Mutant Colorectal Cancer Cells Research Articles

A machine learning and drug repurposing approach to target ferroptosis in colorectal cancer stratified by sex and KRAS.

The landscape of sex differences in Colorectal Cancer (CRC) has not been well characterized with respect to the mechanisms of action for oncogenes such as KRAS. However, our recent study showed that tumors from male patients with KRAS mutations have decreased iron-dependent cell death called ferroptosis. Building on these findings, we further examined ferroptosis in CRC, considering both sex of the patient and KRAS mutations, using public databases and our in-house CRC tumor cohort. Through subsampling inference and variable importance analysis (VIMP), we identified significant differences in gene expression between KRAS mutant and wild type tumors from male patients. These genes suppress (e.g., SLC7A11 ) or drive (e.g., SLC1A5 ) ferroptosis, and these findings were further validated with Gaussian mixed models. Furthermore, we explored the prognostic value of ferroptosis regulating genes and discovered sex- and KRAS-specific differences at both the transcriptional and metabolic levels by random survival forest with backward elimination algorithm (RSF-BE). Of note, genes and metabolites involved in arginine synthesis and glutathione metabolism were uniquely associated with prognosis in tumors from males with KRAS mutations. Additionally, drug repurposing is becoming popular due to the high costs, attrition rates, and slow pace of new drug development, offering a way to treat common and rare diseases more efficiently. Furthermore, increasing evidence has shown that ferroptosis inhibition or induction can improve drug sensitivity or overcome chemotherapy drug resistance. Therefore, we investigated the correlation between gene expression, metabolite levels, and drug sensitivity across all CRC primary tumor cell lines using data from the Genomics of Drug Sensitivity in Cancer (GDSC) resource. We observed that ferroptosis suppressor genes such as DHODH , GCH1 , and AIFM2 in KRAS mutant CRC cell lines were resistant to cisplatin and paclitaxel, underscoring why these drugs are not effective for these patients. The comprehensive map generated here provides valuable biological insights for future investigations, and the findings are supported by rigorous analysis of large-scale publicly available data and our in-house cohort. The study also emphasizes the potential application of VIMP, Gaussian mixed models, and RSF-BE models in the multi-omics research community. In conclusion, this comprehensive approach opens doors for leveraging precision molecular feature analysis and drug repurposing possibilities in KRAS mutant CRC.

A dual gene combination therapy approach by KRAS-G12V gene editing and SLC25A22 gene silencing for KRAS-mutant colorectal cancer

KRAS gene is mutated in 40% of colorectal cancers (CRC), which induces malignant proliferation by regulating cellular nutrient metabolism and biosynthesis. It has been found that malignant proliferation of KRAS-mutant colorectal cancer relies on the upregulation of SLC25A22 protein expression, suggesting that inhibition the expression of both KRAS and SLC25A22 is a potential CRC therapeutics. Stably knocking down the oncogenic KRAS-G12V gene can achieve long-term gene therapy effects, while transient downregulation of SLC25A22, a normal functional gene most of the time, is preferred to kill tumor cells and minimize the side impact on normal cells. Here, two lipid nanoparticles (LNP) were designed to encapsulate KRAS-G12V CRISPR/Cas9 gene editing plasmids (pKRAS-LNPs) and SLC25A22 siRNA (siSLC-LNPs), respectively. Therapeutic effects of both nanoparticles alone and in combination on KRAS-G12V mutant colorectal cancer cells in vitro were first examined. The result showed that delivery of pKRAS-LNPs or siSLC-LNPs alone could effectively achieve KRAS-G12V gene editing or SLC25A22 gene silencing and inhibit tumor cell proliferation, while co-delivery of both LNPs could achieve stronger inhibition of tumor cell proliferation by inducing stronger apoptosis. Furthermore, we found that co-delivery of pKRAS-LNPs and siSLC-LNPs induced stronger apoptosis and cell proliferation inhibition compared to pKRAS&siSLC-LNPs that were constructed by pre-mixing pKRAS and siSLC and then encapsulating them. Finally, we validated that co-delivery of pKRAS-LNPs and siSLC-LNPs can achieve KRAS-G12V colorectal cancer treatment in vivo with a tumor inhibition rate of 61.15%. In summary, the delivery vectors constructed for nucleic acids targeting KRAS and SLC25A22 achieved therapeutic targeting of KRAS-G12V colorectal cancer in vitro and in vivo.

Abstract 5430: Discovery of CYP26A1 as a link between WNT and retinoic acid signaling pathways that modulates retinoid-induced differentiation of APC-mutant colorectal cancer cells

Abstract APC mutation is the main driver mechanism of CRC development and leads to constitutively activated WNT signaling, overpopulation of ALDH+ stem cells (SCs), and incomplete differentiation. We previously reported that retinoic acid (RA) receptors are selectively expressed in ALDH+ SCs, which provides a way to target cancer SCs with retinoids to induce differentiation. We hypothesize that a functional link exists between WNT and RA pathways, and APC mutation generates a WNT:RA imbalance that decreases retinoid-induced differentiation and increases ALDH+ SCs. Accordingly, to restore parity in WNT:RA signaling, we induced wt-APC expression in APC-mutant CRC cells and assessed the ability of all-trans-retinoic acid (ATRA) to induce differentiation. Notably, ATRA substantially increased expression of the WNT-target gene, CYP26A1, and inducing wt-APC reduced this expression by 50%. Thus, RA and WNT pathways crosstalk to modulate CYP26A1 metabolism of retinoids and retinoid agents. We found that inducing wt-APC augments ATRA-induced cell differentiation by: i) decreasing cell proliferation; ii) suppressing ALDH1A1 expression; iii) decreasing ALDH+ SCs; and iv) increasing neuroendocrine cell differentiation. NanoString profiling and bioinformatics also identified a novel CYP26A1-based network that links WNT and RA signaling. Moreover, in an effort to translate manipulation of the WNT/RA imbalance as a therapeutic approach in vitro, we treated HT29, SW480, and HCT116 CRC cells with inhibitors against CYP26A1 enzyme activity and WNT signaling. We found that CYP26A1 inhibitors sensitized CRC cells to the anti-proliferative effect of drugs that downregulate WNT signaling. Furthermore, decreased CYP26A1 expression in tumor vs normal tissue was determined to be a predictor of patient survival in patients who have wt-APC. Thus, by inducing wt-APC expression and exogenously activating RA signaling, we found a way to increase differentiation of SCs along the NEC (and other) lineage(s). Translating this strategy in vivo might lead to new, more effective treatments involving retinoids for CRC patients. Citation Format: Caroline O. Facey, Victoria O. Hunsu, Chi Zhang, Brian T. Osmond, Lynn M. Opdenaker, Bruce M. Boman. Discovery of CYP26A1 as a link between WNT and retinoic acid signaling pathways that modulates retinoid-induced differentiation of APC-mutant colorectal cancer cells [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 5430.

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 1995: DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant colorectal cancer

Abstract The 5-year survival rate for metastatic colorectal cancer (CRC) is less than 14%. Hyperactivation of the ERK/MAPK pathway occurs at a high frequency in CRC, due to mutations (RAS, BRAF) or overexpression/amplification (EGFR) of its signalling components. Aberrant activation of this pathway drives tumour growth and survival and several inhibitors of this pathway are now clinically utilized. The BRAF V600E activating mutation occurs in ~10% of CRC patients and is correlated with an extremely poor prognosis. Use of the small molecule BRAF inhibitor encorafenib in combination with the EGFR inhibitor cetuximab is now approved for BRAF V600E mutant patients, however, the objective response rate is only ~20% and the treatment is rarely curative. Therefore, there is an urgent need to identify predictive biomarkers of response to encorafenib+cetuximab treatment. Dual-specificity phosphatase 4 (DUSP4) is rapidly induced upon activation of ERK/MAPK signaling upon which it acts as a critical negative regulator of signaling output by directly de-phosphorylating ERK. We have observed that DUSP4 is highly expressed in BRAF-mutant CRC cell lines and primary tumours, although there is a range of expression. Whether DUSP4 expression status impacts on response to encorafenib+cetuximab therapy is unknown. The aim of this study therefore was to determine the role of DUSP4 on sensitivity of BRAF-mutant CRC cells to encorafenib+cetuximab treatment in vitro. To examine this, we determined the impact of DUSP4 knock-down, knock-out and re-expression on sensitivity of BRAF-mutant CRC cancer cell lines to encorafenib+cetuximab treatment. Screening a panel of BRAF mutant CRC cell lines for sensitivity to encorafenib+cetuximab revealed a subset of resistant cell lines had either low or undetectable DUSP4 protein expression, while all sensitive cell lines had high DUSP4 expression. siRNA-mediated knockdown of DUSP4 in DUSP4 expressing BRAF-mutant CRC cell lines increased pERK and pFRA1, suggesting activation of ERK/MAPK signalling. DUSP4 knockdown also reduced sensitivity of BRAF mutant CRC cell lines to encorafenib+cetuximab compared to control. These findings were further validated using DUSP4 knock-out with CRISPR-Cas9. Conversely, DUSP4 re-expression in BRAF-mutant CRC cell lines lacking DUSP4 expression, decreased pERK and increased sensitivity to encorafenib+cetuximab treatment. These results demonstrate that DUSP4 regulates ERK/MAPK signalling in BRAF mutant CRC cell lines and that loss of DUSP4 promotes resistance to encorafenib plus cetuximab treatment. These findings warrant investigation of DUSP4 expression status as a predictive biomarker of encorafenib+cetuximab response in patient samples. Citation Format: Kristen Needham, Fiona Chionh, Stan Kaczmarczyk, Connor Kearney, Laura Jenkins, Ian Luk, John M. Mariadason. DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant 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 1995.

Abstract 1250: Activating PIK3CA mutations and hedgehog signaling may confer resistance to KRAS inhibition in colorectal cancer

Abstract Background: Treatment with KRASG12C inhibitors results in an objective response in only small fraction of patients with KRASG12C mutant colorectal cancer (CRC) (7.1% and 22% objective response rates (ORR) in two prospective trials), in contrast to lung cancer where the ORR exceeds 35%. We hypothesize that co-mutation of other oncogenes and/or transcriptomic factors may contribute to the resistance of KRASG12C CRC to KRAS inhibitors. Methods: Mutation profiles of CRC patients were obtained from the AACR Project-GENIE v14 CRC cohort (N = 9441). The impact of KRAS knockout in 59 CRC cell lines was queried from the DepMap database. KRASG12C mutant CRC cell lines SW837 and SW1463 were treated with Sotorasib (KRASG12C inhibitor), Vismodegib and Taladegib (Hedgehog inhibitors), and cell viability was measured up to 96 hours after treatment. Results: In the AACR Project-GENIE CRC (N = 9441) cohort, KRASG12C tumors (2.6%, 245/9441) were significantly co-mutated with PIK3CAmut (19.2%, 47/245; Odds ratio = 1.7 (95% CI: 1.2 - 2.4), Chi-Square test, p-value = 0.002). Dependence on KRAS in CRISPR knockout viability assays was higher in KRASmut vs. wildtype CRC cell lines (median CERES score -1.3 vs -0.5, p-value < 0.001). Interestingly, KRASmut cell lines with PIK3CA co-mutation (n=10) were more resistant to KRAS knockout than PIK3CAwt cells (n=22) (median -1.0 vs -1.4, p-value = 0.03). The SW837 and SW1463 cell lines initially displayed high sensitivity to Sotorasib (IC50: 0.92 µM and 0.90 µM). However, overexpression of PIK3CAE545K mutant induced resistance to the same treatment (IC50: 5.18 µM and 6.45 µM). Furthermore, gene set enrichment analysis (GSEA) showed that Hedgehog signaling was significantly enriched in KRAS knockout resistant KRASmut CRC cell lines. SW837 cell line treated with Sotorasib (S) and a fixed dosage of 25 µM of Vismodegib (V) or Taladegib (T) showed higher sensitivities in combination treatment assays (IC50 decreased: ~11 folds in S+V vs S; and ~15 folds in S+T vs S) as compared to Sotorasib (S) treatment alone. Similar results were seen for the SW1463 cell line (IC50 decreased: ~18 folds in S+V vs S; and ~15 folds in S+T vs S). Conclusion: Resistance in CRC to KRASG12C inhibitors may be attributed to activated PIK3CA mutations and the Hedgehog signaling pathway. The future study will focus on detailed investigations involving comprehensive transcriptome and exome profiles in tumors and PDX models treated with anti-KRAS drugs in monotherapy and combination therapy. Citation Format: Saikat Chowdhury, Jibran Ahmed, Valsala Haridas, David S. Hong, Scott Kopetz, John Paul Shen. Activating PIK3CA mutations and hedgehog signaling may confer resistance to KRAS inhibition 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 1250.

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.

Onvansertib in Combination with FOLFIRI and Bevacizumab in Second-Line Treatment of KRAS-Mutant Metastatic Colorectal Cancer: A Phase Ib Clinical Study.

Onvansertib is a highly specific inhibitor of polo-like kinase 1 (PLK1), with demonstrated safety in solid tumors. We evaluated, preclinically and clinically, the potential of onvansertib in combination with chemotherapy as a therapeutic option for KRAS-mutant colorectal cancer. Preclinical activity of onvansertib was assessed (i) in vitro in KRAS wild-type and -mutant isogenic colorectal cancer cells and (ii) in vivo, in combination with irinotecan, in a KRAS-mutant xenograft model. Clinically, a phase Ib trial was conducted to investigate onvansertib at doses 12, 15, and 18 mg/m2 (days 1-5 and 14-19 of a 28-day cycle) in combination with FOLFIRI/bevacizumab (days 1 and 15) in patients with KRAS-mutant metastatic colorectal cancer who had prior oxaliplatin exposure. Safety, efficacy, and changes in circulating tumor DNA (ctDNA) were assessed. In preclinical models, onvansertib displayed superior activity in KRAS-mutant than wild-type isogenic colorectal cancer cells and demonstrated potent antitumor activity in combination with irinotecan in vivo. Eighteen patients enrolled in the phase Ib study. Onvansertib recommended phase II dose was established at 15 mg/m2. Grade 3 and 4 adverse events (AE) represented 15% of all treatment-related AEs, with neutropenia being the most common. Partial responses were observed in 44% of patients, with a median duration of response of 9.5 months. Early ctDNA dynamics were predictive of treatment efficacy. Onvansertib combined with FOLIFRI/bevacizumab exhibited manageable safety and promising efficacy in second-line treatment of patients with KRAS-mutant metastatic colorectal cancer. Further exploration of this combination therapy is ongoing. See related commentary by Stebbing and Bullock, p. 2005.

The 5-/12-lipoxygenase-BLT2 cascade induces elevated expression of c-Myc, thus mediating the proliferation and migration of KRAS mutant colorectal cancer cells

ABSTRACTColorectal cancer (CRC) is the most common malignant tumor and one of the leading causes of cancer-related death worldwide. Oncogene KRAS is a commonly mutated in CRC and plays crucial roles in the colorectal tumorigenesis. Emerging evidence suggests that increased expression levels of c-Myc are critical for KRAS-mediated CRC progression. However, it is unclear exactly how c-Myc expression is regulated in CRC. In the present study, we found that blockade of 5/12-lipoxygenase (5/12-LO) or leukotriene B4 receptor (BLT2) markedly reduced c-Myc expression in KRAS-mutant LOVO cells, while the control COLO 320DM cells were not affected. When the 5/12-LO products LTB4 and 12(S)-HETE, ligands of BLT2, were added to LOVO cells, c-Myc expression levels were restored, together suggesting that ‘5/12-LO-BLT2 cascade’ regulates c-Myc expression in KRAS-mutant CRC cells. We also observed that ‘5/12-LO-BLT2’-mediated c-Myc upregulation contributes to cell proliferation by regulating the expression of cyclin D1 in LOVO cells. Moreover, ‘5/12-LO-BLT2-cMyc’ cascade regulates the expression of EMT-related proteins in KRAS-mutant CRC cells, and depletion of both c-Myc and BLT2 using siRNA significantly enhanced the level of E-cadherin and reduced the level of Vimentin in LOVO cells. Taken together, our findings suggest that the BLT2-linked cascade promotes KRAS-mutant CRC cell proliferation and migration through the elevated expression of c-Myc. Thus, our results suggest that BLT2 appears to be an effective therapeutic target for KRAS-mutant CRCs.

P53-armed oncolytic adenovirus induces autophagy and apoptosis in KRAS and BRAF-mutant colorectal cancer cells.

Colorectal cancer (CRC) cells harboring KRAS or BRAF mutations show a more-malignant phenotype than cells with wild-type KRAS and BRAF. KRAS/BRAF-wild-type CRCs are sensitive to epidermal growth factor receptor (EGFR)-targeting agents, whereas KRAS/BRAF-mutant CRCs are resistant due to constitutive activation of the EGFR-downstream KRAS/BRAF signaling pathway. Novel therapeutic strategies to treat KRAS/BRAF mutant CRC cells are thus needed. We recently demonstrated that the telomerase-specific replication-competent oncolytic adenoviruses OBP-301 and p53-armed OBP-702 exhibit therapeutic potential against KRAS-mutant human pancreatic cancer cells. In this study, we evaluated the therapeutic potential of OBP-301 and OBP-702 against human CRC cells with differing KRAS/BRAF status. Human CRC cells with wild-type KRAS/BRAF (SW48, Colo320DM, CACO-2), mutant KRAS (DLD-1, SW620, HCT116), and mutant BRAF (RKO, HT29, COLO205) were used in this study. The antitumor effect of OBP-301 and OBP-702 against CRC cells was analyzed using the XTT assay. Virus-mediated modulation of apoptosis, autophagy, and the EGFR-MEK-ERK and AKT-mTOR signaling pathways was analyzed by Western blotting. Wild-type and KRAS-mutant CRC cells were sensitive to OBP-301 and OBP-702, whereas BRAF-mutant CRC cells were sensitive to OBP-702 but resistant to OBP-301. Western blot analysis demonstrated that OBP-301 induced autophagy and that OBP-702 induced autophagy and apoptosis in human CRC cells. In BRAF-mutant CRC cells, OBP-301 and OBP-702 suppressed the expression of EGFR, MEK, ERK, and AKT proteins, whereas mTOR expression was suppressed only by OBP-702. Our results suggest that p53-armed oncolytic virotherapy is a viable therapeutic option for treating KRAS/BRAF-mutant CRC cells via induction of autophagy and apoptosis.

Vitamin C Enhances Anticancer Immunity

The molecular mechanisms underlying vitamin C’s anticancer activity remain unclear. Recently, multiple high-quality studies demonstrated several potentially novel functions of vitamin C in regulating anticancer immunity, including modulating T cell infiltration of tumor microenvironment as well as increasing cancer cell immunogenicity. (First online: June 4, 2023) REFERENCES Creagan ET, Moertel CG, O'Fallon JR, Schutt AJ, O'Connell MJ, Rubin J, et al. Failure of high-dose vitamin C (ascorbic acid) therapy to benefit patients with advanced cancer: a controlled trial. N Engl J Med 1979; 301(13):687–90. doi: https://dx.doi.org/10.1056/NEJM197909273011303 Moertel CG, Fleming TR, Creagan ET, Rubin J, O'Connell MJ, Ames MM. High-dose vitamin C versus placebo in the treatment of patients with advanced cancer who have had no prior chemotherapy: a randomized double-blind comparison. N Engl J Med 1985; 312(3):137–41. doi: https://dx.doi.org/10.1056/NEJM198501173120301 Lin J, Cook NR, Albert C, Zaharris E, Gaziano JM, Van Denburgh M, et al. Vitamins C and E and beta carotene supplementation and cancer risk: a randomized controlled trial. J Natl Cancer Inst 2009; 101(1):14–23. doi: https://dx.doi.org/10.1093/jnci/djn438 Chen Q, Espey MG, Sun AY, Pooput C, Kirk KL, Krishna MC, et al. Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc Natl Acad Sci USA 2008; 105(32):11105–9. doi: https://dx.doi.org/10.1073/pnas.0804226105 Yun J, Mullarky E, Lu C, Bosch KN, Kavalier A, Rivera K, et al. Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH. Science 2015; 350(6266):1391–6. doi: https://dx.doi.org/10.1126/science.aaa5004 Magri A, Germano G, Lorenzato A, Lamba S, Chila R, Montone M, et al. High-dose vitamin C enhances cancer immunotherapy. Sci Transl Med 2020; 12(532). doi: 10.1126/scitranslmed.aay8707. Xu YP, Lv L, Liu Y, Smith MD, Li WC, Tan XM, et al. Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy. J Clin Invest 2019; 129(10):4316–31. doi: https://dx.doi.org/10.1172/JCI129317 Wu X, Zhang Y. TET-mediated active DNA demethylation: mechanism, function and beyond. Nat Rev Genet 2017; 18(9):517–34. doi: https://dx.doi.org/10.1038/nrg.2017.33 Luchtel RA, Bhagat T, Pradhan K, Jacobs WR, Jr., Levine M, Verma A, et al. High-dose ascorbic acid synergizes with anti-PD1 in a lymphoma mouse model. Proc Natl Acad Sci USA 2020; 117(3):1666–77. doi: https://dx.doi.org/10.1073/pnas.1908158117 Morante-Palacios O, Godoy-Tena G, Calafell-Segura J, Ciudad L, Martinez-Caceres EM, Sardina JL, et al. Vitamin C enhances NF-kappaB-driven epigenomic reprogramming and boosts the immunogenic properties of dendritic cells. Nucleic Acids Res 2022; 50(19):10981–94. doi: https://dx.doi.org/10.1093/nar/gkac941

Riok1, A Novel Potential Target in MSI-High p53 Mutant Colorectal Cancer Cells.

The vulnerabilities of cancer cells constitute a promising strategy for drug therapeutics. This paper integrates proteomics, bioinformatics, and cell genotype together with in vitro cell proliferation assays to identify key biological processes and potential novel kinases that could account, at least in part, for the clinical differences observed in colorectal cancer (CRC) patients. This study started by focusing on CRC cell lines stratified by their microsatellite (MS) state and p53 genotype. It shows that cell-cycle checkpoint, metabolism of proteins and RNA, signal transduction, and WNT signaling processes are significantly more active in MSI-High p53-WT cell lines. Conversely, MSI-High cell lines with a mutant (Mut) p53 gene showed hyperactivation of cell signaling, DNA repair, and immune-system processes. Several kinases were linked to these phenotypes, from which RIOK1 was selected for additional exploration. We also included the KRAS genotype in our analysis. Our results showed that RIOK1's inhibition in CRC MSI-High cell lines was dependent on both the p53 and KRAS genotypes. Explicitly, Nintedanib showed relatively low cytotoxicity in MSI-High with both mutant p53 and KRAS (HCT-15) but no inhibition in p53 and KRAS WT (SW48) MSI-High cells. This trend was flipped in CRC MSI-High bearing opposite p53-KRAS genotypes (e.g., p53-Mut KRAS-WT or p53-WT KRAS-Mut), where observed cytotoxicity was more extensive compared to the p53-KRAS WT-WT or Mut-Mut cells, with HCT 116 (KRAS-Mut and p53-WT) being the most sensitive to RIOK1 inhibition. These results highlight the potential of our in silico computational approach to identify novel kinases in CRC sub-MSI-High populations as well as the importance of clinical genomics in determining drug potency.

Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

Constitutive activation of RAS-RAF-MEK-ERK signaling pathway (MAPK pathway) frequently occurs in many cancers harboring RAS or RAF oncogenic mutations. Because of the paradoxical activation induced by a single use of BRAF or MEK inhibitors, dual-target RAF and MEK treatment is thought to be a promising strategy. In this work, we evaluated erianin is a novel inhibitor of CRAF and MEK1/2 kinases, thus suppressing constitutive activation of the MAPK signaling pathway induced by BRAF V600E or RAS mutations. KinaseProfiler enzyme profiling, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), cellular thermal shift assay, computational docking, and molecular dynamics simulations were utilized to screen and identify erianin binding to CRAF and MEK1/2. Kinase assay, luminescent ADP detection assay, and enzyme kinetics assay were investigated to identify the efficiency of erianin in CRAF and MEK1/2 kinase activity. Notably, erianin suppressed BRAF V600E or RAS mutant melanoma and colorectal cancer cell by inhibiting MEK1/2 and CRAF but not BRAF kinase activity. Moreover, erianin attenuated melanoma and colorectal cancer in vivo. Overall, we provide a promising leading compound for BRAF V600E or RAS mutant melanoma and colorectal cancer through dual targeting of CRAF and MEK1/2.

The efficacy and side effects of rigosertib combined with chemotherapy in KRAS mutant colorectal cancer mice

Objective: To investigate the effect of rigosertib (RGS) combined with classic chemotherapy drugs including 5-fluorouracil, oxaliplatin, and irinotecan in colorectal cancer. Methods: Explore the synergy effects of RGS and 5-fluorouracil (5-FU), oxaliplatin (OXA), and irinotecan (IRI) on colorectal cancer by subcutaneously transplanted tumor models of mice. The mice were randomly divided into control group, RGS group, 5-FU group, OXA group, IRI group, 5-FU+ RGS group, OXA+ RGS group and IRI+ RGS group. The synergy effects of RGS and OXA on KRAS mutant colorectal cancer cell lines in vitro was detected by CCK-8. Ki-67 immunohistochemistry and TdT-mediated dUTP nick-end labeling (TUNEL) staining were performed on the mouse tumor tissue sections, and the extracted tumor tissue was analyzed by western blot. The blood samples of mice after chemotherapy and RGS treatment were collected, blood routine and liver and kidney function analysis were conducted, and H&E staining on liver sections was performed to observe the side effects of chemotherapy and RGS. Results: The subcutaneously transplanted tumor models were established successfully in all groups. 55 days after administration, the fold change of tumor size of OXA+ RGS group was 37.019±8.634, which is significantly smaller than 77.571±15.387 of RGS group (P=0.029) and 92.500±13.279 of OXA group (P=0.008). Immunohistochemical staining showed that the Ki-67 index of tumor tissue in control group, OXA group, RGS group and OXA+ RGS group were (100.0±16.8)%, (35.6±11.3)%, (54.5±18.1)% and (15.4±3.9)%, respectively. The Ki-67 index of OXA+ RGS group was significantly lower than that in control group (P=0.014), but there was no significant difference compared to OXA group and RGS group (OXA: P=0.549; RGS: P=0.218). TUNEL fluorescence staining showed that the apoptotic level of OXA+ RGS group was 3.878±0.547, which was significantly higher than 1.515±0.442 of OXA group (P=0.005) and 1.966±0.261 of RGS group (P=0.008). Western blot showed that the expressions of apoptosis related proteins such as cleaved-PARP, cleaved-caspase 3 and cleaved-caspase 8 in the tumor tissues of mice in the OXA+ RGS group were higher than those in control group, OXA group and RGS group. After the mice received RGS combined with chemotherapy drugs, there was no significant effect on liver and kidney function indexes, but the combined use of oxaliplatin and RGS significantly reduced the white blood cells [(0.385±0.215)×10(9)/L vs (5.598±0.605)×10(9)/L, P<0.001] and hemoglobin[(56.000±24.000)g/L vs (153.333±2.231)g/L, P=0.001] of the mice. RGS, chemotherapy combined with RGS and chemotherapy alone did not significantly increase the damage to liver cells. Conclusions: The combination of RGS and oxaliplatin has a stronger anti-tumor effect on KRAS mutant colorectal cancer. RGS single agent will not cause significant bone marrow suppression and hepatorenal injury in mice, but its side effects may increase correspondingly after combined with chemotherapy.

Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

The potential effect of Schisandrin-Bcombination with panitumumab in wild-type and mutant colorectal cancer cell lines: Role of apoptosis and autophagy.

Panitumumab is an approved monoclonal antibody for the treatment of colorectal cancer (CRC); however, mutations in EGFR signaling pathway resulted in poor response. Schisandrin-B (Sch-B) is a phytochemical that was suggested to protect against inflammation, oxidative stress, and cell proliferation. The present study aimed to investigate the potential effect of Sch-B on panitumumab-induced cytotoxicity in wild-type Caco-2, and mutant HCT-116 and HT-29 CRC cell lines, and the possible underlying mechanisms.CRC cell lines were treated with panitumumab, Sch-B, and their combination. The cytotoxic effect of drugs was determined by MTT assay. The apoptotic potential was assessed in-vitro by DNA fragmentation and caspase-3 activity. Additionally, autophagy was investigated via microscopic detection of autophagosomes and quantitative reverse transcription-polymerase chain reaction(qRT-PCR) measurement of Beclin-1, Rubicon, LC3-II, and Bcl-2 expression. The drug pair enhanced panitumumab cytotoxicity in all CRC cell lines where IC50 of panitumumab was decreased in Caco-2 cell line. Apoptosis was induced through caspase-3 activation, DNA fragmentation, and Bcl-2 downregulation. Caco-2 cell line treated with panitumumab showed stained acidic vesicular organelles, contrariwise, all cell lines treated with Sch-B or the drug pair displayed green fluorescence indicating the lack of autophagosomes. qRT-PCR revealed the downregulation of LC3-II in all CRC cell lines, Rubicon in mutant cell lines, and Beclin-1 in HT-29 cell line only.Sch-B at 6.5 µM promoted panitumumab-induced apoptotic cell death, in-vitro, via caspase-3 activation and Bcl-2 downregulation, rather than autophagic cell death. This novel combination therapy against CRC, allows the reduction of panitumumab dose to guard against its adverse effects.

CopA3 peptide inhibits MDM2-p53 complex stability in colorectal cancers and activates p53 mediated cell death machinery

The diverse expression patterns of the tumor suppressor p53 in cancer cells reflect the regulatory efficiency of multiple cellular pathways. By contrast, many human tumors are reported to develop in the presence of wild-type p53. Recently, several oncogene inhibitors have been used clinically to suppress tumor development by functionally reactivating other oncoproteins. On the other hand, p53 reactivation therapies have not been well established, as few of the p53-MDM2 complex inhibitors such as Nutlin-3 induces mutation in p53 gene upon prolonged usage. Therefore, in this study CopA3, a 9-mer dimeric D-type peptide with anticancer activity against the human colorectal cancer cells, was used to explore the efficacy of p53 reactivation in-vitro and in-vivo. The anticancer activity of CopA3 was more selective towards the wild-type p53 expressing cells than the p53 deficient or mutant colorectal cancer cells. In response to this, this study investigated the signaling pathway in vitro and validated its anti-tumor activity in-vivo. The protein-peptide interaction and molecular docking efficiently provided insight into the specific binding affinity of CopA3 to the p53-binding pocket of the MDM2 protein, which efficiently blocked the p53 and MDM2 interaction. CopA3 plays a crucial role in the binding with MDM2 and enhanced the nuclear translocation of the p53 protein, which sequentially activated the downstream targets to trigger the autophagic mediated cell death machinery through the JNK/Beclin-1 mediated pathway. Collectively, CopA3 affected the MDM2-p53 interaction, which suppressed tumor development. This study may provide a novel inhibitor candidate for the MDM2-p53 complex, which could ultimately suppress the growth of colorectal cancer cells without being cytotoxic to the healthy neighboring cells present around the tumor microenvironment.

Oleic acid-induced metastasis of KRAS/p53-mutant colorectal cancer relies on concurrent KRAS activation and IL-8 expression bypassing EGFR activation.

Background: Multigene mutations in colorectal cancer (CRC), including KRAS, BRAF, and p53, afford high metastatic ability and resistance to EGFR-targeting therapy. Understanding the molecular mechanisms regulating anti-EGFR-resistant CRC metastasis can improve CRC therapy. This study aimed to investigate the effects of IL-8 and the activation of KRAS on reactive oxygen species (ROS) production and metastasis of hyperlipidemia-associated CRC harboring mutations of KRAS and p53. Methods: The cytokine array analysis determined the up-expression of secreted factors, including IL-8. The clinical relevance of the relationship between IL-8 and angiopoietin-like 4 (ANGPTL4) was examined in CRC patients from National Cheng Kung University Hospital and TCGA dataset. Expressions of IL-8, ANGPTL4, NADPH oxidase 4 (NOX4), and epithelial-mesenchymal transition (EMT) markers in free fatty acids (FFAs)-treated KRAS/p53 mutant CRC cells were determined. The hyperlipidemia-triggered metastatic ability of CRC cells under treatments of antioxidants, statin, and cetuximab or knockdown of IL-8, KRAS, and EGFR was evaluated in vitro and in vivo. In addition, the effects of antioxidants and depletion of IL-8 and KRAS on the correlation between ROS production and hyperlipidemia-promoted CRC metastasis were also clarified. Results: In this study, we found that free fatty acids promoted KRAS/p53-mutant but not single-mutant or non-mutant CRC cell metastasis. IL-8, the most abundant secreted factor in KRAS/p53-mutant cells, was correlated with the upregulation of NOX4 expression and ROS production under oleic acid (OA)-treated conditions. In addition, the metastasis of KRAS/p53-mutant CRC relies on the ANGPTL4/IL-8/NOX4 axis and the activation of KRAS. The antioxidants and inactivation of KRAS also inhibited OA-induced EMT and metastasis. Although KRAS mediated EGF- and OA-promoted CRC cell invasion, the inhibition of EGFR did not affect OA-induced ANGPTL4/IL-8/NOX4 axis and CRC metastasis. The high-fat diet mice fed with vitamin E and statin or in IL-8-depleted cells significantly inhibited tumor extravasation and metastatic lung growth of CRC. Conclusion: The antioxidants, statins, and targeting IL-8 may provide better outcomes for treating metastatic CRC that harbors multigene mutations and anti-EGFR resistance.

Sensitivities and Dependencies of BRAF Mutant Colorectal Cancer Cell Lines with or without PIK3CA Mutations for Discovery of Vulnerabilities with Therapeutic Potential.

Background: Colorectal cancer represents a common malignancy and remains incurable in the metastatic stage. Identification of molecular alterations that are present in colorectal cancer has led to the introduction of targeted therapies that improve outcomes. BRAF and PIK3CA mutations are observed in a subset of colorectal cancers. Colorectal cancers bearing BRAF mutations may be treated with specific BRAF inhibitors. These drugs benefit patients with BRAF mutant colorectal cancers but responses are rather brief, and progression is the rule. In contrast, no PI3K inhibitors have proven successful yet in the disease. Thus, new treatments to supplement the currently available drugs would be welcome to further improve survival. Methods: Profiled colorectal cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) were examined for BRAF and PIK3CA mutations and were interrogated for molecular characteristics and concomitant alterations that mirror clinical sample alterations. The Genomics of Drug Sensitivity in Cancer (GDSC) project was used for determination of drug sensitivities of BRAF mutated colorectal cell lines with or without concomitant PIK3CA mutations. The Cancer Dependency Map project served as the basis for identification of molecular dependencies and vulnerabilities in these cell lines. Results: CCLE includes 84 colorectal cancer cell lines, which recapitulate the molecular landscape of colorectal cancer. Of these, 23 and 24 cell lines possess BRAF and PIK3CA mutations, respectively. Seven BRAF mutant cell lines have V600E mutations and 14 PIK3CA mutant cell lines have hotspot helical or kinase domain mutations. V600E BRAF mutant cell lines with or without hotspot PIK3CA mutations are heterogeneous in their MSI status and mimic colorectal cancer tissues in other prevalent abnormalities including APC and TP53 mutations. Essential genes for survival include CTNNB1, WRN, and pyrimidine metabolism enzyme CAD. Besides BRAF mutations, BRAF inhibitor sensitivity in colorectal cancer cell lines is conferred by SACS mutations and PRKN locus loss. Conclusions: Colorectal cancer cell lines bearing the frequent BRAF and PIK3CA mutations present many alterations of the parental cancer tissue. Described vulnerabilities represent leads for therapeutic exploration in colorectal cancers with the corresponding alterations.

150 (PB030) - MEKi-based combination strategies for targeting KRAS-driven cancer

Background: KRAS is one of the most frequently mutated oncogenes in human cancer. Recent successes with KRAS G12C-specific inhibitors are changing the treatment paradigm in this subset of cancer patients. An alternative strategy is targeting downstream KRAS effectors, such as MEK. However, MEK inhibitors have historically proven unsuccessful as monotherapy in KRAS mutant cancers, highlighting the need for combinatory approaches. One possibility is the combination of MEK and SOS1 inhibition, which may overcome adaptive resistance to MEKi by blocking SOS1-mediated reactivation of MAPK signaling. Another promising approach is to combine MEKi with inhibitors of Bromodomain and extra-terminal (BET) proteins. While BET inhibitors exhibit broad anti-tumor activity in preclinical cancer models, BETi monotherapy demonstrated only moderate clinical activity. Resistance to BETi has been, at least partially, attributed to MAPK pathway upregulation, suggesting that a combination with MEKi may help to overcome resistance. Material and Methods: Here, we present BI 3011441, a potent and selective inhibitor of human MEK1/2. Using in vitro assays, we compare BI 3011441 to other clinical stage MEK inhibitors. We also combine BI 3011441 with BI 1701963 and BI 894999 - two potent, selective, and orally bioavailable inhibitors of SOS1 and BET, respectively. Both combinations are tested in vitro and/or in vivo across a panel of KRAS mutant pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) models. Results: BI 3011441 efficiently inhibits ERK phosphorylation in cell-based assays and demonstrates the strongest in vitro growth inhibition in a panel of BRAF or KRAS mutant cell lines among the MEK inhibitors tested. In vivo, BI 3011441 leads to a dose-dependent modulation of MAPK pathway, which translates into anti-tumor activity in xenograft models of KRAS-driven cancer. The combination of BI 3011441 with SOS1i BI 1701963 synergistically decreases MAPK signaling resulting in enhanced efficacy in xenograft models, including NSCLC, CRC and PDAC. In NSCLC models this combination results in 10% of partial responses (PR) and 70% of stable disease (SD) as defined by modified Response Evaluation Criteria In Solid Tumors (mRECIST). In PDAC and CRC models the combination leads to 30% and 43% of SD, respectively. When combined with clinically relevant concentrations of BETi BI 894999, BI 3011441 strongly decreases in vitro proliferation and induces apoptosis in KRAS mutant cancer cells. Synergy is observed in 17 out of 44 cell lines across different indications and harboring different KRAS mutations. Conclusions: In summary, we show that combining BI 3011441 with SOS1 or BET inhibitors may lead to improved responses in KRAS mutant tumors, likely by circumventing MAPK pathway-related adaptive resistance. Conflict of interest: Other Substantive Relationships: The authors are either employees or have financial relationships with Boehringer Ingelheim.