Patho-)physiological activation of the IL7-receptor (IL7R) signaling contributes to steroid resistance in pediatric T-cell acute lymphoblastic leukemia (T-ALL). Here, we show that activating IL7R pathway mutations and physiological IL7R signaling activate MAPK-ERK signaling, which provokes steroid resistance by phosphorylation of BIM. By mass spectrometry, we demonstrate that phosphorylated BIM is impaired in binding to BCL2, BCLXL and MCL1, shifting the apoptotic balance toward survival. Treatment with MEK inhibitors abolishes this inactivating phosphorylation of BIM and restores its interaction with anti-apoptotic BCL2-protein family members. Importantly, the MEK inhibitor selumetinib synergizes with steroids in both IL7-dependent and IL7-independent steroid resistant pediatric T-ALL PDX samples. Despite the anti-MAPK-ERK activity of ruxolitinib in IL7-induced signaling and JAK1 mutant cells, ruxolitinib only synergizes with steroid treatment in IL7-dependent steroid resistant PDX samples but not in IL7-independent steroid resistant PDX samples. Our study highlights the central role for MAPK-ERK signaling in steroid resistance in T-ALL patients, and demonstrates the broader application of MEK inhibitors over ruxolitinib to resensitize steroid-resistant T-ALL cells. These findings strongly support the enrollment of T-ALL patients in the current phase I/II SeluDex trial (NCT03705507) and contributes to the optimization and stratification of newly designed T-ALL treatment regimens.

Kinase inhibitors form the largest class of precision medicine. From 2013 to 2017, 17 have been approved, with 8 different mechanisms. We present a comprehensive profiling study of all 17 inhibitors on a biochemical assay panel of 280 kinases and proliferation assays of 108 cancer cell lines. Drug responses of the cell lines were related to the presence of frequently recurring point mutations, insertions, deletions, and amplifications in 15 well-known oncogenes and tumor-suppressor genes. In addition, drug responses were correlated with basal gene expression levels with a focus on 383 clinically actionable genes. Cell lines harboring actionable mutations defined in the FDA labels, such as mutant BRAF(V600E) for cobimetinib, or ALK gene translocation for ALK inhibitors, are generally 10 times more sensitive compared with wild-type cell lines. This sensitivity window is more narrow for markers that failed to meet endpoints in clinical trials, for instance CDKN2A loss for CDK4/6 inhibitors (2.7-fold) and KRAS mutation for cobimetinib (2.3-fold). Our data underscore the rationale of a number of recently opened clinical trials, such as ibrutinib in ERBB2-or ERBB4-expressing cancers. We propose and validate new response biomarkers, such as mutation in FBXW7 or SMAD4 for EGFR and HER2 inhibitors, ETV4 and ETV5 expression for MEK inhibitors, and JAK3 expression for ALK inhibitors. Potentially, these new markers could be combined to improve response rates. This comprehensive overview of biochemical and cellular selectivities of approved kinase inhibitor drugs provides a rich resource for drug repurposing, basket trial design, and basic cancer research.

Cancer cell line panels are important tools to characterize the in vitro activity of new investigational drugs. Here, we present the inhibition profiles of 122 anticancer agents in proliferation assays with 44 or 66 genetically characterized cancer cell lines from diverse tumor tissues (Oncolines). The library includes 29 cytotoxics, 68 kinase inhibitors, and 11 epigenetic modulators. For 38 compounds this is the first comparative profiling in a cell line panel. By strictly maintaining optimized assay protocols, biological variation was kept to a minimum. Replicate profiles of 16 agents over three years show a high average Pearson correlation of 0.8 using IC values and 0.9 using GI values. Good correlations were observed with other panels. Curve fitting appears a large source of variation. Hierarchical clustering revealed 44 basic clusters, of which 26 contain compounds with common mechanisms of action, of which 9 were not reported before, including TTK, BET and two clusters of EZH2 inhibitors. To investigate unexpected clusterings, sets of BTK, Aurora and PI3K inhibitors were profiled in biochemical enzyme activity assays and surface plasmon resonance binding assays. The BTK inhibitor ibrutinib clusters with EGFR inhibitors, because it cross-reacts with EGFR. Aurora kinase inhibitors separate into two clusters, related to Aurora A or pan-Aurora selectivity. Similarly, 12 inhibitors in the PI3K/AKT/mTOR pathway separated into different clusters, reflecting biochemical selectivity (pan-PI3K, PI3Kβγδ-isoform selective or mTOR-selective). Of these, only allosteric mTOR inhibitors preferentially targeted PTEN-mutated cell lines. This shows that cell line profiling is an excellent tool for the unbiased classification of antiproliferative compounds. Mol Cancer Ther; 15(12); 3097-109. ©2016 AACR.

Inhibition of the spindle assembly checkpoint kinase TTK causes chromosome mis-segregation and tumor cell death. However, high levels of TTK correlate with chromosomal instability (CIN), which can lead to aneuploidy. We show that treatment of tumor cells with the selective small molecule TTK inhibitor NTRC 0066-0 overrides the mitotic checkpoint, irrespective of cell line sensitivity. In stable aneuploid cells NTRC 0066-0 induced acute CIN, whereas in cells with high levels of pre-existing CIN there was only a small additional fraction of cells mis-segregating their chromosomes. In proliferation assays stable aneuploid cells were more sensitive than cell lines with pre-existing CIN. Tetraploids are thought to be an intermediate between diploid and unstable aneuploid cells. TTK inhibitors had the same potency on post-tetraploid and parental diploid cells, which is remarkable because the post-tetraploids are more resistant to mitotic drugs. Finally, we confirm that the reference compound reversine is a TTK inhibitor and like NTRC 0066-0, inhibits the proliferation of patient-derived colorectal cancer organoids. In contrast, treatment with TTK inhibitor did not reduce the viability of non-proliferating T cell acute lymphoblastic leukemia cells samples. Consequently, TTK inhibitor therapy is expected to spare non-dividing cells, and may be used to target stable aneuploid tumors.

Background: In epithelial ovarian cancer (EOC), 15-20% of the tumors do not respond to first-line chemotherapy (paclitaxel with platinum-based therapy), and in recurrences this number increases. Our aim is to determine the feasibility of cell proliferation assays of tumor cells isolated from malignant ascites to predict in vitro chemotherapy sensitivity, and to correlate these results with clinical outcome.Materials and Methods: Ascites was collected from twenty women with advanced EOC. Cell samples were enriched for tumor cells and EOC origin was confirmed by intracellular staining of CK7, surface staining of CA125 and EpCAM, and HE4 gene expression. In vitro sensitivity to chemotherapy was determined in cell proliferation assays using intracellular ATP content as an indirect measure of cell number. In vitro drug response was quantified by calculation of the drug concentration at which cell growth was inhibited with 50%. Clinical outcome was determined using posttreatment CA125 level.Results: Cell samples of twenty patients were collected, of which three samples that failed to proliferate were excluded in the analysis (15%). Three other samples were excluded, because clinical outcome could not be determined correctly. In twelve of the fourteen remaining cases (86%) in vitro drug sensitivity and clinical outcome corresponded, while in two samples (14%) there was no correspondence.Conclusions: Our study demonstrates the feasibility of drug sensitivity tests using tumor cells isolated from ascites of advanced EOC patients. Larger observational studies are required to confirm the correlation between the in vitro sensitivity and clinical outcome.