KRASG 12C-inhibitor-based combination therapies for pancreatic cancer: insights from drug screening

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has limited treatment options, emphasizing the urgent need for effective therapies. The predominant driver in PDAC is mutated KRAS proto-oncogene, KRA, present in 90% of patients. The emergence of direct KRAS inhibitors presents a promising avenue for treatment, particularly those targeting the KRAS^G12C mutated allele, which show encouraging results in clinical trials. However, the development of resistance necessitates exploring potent combination therapies. Our objective was to identify effective KRAS^G12C-inhibitor combination therapies through unbiased drug screening. Results revealed synergistic effects with son of sevenless homolog 1 (SOS1) inhibitors, tyrosine-protein phosphatase non-receptor type 11 (PTPN11)/Src homology region 2 domain-containing phosphatase-2 (SHP2) inhibitors, and broad-spectrum multi-kinase inhibitors. Validation in a novel and unique KRAS^G12C-mutated patient-derived organoid model confirmed the described hits from the screening experiment. Our findings propose strategies to enhance KRAS^G12C-inhibitor efficacy, guiding clinical trial design and molecular tumor boards.

Keywords: KRASG12C; SHP2; SOS1; pancreatic cancer.

Fig. 1

Sotorasib response of KRAS^G12C‐mutated MiaPaCa2 cells. (A) MiaPaCa2 cells were treated with the indicated doses of Sotorasib and viability was determined 72 h after the treatment using ATP as a surrogate. n = 3. Gray line reproduced data from Canon et al. [27], Purple line: current manuscript. (B) Phospho‐ERK, ERK, and KRAS western blot 24 h after the treatment of MiaPaCa2 cell with the indicated doses of Sotorasib. HSP90: loading control. One representative blot out of three independent experiments (n = 3) is depicted. (C) Quantification of ERK phosphorylation corresponding to B. (D) Flow cytometry of propidium iodide (PI)‐stained cells 24 h after the treatment of MiaPaCa2 cells with the indicated doses of Sotorasib. n = 3. Two‐way ANOVA, Bonferroni correction: **P < 0.01, ****P < 0.0001. Mean and the standard deviation (SD) is shown.

Fig. 2

Drug Screen to find KRAS^G12C inhibitor combination. (A) Outline of the drug screening experiment. An anchored screen design (anchor: Sotorasib 6 nm) was used. Growth rate corrected analysis was done by the GR metrics algorithm. (B) Left panel: Illustration of the drug library target spectrum. Right panel: Seventy percent of the drugs are in the clinic (FDA‐approved in other tumor entities or at least in phase I trials). (C) Distribution of the delta area under the dose–response curve (AUC) values. A delta AUC < −0.1 was defined as one criterium for a hit. Three compounds with a delta AUC < −0.1 are depicted. (D) Clonogenic growth assay of MiaPaCa2 and PSN1 cells treated with the indicated doses and compounds. One representative clonogenic growth assay out of three is shown. (E) Quantification of clonogenic growth assays in MiaPaCa2 cells (n = 3). *One‐way ANOVA with Tukey's correction for multiple testing *P < 0.05. Mean and the standard deviation (SD) are depicted. (F) Illustration of Bliss synergy values: a score > 10 was considered synergistic, a score < −10 was considered antagonistic. (G) Bliss synergy score of the quantified clonogenic growth assays (E) of MiaPaCa2 cells was calculated with the synergy finder platform.

Fig. 3

Efficacy of the TNO155 and Sotorasib combination in a KRAS ^G12C ‐mutated patient‐derived organoids (PDOs). (A) Case report of a KRAS ^G12C ‐mutated pancreatic ductal adenocarcinoma (PDAC) patient, from which the patient‐derived organoid (PDO) line PDO‐51T was isolated. After the resection of the tumor (pT3, pN2, cM0, R0, G2) the male patient was treated with adjuvant Gemcitabine/Capecitabine for 4 months. Afterwards, the patient was treated with mFOLFIRINOX and Gemcitabine/nab‐Paclitaxel. The overall survival was 15 months. (B) Results of the panel sequencing of PDO‐51T. (C) The top five enriched and depleted signatures of a single sample Gene Set Enrichment Analysis (ssGSEA) of the PDO‐51T line. (D) The indicated Sotorasib doses were used to determine growth over the indicated time points of PDO‐51T, measured by live cell imaging. n = 6. (E, F) PDO‐51T were treated with 41 nm Sotorasib (n = 6), 41 nm TNO155 (n = 6), 41 nm Nintedanib (n = 3), 40 nm Sotorasib/41 nm TNO155 (n = 6), and 40 nm Sotorasib/41 nm Nintedanib (n = 6) or left as vehicle controls (n = 6). Growth curves were determined by live cell imaging. Yellow scale bar: 400 μm. (G) Quantification of the growth after 192 h. Data of the experiment described in F were used to calculate the relative growth (ratio treatment groups versus control). One‐way ANOVA with correction for multiple testing according to Tukey: **P < 0.01, *P < 0.05. Median and quartiles are shown.

Fig. 4

Synergism of Sotorasib with TNO155. (A) 2D pancreatic ductal adenocarcinoma (PDAC) cells (51T‐2D) were generated from PDO‐51T. (B) Phospho‐ERK, pan‐ERK, and KRAS western blot 24 h after the treatment of 51T‐2D cells with the indicated doses of Sotorasib. One representative blot out of three replicates (n = 3) is depicted. (C) Phospho‐ERK Quantification of three independent experiments (n = 3) corresponding to B. (D, E) Clonogenic growth assay of 51T‐2D cells treated with the indicated doses of TNO155 and Sotorasib. (D) One representative experiment. (E) Quantification of three independent experiments (n = 3). One‐way ANOVA with Tukey's correction for multiple testing: ***P < 0.001. (F) Bliss synergy score of the quantified clonogenic growth assays of 51T‐2D cells was calculated with the synergy finder platform. (G, H) MiaPaca2 and 51T‐2D cells were treated as indicated. After 72 h the cells were stained with propidium iodide (PI) and cell cycle FACS was performed. Depicted is the ratio of cells in the S‐phase of the cell cycle to cells in the G1‐phase (n = 3). One‐way ANOVA with correction for multiple testing according to Tukey: *P < 0.05. (I) Staining for Ki67 and cleaved caspase 3 (CC3) in PDO‐51T treated as indicated (n = 1). Scale bar = 10 μm. (J) Phospho‐ERK, pan‐ERK, and KRAS western blot 72 h after the treatment of 51T‐2D cells with the indicated doses of Sotorasib and/or TNO155. One representative blot out of three replicates (n = 3) is depicted. (K) Quantification of three independent experiments (n = 3) corresponding to J. One‐way ANOVA with Tukey's correction for multiple testing: *P < 0.05. Mean and the standard deviation (SD) are shown.