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Supplementary methods providing in depth information about FACT assay, Ba/F3 transformation assay, xenograft in-vivo work and RAS-GTP pulldown experiments.

Frequency and prevalence of KRAS mutations across 8 pooled cohorts

Comparison of (A) cell viability and (B) protein expression levels as assessed by reverse phase protein arrays in Ba/F3 transformation assay demonstrating (C) a lack of correlation between protein level and transformation, supporting that functional status rather than protein level are driving cellular transformation.

Frequency and prevalence of NRAS mutations across 8 pooled cohorts

Western blot of RAS expression and accompanying RAS-GTP pulldown assay for isogenic SW48 cell lines generated for xenograft experiments.

Histogram demonstrating distribution of (A) RAS, (B) KRAS, and (C) NRAS mutations across clinical cohorts with survival data based on activity level.

PURPOSE KRAS p.G12C mutations occur in approximately 3% of metastatic colorectal cancers (mCRC). Recently, two allosteric inhibitors of KRAS p.G12C have demonstrated activity in early phase clinical trials. There are no robust studies examining the behavior of this newly targetable population. METHODS We queried the MD Anderson Cancer Center data set for patients with colorectal cancer who harbored KRAS p.G12C mutations between January 2003 and September 2019. Patients were analyzed for clinical characteristics, overall survival (OS), and progression-free survival (PFS) and compared against KRAS nonG12C. Next, we analyzed several internal and external data sets to assess immune signatures, gene expression profiles, hypermethylation, co-occurring mutations, and proteomics. RESULTS Among the 4,632 patients with comprehensive molecular profiling, 134 (2.9%) were found to have KRAS p.G12C mutations. An additional 53 patients with single gene sequencing were included in clinical data but excluded from prevalence analysis allowing for 187 total patients. Sixty-five patients had de novo metastatic disease and received a median of two lines of chemotherapy without surgical intervention. For the first three lines of chemotherapy, the median PFS was 6.4 months (n = 65; 95% CI, 5.0 to 7.4 months), 3.9 months (n = 47; 95% CI, 2.9 to 5.9 months), and 3.0 months (n = 21; 95% CI, 2.0 to 3.4 months), respectively. KRAS p.G12C demonstrated higher rates of basal EGFR activation compared with KRAS nonG12C. When compared with an internal cohort of KRAS nonG12C, KRAS p.G12C patients had worse OS. CONCLUSION PFS is poor for patients with KRAS p.G12C metastatic colorectal cancer. OS was worse in KRAS p.G12C compared with KRAS nonG12C patients. Our data highlight the innate resistance to chemotherapy for KRAS p.G12C patients and serve as a historical comparator for future clinical trials.

Purpose: Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact, and atypical mutations beyond those in standard guidelines exist. Experimental Design: We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical RAS variants. Using an in vitro cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and in vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations. Results: KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24–6.80; P = 0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results, and activating atypical variants were those that occurred at highest prevalence in clinical cohorts. Conclusions: We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included RAS variants and functionally relevant.

Although KRAS has long been considered undruggable, direct KRAS

PURPOSE: Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact and atypical mutations beyond those in standard guidelines exist. EXPERIMENTAL DESIGN: We reviewed 7 tissue and 1 cfDNA cohorts of 9485 patients to characterize atypical RAS variants. Using an in-vitro cell-based assay (FACT), Ba/F3 transformation and in-vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations. RESULTS: KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K and D33E occurred at prevalence ≥0.1%, while no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR 2.90, 95% CI 1.24–6.80, P=0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results and activating atypical variants were those that occurred at highest prevalence in clinical cohorts. CONCLUSION: We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E and T50I are more prevalent than many guideline included RAS variants and functionally relevant.

PURPOSE: KRAS p.G12C mutations occur in approximately 3% of metastatic colorectal cancers (mCRC). Recently, two allosteric inhibitors of KRAS p.G12C have demonstrated activity in early phase clinical trials. There are no robust studies examining the behavior of this newly targetable population. METHODS: We queried the MD Anderson Cancer Center data set for patients with colorectal cancer who harbored KRAS p.G12C mutations between January 2003 and September 2019. Patients were analyzed for clinical characteristics, overall survival (OS), and progression-free survival (PFS) and compared against KRAS nonG12C. Next, we analyzed several internal and external data sets to assess immune signatures, gene expression profiles, hypermethylation, co-occurring mutations, and proteomics. RESULTS: Among the 4,632 patients with comprehensive molecular profiling, 134 (2.9%) were found to have KRAS p.G12C mutations. An additional 53 patients with single gene sequencing were included in clinical data but excluded from prevalence analysis allowing for 187 total patients. Sixty-five patients had de novo metastatic disease and received a median of two lines of chemotherapy without surgical intervention. For the first three lines of chemotherapy, the median PFS was 6.4 months (n = 65; 95% CI, 5.0 to 7.4 months), 3.9 months (n = 47; 95% CI, 2.9 to 5.9 months), and 3.0 months (n = 21; 95% CI, 2.0 to 3.4 months), respectively. KRAS p.G12C demonstrated higher rates of basal EGFR activation compared with KRAS nonG12C. When compared with an internal cohort of KRAS nonG12C, KRAS p.G12C patients had worse OS. CONCLUSION: PFS is poor for patients with KRAS p.G12C metastatic colorectal cancer. OS was worse in KRAS p.G12C compared with KRAS nonG12C patients. Our data highlight the innate resistance to chemotherapy for KRAS p.G12C patients and serve as a historical comparator for future clinical trials.

Background: Oncogenic KRAS mutations occur in 50% of colorectal cancer (CRC) patients and have long been considered undruggable. Novel covalent inhibitors targeting the KRASG12C mutation have been developed, presenting a unique opportunity to directly target KRAS. However, clinical trials focusing on sotorasib reveal that KRASG12C inhibitors are only modestly active in CRC compared to lung cancer patients with response rates being 7% and 54%, respectively. Adaptive and acquired feedback has contributed to the diminished therapeutic efficacies observed in CRC patients. Recent studies have shown that receptor tyrosine kinases may be the conduit for adaptive feedback to KRASG12C inhibition, with EGFR being the focus of this class. However, while preclinical and clinical studies evaluating combinatorial strategies to maintain the inactivation of KRASG12C have proven that adaptive feedback can be overcome through combinations, the acquired resistance mechanisms remain unclear. In this study, we examine the adaptive and acquired response to KRASG12C inhibition using PDX-CRC models. Methods: We treated three KRASG12C PDX-CRC cell lines (B8182, C1047, and F3008) with sotorasib, panitumumab, and the combo of the two drugs for 6h and 24h. We compare their response to untreated samples to understand the adaptive response over time. Adaptive response to treatment was assessed by measuring the pERK and pEGFR response via western blots. We also treated two KRASG12C PDX-CRC mouse models, B8026 and C1177, with sotorasib, panitumumab, trametinib, as well as doublet and triplet combos with sotorasib. We then assessed treatment response by measuring tumor volume. To develop PDX-CRC models that acquire resistance to sotorasib, we treated the mice continuously with sotorasib over time until we observed noticeable tumor growth. Results: B8182, C1047, and F3008 all show adaptive response to sotorasib. However, while the source of adaptive response is not clear, our data suggests that EGFR may not be the driver in all three cell lines. In our PDX-CRC mouse models, both B8026 and C1177 are responsive to sotorasib and panitumumab. However, C1177 was not responsive to trametinib. Both models however are responsive to doublet combos of sotorasib with either agent, with triplet combo providing the greatest effectiveness in reducing tumor volume. Additionally, B8026 and C1177 PDX-CRC models developed acquired resistance to sotorasib. Conclusion: KRASG12C inhibitors are only modestly active in CRC. Treatment of sotorasib in KRASG12C PDX-CRC cell lines showed that EGFR may not be the primary mediator of adaptive resistance in all models. However, inactivation of KRASG12C can be maintained through combinations targeting the kinases involved in the feedback mechanisms. Finally, while functional characterization of resistance mechanisms is underway, the mechanisms of resistance to KRASG12C appear to be transcriptomic. Citation Format: Oluwadara Coker, Alexey Sorokin, Kelly Gale, Fengqin Gao, John Paul Shen, Lawrence Kwong, Ji Wu, Hey Min Lee, Melanie Woods, Oscar Villareal, Scott Kopetz. Oncogenic KRASG12C dependency in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB264.

110 Background: BRAFV600E mutation identifies mCRC patients with poor prognosis with only little benefit from standard therapy. Analysis from TCGA revealed that 89% of BRAFV600E CRC tumors were associated with a high CpG island methylator phenotype (CIMP-H), which may result in epigenetic silencing of tumor suppressor genes, while only 29% of BRAF wild-type tumors are CIMP-H. In this study, we define key pathways regulated by global DNA hypermethylation in the context of BRAFV600E mutation. Methods: We analyzed the TCGA Illumina 450k array methylation datasets and RNA-sequencing datasets for 97 CIMP-H CRC tumors (27 BRAFV600E; 69 BRAF WT) identified by five universal CIMP annotations ( p14, p16, MLH1, MINT1, MINT2, MINT31). We defined differential methylation profile according to BRAF mutation status and calculated Spearman correlation between methylation and gene expression to identify CIMP-H BRAF-associated genes. Next, pathways enriched with CIMP-H BRAFV600E tumors were defined using PANTHER pathway analysis. Additionally, β-catenin IHC were conducted on 145 MD Anderson CRC patient samples and compared by CIMP and BRAF status. Results: BRAF mutation is associated with lower rates of APC mutation as has previously been shown (32%, 82%). We identified 6,097 differentially methylated probes by BRAF mutation status (FDR = 10-4), and as expected, our data suggests a higher methylation profile in BRAFV600E mutated tumors compared to BRAF WT. Intriguingly, CIMP-H BRAF-associated genes showed enrichment in the Wnt-signaling and cadherin signaling pathways ( p< 0.0001 (FDR < 0.0001)). Despite the epigenetic Wnt-signaling, nuclear β-catenin expression (as a measure of Wnt activity) in CIMP-H and BRAF tumors remains lower than for non-CIMP, and BRAF wild-type ( p= 0.0003 for comparison of CIMP). Conclusions: Genes under methylation regulation in the BRAF-mutant context showed enrichment in Wnt-signaling pathway. Since BRAFV600E CRC tumors have a low association with APC mutation, this data suggests role of epigenetic regulation of the Wnt-pathway activation. However, as measured by nuclear β-catenin, Wnt activation in these tumors is not as high as traditional APC-mutated CRC tumors. CIMP-H tumors with BRAFV600E mutation is a unique subset of CRC tumor that have Wnt-pathway activation regulated by epigenetic modifications more than a β-catenin activation.

3625 Background: Oncogenic KRAS mut drive cancers and confer therapeutic resistance by activating MAPK signaling. Inhibiting KRAS has been elusive until the recent promising phase I trials with KRAS G12C inhibitors (i). We characterized frequencies of KRAS G12C mut and gene co-alt among advanced cancer patients (pts) to identify therapeutic vulnerabilities for combination development. Methods: We analyzed next generation sequencing datasets from MD Anderson Cancer Center (MDACC, n = 42,316) and AACR GENIE (n = 56,970). Genes and individual alterations were annotated for potential actionability with approved or investigational drugs and grouped into 12 oncogenic pathways. Frequencies of potential drug combinations with KRAS G12Ci were estimated per tumor type based on co-occurrence of potentially actionable alterations. Results: KRAS G12C was present in 850/34,801 (2.4%) advanced solid tumor and 22/7698 (0.3%) hematologic malignancy pts in MDACC dataset; and 1422 (2.5%) pts in AACR GENIE. Among solid tumor pts, 798 had histology data and 640 had ≥46 gene profiling. Most common cancers were non-small cell lung (NSCLC, 67%), colorectal (CRC, 24%), other gastrointestinal (oGI, 4%) and gynecologic (gyn, 2%). KRAS G12C prevalence was 19.5% (441/2265) in NSCLC and 4.2% (146/3469) in CRC. Genes most commonly co-altered were TP53 (42%), STK11 (17%) and MET (11%) in NSCLC; TP53 (58%), APC (54%) and PIK3CA (24%) in CRC; TP53 (42%), APC (21%) and ATM (21%) in oGI; TP53 (56%), PIK3CA (25%), and PTEN (19%) in gyn cancers. These co-alt did not impact overall survival. In both datasets, as compared to KRAS wild, KRAS G12C was significantly co-altered with STK11 in NSCLC; PIK3CA and SMAD4 in CRC (P < 0.05 for all). EGFR mut in NSCLC and BRAF mut in CRC rarely co-occurred with KRAS G12C (P < 0.01). Most frequently co-altered oncogenic pathways in NSCLC, CRC, oGI and gyn cancers respectively included PI3K (27, 32, 33, 44 %), receptor tyrosine kinases (13, 16, 42, 13 %) and DNA damage repair (12, 10, 38, 19 %). Potentially actionable co-alt frequencies suggest that combining KRAS G12Ci with mTORi or PI3Ki would be indicated most frequently, in 24% and 13% of all pts respectively. Conclusions: KRAS G12Ci development is most relevant for NSCLC, gastrointestinal and gyn cancers. The co-alt patterns highlight relevant oncogenic pathways and candidate drugs for future combination therapies. Co-inhibition of PI3K-mTOR and MAPK pathways has shown synergism in prior pre-clinical studies but had poor tolerance in pts. There is opportunity to revisit this approach with the new KRAS G12Ci.

180 Background: RAS mutations are found in ~50% of patients (pts) with metastatic colorectal cancer (mCRC) and associated with resistance to anti-EGFR. Circulating tumor DNA (ctDNA) enables detection of resistant RASMUT arising from RASWT. Recently there has been interest in defining the converse: RASMUT tumors that revert to RASWT, with early results suggesting rates of ~7%. Clinical trials in this population are in development, though the incidence has not been validated with robust methodologies. Methods: 1) We identified 74 mCRC pts with baseline RASMUT and longitudinal ctDNA or tissue data enrolled in ATTACC (NCT01196130), a prospective genomic matching protocol utilizing paired tissue/ctDNA samples at baseline. We evaluated serial samples for RAS loss. 2) Using an external cohort of pts with mCRC and serial ctDNA with a targeted NGS assay sequencing all KRAS/ NRAS exons (Guardant360, Guardant Health), we screened pts for baseline RASMUT with no evidence of prior anti-EGFR exposure and evaluated for RAS loss. Results: 74 pts met criteria of RASMUT CRC with serial samples in ATTACC. Of these, 51 retained RASMUT. 22 pts had very low or absent levels of other clonal alterations such as APC or TP53 and are therefore unable to reliably detect RAS loss. One patient had true RAS loss with NRAS G13R, APC and TP53 mutations at baseline and persistent high-level APC and TP53 mutations without a detectable NRAS mutation, for an overall rate of RAS loss of 2% (1/52). In the second cohort we identified 162 pts, 34 of which had insufficient ctDNA to assess RAS loss on the serial sample as defined by loss of clonal alterations like APC and TP53. Of the remaining 128 patients, 11 had RAS loss (8.5%, with 1 NRAS, 10 KRAS). We next compared the relative mutant allele frequency (rMAF) between RAS retainers and RAS loss. The median baseline rMAF for pts who lost RAS was 0.74, compared to 0.86 in pts retaining RAS (p = 0.045). Conclusions: RAS reversion in mCRC from RASMUT to RASWT is uncommon and occurs at a rate between 2-8% in our two cohorts. RAS reversion is associated with a lower rMAF at baseline, suggesting subclonality. Liquid biopsies must be interpreted carefully, such that a determination of RAS mutation status is most informative in the presence of truncal APC and/or TP53 mutations.