Acquisition Of KRAS Mutations Research Articles

Abstract 1926: MRAS the forgotten member of the GTPase superfamily plays a role in KRASG12Ci resistance

Abstract KRASG12C inhibitors have achieved impressive results in the clinic. However, intrinsic or acquired resistance to therapy invariably develops leading to disease progression in most treated patients. Emerging pre-clinical and clinical evidence points to multiple mechanisms being responsible for resistance to KRASG12C inhibitors, including receptor tyrosine kinase (RTK) activation, KRASG12C amplification, activation of alternative signaling pathways including PI3K, mTOR, YAP/TAZ, acquisition of KRAS mutations that would preclude compound binding (e.g. R68S, Y96C/D, H95D/Q/R), conversion of the G12C codon mutation, and additional activating KRAS mutations (G13D, Q61H). In the clinic, many of the described mutations were detected at low frequency, some patients displayed multiple mutations and in 40% of patients no potential driving mutations were identified. It is therefore likely that other non-genetic alterations are also involved in resistance to KRASG12Ci monotherapy. Here, we used preclinical models of KRASG12C non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) to study intrinsic and acquired resistance to KRASG12C inhibitors. Increased expression of the RAS superfamily gene MRAS was observed in most KRASG12C-naïve tumor cells upon treatment with a KRASG12C inhibitors. In line with this, we observed an increase of MRAS-GTP levels in cells treated with KRASG12Ci in vitro. Interestingly, the increase in active MRAS did not lead to concomitant upregulation of MAPK signaling. Long term treatment with adagrasib was used for generating in vitro and in vivo KRASG12C models with acquired resistance to KRASG12Ci. A strong increase in MRAS expression was observed under these conditions, that surpassed the levels seen with short term treatment in naïve models. This data suggests that activation of MRAS-SHOC2-PP1C complex may play a role in compensating for the KRASG12C-driven signaling in both the intrinsic and acquired resistance setting. Knockdown of SHOC2 to baseline levels resulted in re-sensitization to KRASG12Ci monotherapy and to the effects of combinations in adagrasib-resistant NCI-H358 NSCLC cells. Pathway suppression was deepened by combining adagrasib with either a SOS1i (e.g. BI-3406) or SHP2i (e.g. TNO155) resulting in enhanced anti-tumor effects observed in the KRASG12Ci-resistant models. Our findings underscore the role of MRAS in acquired resistance in KRASG12Ci and the potential to overcome this with vertical pathway combinations, such as SOS1i or SHP2i. Citation Format: Venu Thatikonda, Kaja Kostyrko, Sabine Jurado, Simone Lieb, Melanie Hinkel, Donat Alpar, Oliver Bergner, Astrid Jeschko, Mark Pearson, Marco H. Hofmann. MRAS the forgotten member of the GTPase superfamily plays a role in KRASG12Ci resistance [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 1926.

PO-346 Defining microRNA mediated regulation of metabolic pathways involved in colon cancer progression (BST1-microRNA interactions)

IntroductionColorectal cancer (CRC) is the third most common cause of cancer related death in the world. Progressive accumulation of mutations in oncogenic and tumour suppressor pathways (e.g., KRAS and p53) and also microRNA (miR) deregulation are associated with CRC development. Very little has been done to dissect specific CRC pathways involved in miR regulation in response to stress and metabolic changes. Therefore, we previously characterised gene (mRNA) and miR expression as well as metabolic changes in different CRC genotypes. Here, we investigated the interaction between miR-mediated regulation of bone marrow stromal cell antigen 1 (BST1) or CD157, a metabolic gene involved in the conversion of nicotinamide adenine dinuclease (NAD) to paracrine factor cyclic ADP ribose (cADPR), following the acquisition of KRAS mutation in metastatic CRC.Material and methodsA combination of array analysis data, in-silico prediction tools and nuclear magnetic resonance (NMR) system were used to analyse gene and miR expression changes associated with different rounds of knock in/out mutations in Apc, Kras, and p53 CRC mouse models and their tumour-derived organoids (TDOs). Subsequently, we genetically modified human CRC cell lines and patients KRAS mutant (mut) TDOs from CRC metastases to modulate the candidate gene and miR. Organoids formation, growth rate and viability were measured with IncuCyte® Live-Cell Analysis System and Celigo® imaging cytometer.Results and discussionsWe identified mRNA/miR networks involved in cancer metabolic pathways and assessed miRs of potential interest among the candidates. MiR-203 appeared as one of the most significant candidates. Furthermore target validation and analysis of human tissues microarrays showed an association between KRAS mutations, miR-203 down-regulation and over-expression of BST1, which was identified as a direct target of miR-203 regulation in our studies. Repressing BST1 and over-expressing miR-203 had a significant effect on the proliferation and migration abilities of organoids in 3D culture in normal and calorie-deprived conditions.ConclusionThis project aimed at characterising the functional consequences of miR-203 loss and BST1 up regulation in normal and nutrient-deprived conditions in order to find a promising candidate as a therapeutic target in KRAS mut CRC. Although we showed KRAS mut CRC cells lost a growth advantage with miR-203 and BST1 deregulation ex vivo, work is on going to confirm the consequence of their interaction in vivo.

Abstract 2009: Role of kallikrein 6 secretion in metastatic colon cancer

Abstract Growing evidence indicates that serine proteases kallikreins are associated with malignancy and may have potential diagnostic/prognostic applications in cancer. Kallikrein 6 (KLK6) is a member of the family of 15 highly conserved secreted trypsin- or chemotrypsin-like serine proteases. Overexpression of KLK6 has been observed in different pathophysiological states such as neurodegenerative diseases, inflammation and cancer. Activating K-RAS mutations are common in colon cancer progression and contribute to many cancerous phenotypes including increased proliferation, evasion of apoptosis, and increased migration and invasion. We have shown that acquisition of K-RAS mutations in colon cancer cells leads to overexpression of membrane protein caveolin 1 and results in increase of KLK6 expression and secretion. The aim of this study was to elucidate the role of KLK6 expression in metastatic CRC in relation to the status of K-RAS oncogene. Analysis of KLK6 expression in CRC patients by immunohistochemistry shows the statistically significant increase in the staining score for KLK6 protein in the microscopically normal tissue 10 cm away from tumors, which carry mutant K-RAS (G12A mutation) (p=0.05 for crypts staining and p=0.0445 for the surface area). These data suggest a specific role of KLK6 in the invasive phenotype associated with the presence of K-RAS mutation. To investigate the significance of KLK6 expression in metastatic CRC, we developed HCT116 human colon cancer isogenic cell lines (K-RAS G13D mutation) with the genetic knock down of KLK6 expression (shKLK6 clones). The shKLK6 clones had decreased expression and secretion of KLK6 protein with the minimal effect on the cell growth and viability in cell culture. SCID mice injected with shKLK6 cells exhibited a statistically significant increase in the survival rates (p=0.005), decrease in the incidence of distant metastases and the shift in the location of the metastatic foci closer to the cells injection site. Levels of KLK6 protein secreted into the bloodstream were significantly lower in animals injected with shKLK6 clone compared to HCT116 control clone (p<0.04). A comprehensive array-based profiling and paired analysis of mRNA and miRNA expression in HCT116 control and KLK6 knockout cells revealed a group of specific miRNAs- mRNA target interactions involved in KLK6-mediated metastasis formation during CRC. The data reported here underscore the significance of KLK6 overexpression for distant metastasis formation in CRC and present the opportunity for therapeutic interventions. Citation Format: Bethany A. Skovan, Ritu Pandey, Raymond B. Nagle, Haiyan Cui, Julie A. Buckmeier, Robert S. Krouse, Patricia A. Thompson, Natalia A. Ignatenko. Role of kallikrein 6 secretion in metastatic colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2009. doi:10.1158/1538-7445.AM2014-2009

Abstract B4: Everolimus inhibits invasion and angiogenesis in a KRAS-mutated mesenchymal-differentiated colorectal cancer xenografts

Abstract Background: KRAS mutations accounting for 32% to 40% of colorectal cancers and epithelial-to-mesenchymal transition (EMT) have been associated with resistance to targeted therapies in colorectal carcinomas. The growth factor receptor- and/or oncogenic KRAS-dependent AKT-activations may lead mTOR to promote the expression of transcription factors involved in EMT activation in cancer cells. mTOR also plays an important role in survival of cancer cells in response to hypoxia during tumor angiogenesis. In this study, we evaluated the effects of everolimus on KRAS-mutated mesenchymal differentiated colon cancer cells in culture and in xenografts. Methods: The cellular and signaling effects of everolimus was investigated in parental COLO205-S cells (WT KRAS) that display an epithelial phenotype and their derived KRAS mutated (G13D/exon 2) mesenchymal counterparts, COLO205-R cells (Serova, 2010) using MTT assay and Western Blot. COLO205-S and COLO205-R were engrafted in nude mice treated with placebo or 10mg/kg everolimus for 3 weeks. Tumor invasion in mice was evaluated by counting tumor cells invading the skin. Angiogenesis and necrosis were evaluated using CD31 and HE staining, respectively. Results: Acquisition of KRAS mutation and EMT in COLO205-R cells was associated with constitutive AKT activation and resistance to several targeted therapies including lapatinib, and enzastaurin. Inhibition of mTOR with everolimus decreased the proliferation in COLO205-S and COLO205-R cells at high concentrations. KRAS mutation and EMT in COLO205-R tumors were associated with an increased tumor growth rate compared to COLO205-S tumors (mean tumor volume after 17 days of treatment: 1650mm3 vs. 953mm3, respectively, p<0.05). Pathological examinations also showed that COLO205-R cells were more invasive (number of invading cells in surrounding tumor tissues: 321 vs. 202) and more angiogenic than COLO205-S cells in tumors. In COLO205-S and COLO205-R xenografts everolimus significantly reduced tumor growth in nude mice compared to placebo (mean tumor growth inhibition: 35% and 34%, respectively). Everolimus also decreases the invasive potential of cancer cells in tissues surrounding the tumors in COLO205-R xenografts (number of invading cells per field: 63 vs. 321). The number and the size of vessels were significantly reduced (>2-fold) in everolimus treated tumors, suggesting antiangiogenic properties of this mTOR inhibitor. In addition, everolimus also induced larger necrosis area in tumors as compared with placebo (in COLO205-R xenografts: 59% vs. 28%, respectively, p<0.05; in COLO205-S xenografts: 31% vs 15%, respectively, p<0.05). Conclusions: Everolimus displayed effects on cancer cell proliferation and invasion and may also inhibit tumor angiogenesis in KRAS-mutated mesenchymal-differentiated colorectal cancer models. This suggested that odds antiangiogenic effects might be expected using everolimus despite adverse genetic and differentiation profiles of colon cancer cells.

Lifetime History of Tobacco Consumption and K-ras Mutations in Exocrine Pancreatic Cancer

We analyzed the relation between mutations in codon 12 of the K-ras oncogene and lifetime consumption of tobacco in patients with exocrine pancreatic cancer (EPC). Incident cases of EPC were prospectively identified and interviewed during hospital admission about smoking and other factors. Exact logistic regression was used to compare EPC cases (N = 107) with and without K-ras mutations (case-case study). Mutated cases were nonsignificantly less likely to have been smokers than wild-type cases: the odds ratio adjusted by age and sex was 0.54 (95% confidence interval, 0.10-2.69; P = 0.613). With respect to never smokers, adjusted odds ratios for former and current smokers were 0.79 and 0.36, respectively (P = 0.193). Pack-years smoked, years of smoking, and cigarettes smoked per year also tended to be higher in nonmutated than in mutated cases. Neither age at onset of smoking nor the time between quitting and diagnosis were associated with K-ras. Tobacco does not play a major part in the acquisition of K-ras mutations in the pancreatic epithelium. Although both smoking and K-ras mutations have important roles in the etiopathogenesis of EPC, the 2 processes may act independently.