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Supplementary Tables S1-S4 and Figures S1-S3. Tables S1/S2: % tumor response in mice with HCT116 and A2058 tumor xenografts following treatment; Table S3: Diagram showing dose-decision guidelines utilized in the MK-2206 clinical study based on observed toxicities; Table S4: number of patients with drug-related AEs in all courses of treatment; Figure S1/S2: Line graph showing the relative tumor volume and body weight observed in patients over time expressed in terms of days following treatment; Figure S3: Patient presenting with Grade III rash

Purpose:KRAS is mutated in the majority of pancreatic ductal adenocarcinoma. MAPK and PI3K-AKT are primary KRAS effector pathways, but combined MAPK and PI3K inhibition has not been demonstrated to be clinically effective to date. We explore the resistance mechanisms uniquely employed by malignant cells.Experimental Design:We evaluated the expression and activation of receptor tyrosine kinases in response to combined MEK and AKT inhibition in KPC mice and pancreatic ductal organoids. In addition, we sought to determine the therapeutic efficacy of targeting resistance pathways induced by MEK and AKT inhibition in order to identify malignant-specific vulnerabilities.Results:Combined MEK and AKT inhibition modestly extended the survival of KPC mice and increased Egfr and ErbB2 phosphorylation levels. Tumor organoids, but not their normal counterparts, exhibited elevated phosphorylation of ERBB2 and ERBB3 after MEK and AKT blockade. A pan-ERBB inhibitor synergized with MEK and AKT blockade in human PDA organoids, whereas this was not observed for the EGFR inhibitor erlotinib. Combined MEK and ERBB inhibitor treatment of human organoid orthotopic xenografts was sufficient to cause tumor regression in short-term intervention studies.Conclusions:Analyses of normal and tumor pancreatic organoids revealed the importance of ERBB activation during MEK and AKT blockade primarily in the malignant cultures. The lack of ERBB hyperactivation in normal organoids suggests a larger therapeutic index. In our models, pan-ERBB inhibition was synergistic with dual inhibition of MEK and AKT, and the combination of a pan-ERBB inhibitor with MEK antagonists showed the highest activity both in vitro and in vivo.

Purpose: KRAS is the most commonly mutated oncogene in human tumors. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies.Experimental Design: We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumors. Recommended dosing schedules were defined as MK-2206 at 135 mg weekly and selumetinib at 100 mg once daily.Results: Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug–drug interactions. Clinical antitumor activity included RECIST 1.0–confirmed partial responses in non–small cell lung cancer and low-grade ovarian carcinoma.Conclusion: Responses in KRAS-mutant cancers were generally durable. Clinical cotargeting of MEK and AKT signaling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). Clin Cancer Res; 21(4); 739–48. ©2014 AACR.

Supplementary Figures 1-3

G Protein-Coupled Receptor Kinase 2 (Grk2) is a serine-threonine kinase widely expressed in the human body. The GRK family of kinases is known for its role in GPCR phosphorylation and desensitization upon GPCR activation by agonists. Besides its role in GPCR phosphorylation, Grk2 has been shown to phosphorylate non-GPCR receptors and cytoplasmic substrates and has also been demonstrated to play phosphorylation-independent roles in cells. Using pooled CRISPR screening we identified that Grk2 knockout inhibits pancreatic and bladder cancer cell line proliferation in vitro and tumor growth in xenografts in vivo, which has not been demonstrated before. Rescue experiments re-expressing wildtype or kinase inactive Grk2 showed that this effect is dependent on catalytically active Grk2. We developed Grk2 inhibitors with exquisite enzymatic potency and anti-proliferative activity in pancreatic cancer cell lines. On a cellular level Grk2 knockout and Grk2 inhibitors slow down G2/M cell cycle progression in pancreatic cancer cell lines, and Grk2 inhibitor CYG-N-2278 induced a 71% tumor growth inhibition in PAXF1657 pancreatic PDX-derived xenografts. Besides its direct role in cancer cell growth Grk2 also plays a role in immune cell function. Grk2 is highly expressed in immune cells, and we have demonstrated that Grk2 inhibitors upregulate proinflammatory cytokine expression in myeloid cells. In the MC38 colorectal syngeneic tumor model implanted into immunocompetent C57BL6 mice CYG-N-2278 Grk2 inhibitor resulted in a 39% tumor growth inhibition, whereas no effect was observed on MC38 tumor growth in immunodeficient NSG mice, suggesting an immune cell mediated tumor growth inhibition. Analysis of The Cancer Genome Atlas data has showed that Grk2 is amplified across multiple tumor types, including bladder, uterine and cholangiocarcinoma cancer, and that Grk2 amplification is prognostic for tumor outcome, confirming the human relevance and suggesting a translatable patient selection strategy. In conclusion, we identified Grk2 as novel target for oncology, demonstrating a direct effect of Grk2 inhibition on cancer cells through inhibiting G2/M cell cycle progression and an indirect effect on tumor growth through immune cell activation. Cygnal’s Grk2 inhibitors possess superior potency over previous Grk2 inhibitors and have shown promising preclinical anti-tumor efficacy. Citation Format: Alexandra Lantermann, Eugene Chekler, Bruce Lefker, Garmen Yuen, Vanessa Lam, Matthew Strickland, Justyne Pennacchio, Aaron Fulgham, Dara Bree, Thomas Perekslis, Grazia Piizzi, Hongyue Dai, Tim Zheng, John Wagner, Pearl Huang. Identification of Grk2 as novel oncology target and development of potent Grk2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 166.