RAS-targeted therapies Amanda R. Moore, Scott C. Rosenberg, Frank McCormick & Shiva Malek

The RAS (KRAS, NRAS and HRAS) family is the most are being investigated. Such strategies include inhibition frequently mutated gene family in cancers, and, of SOS1 and SHP2, which promote RAS activation, and consequently, investigators have sought an effective RAS inhibition of pathways downstream of RAS. Many of these inhibitor for more than three decades. Now, allele-specific potential therapies are being tried in combinations with covalent inhibitors against the most frequently mutated RAS inhibitors. Mutation-specific biochemical properties, version of RAS in NSCLC, KRAS-G12C, are in late-stage the spectrum of co-mutations, and the tissue of origin are clinical trials, and other strategies to inhibit this pathway likely to affect the effectiveness of such treatments.

RAS mutational spectrum Select single-agent inhibitors in clinical development for RAS-mutant tumours Drug Biomarker Disease setting Study Clinical trial Head and neck Clinical development of potential therapies for RAS-mutant tumours phase number 5% 35% squamous cell 45% KRAS-​G12C inhibitors carcinoma Activation of receptor tyrosine kinases, such as members of KRAS-G12C. GTP-bound RAS activates downstream signalling Sotorasib (AMG 510) KRASG12C mutation NSCLC III NCT04303780 91% G12 17% Lung the EGFR family, leads to a cascade of events at the membrane by binding to the RAS-binding domain of effector proteins, Adagrasib (MRTX849) KRASG12C mutation NSCLC III NCT04685135 32% adenocarcinoma G12A inner leaflet culminating in the activation of RAS. Inhibition at such as RAF and p110, to activate the MAPK and PI3K signalling JNJ-74699157 (ARS-3248) KRASG12C mutation Advanced solid tumours I NCT04006301 Melanoma 29% G12C PDAC various levels — such as EGFR, SOS or SHP2 — decreases the cascades, respectively. Both the MAPK and PI3K signalling GDC-6036 KRASG12C mutation Advanced solid tumours I NCT04449874 Bladder G12D rate at which inactive KRAS–GDP becomes active KRAS–GTP cascades can be inhibited at each kinase tier. Data compiled D-1553 KRASG12C mutation NSCLC, CRC I NCT04585035 urothelial 4% 30% G12F a carcinoma 86% and reduces the GTP-bound RAS population. Mutant RAS from ClinicalTrials.gov and AccessData.FDA.gov. Only effective JDQ433 KRASG12C mutation Advanced solid tumours I/II NCT04699188 G12L b Colorectal 41% proteins accumulate in the GTP-bound state. A number against monomeric BRAF (BRAF-V600E/K). Approved for SOS inhibitors 45% G12R c adenocarcinoma of approaches have been developed to directly inhibit RAS, the treatment of BRAF-mutant melanoma. Approved for the BI-1701963 KRAS mutations Advanced or metastatic solid tumours I NCT04111458 68% G12 G12S including covalent allele-specific inhibitors that bind to treatment of paediatric patients with NF1 mutations. G12V SHP2 inhibitors The most frequently mutated RAS G12Y RMC-4630 Mutations that hyperactivate the MAPK pathway Relapsed or refractory solid tumours I NCT03634982 isoform, codon and amino acid 23% EGFR TNO155 EGFR or KRASG12C mutations Advanced solid tumours I NCT03114319 substitution vary by tissue type family BBP-398 Mutations that hyperactivate the MAPK pathway Advanced solid tumours I NCT04528836 and cancer. Whereas NRAS is the 46% Farnesyltransferase inhibitors 17% most frequently mutated isoform Tipifarnib HRAS mutations HNSCC, NSCLC II NCT02383927, in melanoma, KRAS mutations 85% G12 NCT03496766 dominate in lung, pancreatic and KRAS Q61R Q61H Inhibitors of RAS processing RAF inhibitors colorectal cancers. Even within NRAS Tipifarnib EGFR inhibitors Belvarafenib NRAS mutations Advanced melanoma I NCT04835805 KRAS-mutated cancers, the most HRAS Q61K Cysmethynil Cetuximab LXH254 MAPK pathway mutation Advanced solid tumours I NCT02607813 UCM-1336 Panitumumab frequently mutated codon and the 46% 30% MEK inhibitors distributions of amino acid substitutions at those mutated codons Deltarasin Erlotinib NHTD Gefitinib Binimetinib (MEK162) NRAS mutation Unresectable or metastatic melanoma III NCT01763164 (12 or 61) are different for each tissue type, as shown in pie charts 13% GDP ERK inhibitors beside the relevant organ. Data from TCGA and AACR Project GENIE. 85% Q61 Q61L Allele-specific RAS inhibitors Pan-EGFR family LY3214996 BRAF or NRAS mutations Metastatic melanoma and NSCLC I NCT02857270 Sotorasib (AMG 510) inhibitors Adoptive cell therapies RAS GEF Adagrasib (MRTX849) Afatinib Anti-​RAS-​G12D mTCR HLA-A*11:01 RASG12D mutation Advanced solid tumours I/II NCT03745326 O JNJ-74699157 (ARS-3248) GDP Neratinib Direct RAS inhibitor structures Anti-​RAS-​G12V mTCR HLA-A*11:01 RASG12V mutation Advanced solid tumours I/II NCT03190941 O O GDC-6036 GAP GRB2 G12V F N D-1553 BI-1701963 Anti-RAS-G12V TCR HLA-A*11:01 RAS mutation Advanced solid tumours I/II NCT04146298 P GTP O N N i SOS BAY-293 siRNA strategies H NC JDQ433 N ARS-1620 BI-3406 KRAS-G12D iExosomes KRASG12D mutation PDAC I NCT03608631 N N N N ARS-853 SHP2 JAB-3068 Cancer vaccines N F RM-007 Cl OH Cl F N RMC-4630 mRNA-5671 HLA-A*11:01 and/or HLA-C*08:02; KRASG12C, NSCLC, non-MSI-​ H​ CRC, PDAC I NCT03948763 N OH N N RM-008 RAS TNO155 KRASG12D, KRASG12V or KRASG13D mutations N N N O RLY-1971 N N O KRAS antisense oligonucleotides O GTP Autophagy BBP-398 Select combination therapies in clinical development for RAS-mutant cancers F Cl N OH KRAS-G12D iExosomes F SHP099 Drugs Biomarker Disease setting Study Clinical trial N siG12D LODER phase number AZD4785 RMC-4550 ARS-853 ARS-1620 Adagrasib (MRTX849) Sotorasib (AMG 510) KRAS-​G12C combinations MAPK PI3K Hydroxychloroquine Sotorasib and antibodies to PD1 or PDL1 KRASG12C mutation Advanced NSCLC II NCT03600883 Belvarafenib pathway pathway LXH254 Adagrasib and TNO155 KRASG12C mutation Advanced or metastatic solid tumours I/II NCT04330664 Regions of RAS that could or are being targeted by therapeutics Lifirafenib (BGB-283) GDC-0077 JDQ433 and TNO155 KRASG12C mutation Advanced solid tumours I/II NCT04699188 PLX8394 Taselisib (GDC-0032) GDC-6036 and atezolizumab, cetuximab, KRASG12C mutation Advanced or metastatic solid tumours I NCT04449874 Sorafenib bevacizumab or erlotinib Regions targeted by clinical compounds a RAF p110 Alpelisib Vemurafenib Copanlisib SOS inhibitor combinations Switch-II pocket Switch-II groove BI-2852 Dabrafeniba Duvelisib BI-1701963 and trametinib KRAS mutation Advanced or metastatic solid tumours I NCT04111458 Sotorasib Switch I/II pocket Encorafeniba Switch-II 2C07 Idelalisib (AMG 510) LY3009120 P P BI-1701963 and irinotecan KRAS mutation Metastatic CRC I NCT04627142 AZ-628 SHP2 inhibitor combinations MEK AKT Ipatasertib Switch-I TAK-632 TNO155 and spartalizumab EGFR or ALK WT NSCLC Advanced solid tumours Ib NCT04000529 GppNHp Pimasertib TNO155 and ribociclib EGFR or ALK WT NCSLC, KRAS-mutant​ CRC or NSCLC Advanced solid tumours Ib NCT04000529 RAF inhibitor combinations 90° PD-0325901 P Binimetinib (MEK162)b Everolimus Belvarafenib and cobimetinib RAS or RAF mutations Locally advanced or metastatic tumours Ib NCT03284502 ERK mTOR Temsirolimus HRAS bound to GppNHp, Switch-II pocket (purple) of KRAS- Switch-II groove (purple) bound Switch I/II pocket of KRAS- Cobimetinibb LXH254 and an antibody to PD1 NRAS (melanoma) or KRAS (NSCLC) mutations Advanced solid tumours I NCT02607813 a non-hydrolysable G12C bound to sotorasib (AMG 510), to 2C07, the first covalent G12D (teal) bound to BI-2852, c Selumetinib LXH254 and trametinib, LTT462 or ribociclib KRAS or BRAF (NSCLC) or NRAS (melanoma) mutations Advanced or metastatic solid tumours Ib NCT02974725 nucleotide, as a reference a covalent RAS inhibitor (PDB: 6OIM; RAS inhibitor (PDB: 5VBZ; which disrupts the RAS–SOS1 Trametinibb for orientation (PDB: 5P21). compound identifier: MOV). compound identifier: 92V). interaction (PDB: 6GJ8). BGB-283 and PD-0325901 KRAS-mutant​ NSCLC or endometrial cancer Advanced or refractory solid tumours Ib NCT03905148 LY3214996 MEK inhibitor combinations Ulixertinib (BVD-523) Other potentially targetable regions Cobimetinib and atezolizumab KRAS mutation Advanced and metastatic NSCLC II NCT03600701 GDC-0994 Cobimetinib and RMC-4630 Mutations that hyperactivate the MAPK pathway Relapsed or refractory solid tumours Ib/II NCT03989115 SOS interface KO-947 Clinical compound Selumetinib and MK-8353 RAS or RAF mutations Advanced solid tumours Ib NCT03745989 Dimer SCH-772984 SOS binding Proposed HRAS interface RAF–RAS–RBD FDA approved for other indications Trametinib and ponatinib KRAS mutation Advanced NSCLC I NCT03704688 Vtx-11e (including tumours without RAS mutations) interface (red) dimerization binding interface ERK inhibitor combinations MK-8253 Completed clinical trial of HRAS (PDB: interface (green) NS1 (yellow) of HRAS LY3214996 and midazolam, abemaciclib, or BRAF or RAS mutations Advanced or metastatic solid tumours I NCT02857270 1BKD). bound by NS1 (PDB: 4G0N). Terminated clinical trial nab-paclitaxel + gemcitabine monobody (gold) Cancer vaccine combinations (PDB: 5E95). mRNA-5671 and pembrolizumab HLA-A*11:01 and/or HLA-C*08:02 KRASG12C, KRASG12D, NSCLC, non-MSI-​ H​ CRC, PDAC I NCT03948763 135° 90° RBD interface KRASG12V or KRASG13D mutations

Abbreviations References Affiliations Competing interests statement Reaction Biology Reaction Biology’s KRAS Drug Discovery Platform offers: CRC, colorectal cancer; DCAI, 4,6-dichloro-2- 1. Canon, J. et al. The clinical KRAS(G12C) inhibitor 5. Moore, A. R., Rosenberg, S. C., McCormick, F. & Malek, S. • Amanda R. Moore, Scott C. F.McC. is a consultant for the following companies: Amgen, Pfizer Reaction Biology provides scientists worldwide with a trusted • Nucleotide exchange assays to monitor GDP to GTP exchange (KRAS and mutants, SOS1, SOS2) methyl-3-aminoethyl-indole; EGFR, epidermal AMG 510 drives anti-tumour immunity. Nature 575, RAS-targeted therapies: is the undruggable drugged? Nat. Rosenberg and Shiva Malek are at the Inc., and Quanta Therapeutics; is a consultant and co-founder with • Direct binding assays via biophysical methods (KRAS and mutants) growth factor receptor; mAb, monoclonal 217–223 (2019). Rev. Drug Discov. 19, 533–552 (2020). Department of Discovery Oncology, ownership interest including stock options of BridgeBio Pharma, research partner supporting all phases of preclinical drug discovery G12C Genentech Inc., South San Francisco, Inc; and is Scientific Director of the NCI Ras Initiative at Frederick • Protein–protein interaction assays via biochemical and cell-based methods (KRAS and mutants antibody; MSI-H, microsatellite instability- 2. Hallin, J. et al. The KRAS inhibitor MRTX849 6. Ostrem, J. M., Peters, U., Sos, M. L., Wells, J. A., & Shokat, K. M. from target validation through hit identification, hit to lead and lead high; mTCR, murine T cell receptor; provides insight toward therapeutic susceptibility K-Ras(G12C) inhibitors allosterically control GTP affinity CA, USA. National Laboratory for Cancer Research/Leidos Biomedical optimization. with SOS or RAF) nab-paclitaxel, nanoparticle albumin-bound of KRAS-mutant cancers in mouse models and and effector interactions. Nature 503, 548–551 (2013). • Frank McCormick is at Helen Diller Research Inc. S.M. is an employee of Genentech/Roche. A.R.M. Family Comprehensive Cancer and S.C.R. are also post-doctoral fellows employed by Genentech/ From labs in the USA and Europe, Reaction Biology performs • Recombinant proteins (KRAS and mutants, GEFs, GAP, and others) paclitaxel; NSCLC, non-small-cell lung patients. Cancer Discov. 10, 54–71 (2019) 7. The AACR Project GENIE Consortium. AACR Project G12C Center, University of California San Roche. • Cellular phosphorylation assays (BRAF) cancer; PDAC, pancreatic ductal 3. Hong, D. S. et al. KRAS Inhibition with sotorasib GENIE: Powering Precision Medicine through an research projects for customers around the globe with collaborative Francisco, San Francisco, CA, USA. • Cellular detection assays (phospho-STAT, phospho-ERK, phospho-MEK) adenocarcinoma; PDB, protein data bank; in advanced solid tumors. N. Engl. J. Med. 383, International Consortium. Cancer Discov. 7, 818–831 The poster content is peer reviewed, editorially independent and support and quality data for a superior research result. We would be 1207–1217 (2020). (2017). the sole responsibility of Springer Nature Limited. Edited by Megan • Profiling KRAS-mutated cell lines in 2D and 3D setups RBD, RAS-binding domain; SOS, Son of delighted to support your research needs, too. Sevenless; TCGA, the cancer genome atlas; 4. Janes, M. R. et al. Targeting KRAS mutant cancers 8. Kessler, D. et al. Drugging an undruggable pocket on Cully; copyedited by George Booth; designed by Susanne Harris. Let’s discover together. Reaction Biology • USA / Europe • www.reactionbiology.com WT, wild type. with a covalent G12C-specific inhibitor. Cell 172, KRAS. Proc. Natl Acad. Sci. U.S.A. 116, 15823–15829 © 2021 Springer Nature Limited. All rights reserved. 578–589 (2018). (2019). https://www.nature.com/articles/s41573-021-00220-6