFU-GM and established IWG-MRT-ECNM response criteria (e.g. mast cell infiltration in BM, serum tryptase level) and KIT D816V allele burden in peripheral blood. Midostaurin- naive CFU-GM from 7/10 (70%) patients were also incubated with different concentrations of BLU-285 ranging from 0 to 75 nM. A dose-dependent, significant relative reduction (≥50%) of KIT D816V+ CFU-GM colonies was observed at concentrations between 45 and 75nM in 5/7 (71%) patients. Of interest, 3/5 (60%) in vitro responders to BLU-285 were resistant to midostaurin (in vivo and in vitro ) while CFU-GM colonies from 2 patients resistant to BLU-285 were also resistant to midostaurin. In addition to KIT D816V, recurrent molecular aberrations (median 2/patient, range 1-3) were identified in all patients, most frequently in SRSF2 (n=9), TET2 (n=7) and ASXL1 (n=4). Neither drug had an effect on the relative frequency of additional mutations in CFU-GM colonies. In summary, we conclude that a) the relative reduction of KIT D816V+ CFU-GM colonies between month 0 and month 6 on midostaurin correlates with clinical response, b) the CFU-GM colony assays may provide useful information for prediction of response to midostaurin, c) the highly selective KIT D816V inhibitor BLU-285 has significant activity against KIT D816V, even in cases which are resistant to midostaurin, and d) neither drug had an effect on the prognostically relevant additional mutations. Disclosures Evans: Blueprint Medicines: Employment, Equity Ownership. Gardino: Blueprint Medicines Corporation: Employment. Lengauer: Blueprint Medicines Corporation: Employment.