Tumors Harboring KRAS Mutations Research Articles

Abstract B085: The HIF-2 transcription factor mediates resistance to ferroptosis in pancreatic adenocarcinoma

Abstract Ferroptosis is an iron-dependent form of cell death converging on lipid peroxidation, first identified by examining compounds with enhanced lethality to KRAS mutant cells. Two compounds widely used as ferroptosis inducers are erastin, which acts as an inhibitor of cystine import and thereby depletes cellular glutathione, and RSL-3, which inhibits glutathione peroxidase 4. Despite over 90% of pancreatic adenocarcinoma (PDAC) tumors harboring KRAS mutations, PDAC exhibits relative resistance to ferroptosis compared to other tumor types, and the mechanisms behind this resistance remain unclear. The pancreatic tumor environment is marked by severe hypoxia, due to poor vascularity and a dense fibrotic stroma, and so we hypothesized that hypoxia might mediate resistance to ferroptosis. Here we report that across a panel of human and murine PDAC cell lines, hypoxia induces resistance to both ferroptosis inducing agents, erastin and RSL-3. The effect of hypoxia was synergistic with exposure to pancreatic cancer tumor interstitial fluid, which induces a hypoxic-like gene expression profile even under normoxia and enhances hypoxic gene expression changes when combined with hypoxia. Our findings reveal that hypoxia-mediated resistance to ferroptosis is orchestrated by the hypoxia-inducible transcription factor HIF-2, as evidenced by its complete abolition in HIF-2 knockout cell lines, while the protective effect of hypoxia was maintained in HIF-1 knockout lines. RNA-seq analyses and chromatin immunoprecipitation studies (chIP) identified several HIF-2 target genes responsible for coordinating ferroptosis resistance mechanisms. Cysteine is a precursor for glutathione, and HIF-2 increased cystine import through upregulation of both components of the cystine-glutamate antiporter system Xc (SLC7A11 and SLC3A2) and increased cysteine biosynthesis via upregulation of transsulfuration pathway enzymes (CBS and CTH). In addition, HIF-2 upregulated Parkin, which we identify as a novel regulator of ferroptosis, thereby increasing mitophagy and decreasing reactive oxygen species. In vivo studies using human and murine PDAC xenograft models confirmed that HIF-2 knockout impaired tumor growth and increased susceptibility to the pro-ferroptotic agent sulfasalazine. Collectively, these data identify a mechanism by which PDAC evades ferroptosis and identifies a possible therapeutic strategy of combining HIF-2 inhibitors with pro-ferroptotic agents in pancreatic cancer. Citation Format: Maimon E Hubbi, Catherine Wang, Erin Hollander, Yasir Suhail, Kshitiz, Alexander Muir, Ben Z Stanger, Chi V Dang. The HIF-2 transcription factor mediates resistance to ferroptosis in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B085.

Abstract C027: Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models

Abstract Background/Objective: Prognosis remains grim for localized or metastasized pancreatic ductal adenocarcinoma (PDAC) with 5-year survival rates of 3-16%. More than 90% of PDAC cases harbor KRAS mutations. KRASG12C inhibitors recently developed have limited use for PDAC because only 3% of cases express this mutation. We developed a novel pan-RAS inhibitor, ADT-1004 and evaluated antitumor activity in tumor models established from murine or human PDAC cell lines or patient-derived xenografts (PDX). ADT-1004 is an orally bioavailable prodrug of ADT-007, a highly potent and selective pan-RAS inhibitor (doi.org/10.1101/2023.05.17.541233). Methods: Murine PDAC cells harboring the KRASG12D mutation (KPC-Luc or 2838C3-Luc) were orthotopically implanted into the pancreas of C57BL/6J mice. Four PDX PDAC tumors harboring KRAS mutations were implanted orthotopically in NSG mice. To assess potential to overcome RAS inhibitor resistance, parental KRASG12C MIA PaCa-2 PDAC cells and a resistant derivative of MIA PaCa-2 cells were implanted subcutaneously. To assess selectivity, mice were implanted with RASWT BxPC-3 cells SC. Mice were treated orally with ADT-1004 (40mg/kg) 5x/week for 4 weeks. Sotorasib and adagrasib KRASG12C inhibitors were administered at the same dose, route, and schedule as ADT-1004. Results: ADT-007 potently and selectively inhibited the growth of human and mouse PDAC cell lines in vitro. For example, the IC50 value of ADT-007 for KRASG12C MIA PaCa-2 PDAC cells was 2 nM compared to 2500 nM for RASWT BxPC-3 PDAC cells. Growth inhibition by ADT-007 was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-1004 was well tolerated in mice at doses up to 175 mg/kg bid orally with sustained plasma levels of ADT-007 exceeding growth IC50 values. ADT-1004 displayed robust antitumor activity in mouse tumor models using human- or mouse-derived PDAC cell lines with G12D, G12V, G12C, or G13Q KRAS mutations, causing selective inhibitory effects on ERK phosphorylation in tumors but not in normal tissues. ADT-1004 also inhibited growth of KRASG12C mutant MIA PaCa-2 tumors to a comparable extent as sotorasib and adagrasib. Sotorasib and adagrasib were ineffective against xenografted tumors of resistant cells derived from MIA PaCa-2 cells, while ADT-1004 strongly inhibited tumor growth. Target selectivity of ADT-1004 was confirmed in vivo, as ADT-1004 did not affect the growth of RASWT BxPC-3 tumors. Conclusions: ADT-1004 inhibited tumor growth of KRAS mutant PDAC tumors by blocking activated RAS to disrupt downstream MAPK/AKT signaling. Side-by-side comparisons reveal antitumor efficacy advantages of ADT-1004 over mutant-specific KRAS inhibitors. These results highlight the promise of ADT-1004 for treatment of PDAC and other RAS-driven cancers by addressing the complex genetic landscape of many cancers and limitations from resistance to mutant specific KRAS inhibitions. Citation Format: Dhana Sekhar Reddy Bandi, Ganji P Nagaraju, Jeremy B Foote, Adam B Keeton, Xi Chen, Kristy L Berry, Yulia Y Maxuitenko, Upender Manne, Donald J Buchsbaum, Gary A Piazza, Bassel F El-Rayes. Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C027.

Abstract 1083: MEKi and SOS1i as a combination treatment option for KRAS mutant endometrial cancer

Abstract Endometrial cancer (EC) is the most common cancer of the female genital tract, resulting in over 75,000 deaths per year worldwide. Despite recent advances in medical oncology research, chemo- and radiotherapy are still the standard of care treatments for recurrent or metastatic EC and prognosis for these patients remains poor, with median survival rates of less than one year. Approximately 15-30% of the more common type I EC harbors oncogenic KRAS mutations, which have been linked to disease progression. Preclinical studies have shown that MEK inhibitors exhibit strong anti-tumor activity in KRAS mutant EC models. In the clinical setting however, MEKi monotherapy has proven unsuccessful in KRAS mutant EC patients, suggesting that combinations with other targeted agents may be needed to achieve increased signaling modulation and enhanced therapeutic activity. As suggested by our previous work in KRAS mutant lung, colorectal and pancreatic cancer, the combined inhibition of MEK and SOS1 may overcome adaptive resistance to MEKi by blocking SOS1-mediated reactivation of MAPK signaling. We evaluated whether this hypothesis can also be applied in the context of EC. Here we treated preclinical models of EC with a combination of clinically relevant doses of a MEKi (trametinib) and BI-3406, a potent and selective orally bioavailable SOS1 inhibitor. Treatment of KRAS mutant EC cells resulted in decreased activity of the KRAS-MAPK pathway, as measured by ERK phosphorylation. This effect was further enhanced by co-treatment with BI-3406. In vivo, the combination of MEKi with SOS1i synergistically decreased tumor growth in two KRAS G12D mutant orthotopic patient-derived xenograft models of EC. The anti-tumor effect of the combination was comparable to the effect of paclitaxel, the standard of care treatment in EC. This suggests that the combination of MEKi with SOS1i may be a potential therapeutic approach for the treatment of EC patients with tumors harboring KRAS mutations. Citation Format: Kaja Kostyrko, Melanie Hinkel, Marco H. Hofmann, Francesca Trapani. MEKi and SOS1i as a combination treatment option for KRAS mutant endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1083.

Comprehensive characterization of KRAS mutations and inter-relation with primary tumor location in colorectal cancers.

231 Background: The recent development of KRAS G12C inhibitors underscores the potential to target KRAS mutations. Right-sided and left-sided colon tumors (RT and LT) exhibit different molecular features. We characterize the prevalence of KRAS-variants, interrelation with primary tumor location, and association with immune biomarkers in CRC. Methods: We retrospectively reviewed CRC tumors of all stages (with known sidedness) that underwent NGS with the Tempus xT assay (DNA-seq of 648 genes at 500x coverage, full transcriptome RNA-seq). Bivariate analyses were performed to compare KRAS alterations, immune biomarkers, and co-mutations by tumor location. P-values comparing individual co-mutations between groups were adjusted for false discovery (FDR). Results: A total of 3,391 CRC were analyzed (RT: n = 442 [13%], transverse: n = 116 [3%], LT; n = 2,833 [84%]) of which 1486 (44%) tumors harbored KRAS mutations. Overall, KRAS mutations were more frequent in RT compared to transverse tumors and LT (52% vs 41% vs 43%, p<0.001, respectively). The most frequent KRAS mutation variants observed were G12D (29 %), G12V (22%), G13D (16%), and G12C (5.7%). There was no significant difference in the prevalence of KRAS variant types between LT and RT (p=0.5). Significant differences in genomic co-mutations with various KRAS variants were observed in the following genes: TP53, FBXW7, and NF1 (FDR- P<0.05). RT and transverse tumors were more likely have MSI-H and TMB-H (>10 mut/mb) status than LT (MSI-H: 18% vs 22% vs 2.2% and TMB-H (20% vs 22% vs 3%, P<0.001), respectively. CRC tumors harboring G13D variants were more likely to be associated with and MSI-H and TMB-H status (and 7.7% and 8.5%) compared to G12D (2.8 % and 3.9 %), G12V (1.8 % and 2.1%), and G12C (0% and 2.4%); P = 0.003 and 0.001. Conclusions: The most frequent KRAS mutation variants observed in CRC tumors were G12D, G12V, G13D, and G12C. There was no significant difference in the prevalence of KRAS variant types between tumors of the left vs right colon. CRC tumors that harbored G13D variants were significantly more likely to be associated with MSI-H and TMB-H status. [Table: see text]

Impact of KRAS mutation variants on clinicopathological features and outcomes in patients with metastatic pancreatic adenocarcinoma.

749 Background: Most pancreatic ductal adenocarcinoma (PDAC) exhibit KRAS mutations. A subset of PDACs harbor KRAS wild-type (WT) and appear to carry better prognosis. Given the recent development of KRAS G12C inhibitors, it is imperative to better understand KRAS variants in contemporary cohorts. Herein, we characterized the prevalence of the different KRAS variants and its impact on clinicopathological features and outcomes. Methods: A retrospective review of patients with metastatic PDAC and a known KRAS mutation status who underwent next-generation sequencing (NGS) from liver biopsy was performed. Descriptive statistics analyzed the differences in clinicodemographic features by presence and type or absence of KRAS variants. Kaplan-Meier method was used to assess overall survival (OS). Cox regression was used to study the relationship between KRAS variants and OS when confounding factors were adjusted. Results: 50 pts with PDAC diagnosed at a single community-based institution between 2011-2018 were evaluated. Median age at diagnosis was 68 years (24-80 range), 68% were male, and 76% presented with metastases. Most (86%) tumors harbored KRAS mutations. The most frequent variants were G12V (44%), G12D (37%), G12R (9%), Q61R (5%), G12C (2%), Q61H (2%). Sex (p=0.03) and tumor differentiation (p=0.04) varied according to mutation status and KRAS variant subtype. However, no significant difference was observed in age, race, smoking status, location of primary, and performance status (PS) in KRAS variants. Overall, median OS in months was 8.9 (95% CI 6.7-14.3) for KRAS mutated patients and 11.2 (95% CI 2.1-33.5) for patients with WT. Among patients with KRAS mutations, median OS in months by variant was as following: G12V 9.6 (95%CI 6.6-17.2), G12D 8.4 (95%CI 3.9-15.4), G12R 8.2 (95%CI 1-20.8), and Q61R 11.8 (95%CI 1.3-22.3). However, these differences did not reach statistical significance (p=0.72). In multivariable analyses, female sex (HR 0.16, 95% CI 0.03-0.81, p=0.03) and no receipt of chemotherapy (38.5 95% CI 2.1-698.50, p=0.01) were significant predictors of death. KRAS mutation variant type was not an independent predictor of death. Conclusions: No significant difference was observed in metastatic PDAC with KRAS mutations compared to WT. However, numerical difference in OS were observed among the various KRAS mutation type. Hence, larger studies are needed to better define the effect of KRAS type on outcomes.

Extensive genomic analysis in patients with KRAS-mutated solid tumors shows high frequencies of concurrent alterations and potential targets but has limited clinical impact

Background This study aimed to investigate the distribution and frequency of concurrent alterations in different cancers across KRAS subtypes and in different KRAS subtypes across cancers, and to identify potentially actionable targets and patients who received targeted treatment matched to their genomic profile (GP). Materials and Methods In this descriptive and single-center study, we included 188 patients with solid tumors harboring KRAS mutations in codon 12, 13, 61, 117, or 146, referred to the Phase 1 Unit, Rigshospitalet, Copenhagen, Denmark from mid-2016 to 2020. Genomic co-alterations were detected with whole-exome sequencing, RNA sequencing, SNP array, and mRNA expression array on fresh biopsies. The study is part of the Copenhagen Prospective Personalized Oncology study (NCT02290522). Results The majority of patients had colorectal cancer (60.1%), non-small cell lung cancer (11.2%), or pancreatic cancer (10.6%). Most tumors were KRAS-mutated in codon 12 or 13 (93.7%) including G12D (27.1%), G12V (26.6%), G12C (11.7%), and G13D (11.2%). A total of 175 different co-alterations were found, most frequently pathogenic APC and TP53 mutations (55.9% and 46.4%, respectively) and high expression of CEACAM5 (73.4%). Different cancers and KRAS subtypes showed different patterns of co-alterations, and 157 tumors (83.5%) had potentially actionable targets with varying evidence of targetability (assessed using ESMO Scale for Clinical Actionability of molecular Targets). Of the 188 patients included in the study, 15 (7.4%) received treatment matched to their GP (e.g., immunotherapy and synthetic lethality drugs), of whom one had objective partial response according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Conclusion Performing extensive genomic analysis in patients with known KRAS-mutated solid tumors may contribute with information to the genomic landscape of cancers and identify targets for immunotherapy or synthetic lethality drugs, but currently appears to have overall limited clinical impact, as few patients received targeted therapy matched to their GP.

EP16.03-025 Immunogenomics of Clinical Resistance to KRAS G12C Inhibition

Activating mutations in KRAS are a frequent source of oncogenesis but have lacked clinical targeted therapies despite being first identified four decades ago. Selective drugs for the G12C KRAS mutation have recently been approved for use in the United States, and have been granted conditional marketing authorization in the EU. Pre-clinical data indicate that tumors harboring KRAS mutations can rapidly develop resistance to targeted inhibition, and non-durable responses have been observed in clinical use.

The prevalence of common KRAS variants and associated outcomes in patients with metastatic colorectal cancer (mCRC).

173 Background: KRAS is the most common driver oncogene in mCRC. Comprehensive analysis of KRAS variants prevalence and the relationship between variants and outcomes are lacking. Herein, we aimed to examine the impact of KRAS variants on outcomes in patients (pts) with mCRC. Methods: A retrospective review of pts with mCRC with known KRAS mutation status was performed. Fisher’s exact test was used to analyze the association between KRAS variants. Cox Proportional Hazard modeling was used to study the relationship between KRAS variants and overall survival (OS). Kaplan-Meier method was used to assess OS. Results: A total of 423 pts diagnosed with mCRC from 2014-2020 with available extended KRAS status were evaluated. Median age at diagnosis was 59.8 yrs (22.3-92 range), 57% were male, 22% were Black, and 75% presented with de novo metastatic disease. A majority (56%) of tumors harbored KRAS mutations. The most frequent KRAS variants were G12D 47% (111), G12V 12% (28), G12C 13% (31), G13D 9% (21), and 20% (47) were other variants. In univariate analyses, the presence of a KRAS mutation, Black race, de novo metastatic disease, age, receipt of chemotherapy, and receipt of surgery were associated with OS. Tumor location was not associated with OS. In multivariable analyses, mutation type was a significant predictor of death and the presence of G12D was associated with an increased risk of death compared to G12V and KRAS wildtype. There was no increased risk of death in pairwise comparisons of G12D and G13D or other KRAS variants. Black race, de novo metastatic disease, and no receipt of surgery were additional independent predictors of death (Table). With a median follow-up of 25.7 months (mo.), median OS was 35.5 mo. (95% CI 10.5-50.9) with G12C, Not Reached (NR) (95% CI 21-NR) with G12V, 36.2 mo. (95%CI 14.9-58.5) with G13D, 26.2 mo. (95% CI 21.8-37) with G12D, 39.6 mo. (95% CI 22.4-47.9) for other KRAS mutations, and 59.6 mo. (95%CI 48.2-NA) for KRAS wildtype, respectively (p=0.0003). Conclusions: Our findings highlight differences in unadjusted overall survival when comparing G12D to some other KRAS variants. Codon and amino acid-specific mutations of KRAS should be considered when evaluating prognosis and further study on treatment effects and sequencing is warranted. [Table: see text]

Mesonephric-like Differentiation of Endometrial Endometrioid Carcinoma: Clinicopathological and Molecular Characteristics Distinct from Those of Uterine Mesonephric-like Adenocarcinoma.

When diagnosing endometrial carcinoma cases, we encountered histological features that strikingly resembled uterine mesonephric-like adenocarcinoma (MLA), but the differential diagnosis remained challenging after performing immunostaining. Considering the aggressive biological behavior and poor prognosis of uterine MLA, we believe that the accurate recognition of mesonephric-like differentiation (MLD) is important in the diagnosis of endometrial carcinoma. We aimed to investigate the clinicopathological and molecular characteristics of such cases and compared them with those of uterine MLAs. Five patients diagnosed with endometrioid carcinoma (EC) with MLD were included in this study. Histological evaluation, immunostaining, and targeted sequencing were performed. All five tumors showed typical morphological features of MLA, including densely aggregated tubular structures, deep basophilia under low-power magnification microscopy, eosinophilic intraluminal secretions, and diverse growth patterns. Immunostaining revealed moderate-to-strong nuclear immunoreactivity for estrogen and progesterone receptors in more than 50% tumor cells. The staining intensities and proportions of PAX2 and GATA3 were variable. None of the tumors harbored KRAS mutations. Considering the prognostic implications, ancillary tests, including immunostaining and targeted sequencing, should be performed to accurately differentiate between endometrial EC-MLD and uterine MLA.

KRAS mutations in patients with colorectal cancer in Libya.

Large prospective clinical trials have demonstrated that colorectal cancers (CRCs) with wild-type KRAS respond favorably to anti-epidermal growth factor receptor treatment, thus making mutational analysis obligatory prior to treatment. In our study, frozen CRC tissues from Libyan patients were analyzed for KRAS and HRAS mutations in codons 12/13 by direct sequencing and the correlations with clinical and pathological parameters were investigated. A total of 34 CRC cases, comprising 19 men and 15 women (age range, 24-87 years), were subjected to systematic analysis for RAS mutations. Although HRAS mutations were not detected in any of the patients in the study group, KRAS codon 12/13 mutations were present in 38.2% (13/34) of the patients. The frequent types of codon 12 mutations were glycine to aspartate (G12D, 46.1%); glycine to valine (G12V, 30.8%) and glycine to cysteine (G12C, 15.4%), while the codon 13 mutations were glycine to aspartate (G13D, 7.7%). G→A mutations occurred in 53.8% (7/13) of the patients, while G→T mutations occurred in 46.2% (6/13) of the patients. Mutations occurred at the first base of codon 12 or 13 in 2/13 (15.4%) and at the second base in 11/13 (84.6%) patients. There was no significant association between clinicopathological characteristics and KRAS mutation status, except the site of the tumors harboring KRAS mutations, which was as follows: The frequency was higher among tumors located in the left colon (8/13, 61.5%) compared to other sites (P=0.027). KRAS mutations were correlated with advanced age, with 10/13 being aged >50 years and affected 8/15 female patients (53%) compared with 5/19 male patients (26%). The highest frequency of KRAS mutations was observed in highly differentiated CRCs (8/13).

Landscape of KRASG12C, associated genomic alterations, and interrelation with immuno-oncology (IO) biomarkers.

3127 Background: Sotorasib has shown promising activity in cancer patients (pts) specifically harboring the KRASG12C mutation. Response rates vary significantly by tumor type, suggesting KRASG12C pathogenesis may be cancer-type-dependent. Methods: We retrospectively analyzed de-identified records of 79,004 pts with various cancer types that underwent Tempus xT and xF next generation sequencing assays. Fisher’s exact test was used to analyze the association between cancer subtypes and KRAS variants. Logistic regression was used to study co-mutations between KRASG12C and other oncogenes, as well as the association between KRAS variants and IO biomarkers. False discovery rate-adjusted P-value (FDR P) was used for multiple testing. Results: In total, 13,578 (17.4%) tumors harbored KRAS mutations, of which 1,632 were G12C; 750 KRAS wild-type (WT) tumors gained KRAS mutation on follow-up testing, with 79 harboring G12C. The most frequent KRAS variants across all cancers were G12D, G12V, G12C, and G13D (see Table). The distribution of KRAS variants significantly varied by cancer type, with G12C and G12D being the most prevalent in non-small cell lung (NSCLC) and colorectal (CRC) cancers, respectively. G12C was most prevalent in NSCLC (9%), appendiceal cancer (3.9%), CRC (3.2%), tumors of unknown origin (1.6%), and pancreatic cancer (1.3%). Compared to non-G12C, G12C was more frequently identified in females (56% vs. 51%, FDR P = 0.0005), smokers/prior smokers (85% vs. 56%, FDR P < 0.0001), and pts > 60 years of age (73% vs. 63%, FDR P = 0.0006). While no G12C tumors exhibited BRAFV600E co-mutations, BRAFnon-V600E co-mutations were seen in 3.1% of pts. Significant differences were observed in genomic alterations co-occurring with G12C compared to non-G12C (e.g., STK11 (20.6% vs 6%), KEAP1 (15.4% vs 4.6%), SMAD4 (7.2% vs 19%), and PDGFRA (5.8% vs 3%); FDR P < 0.0001). However, G12C and oncogene co-mutations were not significantly different between NSCLC and CRC, except for KEAP1 (FDR_ P = 0.04). Compared to non-G12C and WT, G12C tumors were associated with TMB-High and PD-L1 over expression but were less likely to have MSI-H status (FDR P < 0.0001; Table). Conclusions: Our data suggest that KRAS variants significantly differ by cancer type. Tumors harboring KRASG12C were significantly associated with high TMB and PD-L1 overexpression. KRASG12C mutation appeared to be associated with smoking status. These results may guide future therapeutic strategies.[Table: see text]

A phase I, open-label, dose-escalation trial of BI 1701963 as monotherapy and in combination with trametinib in patients with KRAS mutated advanced or metastatic solid tumors.

TPS3651 Background: Activating mutations of KRAS drive many types of cancer. Activation of KRAS relies on guanine nucleotide exchange factors, such as SOS1, to mediate exchange of GDP for GTP. BI 1701963 is a small-molecule protein–protein interaction inhibitor that prevents the interaction between KRAS and SOS. Binding of BI 1701963 to the catalytic site of SOS1 inhibits binding of SOS1 to RAS–GDP, thereby hindering the exchange from RAS–GDP (inactive form) to RAS–GTP (active form). In preclinical studies this has been shown to lead to cytostasis in cancer cells addicted to KRAS signaling. Methods: NCT04111458 is a first-in-human trial of BI 1701963 in patients aged ≥18 years with tumors harboring KRAS mutations. Primary objectives are to determine the maximum tolerated dose (MTD) and recommended Phase II dose of BI 1701963 as monotherapy and in combination with trametinib, based on dose-limiting toxicities (DLTs). Secondary objectives are to evaluate safety, tolerability, pharmacokinetics/-dynamics and preliminary efficacy. The study will have two arms (mono- and combination therapy), and be divided into dose escalation (Part A), confirmation (Part B) and expansion (Part C, combination only) phases. Inclusion criteria include activating KRAS mutation, ≥1 evaluable lesion (RECIST 1.1), ECOG PS ≤1 and adequate organ function. Exclusion criteria include history of: RAS, MAPK or SOS1 targeting therapies; retinal vein occlusion; retinal pigment epithelial detachment; and decreased cardiac function. Parts B and C will be conducted in patients with advanced NSCLC. Treatment will continue until confirmed clinical benefit, defined toxicities, or withdrawal of consent. The primary endpoints are: Part A, the MTD, and the number of patients with DLTs during Cycle 1; Part B (monotherapy), the number of patients with DLTs; Part C, objective response (OR, RECIST 1.1). Starting doses of BI 1701963 in Part A will be 50 mg once daily (QD) orally (monotherapy) and 100 mg QD (combination, once proved safe as monotherapy) and will be escalated until the MTD is reached. The starting dose of trametinib will be 1 mg QD, escalated to the MTD or a max. of 2 mg QD. Dose cohorts will include ≥3 patients, with two therapeutic relevant doses (TRDs) established in each arm. In Part B, patients will be randomized to groups receiving one of the TRDs. If an OR is observed at a TRD in the combination arm, additional patients will be recruited into expansion cohorts receiving the relevant dose. As of Feb 11, 2020 three patients have been treated. Clinical trial information: NCT04111458 .

A Ras destabilizer KYA1797K overcomes the resistance of EGFR tyrosine kinase inhibitor in KRAS-mutated non-small cell lung cancer

The epidermal growth factor receptor (EGFR) inhibitors such as erlotinib and gefitinib are widely used for treatment of non-small cell lung cancer (NSCLC), but they have shown limited efficacy in an unselected population of patients. The KRAS mutations, which are identified in approximately 20% of NSCLC patients, have shown to be associated with the resistance to the EGFR tyrosine kinase inhibitors (TKIs). Currently, there is no clinically available targeted therapy which can effectively inhibit NSCLC tumors harboring KRAS mutations. This study aims to show the effectiveness of KYA1797K, a small molecule which revealed anti-cancer effect in colorectal cancer by destabilizing Ras via inhibiting the Wnt/β-catenin pathway, for the treatment of KRAS-mutated NSCLC. While erlotinib fail to have anti-transforming effect in NSCLC cell lines harboring KRAS mutations, KYA1797K effectively inhibited the Ras-ERK pathway in KRAS-mutant NSCLC cell lines. As a result, KYA1797K treatment suppressed the growth and transformation of KRAS mutant NSCLC cells and also induced apoptosis. Furthermore, KYA1797K effectively inhibited Kras-driven tumorigenesis in the KrasLA2 mouse model by suppressing the Ras-ERK pathway. The destabilization of Ras via inhibition of the Wnt/β-catenin pathway is a potential therapeutic strategy for KRAS-mutated NSCLC that is resistant to EGFR TKI.

Abstract 2987: Identification of new combination therapies for lung tumors harboring KRAS mutations

Abstract Oncogenic mutations in KRAS are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor prognosis and resistance to existing therapies. Thus, new therapeutic strategies are needed to treat these tumors. We have previously shown that NSCLC cells harboring KRAS mutations are more sensitive to inhibition of KRAS downstream effectors MEK and RAF and to treatment with IGF1R inhibitors than their wild-type counterparts. In order to identify complementary targets for the improvement of IGF1R and/or MEK targeting therapies, we performed a whole-genome shRNA screen in KRAS-mutant NSCLC cells. Interestingly, the list of sensitizers to IGF1R inhibitors included several genes encoding components of the mTOR pathway. Viability assays in a panel of lung cancer cell lines confirmed that combinations of IGF1R inhibitors with mTOR inhibitors, both rapalogs and kinase inhibitors, resulted in a synergistic anti-proliferative effect in KRAS-mutant NSCLC cells. Mechanistic investigations demonstrated some differences between the effects of rapalogs and mTOR kinase inhibitors although, in both cases, mTOR inhibitors increased IGF1R and AKT phosphorylation. Combination with IGF1R inhibitors blocked the reactivation of the PI3K pathway resulting in a robust suppression of PI3K and mTORC1 signaling. Notably, the inhibition of these signaling pathways was stronger in KRAS-mutant cells than in wild-type cells. Addition of a MEK inhibitor to the combination produced a more profound and durable suppression of cell proliferation and a stronger induction of apoptosis. Finally, we validated the different drug combinations in mouse models of KRAS-induced NSCLC. Results showed that the three-drug combination produced a marked tumor regression in a NSCLC mouse model driven by mutant Kras and p53 loss-of-function and also in urethane-induced lung tumors. These findings suggest potential novel therapeutic strategies for NSCLC tumors harboring KRAS mutations. Citation Format: Miriam Molina-Arcas, Christopher Moore, Sareena Rana, Febe van Maldegem, Stuart Horswell, David Hancock, Julian Downward. Identification of new combination therapies for lung tumors harboring KRAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2987.

A KRAS wild type mutational status confers a survival advantage in pancreatic ductal adenocarcinoma.

The KRAS oncogene is a driver mutation and is present in greater than 90% of pancreatic ductal adenocarcinomas (PDAC). A subset of these tumors, however, do not harbor mutations in KRAS (wild type KRAS). Studies have shown that patients with mutated KRAS have a poorer survival on first-line gemcitabine-based chemotherapy compared to wild type KRAS. In this study, we examined a cohort of patients with PDAC at our institution who were either wild type or mutant for the KRAS gene and assessed for differences in survival and response to different chemotherapeutic regimens. We examined clinical records of patients treated at the Abramson Cancer Center of the University of Pennsylvania from 2013 to 2017. Patients with a pancreatic mass and a histologic diagnosis of pancreatic or pancreaticobiliary adenocarcinoma were identified. Thirty-nine patients with PDAC who underwent tumor sequencing at Penn Medicine's Center for Personalized Diagnostics (CPD) were selected for further study. Twelve patients were identified whose tumors were KRAS wild type. Twenty-seven patients with PDAC whose tumors harbored KRAS mutations were selected as controls (KRAS mutant). We noted a longer overall survival (OS) among KRAS wild type patients compared to KRAS mutant patients (P=0.026). This was independent of the age at diagnosis, patient gender, stage of diagnosis, tumor morphology, mismatch repair (MMR) status, and chemotherapeutic regimen. Similar to previously reported studies, PDAC with a KRAS wild type mutational profile has a better prognosis with a longer OS. This improved prognosis is independent of the protocol utilized in therapy for these patients. Our findings suggest that future clinical trials in pancreatic cancer should take into consideration the presence of KRAS mutations in their pre-planned analysis when assessing the efficacy of a novel therapeutic approach. This may be a crucial factor in trial concepts and outcomes.

Targeted next generation sequencing in Chinese colorectal cancer patients guided anti-EGFR treatment and facilitated precision cancer medicine.

ObjectiveColorectal cancer (CRC) patients with both RAS and BRAF wild-type tumors determined by non-next generation sequencing (NGS) testing may still not respond due to the presence of additional mutated genes such as PIK3CA or PTEN. In this study, a broad, hybrid capture-based NGS assay was used to identify RAS, BRAF and additional targetable genetic alterations from Chinese CRC tissues.MethodsFifty-seven cases of CRC were enrolled, and all the patients signed the informed consent. In total, 7708 exons of 508 tumor-related genes and 78 introns of 19 frequently rearranged genes were assessed for base substitutions, INDELs, copy number alterations, and gene fusions.ResultsThe study found that 50.9% (29/57) of the tumors harbored KRAS mutations, 3.5% (2/57) harbored NRAS mutations and 3.5% (2/57) harbored BRAF mutations. More specifically, 89.7% (26/29) of RAS mutations were located in codon 12. Except for RAS and RAF, anti-EGFR therapy response genetic mutations in PTEN (n=2) and PIK3CA (n=1) were found in 4.7% (3/64) of the samples. Actionable alterations were found in HER2 (n = 7), CCND2 (n = 2), NF1 (n = 1), and BRCA1 (n = 1).ConclusionsOur results illustrated that 82.5% (47/57) of the samples harbored at least one actionable genetic alteration identified by NGS. HER2 amplifications or mutations, which were identified in 12.3% of the tissues, defined a unique molecular subtype of CRC. The study suggests that high-throughput NGS testing in CRC tissues is a comprehensive and efficient genomic profiling assay to guide personalized therapy.

Abstract 2799: Development of a mouse model of KRAS mutated colorectal cancer tumorigenesis

Abstract Introduction Development of a preclinical animal model that mimics KRAS mutated colorectal cancer disease etiology is essential for validation of new therapeutic interventions. Colorectal cancer patients with tumors harboring KRAS mutation are excluded from receiving anti EGFR monoclonal antibodies, and currently have no alternate FDA approved treatment options available. Ongoing research requires validations of different modalities on a robust preclinical study platform that will resemble the disease phenotype and genetic profile. In this context we have developed viable animal mouse model of colorectal cancer bearing KRAS mutation that authentically serves as a model for the disease. Methodology: The C57 BL/6 mice with truncated APC floxed allele was crossed with heterozygous KRAS floxed outbred mice to generate APC f/f KRAS +/f mouse colony. In another set of breeding APC floxed mice were crossed with CDX2-Cre-ERT2 mice and selected for APCf/f CDX2-Cre-ERT2 after second round of inbreeding. The final model of the disease was generated by the cross of the two aforementioned colonies when viable APC f/f KRAS +/f CDX2-Cre-ERT2 were genotyped and characterized. The model animals were tamoxifen induced to generate tumors. Micro-PET scan was used to detect and measure tumor volume. H & E staining was done to establish neoplasm and immunohistochemistry was performed to determine histological similarities with human FFPE biopsies. The MSI/MSS status was also determined. Results Ø The newly developed animal model develops colonic tumors upon induction with Tamoxifen. Ø The tumors were malignant as confirmed by H and E staining. Ø The tumors showed a higher radioactive FDG uptake (SUV) in micro-PET scan Ø The tumors resembled human colorectal cancer tissue Ø Once tumor is generated the animals died of cachexia and rectal bleeding Conclusion: Heterozygous mutation of KRAS oncogene along with deletion mutation of Adenomatous polyposis coli (APC) gene is embryonically lethal in animal models. We have successfully developed a viable animal model of KRAS mutated colorectal cancer with tamoxifen inducible tumors harboring KRAS and APC mutations. The tumors mimic the human disease type both genetically and immunohistochemically. This model can serve as a robust preclinical platform for understanding the disease mechanism at the molecular level as well for evaluation of various therapeutic interventions. Citation Format: Radhashree Maitra, Madhu Kumar Venkatesh, Titto Alby Augustine, Carol Chandy, Qiang Liu, Sanjay Goel. Development of a mouse model of KRAS mutated colorectal cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2799. doi:10.1158/1538-7445.AM2017-2799

Abstract CT046: Phase I dose escalation study of the CDK4/6 inhibitor palbociclib in combination with the MEK inhibitor PD-0325901 in patients with RAS mutant solid tumors

Abstract Background: Both MEK and CDK4/6 have been investigated as therapeutic targets in preclinical models of RAS mutant solid tumors. Multiple mechanisms contribute to synergism including the development of MAP kinase-dependence in RAS mutant cells exposed to CDK4/6 inhibition, leading to increased apoptosis, as well as enhanced induction of senescence with combinatorial vs. monotherapy treatment. We conducted a Phase 1 study of combined palbociclib and PD-0325901 in patients with RAS mutant solid tumors to assess safety, tolerability, and MTD, as well as pharmacokinetic parameters, preliminary efficacy and effects on mutant RAS allelic burden in plasma. Methods: Patients with RAS mutant solid tumors were enrolled utilizing a 3 + 3 design. Palbociclib and PD-0325901 were given orally once and twice daily, respectively for three of every four weeks. The maximum planned administered doses were 125 mg palbociclib daily and 8 mg PD-025901 twice daily. Pharmacokinetic parameters were measured on cycle 1 day 21 and plasma samples to measure RAS allelic burden were serially collected. Results: Twenty-five patients (17 with KRAS mutant NSCLC) who received at least one dose of each study drug were enrolled over 5 dose levels including (palbociclib/PD-0325901) 75/2, 75/4, 100/4, 125/4 and 125/8. The maximum administered dose of 125 mg palbociclib daily and 8 mg twice daily PD-0325901 was tolerable. One DLT of pneumonitis occurred at the 100/4 dose level. The most frequent (>10%) drug-related toxicities were leukopenia (72%), anemia (72%), thrombocytopenia (72%), neutropenia (64%), acneiform rash (64%), diarrhea (52%), fatigue (44%), lower extremity edema (32%), vomiting (28%), nausea (28%), oral mucositis (24%), increased AST (20%), increased creatinine (12%), epistaxis (12%), and blurred vision (12%). The median number of cycles completed was 3 (range: 1 - 28+). Across all patients, 1 patient (4%) with KRAS mutant NSCLC achieved partial response and 18 (72%) had stable disease as the best response. Eleven patients were progression-free > 3 months, and 6 were progression-free > 6 months, including 5 with KRAS mutant NSCLC, two of whom received prior immune checkpoint blockade. Among patients with KRAS mutant NSCLC, clinical benefit was seen among those with tumors harboring KRAS mutation alone, as well as those with tumors demonstrating concomitant loss of TP53 or CDKN2AB. A dose-dependent decrease in plasma RAS allelic burden was observed across dose levels. Conclusions: Administration of combined palbociclib and PD-0325901 was tolerable and produced promising progression-free survival among patients with KRAS mutant NSCLC. Additional dose levels utilizing continuous MEK inhibition are being evaluated. Citation Format: Geoffrey I. Shapiro, John Hilton, Leena Gandi, Nicole Chau, James Cleary, Andrew Wolanski, Adrienne Anderson, Brian Beardslee, Faith Hassinger, Ketki Bhushan, Elizabeth Downey, Joseph Gibson, Solida Pruitt-Thompson, Alona Muzikansky, Suzanne Barry, Nora Feeney, Cloud Paweletz, Geoffrey Oxnard, Jeffrey Supko, Pasi Jänne, Kwok-Kin Wong, Bruce Johnson. Phase I dose escalation study of the CDK4/6 inhibitor palbociclib in combination with the MEK inhibitor PD-0325901 in patients with RAS mutant solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT046. doi:10.1158/1538-7445.AM2017-CT046

Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement

Mutations in the KRAS gene, which persistently activate RAS function, are most frequently found in many types of human cancers. Here, we proposed and verified a new approach against cancers harboring the KRAS mutation with high cancer selectivity and efficient anti-cancer effects based on targeted RNA replacement. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically target and reprogram the mutant KRAS G12V transcript to induce therapeutic gene activity in cells. Adenoviral vectors containing the specific ribozymes with downstream suicide gene were constructed and then infection with the adenoviruses specifically downregulated KRAS G12V expression and killed KRAS G12V-harboring cancer cells additively upon pro-drugtreatment, but it did not affect the growth of wild-type KRAS-expressing cells. Minimal liver toxicity was noted when the adenoviruses were administered systemically invivo. Importantly, intratumoral injection of the adenoviruses with pro-drug treatment specifically and significantly impeded the growth of xenografted tumors harboring KRAS G12V through a trans-splicing reaction with the target RNA. In contrast, xenografted tumors harboring wild-type KRAS were not affected by the adenoviruses. Therefore, RNA replacement with a mutant KRAS-targeting trans-splicing ribozyme is a potentially useful therapeutic strategy to combat tumors harboring KRAS mutation.

STK11/LKB1 Deficiency Promotes Neutrophil Recruitment and Proinflammatory Cytokine Production to Suppress T-cell Activity in the Lung Tumor Microenvironment.

STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer (NSCLC), especially in tumors harboring KRAS mutations. Many oncogenes promote immune escape, undermining the effectiveness of immunotherapies, but it is unclear whether the inactivation of tumor suppressor genes, such as STK11/LKB1, exerts similar effects. In this study, we investigated the consequences of STK11/LKB1 loss on the immune microenvironment in a mouse model of KRAS-driven NSCLC. Genetic ablation of STK11/LKB1 resulted in accumulation of neutrophils with T-cell-suppressive effects, along with a corresponding increase in the expression of T-cell exhaustion markers and tumor-promoting cytokines. The number of tumor-infiltrating lymphocytes was also reduced in LKB1-deficient mouse and human tumors. Furthermore, STK11/LKB1-inactivating mutations were associated with reduced expression of PD-1 ligand PD-L1 in mouse and patient tumors as well as in tumor-derived cell lines. Consistent with these results, PD-1-targeting antibodies were ineffective against Lkb1-deficient tumors. In contrast, treating Lkb1-deficient mice with an IL6-neutralizing antibody or a neutrophil-depleting antibody yielded therapeutic benefits associated with reduced neutrophil accumulation and proinflammatory cytokine expression. Our findings illustrate how tumor suppressor mutations can modulate the immune milieu of the tumor microenvironment, and they offer specific implications for addressing STK11/LKB1-mutated tumors with PD-1-targeting antibody therapies.