Identification of Wee1 as Target in Combination with Avapritinib for the Treatment of Gastrointestinal Stromal Tumor

Abstract

Gastrointestinal stromal tumor (GIST) management has been revolutionized by the identification of activating mutations in KIT and PDGFRA and clinical application of receptor tyrosine kinase (RTK) inhibitors in advanced disease. Stratification of GIST into molecularly defined subsets provides insight into clinical behavior and response to approved targeted therapies. Although these RTK inhibitors are effective in most GIST, resistance remains a significant clinical obstacle.Development of effective strategies for refractory GIST requires identification of novel targets to provide additional therapeutic options. Global kinome profiling has potential to identify critical signaling networks and reveal protein kinases that are essential in GIST. Using Multiplexed Inhibitor Beads and Mass Spectrometry paired with a super-SILAC kinome standard, we explored the majority of the kinome in GIST specimens from the three most common molecular subtypes to identify novel kinase targets. Kinome profiling revealed distinct signatures in GIST subtypes.PDGFRA-mutant GIST had elevated tumor associated macrophage (TAM) kinases and immunohistochemical analysis confirmed increased TAMs present in these tumors. Kinome profiling with loss-of-function assays revealed a significant role for G2-M tyrosine kinase, Wee1, in GIST survival. In vitro and in vivo studies revealed significant efficacy of MK-1775 (Wee1 inhibitor) in combination with avapritinib in KIT and PDGFRA-mutant GIST cell lines, and notable efficacy of MK-1775 as a monotherapy in the PDGFRA-mutant line. These studies provide strong preclinical justification for the use of MK-1775 in GIST.

Combined Inhibition of AKT and KIT Restores Expression of Programmed Cell Death 4 (PDCD4) in Gastrointestinal Stromal Tumor

“…Wells were then treated with varying doses of MK-4440 and/or IM. Cell proliferation and viability were measured at 72 hours post treatment using the CellTiter Blue Viability Assay (Promega, Fitchburg, WI, USA) as described previously (24). Assays were performed as three independent biological replicates, with minimum of six technical replicates in each treatment arm.…”

“…Assays were performed as three independent biological replicates, with minimum of six technical replicates in each treatment arm. Combination indexes of CILD50 (GIST-T1) and CILD30 (GIST430, GIST-T1/829) were quantified using the previously described approach (24).…”

“…BrdU incorporation assay was used to measure cell proliferation. GIST-T1 cells were labeled with BrdU for 4 hours and GIST430 cells were labeled for 20h, and then analyzed with BrdU-APC Flow Kit (BD Biosciences) as described previously (24).…”