Fig. 7 | Nature Communications

Fig. 7

From: Survival of pancreatic cancer cells lacking KRAS function

Fig. 7

Combined KRAS and PI3K inhibition as a therapeutic approach in PDAC cells. a Schematic of lentiviral constructs to express Cas9 and a doxycycline (DOX)-inducible sgRNA targeting KRAS (sgKRAS). PeF1a ubiquitously expressed elongation factor 1a promoter. 2A self-cleaving peptide. Blast blasticidin resistance gene. TRE tetracycline-responsive element. PH1/TO H1 promoter with Tet-operator sites. PUb-P ubiquitin promoter. tetR Tet-repressor. DOX treatment relieves tetR repression of H1 promoter to permit sgKRAS expression. Western blot showed complete KRAS protein ablation in two different A13 mmKras.366 clones after 7 days of DOX treatment in vitro. b Combined KRAS (by DOX-inducible mmKras.366) and PI3K (by GDC-0941) inhibition in established subcutaneous tumors effectively inhibited tumor growth, whereas inhibition of KRAS or PI3K alone was insufficient to suppress tumor growth long term. Average tumor volume fold increase (normalized to start of DOX treatment, pooled for both clones in a) ± s.e.m. (n = 10 tumors per group) is plotted. Dashed line denotes bliss independence for additive effect of DOX and GDC-0941, consistent with synergism starting at day 11. **p < 0.01, two-tailed Student’s t test, DOX + GDC-0941 vs. Vehicle only at end of experiment. ***p < 0.001, two-tailed Student’s t test, DOX + GDC-0941 vs. DOX + Vehicle at end of experiment. c Western blot confirmed loss of KRAS protein in knockout clones derived from PACO19 (H11, H13, H17) and PACO9 (H12) compared to intact clones (PACO19-E6,E9; PACO9-E5,E20). HSP90 is loading control. d KRAS alleles from PACO19 and PACO9 clones showed out-of-frame indels in KRAS deficient clones. Reference corresponds to UCSC hg19 sequence. All clones retained a single indel allele except for PACO19 H13 for which two different indels were identified, one of which was a 95 bp deletion that is not pictured in full. The purple and orange bars denote the sgRNA and PAM sequences, respectively. e Dose-response curves of PACO19 and PACO9 KRAS intact (gray) and deficient (purple) clones to the pan-PI3K inhibitor GDC-0941. Each replicate (n = 3 for each dose) and curve fit are shown. f Western blot showed a decrease in pERK1/2 levels in KRAS deficient (H13) but not intact (E6) clones derived from PACO19 cells following GDC-0941 treatment

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