Dual Inhibition of KRASG12D and Pan-ERBB Is Synergistic in Pancreatic Ductal Adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a low survival rate. Recently, new drugs that target KRASG12D, a common mutation in PDAC, have been developed. We studied one of these compounds, MRTX1133, and found it was specific and effective at low nanomolar concentrations in patient-derived organoid models and cell lines harboring KRASG12D mutations. Treatment with MRTX1133 upregulated the expression and phosphorylation of EGFR and HER2, indicating that inhibition of ERBB signaling may potentiate MRTX1133 antitumor activity. Indeed, the irreversible pan-ERBB inhibitor, afatinib, potently synergized with MRTX1133 in vitro, and cancer cells with acquired resistance to MRTX1133 in vitro remained sensitive to this combination therapy. Finally, the combination of MRTX1133 and afatinib led to tumor regression and longer survival in orthotopic PDAC mouse models. These results suggest that dual inhibition of ERBB and KRAS signaling may be synergistic and circumvent the rapid development of acquired resistance in patients with KRAS mutant pancreatic cancer.

Significance: KRAS-mutant pancreatic cancer models, including KRAS inhibitor-resistant models, show exquisite sensitivity to combined pan-ERBB and KRAS targeting, which provides the rationale for testing this drug combination in clinical trials.

Graphical abstract

Figure 1.

MRTX1133 specifically targets oncogenic KRAS^G12D human and mouse PDAC models and leads to the upregulation of ERBB activation. A, Dose–response analysis of PDOs harboring the KRAS^WT, KRAS^G12V, or KRAS^G12D alleles 5 days after treatment with MRTX1133. B, Immunoblot analyses of the upstream and downstream targets of KRAS signaling in hT102 (KRAS^WT) and hM1F (KRAS^G12D) at 0, 3, 24, and 72 hours using 60 nmol/L MRTX1133. C, Quantification of B. D, Dose–response analysis of PDAC cell lines harboring the KRAS^WT or KRAS^G12D alleles 3 days after treatment with MRTX1133. E, Immunoblot analyses of the upstream and downstream targets of KRAS in BXPC3 (KRAS^WT) and SUIT2 (KRAS^G12D) at 0, 3, 24, and 72 hours using 60 nmol/L MRTX1133 for BXPC3 and SUIT2. F, Quantification of E. G, Dose–response analysis of mouse KRAS^G12D KPC organoids 3 days after treatment with MRTX1133. H, Immunoblot analyses of the upstream and downstream targets of KRAS in two different KPC mouse organoids (mT3 and mT9) at 0, 3, 24, and 72 hours using 60 nmol/L MRTX1133. I, Quantification of H. J, Dose–response analysis of mouse KRAS^G12D KPC cell lines 3 days after treatment with MRTX1133. K, Immunoblot analyses of the upstream and downstream targets of KRAS in two different KPC lines at 0, 3, 24, and 72, hours using 60 nmol/L MRTX1133. L, Quantification of K. Data, mean values ± SD. Immunoblots are representative of at least three independent experiments.

Figure 2.

Irreversible Pan-ERBB inhibitors work synergistically with MRTX1133 in vitro. A, Evaluation of the synergistic effect of MRTX1133 with afatinib (panERBB inhibitor), lapatinib (EGFR/HER2 inhibitor), erlotinib (EGFR inhibitor), trametinib (MEK1/2 inhibitor), and ulixertinib (ERK1/2 inhibitor) using the BLISS synergy model in hF44 PDO, B and C, SUIT2 (B), and ASPC1 (C) show the potent and consistent synergy of MRTX1133 with afatinib. D–F, Western blot analyses of the upstream and downstream pathway targets of KRAS show a synergistic downregulation of the expression of EGFR and HER2 in hM1F (D), SUIT2 (E), and ASPC1 (F). G–I, Quantification of D, E, and F, respectively. Data, mean values ± SD. Immunoblots are representative of at least three independent experiments.

Figure 3.

MRTX1133 and afatinib combination remains effective in cells that have acquired MRTX1133 resistance. A and B, Cell viability of parental and MRTXR SUIT2 lines after treatment with MRTX1133 (A) or afatinib (B). C, Evaluation of the synergistic effect of MRTX1133 with afatinib using the BLISS and HSA synergy models in MRTXR SUIT2. D and E, Immunoblot analysis for the upstream and downstream signaling of KRAS after single-agent (D) or combination therapy with MRTX1133 and afatinib (E). F, qRT-PCR analysis KRAS expression in parental and MRTXR SUIT2 cells. G, Immunoblot analysis for RAS^G12D in parental or MRTXR SUIT2. H, Immunoblot analysis of RAS^G12D and pan-RAS in parental SUIT2, untreated MRTXR SUIT2, and MRTX1133-treated MRTXR SUIT2 after RAS-GTP pulldown. I, Quantification of H. Values are quantifications from two different Western blots normalized to 0.6% GAPDH input. Data, mean values ± SD. Immunoblots are representative of at least three independent experiments.

Figure 4.

Irreversible Pan-ERBB inhibitor, afatinib, potentiates MRTX1133 in vivo. A, Posttreatment tumor volumes normalized to pretreatment volumes of NSG mice orthotopically injected with SUIT2 and treated for 10 days with vehicle, MRTX1133, afatinib, or combo. B, Tumor weights of mice in A. C, Tumors of mice in A imaged at necropsy. D, Posttreatment tumor volumes normalized to pretreatment volumes of NSG mice orthotopically injected with MRTXR SUIT2 and treated for 10 days with vehicle, MRTX1133, afatinib, or combo. E, Tumor weights of mice in D. F, Tumors of mice in D imaged at necropsy. G, Posttreatment tumor volumes normalized to pretreatment volumes of C57BL/6J mice orthotopically injected with parental KPC 1242 cells and treated for 10 days with vehicle, MRTX1133, afatinib, or combo. H, Tumor weights of mice in G. I, Tumors of mice in G imaged at necropsy. J, Posttreatment tumor volumes normalized to pretreatment volumes of C57BL/6J mice orthotopically injected with MRTXR KPC 1242 cells and treated for 10 days with vehicle, MRTX1133, afatinib, or combo. K, Tumor weights of mice in J. L, Tumors of mice in J imaged at necropsy. M, Survival study of C57BL/6J mice orthotopically injected with parental KPC 1242 cells and treated with vehicle, MRTX1133, afatinib, or combo. †, censored data; *, P < 0.05; **, P < 0.01; ***, P < 0.001, log-rank test. Data, mean values ± SD.