KRASG12C-independent feedback activation of wild-type RAS constrains KRASG12C inhibitor efficacy

Abstract

Although KRAS has long been considered undruggable, direct KRAS^G12C inhibitors have shown promising initial clinical efficacy. However, the majority of patients still fail to respond. Adaptive feedback reactivation of RAS-mitogen-activated protein kinase (MAPK) signaling has been proposed by our group and others as a key mediator of resistance, but the exact mechanism driving reactivation and the therapeutic implications are unclear. We find that upstream feedback activation of wild-type RAS, as opposed to a shift in KRAS^G12C to its active guanosine triphosphate (GTP)-bound state, is sufficient to drive RAS-MAPK reactivation in a KRAS^G12C-independent manner. Moreover, multiple receptor tyrosine kinases (RTKs) can drive feedback reactivation, potentially necessitating targeting of convergent signaling nodes for more universal efficacy. Even in colorectal cancer, where feedback is thought to be primarily epidermal growth factor receptor (EGFR)-mediated, alternative RTKs drive pathway reactivation and limit efficacy, but convergent upstream or downstream signal blockade can enhance activity. Overall, these data provide important mechanistic insight to guide therapeutic strategies targeting KRAS.

Keywords: CP: Cancer; KRAS; KRASG12C; adagrasib; adaptive resistance; sotorasib.

Figure 1.. KRAS^G12C inactive GDP-state inhibitors are prone to adaptive feedback reactivation of the MAPK pathway that is not driven by KRAS

(A) KRAS-^G12C mutant cell lines were treated with AMG 510 (100 nM) for 0, 4, 24, 48, and 72 h. Blot analysis was performed for phospho- (p)MEK, pERK, pRSK, pAKT, and total MYC with GAPDH as a loading control. (B) Densitometry of pERK normalized to GAPDH for blots in (A) and cell lines treated with ARS-1620 (10 μM) or MRTX849 (100 nM) for 4, 24, 48, and 72 h; results represent an average of pERK across all eight cell lines). (C and G) Cell lines were treated with 10 μM ARS-1620 or 100 nM AMG 510 for 4, 24, 48, or 72 h either refreshed at each time point or not refreshed throughout the time course, and lysates were subject to a RAF-RBD pull-down and blot analysis of KRAS, NRAS, HRAS, and total RAS as well as pERK, pRSK, and GAPDH for input samples. (D and H) Densitometry of pERK normalized to GAPDH for blots in (C) and (G). (E and I) Densitometry analysis of KRAS-GTP levels normalized to input KRAS and GAPDH loading control for blots in (C) and (G). (F and J) LC/MS analysis of ARS-1620 (10 μM) or AMG 510 (100 nM) drug levels in media over time incubated either alone at 37°C or with the H358 cell line.

Figure 2.. Wild-type RAS drives adaptive feedback reactivation of the RAS MAPK pathway

(A) SW1463, MIA PaCa-2, and H358 cell lines were treated with 1 or 10 μM ARS-1620 or 0.1 or 0.3 μM AMG 510 for 4 h or 7 days with drug refreshed every 2 days, and lysates were subject to a RAF-RBD pull-down and blot analysis of KRAS, NRAS, HRAS, and total RAS as well as pERK, pRSK, and GAPDH for input samples. (B) Densitometry analysis of KRAS-GTP levels normalized to input KRAS and GAPDH loading control for blots in (A). (C) Densitometry analysis of KRAS-, NRAS-, and HRAS-GTP levels normalized to input KRAS and GAPDH loading control for blots in (A). (D and E) Densitometry analysis of KRAS-GTP, NRAS-GTP, and HRAS-GTP levels normalized to input RAS and GAPDH loading control of blots of cell lines treated with AMG 510 (100 nM) for 4, 24, 48, or 72 h in Figure S1C. (F and H) SW1463, MIA PaCa-2, and H358 cell lines were subject to siRNA knockdown of NRAS, HRAS, and NRAS and HRAS and treated with AMG 510 (100 nM) or RM-018 (100 nM) for 24, 48, and 72 h. Blot analysis was performed for pMEK, pERK, pRSK, pAKT, and total NRAS, HRAS, KRAS, and MYC with GAPDH as a loading control. (G and I) Densitometry of pERK normalized to GAPDH for blots in (F) and (H); results represent an average of pERK across three cell lines. Statistical significance was evaluated by Student’s t test, where *p < 0.05 and **p < 0.01. ns, not significant.

Figure 3.. Vertical combination strategies abrogate adaptive response to KRAS^G12C inhibition

(A) MIA PaCa-2 cells were treated with AMG 510 (100 nM) or RM-018 (100 nM) alone or in combination with the SHP2 inhibitor RMC-4550 (1 μM) for 4, 24, 48, or 72 h, and lysates were subject to a RAF-RBD pull-down and blot analysis of KRAS, NRAS, HRAS, and total RAS as well as pERK, pRSK, and GAPDH for input samples. (B) Densitometry analysis of KRAS-GTP levels normalized to input KRAS and GAPDH loading control (bar) for blots and pERK normalized to GAPDH loading control (line) in (A). Data represent combined densitometry for MIA PaCa-2 in (A) and SW1463 and H358 in Figure S2A. (C) Densitometry analysis of KRAS-GTP levels to input KRAS and GAPDH loading control and densitometry analysis of KRAS-GTP, NRAS-GTP, and HRAS-GTP levels normalized to input RAS and GAPDH loading control of blots of cell lines treated with AMG 510 alone or in combination with RMC-4550 or the MEK inhibitor trametinib (10 nM) in Figure S2C. (D and E) Densitometry analysis of pERK normalized to loading control GAPDH for blots of KRAS12C mutant non-CRC and CRC subjected to indicated treatments in in Figures S2D and S2E. (F and G) Densitometry analysis of KRAS-GTP, NRAS-GTP, and HRAS-GTP levels normalized to input RAS and GAPDH loading control of blots of KRAS^G12C-mutant CRC cell lines treated with AMG 510 alone or in combination with RMC-4550 or the EGFR inhibitor panitumumab (30 μg/mL) for 4 or 48 h in Figure S3A. (H) Quantification of crystal violet stain of CRC cell lines treated with AMG 510 (100 nM), RMC-4550 (1 μM), panitumumab (30 μg/mL), trametinib (10 nM), or a combination for 10–14 days in Figure S3B.

Figure 4.. EGFR and MEK doublet and triplet therapies enhance the efficacy of KRAS^G12C inhibition in vivo

(A and C) B8182 and F3008 PDX-derived KRAS^G12C-mutant CRC lines were treated with AMG 510 alone or in combination with panitumumab, RMC-4550, or trametinib for 24, 48, and 72 h. Blot analysis was performed for pMEK, pERK, pRSK, pAKT, and total MYC with GAPDH as a loading control. (B and D) Densitometry of pERK normalized to GAPDH for blots in (A) and (C) and Figures S4A and S4B. (E) Indicated KRAS^G12C CRC PDX models were treated daily with AMG 510 (100 mg/kg) and trametinib (1 mg/kg) and twice weekly with panitumumab (0.5 mg) alone or in combination for 21 days. (F) Waterfall plots of endpoint tumors in (E); statistical significance was evaluated by Mann-Whitney test, where **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 5.. Heterogeneous RTK expression limits the durability of EGFR treatment in KRAS^G12C, and SHP2 inhibition leads to deeper more durable response in vivo

(A) Combined RPPA analysis of KRAS^G12C CRC PDX tumors treated with AMG 510 (100 mg/kg), panitumumab (0.5 mg), or trametinib (1 mg/kg) alone or in combination for 3 days. (B) RPPA analysis of SW837 and SW1463 cell lines treated with the indicated inhibitors for 24 h, 72 h, or 7 days. (C) RTK array expression analysis of CRC cell lines in Figures S5A and S5B. (D) B8182, LIM2099, and SW837 cell lines were treated with AMG 510 alone or in combination with panitumumab for 24, 48, or 72 h. Blot analysis was performed for pEGFR, pHER2, pFGFR3, pSHP2, pMEK, pERK, pRSK, pAKT, and total MYC with GAPDH as a loading control. (E–G) B8182, LIM2099, and SW827 cell lines were treated with AMG 510 alone or in combination with panitumumab or the FGFR inhibitor BGJ398 (1 μM), and densitometry analysis was performed for pERK normalized to GAPDH. (H–J) B8182, LIM2099, and SW827 cell lines were treated with AMG 510 alone or in combination with panitumumab or RMC-4550, and densitometry analysis was performed for pERK normalized to GAPDH. (K–M) B8182 PDX, LIM2099, and SW837 xenograft models were treated daily with AMG 510 (100 mg/kg) or RMC-4550 (30 mg/kg) or twice weekly with panitumumab (0.5 mg/kg) alone or in combination for 28, 66, and 35 days, respectively. (N–P) Waterfall plots of endpoint tumors in xenograft models in (K)–(M). Statistical significance was evaluated by Mann-Whitney test, where *p < 0.05, **p < 0.01, and ****p < 0.0001.