The PI3K-AKT-mTOR axis persists as a therapeutic dependency in KRASG12D-driven non-small cell lung cancer

Abstract

Introduction: KRAS^G12C and KRAS^G12D inhibitors represent a major translational breakthrough for non-small cell lung cancer (NSCLC) and cancer in general by directly targeting its most mutated oncoprotein. However, resistance to these small molecules has highlighted the need for rational combination partners necessitating a critical understanding of signaling downstream of KRAS mutant isoforms.

Methods: We contrasted tumor development between Kras^G12C and Kras^G12D genetically engineered mouse models (GEMMs). To corroborate findings and determine mutant subtype-specific dependencies, isogenic models of Kras^G12C and Kras^G12D initiation and adaptation were profiled by RNA sequencing. We also employed cell line models of established KRAS mutant NSCLC and determined therapeutic vulnerabilities through pharmacological inhibition. We analysed differences in survival outcomes for patients affected by advanced KRAS^G12C or KRAS^G12D-mutant NSCLC.

Results: KRAS^G12D exhibited higher potency in vivo, manifesting as more rapid lung tumor formation and reduced survival of KRAS^G12D GEMMs compared to KRAS^G12C. This increased potency, recapitulated in an isogenic initiation model, was associated with enhanced PI3K-AKT-mTOR signaling. However, KRAS^G12C oncogenicity and downstream pathway activation were comparable with KRAS^G12D at later stages of tumorigenesis in vitro and in vivo, consistent with similar clinical outcomes in patients. Despite this, established KRAS^G12D NSCLC models depended more on the PI3K-AKT-mTOR pathway, while KRAS^G12C models on the MAPK pathway. Specifically, KRAS^G12D inhibition was enhanced by AKT inhibition in vitro and in vivo.

Conclusions: Our data highlight a unique combination treatment vulnerability and suggest that patient selection strategies for combination approaches using direct KRAS inhibitors should be i) contextualised to individual RAS mutants, and ii) tailored to their downstream signaling.

Keywords: KRAS; KRASG12D inhibition; NSCLC; PI3K-AKT-mTOR pathway.

Fig. 1

KRAS^G12D is more potent than KRAS^G12C in driving NSCLC initiation in vivo. A Schematic illustrating the use of GEMM models to study the impact of KRAS mutant isoforms on NSCLC initiation. Green star = KRAS mutation. Created with BioRender.com/b49e578. B Schematic illustrating KRAS mutant oncogenes silenced by the insertion of a STOP codon flanked by LoxP sites. AdCre administration by inhalation leads to LoxP site recombination, removing the STOP codon allowing KRAS mutant isoform expression in GEMM mice lungs. Conditional KRAS mutant mice were crossed with mice in which tp53 is also flanked by LoxP sites. AdCre induces LoxP recombination and loss of p53 protein expression. C (Above) Timeline of experiment. (Below) Representative H&E sections and HALO quantification of lung tumor area and number per mouse comparing Kras^G12C/tp53^WT and Kras^G12D/tp53^WT mice 11 months after intranasal AdCre exposure (n = 9 Kras^G12C/tp53^WT mice and 5 Kras^G12D/tp53^WT mice); scale bar = 5 mm. D (Above) Timeline of experiment. (Below) Representative H&E sections and HALO quantification of lung tumor area and number comparing Kras^G12C/tp53^KO and Kras^G12D/tp53^KO mice 4 months after intranasal AdCre exposure (n = 7–8 mice per genotype); scale bar = 5 mm. E (Left) Timeline of experiment. (Right) Survival analysis for Kras^G12C/tp53^KO and Kras^G12D/tp53^KO mice after intranasal delivery of AdCre (n = 5 mice per genotype, Log-Rank (Mantel-Cox) test). F (Top left) Representative H&E images, (Bottom left) HALO mark-up and (Right) HALO quantification of tumor area and number comparing Kras^G12C/tp53^KO and Kras^G12D/tp53^KO mice from survival study (n = 5 mice per genotype); scale bar = 5 mm. C, D and F depict mean ± s.e.m and statistical analysis carried out using unpaired Student’s t-test. ****P < 0.0001, ***P < 0.001, **P < 0.01, ns > 0.05

Fig. 2

KRAS^G12D co-opts the PI3K-AKT-mTOR pathway to promote tumor initiation in NSCLC. A Schematic illustrating using GEMM models and isogenic MLE-12 cells to compare the impact of KRAS mutant isoforms on NSCLC initiation and signaling. Green star = KRAS mutation. Created with BioRender.com/b26f425. B Western blot analysis of KRAS and FLAG-tagged KRAS upon 24 h exposure of isogenic MLE-12 cells to 100 ng/mL doxycycline. PAR = parental. C (Left) MLE-12 spheroid viability upon 24 h 100 ng/mL doxycycline exposure measured by CellTiter-Glo 3D and (Right) MLE-12 spheroid area upon 96 h 100 ng/mL doxycycline exposure measured by ImageJ. Data normalised to untreated (no doxycycline) control (n = 3 at 24 h and n = 4 at 96-h). D GSEA showing that mTORC1 signaling genes are positively correlated with KRAS^G12D expression compared to KRAS^WT. E Heatmap showing DEGs belonging to mTORC1 signaling gene-set when comparing KRAS^G12D to KRAS^WT MLE-12 spheroids 24 h after 100 ng/mL doxycycline exposure (n = 3). F Western blot analysis of ERK, AKT, S6 and 4E-BP1 phosphorylation (Ser65) 24 h after exposure of isogenic MLE-12 spheroids to 100 ng/mL doxycycline. Representative of 3 independent experiments. G Flow cytometric quantification of S6 phosphorylation levels upon 24 h exposure of isogenic MLE-12 spheroids to 100 ng/mL doxycycline. Data normalised to untreated (no doxycycline) control (n = 3). H (Above) Representative immunohistochemical staining and (Below) quantification of ERK and S6 phosphorylation in early lung lesions of Kras^G12C/tp53^KO and Kras^G12D/tp53^KO mice (n = 6–9 mice per genotype); scale bar = 50 µm. I Flow cytometric quantification of surface CD44 and EpCAM in isogenic MLE-12 spheroids upon 24 h exposure to 100 ng/mL doxycycline. Data normalised to untreated (no doxycycline) control (n = 3). J Flow cytometric quantification of surface CD44 and EpCAM in isogenic MLE-12 spheroids upon 24 h exposure to 100 ng/mL doxycycline in the presence of (Left) 1 µM capivasertib (AKTi) or (Right) 1 µM ulixertinib (ERKi). Data normalised to DMSO control (n = 4). C, G, I and J depict mean ± s.e.m and statistical analysis carried out using one-way ANOVA test. (H) depicts mean ± s.e.m and statistical analysis carried out using unpaired Student’s t-test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns > 0.05. DOX = doxycycline

Fig. 3

Long-term KRAS^G12D-exposed cells display specific PI3K-AKT-mTOR pathway dependency. A Schematic illustrating using isogenic KRAS MEFs to determine growth rates, signaling differences and therapeutic vulnerabilities conferred by long-term expression of KRAS mutant isoforms. Green star = KRAS mutation. Created with BioRender.com/b26f425. B Spheroid area of isogenic KRAS MEFs quantified using ImageJ. Spheroid areas over time were normalised to spheroid area at day 1 (n = 3). Mean ± s.e.m. depicted. C BrdU/PI staining of isogenic KRAS MEFs at 72 h and 8 days (d8) in 3D. Proliferating cells are expressed as % BrdU-positive. Representative of three independent experiments. D Western blot analysis of ERK, AKT and S6 phosphorylation levels in isogenic KRAS MEFs at 24 h in 3D. Representative of three independent experiments. E Viability of isogenic KRAS MEFs in response to 100 nM sotorasib (G12Ci), 100 nM MRTX1133 (G12Di), 1 µM buparlisib (PI3Ki), 10 µM capivasertib (AKTi), 1 µM everolimus (mTORi) and rapamycin (mTORC1i) in 3D. Viability was measured after 72 h of drug exposure by CellTiter-Glo 3D. Viability expressed as % of DMSO control (n = 4). Mean depicted. F GSEA showing that mTORC1 signaling genes are positively correlated with KRAS^G12D expression and KRAS Signaling Up genes are positively correlated with KRAS^G12C expression. G Heatmaps showing DEGs belonging to mTORC1 Signaling and KRAS Signaling UP gene-sets comparing Kras^G12C to Kras^G12D MEFs (n = 3 per genotype). Statistical significance analysed using one-way ANOVA (between each time point for B or between drug treatment for E) but only significance between Kras^G12C and Kras^G12D MEFs presented. ***P < 0.001, **P < 0.01, *P < 0.05, ns > 0.05

Fig. 4

KRAS^G12C and KRAS^G12D NSCLC cells exhibit RAS effector-specific dependencies. A Schematic illustrating using GEMM-derived NSCLC cell lines, human NSCLC cell lines and patient data to determine growth rates, signaling differences and therapeutic vulnerabilities imposed by KRAS mutant isoforms in advanced disease. Green star = KRAS mutation. Created with BioRender.com/t58e004. B (Above) Representative immunohistochemical staining and (Below) quantification of Ki67 and Cyclin D1 expression in established lung tumors of Kras^G12C/tp53^KO and Kras^G12D/tp53^KO mice (n = 5 per genotype). Scale bar = 200 µm. C (Above) Representative immunohistochemical staining and (Below) quantification of ERK and S6 phosphorylation in established lung tumors of Kras^G12C/tp53^KO and Kras^G12D/tp53^KO mice (n = 5 per genotype). Scale bar = 200 µm. D Western blot analysis of ERK, AKT and S6 phosphorylation in Kras^G12C and Kras^G12D mTCLs at 48 h in 3D. Representative of three independent experiments. E Viability of Kras^G12C and Kras^G12D mTCLs in response to 1 µM sotorasib (G12Ci), 10 µM U0126 (MEKi), 10 µM ulixertinib (ERKi), 1 µM buparlisib (PI3Ki), 10 µM capivasertib (AKTi) and 10 nM everolimus (mTORi). Viability was measured after 48 h of drug exposure by crystal violet staining. Viability expressed as % of DMSO control (n = 3). F Western blot analysis of ERK, AKT, S6 and 4E-BP1 phosphorylation of Kras^G12C and Kras^G12D human NSCLC cell lines 48 h in 3D. Representative of three independent experiments. G Viability of human KRAS^G12C and KRAS^G12D NSCLC cell lines in response to 10 µM ulixertinib (ERKi) and 10 µM capivasertib (AKTi) in 3D. Viability was measured after 72 h of drug exposure by CellTiter-Glo 3D. Viability expressed as % of DMSO control (n = 3). H Cell death analyses of human (Left) KRAS^G12C and (Right) KRAS.^G12D NSCLC cell lines in response to 10 µM ulixertinib (ERKi) and 10 µM capivasertib (AKTi) in 3D. Cell death was measured after 48 h of drug exposure by flow cytometric quantification of PI staining. Data normalised to DMSO control (n = 3). B, C, E and G depict mean ± s.e.m and statistical analysis carried out using unpaired Student’s t-test. (H) depicts mean ± s.e.m and statistical analysis carried out using one-way ANOVA ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns > 0.05

Fig. 5

KRAS^G12D inhibition and PI3K-AKT-mTOR inhibition synergise in KRAS^G12D cells. A Isogenic MEFs were treated with increasing concentrations of either sotorasib (G12Ci) or MRTX1133 (G12Di) and capivasertib (AKTi) in 3D. 72 h later, viability was measured by CellTiter-Glo 3D. Viability expressed as % of DMSO control. Drug interaction was calculated using Bliss synergy scoring (n = 3). B The same conditions and analysis as in 5A in H358 and HCC1171 (KRAS^G12C) and SKLU-1 and HCC461 (KRAS^G12D) NSCLC cell lines (n = 3). C Human NSCLCs were treated with either 1 nM (for H358, HOP62, H2030 and HCC1171), 5 nM (H1792) or 10 nM (H23) sotorasib (G12Ci) or 10 nM MRTX1133 (G12Di) (for A427, SKLU-1 and HCC461) or 10 µM capivasertib (AKTi) and a combination of both in 3D. 48 and 120 h later, viability was measured by CellTiter-Glo 3D. CI values were calculated and presented per cell line and as a mean of all cell lines per genotype (n = 3). D (Left) Timeline of experiment. (Right) Tumor weights after HCC461 cells were inoculated onto the chorioallantoic membrane of 10-day old chicken embryos and exposed to two rounds of treatment with 5 nM MRTX1133 (G12Di), 50 μM capivasertib (AKTi) or 5 μM ulixertinib (ERKi) individually or as part of combination treatments. (n = 7–16 tumors per condition). g = grams. E (Left) Timeline of experiment. (Right) Relative tumor volumes of KPAR^G12D tumors after inoculation onto the flanks of C57BL/6 mice which were exposed to 6 daily treatments of 10 mg/kg MRTX1133 (G12Di) or 100 mg/kg capivasertib (AKTi) individually or in combination. Tumors represented as % change in volume relative to day 1 measurements (n = 3–4 tumors per condition). D depicts mean ± s.e.m and statistical analysis carried out using a one-way ANOVA with Tukey’s post-test and a t-test for comparisons between G12Di and the combination treatment. E depicts mean ± s.e.m and statistical analysis carried out using a two-way ANOVA with Tukey’s multiple comparison’s test. ****P < 0.0001, ***P < 0.001, **P < 0.01, **P < 0.05, ns > 0.05. Note: CI = combination index. For CI analysis, points appearing above the top dotted line signify drug antagonism. Points appearing between the top and bottom dotted line signify drug additivity. Points appearing below the bottom dotted line signify drug synergism. For bliss synergy scoring (BSS), a value of less than -10 signifies drug antagonism, a value of between -10 and 10 signifies drug additivity and a value of above 10 signifies drug synergy

Fig. 6

Schematic illustrating the progression of KRAS mutant-specific NSCLC. Upon initiation, the greater potency of KRAS^G12D induces rapid tumorigenesis relative to KRAS^G12C likely via the PI3K-AKT-mTOR pathway. During progression, the KRAS mutant isoform-specific differences in potency are not evident, underpinning the equivalent survival outcomes in patients. However, KRAS^G12D tumors maintain reliance on the PI3K-AKT-mTOR pathway, while KRAS^G12C increases oncogenic potential through other means, conferring therapeutic vulnerabilities which can be exploited. Green star = KRAS mutation. Created with BioRender.com/x11h794