Targeting the cell cycle in head and neck cancer by Chk1 inhibition: a novel concept of bimodal cell death

a Overview of the workflow presented in this manuscript. b Heatmap representing the lethality score20 of ATM, ATR, CHEK1, CHEK2 from the individual replicates of the genome-wide siRNA screen, independently performed in HNSCC cell lines VU-SCC-1131 and VU-SCC-120. Blue represents no effect on viability, yellow represents the decrease in viability. FDR corrected p-value cutoff: 0.005. c Transfections with SMARTpools containing four pooled siRNA sequences targeting either ATM, ATR, CHEK1, or CHEK2 demonstrated that only siCHEK1 decreased cell viability for ≥50% (UM-SCC-22A and VU-SCC-120 relative viability 0.34 and 0.45, respectively). Knockdown of siATM, siATR, and siCHEK2 did not reduce cell viability in tested cell lines (relative average viability UM-SCC-22A, respectively, 0.86, 1.06, 0.96; for VU-SCC-120, respectively, 0.97, 1.30, 1.20). siCONTROL#2 was transfected as negative control, siUBB targeting Ubiquitin B as positive control. d Knockdown of ATM, ATR, CHEK1, and CHEK2 was analyzed 24？h post transfection in VU-SCC-120 by RT-qPCR. Expression was normalized for GUSB and relative to the siCONTROL#2. Values were 0.49, 0.25, 0.21, and 0.40, respectively. e Microarray gene expression data of 22 tumors (red boxplots) with paired normal mucosa (green boxplots) revealed a significant increase of ATR, CHEK1, and CHEK2 expression in tumors at the RNA level, but not for ATM. Data are represented as boxplots with on the y-axis the relative expression against the reference RNA34. The horizontal line represents the median value. f Basal CHEK1 mRNA expression levels were compared between primary oral keratinocytes and fibroblasts and tumor cell lines UM-SCC-22A and VU-SCC-120. A relative fold change expression ratio was calculated towards the basal CHEK1 expression in the keratinocytes. Fibroblasts expressed a two-fold increase in CHEK1, VU-SCC-120 a 3.4-fold increase, and UM-SCC-22A an 8.3-fold increased expression. g Deconvolution of the four individual SMARTpool CHEK1 siRNAs on two HNSCC cell lines (red bars) and primary oral keratinocytes and fibroblasts (both represented in green). A significant decrease in cell viability was observed in the HNSCC cell lines (two-sided t-test p-values versus siCONTROL#2 viability for UM-SCC-22A: siCHEK1 pool: 0.0002, siCHEK1 #6: 0.0002, siCHEK1 #7: 0.0003, siCHEK1 #8: 0.0004, siCHEK1 #26: 0.0092. For VU-SCC-120: siCHEK1 pool: 0.0005, siCHEK1 #6: 0.0002, siCHEK1 #7: 0.0003, siCHEK1 #8: 0.0276, siCHEK1 #26: 0.0002.). No significant reduction in viability was obtained upon CHEK1 knockdown in the primary mucosal cells, while