KRAS Wild-type Cells Research Articles

Metabolic Response to Small Molecule Therapy in Colorectal Cancer Tracked with Raman Spectroscopy and Metabolomics.

Despite numerous screening tools for colorectal cancer (CRC), 25 % of patients are diagnosed with advanced disease. Novel diagnostic technologies that are early, accurate, and rapid are imperative to assess the therapeutic efficacy of clinical drugs and identify new biomarkers of treatment response. Here Raman spectroscopy (RS) was used to track metabolic reprogramming in KRAS-mutant HCT116 and SW837 cells, and KRAS wild-type CC cells. RS combined with multivariate analysis methods distinguished nonresponsive, partially responsive, and responsive cells treated with cetuximab, a monoclonal antibody for EGFR inhibition, sotorasib, a clinically approved KRAS inhibitor, and various doses of trametinib, an inhibitor of the MAPK pathway. Cells treated with a combination of subtoxic doses of trametinib and BKM120, an inhibitor of the PI3K pathway, showed a synergistic response between the two pathways. Using a supervised machine learning regression model, we established a scoring methodology trained to a priori predict therapeutic response to new treatment combinations. RS metabolites were verified with mass spectrometry, and enrichment pathways were identified, including amino acid, purine, and nicotinate and nicotinamide metabolism that differentiated monotherapy from combination therapy. Our approach may ultimately be applicable to patient-derived primary cells and cultures of patient tumors to predict effective drugs for individualized care.

Asparagine synthetase and G-protein coupled estrogen receptor are critical responders to nutrient supply in KRAS mutant colorectal cancer.

Survival differences exist in colorectal cancer (CRC) patients by sex and disease stage. However, the potential molecular mechanism(s) are not well understood. Here we show that asparagine synthetase (ASNS) and G protein-coupled estrogen receptor-1 (GPER1) are critical sensors of nutrient depletion and linked to poorer outcomes for females with CRC. Using a 3D spheroid model of isogenic SW48 KRAS wild-type (WT) and G12A mutant (MT) cells grown under a restricted nutrient supply, we found that glutamine depletion inhibited cell growth in both cell lines, whereas ASNS and GPER1 expression were upregulated in KRAS MT versus WT. Estradiol decreased growth in KRAS WT but had no effect on MT cells. Selective GPER1 and ASNS inhibitors suppressed cell proliferation with increased caspase-3 activity of MT cells under glutamine depletion condition particularly in the presence of estradiol. In a clinical colon cancer cohort fromThe Cancer Genome Atlas, both high GPER1 and ASNS expression were associated with poorer overall survival for females only in advanced stage tumors. These results suggest KRAS MT cells have mechanisms in place that respond to decreased nutrient supply, typically observed in advanced tumors, by increasing the expression of ASNS and GPER1 to drive cell growth. Furthermore, KRAS MT cells are resistant to the protective effects of estradiol under nutrient deplete conditions. The findings indicate that GPER1 and ASNS expression, along withthe interaction between nutrient supply and KRAS mutations shed additional light on the mechanisms underlyingsex differences in metabolism and growth in CRC, and have clinical implications in the precision management of KRAS mutant CRC.

Erlotinib Treatment in Colorectal Cancer Suppresses Autophagy Based on KRAS Mutation.

The KRAS gene is mutated in approximately 45% of colorectal cancer patients. There are currently very few targeted treatments or therapies equipped to directly inhibit KRAS due to its unusual structural intricacies. Erlotinib, an EGFR inhibitor, has previously been demonstrated to reduce cell viability by inducing autophagy in lung cancer cell lines with varying EGFR mutations. In contrast to lung cancer cells, evidence is provided herein for the first time that erlotinib treatment in colorectal cancer (CRC) cell lines reduces autophagy and still results in decreased cell viability. However, the effects of erlotinib in CRC cell lines containing a wildtype KRAS gene were different than in cells carrying a mutant KRAS gene. We show that there is significantly more downregulation of autophagy in KRAS mutant CRC cells compared to KRAS wildtype cells, both at transcriptional and translational levels, suggesting that the KRAS mutation is advantageous for cancer growth, even in the presence of erlotinib. Cell viability results determined that KRAS wildtype CRC cells had significantly more cell death compared to KRAS mutant cells. Using patient mRNA datasets, we showed that there was a significant correlation between the presence of the KRAS mutation and the expression of autophagy proteins. Additionally, through molecular dynamics simulations, we develop a blueprint for KRAS and autophagy protein interaction and the impact of the KRAS mutation on autophagy protein regulation. Overall, this is the first report of erlotinib treatment in CRC cells that assesses autophagy, and we demonstrate that autophagy activity is downregulated in these cells. This effect is not only greater in cells carrying a KRAS mutation compared to wildtype cells, but the KRAS mutant cells also have increased cell viability compared to wildtype cells. We hypothesize that the difference in cell viability and autophagy expression between KRAS mutant and KRAS wildtype cells after treatment with erlotinib can be of therapeutic value to treat CRC patients carrying KRAS mutations.

CX-4945 (Silmitasertib) Induces Cell Death by Impairing Lysosomal Utilization in KRAS Mutant Cholangiocarcinoma Cell Lines.

Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.

Abstract 5912: Rational design of a small-molecule inhibitor targeting the allosteric site of sos1 in oncogenic k-ras pancreatic cancer

Abstract Oncogenic Kras signaling is a key therapeutic target in human cancers. Ras activation is catalyzed by guanine nucleotide exchange factors (GEFs), of which SOS1 is a major player that transduces receptor tyrosine kinase signaling to Ras. In addition to recently devised oncogenic Kras targeting small molecule inhibitors, disrupting the catalytic interaction between Ras and SOS1 has emerged as an attractive therapeutic strategy in working synergistically with Kras inhibitors and overcoming resistance. Previous studies have demonstrated an active “feed forward” loop of active Kras binding to the allosteric site of SOS1 that further stimulates SOS1 catalytic activation of wild type H/N/Kras, which is required for oncogenic Kras driven tumorigenesis. Here we have developed a rational approach that combines virtual screening with experimental screening to identify small molecule inhibitors that target the allosteric site of SOS1 to suppress oncogenic Kras mediated SOS1-regulated wild-type Ras activities. Unlike SOS1 catalytic site inhibitors which can have significant toxicity due to their ability to inhibit normal Ras signaling function, the lead SOS1 allosteric inhibitors developed in our laboratory binds to the allosteric site of SOS1 and blocks active Ras interaction with SOS1: we found these inhibitors can competitively suppresses SOS1-Ras interaction at the SOS1 allosteric site and dose-dependently inhibits the feed-forward SOS1 GEF activity. Mutagenesis and structure-activity relationship studies mapped the site of action to the SOS1 allosteric site, and preliminarily defined the chemical moieties in the inhibitor essential for the activity. The inhibitor showed dose-dependent and selective efficacy in inhibiting WT Ras activity, downstream signaling activities, and associated cell proliferation in KRAS mutation cells but not KRAS wild-type cells. Compared to the SOS1 catalytic inhibitor, BI3406, our inhibitors showed better selectivity for KRAS mutant pancreatic cancer cells vs. WT KRAS cells. These studies establish a proof of principle for the rational design of small molecule inhibitors targeting an oncogenic Kras specific allosteric site of SOS1 and provide a novel therapeutic concept to target KRAS-driven tumors more effectively. Citation Format: Xin Duan, Chris R Evelyn, Jacek Biesiada, James F Johnson, William L. Seibel, Jaroslaw Meller, Yi Zheng. Rational design of a small-molecule inhibitor targeting the allosteric site of sos1 in oncogenic k-ras pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5912.

Abstract 5970: Carbamazepine mediated SMARCA4 inhibition as a therapeutic target for KRAS-mutated CRC

Abstract Introduction: Carbamazepine (CBZ) is an autophagy inducer along with its other therapeutic properties. KRAS-mutated CRC has a worse prognosis and limited treatment options when compared to KRAS wild-type CRC. In this study, we followed the effects of CBZ treatment in the mutant and wild-type CRC cells. We herein report that CBZ exhibits mutant selectivity in efficacy and significantly modulates SMARCA4 levels, a protein heavily implicated in both tumor suppression and proliferation, presenting a promising method for KRAS-mutated CRC treatment. Methodology: 2 cell lines, HCT116 (KRAS mutant) and Hke3 (KRAS wild-type), were used in this study. These cells were treated with Carbamazepine (CBZ) for either 6 or 24 hours. Subsequently, RNA and protein extractions were performed to assess the expression levels of SMARCA4 through quantitative polymerase chain reaction (qPCR) and Western blot analysis, respectively. Furthermore, the TCGA COAD-READ patient dataset was accessed using Xena. The data is used to assess the correlation of KRAS and SMARCA4 wt-tumor vs. mutant-tumor. The dataset was also used to visualize the effect of high SMARCA4 mRNA on the different patient cohorts. Results: Basal levels of SMARCA4 are higher in CRC patients than control (p = 1.81e-16). Patients with a KRAS mutation has higher levels than KRAS-wt (p = 0.000005). KRAS mutated cells treated with CBZ expressed less BRG1 (SMARCA4 protein) at 24 hours (Fold Change = 0.68, p= 0.041). KRAS wildtype cells treated with CBZ expressed more BRG1 at 6 hours (Fold Change = 1.23, p= 0.006). The fold change of BRG1 in KRAS-mutant was significantly lower than in KRAS-wildtype at both 6 and 24 hours (p= 0.013 and p= 0.002). KRAS mutated cells treated with CBZ expressed less SMARCA4 mRNA at 24 hours (Fold Change = 0.69, p= 0.008). KRAS wildtype cells treated with CBZ expressed more SMARCA4 at 6 hours (Fold Change = 1.62, p= 0.006) and 24 hours (Fold Change = 1.31, p= 0.06). The fold change of SMARCA4 in KRAS-mutant was significantly lower than in KRAS-wildtype at both 6 and 24 hours (p= 0.0032 and p= 0.00009). BRG1 expression affected overall survival for the first 1500 days - only in patients with a KRAS-mutant (p= 0.015). Conclusion: This study sheds light on the potential therapeutic role of CBZ in colorectal cancer, particularly in the context of KRAS-mutated CRC. Our findings indicate that CBZ treatment has distinct effects on SMARCA4 expression in CRC depending on KRAS mutation status. In KRAS-mutated cells, CBZ reduced SMARCA4 protein and mRNA levels. Conversely, in KRAS wild-type cells, CBZ resulted in an increase in SMARCA4 expression. Furthermore, our analysis of patient datasets reaffirmed the association between elevated SMARCA4 and KRAS, since high SMARCA4 expression led to worse overall survival rates, specifically in KRAS-mutant CRC patients. These findings emphasize the need for further exploration of CBZ as a potential treatment strategy for CRC. Citation Format: Aaron Shaykevich, Danbee Chae, Isaac Silverman, Jeremy Bassali, Netanel Louloueian, Alexander Siegman, Gargi Bandyopadhyaya, Sanjay Goel, Radhashree Maitra. Carbamazepine mediated SMARCA4 inhibition as a therapeutic target for KRAS-mutated CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5970.

Abstract 4328: Erlotinib suppresses autophagy in colorectal cancer

Abstract Introduction: Oncogenic KRAS mutation is prevalent in approximately 45% of colorectal cancer (CRC) patients. Limited targeted therapies are available to inhibit the detrimental effects of KRAS mutation primarily pertaining to its unique structural intricacies. Erlotinib is an EGFR inhibitor which inducing autophagy in lung cancer cells. In this study, we followed the effects of Erlotinib treatment in mutant and wild-type colorectal cancer (CRC) cells. We report that Erlotinib exhibits selectivity between mutated KRAS and wild type, significantly modulating the expression of autophagy proteins ATG5, ULK1, Beclin1, and LC3A. Methodology: The 2 cell lines used in this study were HCT116 (KRAS mutant) and Hke3 (KRAS wild type). These cells were treated with Erlotinib for 24 hours and 48 hours, A cell viability assay was performed and subsequently followed by protein extraction. The expression of autophagy proteins ATG5, ULK1, Beclin1, and LC3A was determined. The TCGA COAD-READ patient dataset was utilized using GEPIA 2 software. A correlation was found between the autophagy proteins and the KRAS gene expression. Results: The cell viability assays showed that there was significantly more cell death in KRAS wild-type cells compared with KRAS mutant at time points ranging from 24 to 48 hours (p = 0.036). The experiment also showed that treatment of Erlotinib with concentrations of 20 uM, 35 uM, and 50 uM and significant down regulatory fold changes (p < 0.05). Both KRAS mutated and wild-type cells treated with Erlotinib expressed significant downregulation of ATG5, ULK1, Beclin1, and LC3A proteins at 48 hours (p < 0.05). The 20 uM treatment demonstrated a significance of p < 0.05 for all proteins. The 20 uM treatment resulted in fold changes of the KRAS mutated cells for ATG5, ULK1, Beclin1, and LC3A to be 1.42, 1.42, 1.81, and 2.09 respectively. A strong KRAS correlation was found with ATG5, ULK1, Beclin1, and LC3A using COAD & READ tumor and COAD & READ normal (R > 0.75, p < 0.05). Conclusion: This study highlights the effects of Erlotinib as a treatment for KRAS mutated CRC. Our results confirmed that Erlotinib had the opposite effect on CRC cells in relation to the expression of the autophagy proteins when compared to lung cancer cells. There were distinct effects of Erlotinib between KRAS wild-type and mutant cells in both cell viability and the expression of autophagy-related proteins. In both KRAS cell types, there was significant downregulation of autophagy proteins, however, the KRAS wild type had significantly more downregulation than the KRAS mutant. This data suggests that there is a relationship between KRAS mutation and the expression of the autophagy proteins. Our analysis of the patient datasets confirmed the strong correlation between KRAS mutation and the expression of autophagy proteins. These findings emphasize the need to explore further how Erlotinib can be augmented into the current treatment regimen as a benefit to CRC patients. Citation Format: Alexander Siegman, Aaron Shaykevich, Isaac Silverman, Sanjay Goel, Radhashree Maitra. Erlotinib suppresses autophagy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4328.

Abstract 3316: LY3962673, an oral, highly potent, mutant-selective, and non-covalent KRAS G12D inhibitor demonstrates robust anti-tumor activity in KRAS G12D models

Abstract KRAS G12D mutations are activating oncogenic events that occur in approximately 35%, 13%, and 4% of pancreatic, colorectal, and non-small cell lung cancers, respectively, and less commonly in other cancers. We previously demonstrated that LY3962673 is a highly potent inhibitor of KRAS G12D and is selective against wild-type (WT) KRAS in mutant -cell lines and -in vivo models. Here, we describe the mechanism by which LY3962673 inhibits KRAS G12D and report a more comprehensive evaluation of LY3962673 activity across a panel of genetically and histologically diverse cancer cell lines, as well as in multiple patient-derived xenograft (PDX) models. LY3962673 is a non-covalent KRAS G12D inhibitor with high affinity binding to KRAS G12D-GDP (Kd 0.071 nM) compared to KRAS G12D-GTPγS (Kd 26.7 nM). In a panel of cancer cell lines with KRAS G12D mutations, non-G12D mutations or KRAS WT, LY3962673 selectively suppressed MAPK signaling and inhibited the growth of KRAS G12D mutant cancer cells while sparing KRAS WT and non-G12D mutant cells. Sensitivity to LY3962673 varied among the KRAS G12D-mutant cells tested, suggesting that not all cell lines share the same dependence on KRAS G12D for their growth and survival. Furthermore, in multiple KRAS G12D-mutant PDX models representing diverse tumor types, LY3962673 demonstrated anti-tumor activities, ranging from tumor growth inhibition to robust tumor regression. LY3962673 also showed enhanced efficacy when combined with other anti-cancer agents. Taken together, the findings underscore the potential of LY3962673 as a monotherapy or in combination with other anti-cancer agents, as a promising oral therapeutic option for a range of cancer types with KRAS G12D mutations. Citation Format: Xueqian Gong, Hong Gao, Mark H. Bender, Wenyu Ming, Youyan Zhang, Trent R. Stewart, Chun Ping Yu, Wei Guo Xu, Aurthur Xintian You, Wen Ting Bian, Binghui Li, Tao Wang, Huimin Bian, Manuj Tandon, Andrew Capen, Rachel N. Cavitt, Bryan D. Anderson, Wayne Bocchinfuso, Anke Klippel, Chandrasekar Iyer. LY3962673, an oral, highly potent, mutant-selective, and non-covalent KRAS G12D inhibitor demonstrates robust anti-tumor activity in KRAS G12D models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3316.

Effectiveness of 225Ac-Labeled Anti-EGFR Radioimmunoconjugate in EGFR-Positive Kirsten Rat Sarcoma Viral Oncogene and BRAF Mutant Colorectal Cancer Models.

Eighty percent of colorectal cancers (CRCs) overexpress epidermal growth factor receptor (EGFR). Kirsten rat sarcoma viral oncogene (KRAS) mutations are present in 40% of CRCs and drive de novo resistance to anti-EGFR drugs. BRAF oncogene is mutated in 7%-10% of CRCs, with even worse prognosis. We have evaluated the effectiveness of [225Ac]Ac-macropa-nimotuzumab in KRAS mutant and in KRAS wild-type and BRAFV600E mutant EGFR-positive CRC cells invitro and invivo. Anti-CD20 [225Ac]Ac-macropa-rituximab was developed and used as a nonspecific radioimmunoconjugate. Methods: Anti-EGFR antibody nimotuzumab was radiolabeled with 225Ac via an 18-membered macrocyclic chelator p-SCN-macropa. The immunoconjugate was characterized using flow cytometry, radioligand binding assay, and high-performance liquid chromatography, and internalization was studied using live-cell imaging. In vitro cytotoxicity was evaluated in 2-dimensional monolayer EGFR-positive KRAS mutant DLD-1, SW620, and SNU-C2B; in KRAS wild-type and BRAFV600E mutant HT-29 CRC cell lines; and in 3-dimensional spheroids. Dosimetry was studied in healthy mice. The invivo efficacy of [225Ac]Ac-macropa-nimotuzumab was evaluated in mice bearing DLD-1, SW620, and HT-29 xenografts after treatment with 3 doses of 13 kBq/dose administered 10 d apart. Results: In all cell lines, invitro studies showed enhanced cytotoxicity of [225Ac]Ac-macropa-nimotuzumab compared with nimotuzumab and controls. The inhibitory concentration of 50% in the DLD-1 cell line was 1.8 nM for [225Ac]Ac-macropa-nimotuzumab versus 84.1 nM for nimotuzumab. Similarly, the inhibitory concentration of 50% was up to 79-fold lower for [225Ac]Ac-macropa-nimotuzumab than for nimotuzumab in KRAS mutant SNU-C2B and SW620 and in KRAS wild-type and BRAFV600E mutant HT-29 CRC cell lines. A similar trend was observed for 3-dimensional spheroids. Internalization peaked 24-48 h after incubation and depended on EGFR expression. In the [225Ac]Ac-macropa-nimotuzumab group, 3 of 7 mice bearing DLD-1 tumors had complete remission. Median survival was 40 and 34 d for mice treated with phosphate-buffered saline and [225Ac]Ac-macropa-rituximab (control), respectively, whereas it was not reached for the [225Ac]Ac-macropa-nimotuzumab group (>90 d). Similarly, median survival of mice bearing HT-29 xenografts was 16 and 12.5 d for those treated with [225Ac]Ac-macropa-rituximab and phosphate-buffered saline, respectively, and was not reached for those treated with [225Ac]Ac-macropa-nimotuzumab (>90 d). One of 7 mice bearing HT-29 xenografts and treated using [225Ac]Ac-macropa-nimotuzumab had complete remission. Compared with untreated mice, [225Ac]Ac-macropa-nimotuzumab more than doubled (16 vs. 41 d) the median survival of mice bearing SW620 xenografts. Conclusion: [225Ac]Ac-macropa-nimotuzumab is effective against KRAS mutant and BRAFV600E mutant CRC models.

KRAS-mutant colorectal cancer cell lines cause a prothrombotic state through the upregulation of thrombin: experimental study

Background: The KRAS genotype status is strongly associated with a prothrombotic state in colorectal cancer, and hypercoagulability and cancer-related thrombosis are among the significant events leading to poor prognosis. However, this correlation has not been confirmed at the cellular level. This study aimed to assess the maximum platelet aggregation rate and thrombin expression induced by colorectal cancer cells under different KRAS genotypes. Materials and methods: Platelet aggregation rate assay and western blotting analysis were used to detect platelet aggregation and thrombin expression induced by four colorectal cancer cells with different KRAS genotypes, including RKO, HCT116, SW480, and SW620. FVIIa/tissue factor and thrombin inhibitors were added to explore changes in platelet aggregation rates induced by colorectal cancer cells and the association between KRAS genotype status and hypercoagulable state. Results: KRAS-mutant cells were more likely to increase maximal platelet aggregation, with RKO, HCT116, SW480, and SW620 inducing 34.7%, 55.4%, 44.4%, and 63.8% of platelet aggregation, respectively. The maximum platelet aggregation rate was higher in the metastatic rectal cancer tumour strain SW620 than in the primary rectal cancer strain SW480. RKO cells had lower thrombin expression than the other three cells. Furthermore, the addition of thrombin inhibitors caused a more significant decrease in the platelet aggregation rate in KRAS-mutant cell lines compared to KRAS wild-type cell lines. Conclusion: Compared to KRAS wild-type colorectal cancer cells, KRAS-mutant colorectal cancer cell lines were more likely to be hypercoagulable through the upregulation of thrombin expression, which was mainly achieved through the TF-thrombin pathway.

Abstract B115: Preclinical characterization of LY3962673, an orally bioavailable, highly potent, and selective KRAS G12D inhibitor

Abstract KRAS G12D mutations are activating oncogenic events that occur in approximately 35%, 13%, and 4% of pancreatic, colorectal, and non-small cell lung cancers, respectively and less commonly in other cancers. Here, we describe LY3962673, a highly potent inhibitor of KRAS G12D that is selective against wild-type (WT) KRAS and orally bioavailable. Compound potency and selectivity were measured using surface plasmon resonance (SPR) and cell-based assays monitoring the inhibition of pERK (Thr202/Tyr204) and cell growth. In SPR assays, LY3962673 had Kd values of 0.058 and 4.7 nM for GDP-bound KRAS G12D and WT KRAS, respectively (81-fold selective). In KRAS G12D-mutant HPAC, LY3962673 inhibited ERK1/2 phosphorylation with an IC50 of 4.2 nM contrasting 231 nM IC50 in WT KRAS MKN45 cells (55-fold selective). Similarly, LY3962673 inhibited cell growth in KRAS G12D and WT KRAS cell lines with IC50 values of 7.7 nM and 1800 nM, respectively (233-fold selective). LY3962673 shows a clean safety profile in a 133 off-target panel screen. LY3962673 demonstrates favorable in vitro ADME properties. LY3962673 shows oral bioavailability across preclinical species when dosed either as freebase or salt form. Tumor growth inhibition and PK/PD studies were performed in mice. In vivo, LY3962673 administered orally demonstrated tumor regressions in KRAS G12D-driven tumor models, without body weight loss. This wide therapeutic index is predicted to allow for maximizing dose intensity and efficacy in patients. These data demonstrate that LY3962673 potently inhibits KRAS G12D while sparing WT KRAS and other off-targets with its selectivity. We hypothesize that LY3962673’s low nanomolar potency, desirable selectivity profile, and oral bioavailability will provide efficacy and tolerability in patients with KRAS G12D-driven cancers. An IND submission is planned in the first half of 2024. Citation Format: Chandrasekar Iyer, Binghui Li, Trent R Stewart, Tao Wang, Andrew Capen, Rachel Cavitt, Bryan D Anderson, Wayne Bocchinfuso, Gaiying Zhao, Michael J Rodriguez, Santiago Carballares, Andrew Cooke, Robert Bondi, Lee Burns, Lakshmi Kelamangalath, Ross Wallace, Gabrielle Kolakowski, James R Henry, Chong Si. Preclinical characterization of LY3962673, an orally bioavailable, highly potent, and selective KRAS G12D inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B115.

An integrated model for predicting KRAS dependency.

The clinical approvals of KRAS G12C inhibitors have been a revolutionary advance in precision oncology, but response rates are often modest. To improve patient selection, we developed an integrated model to predict KRAS dependency. By integrating molecular profiles of a large panel of cell lines from the DEMETER2 dataset, we built a binary classifier to predict a tumor's KRAS dependency. Monte Carlo cross validation via ElasticNet within the training set was used to compare model performance and to tune parameters α and λ. The final model was then applied to the validation set. We validated the model with genetic depletion assays and an external dataset of lung cancer cells treated with a G12C inhibitor. We then applied the model to several Cancer Genome Atlas (TCGA) datasets. The final "K20" model contains 20 features, including expression of 19 genes and KRAS mutation status. In the validation cohort, K20 had an AUC of 0.94 and accurately predicted KRAS dependency in both mutant and KRAS wild-type cell lines following genetic depletion. It was also highly predictive across an external dataset of lung cancer lines treated with KRAS G12C inhibition. When applied to TCGA datasets, specific subpopulations such as the invasive subtype in colorectal cancer and copy number high pancreatic adenocarcinoma were predicted to have higher KRAS dependency. The K20 model has simple yet robust predictive capabilities that may provide a useful tool to select patients with KRAS mutant tumors that are most likely to respond to direct KRAS inhibitors.

Abstract B014: Phenotypic CRISPR genome-wide screening for the discovery of genetic regulators of allele-specific mutant KRAS

Abstract Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most frequently mutated oncogene detected in 30% of all human cancers. Drugs that can impede the function of oncogenic KRAS offer exceptional therapeutic value. However, direct targeting KRAS is challenging due to its protein structure that lacks deep binding pockets for small-molecule inhibitors. Despite the recent success in targeting the G12C allele, targeted therapy for another hotspot mutant – G12V mutant KRAS has not been described. Here, we utilize CRISPR-Cas9 based genome-wide knockout screens and leverage a high-throughput microfluidic cell sorting platform to identify genes that regulate mutant KRAS harboring G12V mutation. The screens were conducted in two colorectal cancer cell lines HT29 and SW480, expressing wild-type KRAS and homozygous G12V KRAS, respectively. We have identified a list of genes whose loss-of-function reduces wild-type or mutant G12V KRAS protein expression. Our top gene identified from the mutant KRAS screen - denoted as selective-mutant KRAS modulator (SMKM) has shown promising results in selectively decreasing G12V KRAS protein expression. Through multiple assays including live cell imaging, western blot, flow cytometry, and proteomic analysis in SMKM knockout cells and SMKM inhibitor-treated cells. We found highly selective reduction in G12V KRAS protein expression. More importantly, genetically or pharmacologically inhibiting SMKM has demonstrated anti-tumor activity. We conducted cell viability assays including CellTiter-glo, MTT, and clonogenic assay. We treated a panel of G12V KRAS mutant and wild-type KRAS cell lines with SMKM inhibitor and found significantly reduced cell viability in G12V KRAS mutant cell lines across colorectal and lung cancer. To further demonstrate the downstream impact on mutant KRAS signaling, secondary assays including western blot and Lumit immunoassay were conducted. We observe a reduction in the aberrant phospho-ERK and phospho-AKT signaling in SMKM inhibitor treated cells. Our in vivo efficacy trial using heterozygous G12V KRAS lung cancer NCI-H441 xenograft model has shown SMKM inhibition significantly reduces tumor growth and increases the survival rate. Together, these data indicate the feasibility of selectively targeting G12V KRAS with SMKM inhibition. Lastly, our results provide a first-in-class small molecule oral inhibitor based selective protein clearance as an effective anti-G12V mutant KRAS therapeutic strategy. Citation Format: Xiyue Hu, Randy Singh, Shana Kelley. Phenotypic CRISPR genome-wide screening for the discovery of genetic regulators of allele-specific mutant KRAS [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B014.

Abstract 2839: Methionine restriction radiosensitizes KRAS mutant rectal cancer

Abstract Purpose/Objective(s): Radiation therapy (RT) is the standard of care in patients with locally advanced rectal cancer, but not all patients respond equally to RT. KRAS-mutated (KRASmut) cancer cells are highly radioresistance and require higher levels of methionine, an essential amino acid, compared to KRAS wildtype (KRASwt) and normal cells, and diet restriction of methionine has been shown to potentiate the effects of radiation in vivo. We hypothesized that methionine restriction (MR) could radiosensitize KRASmut rectal cancer cells. Materials/Methods: HCT116 KRASG13D/+ and SW48 KRASwt colorectal carcinoma isogenic cell lines and their isogenic derivative cell lines HCT 116 KRAS+/- and SW48 KRASG13D were obtained from Horizon Discovery Ltd (Cambridge, UK). Radiation clonogenic assays, immunofluorescence, and western blot analysis of key DNA damage response proteins were performed in the KRASmut vs. KRASwt colorectal cells treated with ionizing radiation (IR, 2-6 Gy) at increasing time points following pre-treatment with methionine control (MC) (200 µM) or MR (40-fold reduction in methionine, 5 µM) media for 48 hours. One hundred thousand luciferase-expressing KRASwt or KRASmut SW48 cells were implanted orthotopically in the rectum of athymic nude mice. After confirmation of tumor formation at one-week post-injection, the mouse diet was changed to either an MC (0.84% methionine w/w) or MR (0.12% methionine w/w) diet for a total of two weeks. Rectal tumors were treated on an Xstrahl small animal radiation research platform (SARRP) to a dose of 25 Gy in 5 daily fractions (1 cm x 1 cm collimator, isocenter at anal verge) during the second week of diet modification. Tumor growth was measured via bioluminescence on IVIS Lumina every two weeks for four weeks post-radiation. Results: MR significantly increased radiosensitization compared to MC in both HCT116 and SW48 cells in vitro. KRASmut cells showed a 20-30% increase in radiation in the dose enhancement ratio compared to the KRASwt cells. The protein expression and nuclear foci per cell of the DNA damage marker γH2AX were significantly higher in the KRASmut cells compared to KRASwt cells following 5 Gy of RT at 6 hours. Protein expression of homologous recombination pathway proteins RAD51 and the CtBP interacting protein (CtIP) were higher at baseline in the KRASmut cells than KRASwt. MR substantially reduced the expression of these proteins following RT. Mice harboring SW48 KRASmut rectal tumors treated with 5 Gy x 5 RT and MR showed a significant increase in the complete response rate compared to MC (80% vs. 10%, p=0.02). In the mice harboring SW48 KRASwt rectal tumors, there was no significant difference in response rate between MR vs. MC (40% vs. 30%, p=NS). Conclusion: Our study indicates a novel strategy to radiosensitize resistant KRAS-mutated rectal cancer through MR. Studies testing the mechanism of KRAS-dependent methionine-mediated DNA repair is ongoing. Citation Format: Adam R. Wolfe, Sarita Garg, Stetson Van Matre, Robin Eluvathingal, Oscar Zuniga, Henrique Rodrigues, Nükhet Aykin-Burns, Isabelle Racine Miousse. Methionine restriction radiosensitizes KRAS mutant rectal 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 2839.

Abstract 2627: The small molecule KRAS inhibitor, TEB-17231, blocks tumor progression and overcomes KRASG12C inhibitor mediated resistance

Abstract KRAS pathways are frequently upregulated in cancers bearing KRAS mutations or by other signaling modifications. Also, resistance to KRASG12C inhibitors and other KRAS/RAF/MEK pathway drugs is commonly observed in the treated cancer patients. A vital unmet need is the development of agents that potently and safely inhibit the altered KRAS signaling in these settings. A broadly acting KRAS mutation small molecule inhibitor lead series was developed using KRAS domain structure-guided scaffold modifications starting from a group of KRASG12C inhibitors. Optimized properties of these compounds were evaluated by tumor cell selectivity in vitro and pharmacophore elaboration. A novel lead series was identified with robust tumor cell growth inhibition, exemplified with the TEB-17112 lead candidate, having IC50 of inhibition against multiple KRAS-altered cell lines: NCI-H358 (KRAS-G12C, 33.6nM), AGS (KRAS-G12D, 23.5 nM), SW480 (KRAS-G12V, 22.5 nM) and A375 (KRAS-wildtype, 58.9 nM) respectively. TEB-17112 demonstrated inhibition of SW480 and H358 mouse xenografts at 25 or 50 mg/kg PO QD (21 days) without significant body weight loss. On further optimization of the lead series, TEB-17231 had on average a higher potency against KRAS mutant tumor cell lines, including H358 (12 nM), AGS (5.4 nM), SW480 (3.6 nM), and A427 (KRAS-G12D, 9.7 nM). With a candidate group of 7 compounds from this lead series, there was consistently a 5- to 6-fold selectivity of inhibition for KRAS mutant over KRAS wildtype cell lines. The lead candidate TEB-17231 was also observed to inhibit cell lines with other KRAS mutations: NCI-H1734 (KRAS-G13C, 6.5 nM), MDA-MB-231 (KRAS-G13D,11.2 nM), RPMI-8226 (KRAS-G12A, 11.3 nM), Calu-6 (KRAS-Q61K,19.8 nM), KP-2 (KRAS-G12R, 24.1 nM), LS123 (KRAS-G12S, 21 nM), as well as HRAS and NRAS mutant cell lines. TEB-17231 inhibits phospho-ERK downstream signaling in several tumor cell lines. Further, TEB-17231 exhibited tumor growth inhibition (IC50) in KRAS-G12C Ba/F3 (14 nM) and derivative cell lines that acquired resistance to G12C inhibitor MRTX849 by the additional KRAS domain mutations as follows: H95Q (32 nM), H95D (40 nM), Y96C (39 nM), Y96D (20 nM) and R68S (22 nM), compared with greater than 1μM for MRTX849. TEB-17231 induces late apoptosis of NCI-H358 cells in a manner that is time and concentration dependent. In addition to the improved selectivity and potency, TEB-17231 demonstrated satisfactory pharmacokinetics and oral bioavailability in mice (55%). Furthermore, with the SW480 (G12V) in vivo xenograft model, TEB-17231 showed clear tumor growth inhibition with QD dosing for 4-8 mg/kg PO without body weight loss. These properties highlight TEB-17231 as a potential clinically actionable inhibitor for the KRAS-modified tumor population and warrant investigation of TEB-17231 in combination with other agents in the future. Citation Format: Zusheng Xu, David T. Weaver, Robert Drakas, Yangtong Lou. The small molecule KRAS inhibitor, TEB-17231, blocks tumor progression and overcomes KRASG12C inhibitor mediated resistance [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 2627.

Exosomes derived from MDR cells induce cetuximab resistance in CRC via PI3K/AKT signaling‑mediated Sox2 and PD‑L1 expression.

The anti-EGFR antibody cetuximab is used as a first-line targeted therapeutic drug in colorectal cancer. It has previously been reported that the efficacy of the EGFR antibody cetuximab is limited by the emergence of acquired drug resistance. In our previous study the transmissibility effect of exosomes from drug resistant tumor cells to sensitive tumor cells was identified. It can therefore be hypothesized that drug resistant cells might affect neighboring and distant cells via regulation of exosome composition and behavior. However, the mechanism of exosomes in KRAS-wild-type colorectal cancer (CRC) remains unknown. In the present study, functional analysis of overall survival post-diagnosis in patients with KRAS wild-type and those with mutant CRC was performed using human CRC specimens. Furthermore, it was demonstrated that multidrug resistance (MDR) cancer cell-derived exosomes were potentially a key factor, which promoted cetuximab-resistance in CRC cells and reduced the inhibitory effect of cetuximab in CRC xenograft models. The Cell Counting Kit-8 and colony formation assays were performed to assess the effects of exosomes derived from CRC/MDR cells on cetuximab resistance. Sphere formation assay results demonstrated that exosomes derived from CRC/MDR cells altered the self-renewal and multipotential ability of stem-cell-associated markers and facilitated resistance to cetuximab in cetuximab-sensitive cells. Furthermore, exosomes derived from CRC/MDR cells decreased sensitivity to cetuximab via the activation of PI3K/AKT signaling, which promoted Sox2 and programmed death-ligand 1 (PD-L1) mRNA and protein expression according to reverse transcription-quantitative PCR, western blotting and immunohistochemistry analyses, as well as apoptosis resistance both in vitro and in vivo according to a TUNEL assay. In conclusion, the results of the present study demonstrated that exosomes derived from CRC/MDR cells may promote cetuximab resistance in KRAS wild-type cells via activation of the PI3K/AKT signaling pathway-mediated expression of Sox2 and PD-L1, which will be useful for investigating a potential clinical target in predicting cetuximab resistance.

KRAS G12D mutation eliminates reactive oxygen species through the Nrf2/CSE/H 2S axis and contributes to pancreatic cancer growth.

In pancreatic cancer, KRAS G12D can trigger pancreatic cancer initiation and development. Rapid tumor growth is often accompanied by excess intracellular reactive oxygen species (ROS) production, which is unfavorable to tumor. However, the regulation of intracellular ROS levels in KRAS mutant pancreatic cancer remains unclear. In this study, we establish BxPC3 stable cell strains expressing KRAS wild type (WT) and G12D mutation and find unchanged ROS levels despite higher glycolysis and proliferation viability in KRAS mutant cells than KRAS WT cells. The key hydrogen sulfide (H 2S)-generating enzyme cystathionine-γ-lyase (CSE) is upregulated in KRAS mutant BxPC3 cells, and its knockdown significantly increases intracellular ROS levels and decreases cell glycolysis and proliferation. Nuclear factor erythroid 2-related factor 2 (Nrf2) is activated by KRAS mutation to promote CSE transcription. An Nrf2 binding site (‒47/‒39 bp) in the CSE promoter is verified. CSE overexpression and the addition of NaHS after Nrf2 knockdown or inhibition by brusatol decreases ROS levels and rescues cell proliferation. Our study reveals the regulatory mechanism of intracellular ROS levels in KRAS mutant pancreatic cancer cells, which provides a potential target for pancreatic cancer therapy.

Efficacy of Combined Use of Everolimus and Second-Generation Pan-EGRF Inhibitors in KRAS Mutant Non-Small Cell Lung Cancer Cell Lines.

Background: EGFR mutations are present in approximately 15–50% of non-small cell lung cancer (NSCLC), which are predictive of anti-EGFR therapies. At variance, NSCLC patients harboring KRAS mutations are resistant to those anti-EGFR approaches. Afatinib and allitinib are second-generation pan-EGFR drugs, yet no predictive biomarkers are known in the NSCLC context. In the present study, we evaluated the efficacy of pan-EGFR inhibitors in a panel of 15 lung cancer cell lines associated with the KRAS mutations phenotype. Methods: KRAS wild-type sensitive NCI-H292 cell line was further transfected with KRAS mutations (p.G12D and p.G12S). The pan-EGFR inhibitors’ activity and biologic effect of KRAS mutations were evaluated by cytotoxicity, MAPK phospho-protein array, colony formation, migration, invasion, and adhesion. In addition, in vivo chicken chorioallantoic membrane assay was performed in KRAS mutant cell lines. The gene expression profile was evaluated by NanoString. Lastly, everolimus and pan-EGFR combinations were performed to determine the combination index. Results: The GI50 score classified two cell lines treated with afatinib and seven treated with allitinib as high-sensitive phenotypes. All KRAS mutant cell lines demonstrated a resistant profile for both therapies (GI50 < 30%). The protein array of KRAS edited cells indicated a significant increase in AKT, CREB, HSP27, JNK, and, importantly, mTOR protein levels compared with KRAS wild-type cells. The colony formation, migration, invasion, adhesion, tumor perimeter, and mesenchymal phenotype were increased in the H292 KRAS mutated cells. Gene expression analysis showed 18 dysregulated genes associated with the focal adhesion-PI3K-Akt-mTOR-signaling correlated in KRAS mutant cell lines. Moreover, mTOR overexpression in KRAS mutant H292 cells was inhibited after everolimus exposure, and sensitivity to afatinib and allitinib was restored. Conclusions: Our results indicate that allitinib was more effective than afatinib in NSCLC cell lines. KRAS mutations increased aggressive behavior through upregulation of the focal adhesion-PI3K-Akt-mTOR-signaling in NSCLC cells. Significantly, everolimus restored sensibility and improved cytotoxicity of EGFR inhibitors in the KRAS mutant NSCLC cell lines.

Abstract 4107: Discovery and preclinical evaluation of VRTX126, a novel, highly selective and potent KRASG12Cinhibitor

Abstract KRASG12C is an important oncogenic driver mutation and occurs in about 13% of NSCLC and 1-3% of colorectal and other solid tumors. Lumakras࣪ (sotorasib), a KRASG12C inhibitor was recently approved for treatment of NSCLC, and others, such as, Adagrasib have shown relatively modest activity for KRASG12C.This modest activity may be partly due to incomplete target occupancy or due to unavailability of the drug at the target site, to elicit maximum clinical response. Clearly, this gives path for development of inhibitors which are potent, but also have desired drug like properties to achieve maximal clinical benefit in patients harboring KRASG12C mutation. Here, we report the identification of VRTX126, a novel, highly selective and potent inhibitor of the KRASG12C, discovered using fragment-based drug design. VRTX126 exhibited an IC50 of 10 nM in H358. VRTX126 also inhibited phospho-ERK with an IC50 of &amp;lt;20 nM and &amp;lt;10 nM in H358 and Mia-Paca-2 cell lines respectively, while demonstrating multi fold selectivity over KRAS wild-type or nonG12C mutant cells lines. In xenograft model harboring KRASG12C mutation, VRTX126 exhibited tumor growth inhibition at therapeutically relevant doses. Combination strategies with other targeted therapies such as, our proprietary SOS1 inhibitor, and other marketed or available MEK1/2, SHP2 inhibitors targeting Ras/Raf/MEK pathway is being explored. VRTX126 showed favourable oral properties with dose proportional pharmacokinetics. VRTX126 was found to have no adverse effect on hERG (10 uM). Additionally, early evaluation in acute (&amp;gt;1 g/kg (p.o.) and &amp;gt;0.2 g/kg (i.v.) was well tolerated, with no elevation in liver enzymes and repeat does studies indicated good safety margins, thus supporting further evaluation of VRTX126 in IND enabling studies. A first-in-human Phase 1 clinical study in KRASG12C mutated patients of NSCLC and mCRC will be planned upon completion of through safety evaluations. Citation Format: Prashant Kashinath Bhavar, Uday Kumar Surampudi, Partha Pratim Sarma, Mahesh Mohan Shidore, Anuj Ramesh Kshirsagar. Discovery and preclinical evaluation of VRTX126, a novel, highly selective and potent KRASG12Cinhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4107.

Abstract 1057: Significant efficacy demonstrated with the combination of ulixertinib (ERK1/2 inhibitor) and CDK4/6 inhibitors in MAPK altered models

Abstract Ulixertinib (BVD-523) is a first-in-class small molecule inhibitor of ERK1/2 currently being investigated in several oncology clinical trials, both as a single agent and in combination with other anti-cancer therapeutics. Palbociclib and ribociclib are FDA approved orally active, potent, and highly selective reversible inhibitors of the CDK4 and CDK6 kinases. As it is well established that ERK activation increases cyclin D levels and entry into the cell cycle, we hypothesized that the combination of ERK1/2 and CDK4/6 inhibition would have synergistic antitumor activity and cause tumor regression in vivo. Initial in vitro work combined ulixertinib with CDK4/6 inhibitors across a small panel of lung cancer cell lines. Cell lines carrying a KRAS mutation were more sensitive to ulixertinib relative to KRAS wild type cell lines based on single agent IC50 values. The single agent IC50 values for the CDK4/6 inhibitors were dependent on whether a metabolic or non-metabolic readout for cell viability was used. The combination interactions across a dose matrix of concentrations were determined by the Loewe Additivity and Bliss Independence models. The results of combining ulixertinib and CDK4/6 inhibitors ranged from additive to potentially synergistic. The combination of ulixertinib and palbociclib was then assessed against four xenograft models representing colorectal, melanoma, and pancreatic tumor types, each harboring an alteration within a component of the MAPK pathway. Palbociclib monotherapy across all models showed limited tumor growth inhibition (TGI) while ulixertinib monotherapy demonstrated modest TGI across all models. The combination groups demonstrated significant responses ranging from 75% - 90% TGIs. All treatment regimens were well tolerated across all models. Downstream assays were completed including reverse phase protein arrays (RPPA). Using RPPA, treatment effects on protein signaling was evaluated in the MAPK family, cell cycle regulation, and other associated feedback and compensatory pathways. Notably, suppression of protein targets downstream of ERK1/2 were seen with both ulixertinib monotherapy and combination therapy. Similarly, the combination therapy group reduced protein levels involved in cell cycle progression, which was not seen in either monotherapy group alone. The efficacy demonstrated with this preclinical work has proven to be translatable to the clinic as the combination of ulixertinib and palbociclib recently achieved MTD in a Phase I trial in advanced solid tumors including pancreatic cancer (NCT 03454035). Citation Format: Caroline M. Emery, Brian Corgiat, Justin Davis, David Sorrell, Mitch Johnson, Brent Kreider, Deborah Knoerzer. Significant efficacy demonstrated with the combination of ulixertinib (ERK1/2 inhibitor) and CDK4/6 inhibitors in MAPK altered models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1057.