Divide and conquer: towards isoform-specific diagnosis and therapy of KRAS-mutant lung cancer

The identification of oncogenes in animal cells by experiments with retroviruses, and the discovery that similar genetic alterations can cause tumours in humans, laid the foundation for modern cancer research. From that pioneering work to the current genomic era, RAS genes marked the beginning of molecular oncology, and remained at the forefront of cancer research ever since.

In humans, the three genes HRAS, NRAS and KRAS encode four proteins: H-Ras (Harvey-Ras), N-Ras (Neuroblastoma-Ras) and two isoforms of K-Ras (Kirsten-Ras), K-Ras4A and K-Ras4B, which in turn derive from alternative splicing isoforms (1). These oncoproteins are members of a larger superfamily of small guanosine triphosphatase (GTPase), containing over 170 proteins, which are divided into five main branches on the basis of protein structure, function or both: Ras, Rho, Rab, Ran and Arf (2,3). All Ras isoforms are characterised by two main domains: a highly conserved catalytic domain, and a farnesylated hypervariable region that modulates membrane interaction for distinctive localizations. The catalytic domain contains the guanosine triphosphate (GTP)-guanosine diphostate (GDP) binding site and interaction sites with effector proteins: because it is identical in all Ras isoforms, all the proteins can interact with the same set of downstream effectors (4). Similar to all the RAS proteins, Kras functions as binary molecular switch in the regulation of pathways responsible for cell proliferation and survival. When stimulated by mitogenic signals, Kras binds to GTP, and activates downstream molecules and effectors. When the stimulation is terminated, Kras-GTP switches to Kras-GDP. Given the slow intrinsic hydrolysis rate of Kras, deactivation of the signal depends critically on GTPase activating protein (GAP), that enhances the GTPase activity and leads Kras to an inactive state (1,4).

Due to differences in post-translational modifications of Ras, including farnesylation, geranylgeranylation and palmitoylation, and because of different intracellular localizations at the plasma membrane and the endomembranes, Ras proteins have access to different effector pools, and are thus able to generate distinct signals (1,5). More than 20 downstream effector signalling pathways responsible for basic cellular process have been identified, including the serine/threonine protein kinase Raf, MAPK and ERK kinase (MEK), extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway, and the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (PKB/AKT) pathway.

Mutations in RAS proto-oncogenes are present in about 20% of all human cancers. KRAS mutations are responsible for 75% of adenocarcinomas, while NRAS and HRAS are mutated more often in melanomas and squamous epithelial carcinomas, respectively. The highest prevalence of KRAS mutations are found in pancreatic adenocarcinomas (90%), appendiceal adenocarcinomas (60%), small bowel adenocarcinomas (50%), and colorectal carcinomas (50%) (6,7). KRAS mutated lung adenocarcinomas represent about 25% to 30% of all lung cancers, but cannot be considered as a homogeneous entity anymore. Indeed, different isoforms are known: 80% of KRAS mutations occur at codon 12 and 13 of exon 2, with G12C the most common form (40%), followed by G12V (about 20%) and G12D (about 15%). Very few mutations are observed at codon 61 of exon 3, which instead is common in NRAS mutated cancer. Mutational heterogeneity can be in part explained by tissue exposure to mutagenic agents and specific molecular regulatory mechanisms. For example, exposure to tobacco smoke shows a distinctive coupling to KRAS-driven non-small cell lung cancer (NSCLC), especially to G12C variants. This specific mutation is common in former and current smokers, and rarely exists in never-smokers (4,8,9). KRAS-mutated lung cancers are characterised by high tumor mutation burden (TMB), a genetic signature of direct tobacco smoke exposure with predominant C>A (G>T) transversion mutations and elevated markers of immune evasion (high PD-L1 expression) (8,10,11). Patients are predominantly female (58%), median age is 65 years, and with history of smoking (93%) (8,12). KRAS mutations have been linked to a poor prognosis compared to epidermal growth factor receptor (EGFR)-mutated and KRAS-wild type lung cancer. Isoform specific outcomes are not consistent across studies. Patients with KRAS codon 13 mutations appear to have shorter overall survival compared with patients with codon 12 mutations, G12C and G12V are associated with worse progression-free survival compared with other G12X mutations or wild-type KRAS (13-16).

Although no significant differences in survival and demographic data were observed according to the different KRAS G12X variants, distinct genomic and transcriptomic features, location and variant type are important factors for oncogenic potential, activation of distinct signalling pathways, and allele-specific genomic landscapes (10,12).

In their article “Comparative Analysis and Isoform-Specific Therapeutic Vulnerabilities of KRAS Mutations in Non-Small Cell Lung Cancer”, Ricciuti et al. show that KRAS mutational isoforms correlate with distinct biological and genomic profiles, clinical phenotypes and therapeutic outcomes (17). Based on in vitro and in vivo analysis the authors demonstrate that KRAS G12D has the highest oncogenic potential of all the variants analysed. Although no difference in PD-L1 expression was observed between the isoforms, KRAS G12D was associated with the lowest tumour mutational burden. These results are important and consistent with previous data published recently (10).

KRAS mutations may confer insensitivity to inhibitors of upstream and downstream signalling pathways. Ricciuti et al. observe that the MEK-inhibitor trametinib presents similar anti-proliferative effects in all KRAS-isoforms, whereas sensitivity to selumetinib, another MEK-inhibitor, is different across the isoforms, with 12C and Q61H being the most responsive. Selumetinib in vivo exerts a better response in G12C than sotorasib. The authors postulate delayed feedback mechanisms for MEK inhibitors compared with KRAS G12C inhibitors as a single agent. Interestingly, the combined treatment with selumetinib and sotorasib is more effective than either drug alone, leading to significant size reduction of KRAS mutated tumours in mice. Also, the combined SH2 containing protein tyrosine phosphatase-2 (SHP2)/MEK inhibition is efficacious, principally in G12C, G12D and G12V mutants compared with other KRAS mutants. This observation is consistent with previous experiments and is relevant because SHP2 play an important role in several types of cancer (18). Numerous combination trials with KRAS and other MAPK pathway (SHP2/MEK/ERK) inhibitors are in progress (NCT04185883, NCT05480865).

In the analysis of the co-mutational landscape and gene-expression profiles, Ricciuti et al. demonstrate that each genomic variant presents specific patterns: mutations in serine/threonine kinase 11 (STK11) and ataxia telangiectasia mutated (ATM) genes were significantly enriched in tumours harbouring G12C, G12A, or G12V, while G13X mutations were associated with the highest rate of concomitant STK11 and Kelch-like ECH-associated protein 1 (KEAP1)-mutations. STK11/KEAP1 co-mutations downregulate pathways of innate and adaptive immunity, which in turn are associated with reduced response to PD(L)-blockade (19,20). These results are in accordance with previous observations, suggesting a link between KRAS, the genomic landscape, and the immune system (20,21).

The recent approval by Food and Drug Administration (FDA) of sotorasib and adagrasib, two selective and irreversible inhibitors of KRAS G12C, is an important milestone in the treatment of patients with metastatic lung cancer (22,23). Further advances in the field of KRAS mutant cancers can be expected from combination therapies, as evidenced by recently published results about the combination of adagrasib and cetuximab in colorectal carcinoma (24). In consonance with the results by Ricciuti et al., broader genomic and transcriptomic characterisation of KRAS mutant tumours is an important basis for the research of further combination therapies. This, and the development of compounds targeting KRAS mutations other than G12C, necessitates an accurate molecular sub-classification and a broader view on the genomic landscape in KRAS mutated cancers.

Acknowledgments

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Translational Lung Cancer Research. The article did not undergo external peer review.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-23-72/coif). ATA participated in an Advisory Board by Janssen, presented a lecture for Janssen and for Astra Zeneca. All honoraria were paid to institution. ATA received support for attending meeting by Janssen. OG was a consultant for AMGEN, participated in advisory boards by Amgen, Roche and Pfizer, and is a member of data safety monitoring board for Boehringer Ingelheim. All honoraria were paid to institution. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Li S, Balmain A, Counter CM. A model for RAS mutation patterns in cancers: finding the sweet spot. Nat Rev Cancer 2018;18:767-77. [Crossref] [PubMed]
2. Wennerberg K, Rossman KL, Der CJ. The Ras superfamily at a glance. J Cell Sci 2005;118:843-6. [Crossref] [PubMed]
3. Colicelli J. Human RAS superfamily proteins and related GTPases. Sci STKE 2004;2004:RE13. [Crossref] [PubMed]
4. Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res 2012;72:2457-67. [Crossref] [PubMed]
5. Hancock JF. Ras proteins: different signals from different locations. Nat Rev Mol Cell Biol 2003;4:373-84. [Crossref] [PubMed]
6. Prior IA, Hood FE, Hartley JL. The Frequency of Ras Mutations in Cancer. Cancer Res 2020;80:2969-74. [Crossref] [PubMed]
7. Lee JK, Sivakumar S, Schrock AB, et al. Comprehensive pan-cancer genomic landscape of KRAS altered cancers and real-world outcomes in solid tumors. NPJ Precis Oncol 2022;6:91. [Crossref] [PubMed]
8. Salem ME, El-Refai SM, Sha W, et al. Landscape of KRAS(G12C), Associated Genomic Alterations, and Interrelation With Immuno-Oncology Biomarkers in KRAS-Mutated Cancers. JCO Precis Oncol 2022;6:e2100245. [Crossref] [PubMed]
9. Le Calvez F, Mukeria A, Hunt JD, et al. TP53 and KRAS mutation load and types in lung cancers in relation to tobacco smoke: distinct patterns in never, former, and current smokers. Cancer Res 2005;65:5076-83. [Crossref] [PubMed]
10. Judd J, Abdel Karim N, Khan H, et al. Characterization of KRAS Mutation Subtypes in Non-small Cell Lung Cancer. Mol Cancer Ther 2021;20:2577-84. [Crossref] [PubMed]
11. Alexandrov LB, Ju YS, Haase K, et al. Mutational signatures associated with tobacco smoking in human cancer. Science 2016;354:618-22. [Crossref] [PubMed]
12. El Osta B, Behera M, Kim S, et al. Characteristics and Outcomes of Patients With Metastatic KRAS-Mutant Lung Adenocarcinomas: The Lung Cancer Mutation Consortium Experience. J Thorac Oncol 2019;14:876-89. [Crossref] [PubMed]
13. Ihle NT, Byers LA, Kim ES, et al. Effect of KRAS oncogene substitutions on protein behavior: implications for signaling and clinical outcome. J Natl Cancer Inst 2012;104:228-39. [Crossref] [PubMed]
14. Yu HA, Sima CS, Shen R, et al. Prognostic impact of KRAS mutation subtypes in 677 patients with metastatic lung adenocarcinomas. J Thorac Oncol 2015;10:431-7. [Crossref] [PubMed]
15. Johnson ML, Sima CS, Chaft J, et al. Association of KRAS and EGFR mutations with survival in patients with advanced lung adenocarcinomas. Cancer 2013;119:356-62. [Crossref] [PubMed]
16. Muthiah A, Chudasama R, Mingrino J, et al. Clinical characteristic and outcomes of KRAS G13 mutant non-small cell lung cancers. J Clin Oncol 2022;40:abstr e21017.
17. Ricciuti B, Son J, Okoro JJ, et al. Comparative Analysis and Isoform-Specific Therapeutic Vulnerabilities of KRAS Mutations in Non-Small Cell Lung Cancer. Clin Cancer Res 2022;28:1640-50. [Crossref] [PubMed]
18. Ruess DA, Heynen GJ, Ciecielski KJ, et al. Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphatase. Nat Med 2018;24:954-60. [Crossref] [PubMed]
19. Ricciuti B, Arbour KC, Lin JJ, et al. Diminished Efficacy of Programmed Death-(Ligand)1 Inhibition in STK11- and KEAP1-Mutant Lung Adenocarcinoma Is Affected by KRAS Mutation Status. J Thorac Oncol 2022;17:399-410. [Crossref] [PubMed]
20. Skoulidis F, Byers LA, Diao L, et al. Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities. Cancer Discov 2015;5:860-77. [Crossref] [PubMed]
21. Scharpf RB, Balan A, Ricciuti B, et al. Genomic Landscapes and Hallmarks of Mutant RAS in Human Cancers. Cancer Res 2022;82:4058-78. [Crossref] [PubMed]
22. Jänne PA, Riely GJ, Gadgeel SM, et al. Adagrasib in Non-Small-Cell Lung Cancer Harboring a KRAS(G12C) Mutation. N Engl J Med 2022;387:120-31. [Crossref] [PubMed]
23. Skoulidis F, Li BT, Dy GK, et al. Sotorasib for Lung Cancers with KRAS p.G12C Mutation. N Engl J Med 2021;384:2371-81. [Crossref] [PubMed]
24. Yaeger R, Weiss J, Pelster MS, et al. Adagrasib with or without Cetuximab in Colorectal Cancer with Mutated KRAS G12C. N Engl J Med 2023;388:44-54. [Crossref] [PubMed]