KRAS Mutant Colorectal Cancer Cells Research Articles

Identification and verification of KEAP1‐related genes and targets regulated by potential ingredients in KRAS mutant colorectal cancer

AbstractAs one of the most prevalent tumors, colorectal cancer (CRC) owns complex pathogenesis and the high recurrence rate significantly influence the prognosis of patients with KRAS mutant CRC, to discovery the crosstalk among KEAP1‐NFE2L2 and oncogenic KRAS as the potential prognostic biomarkers in CRC, which might be used to develop novel and effective therapeutics. By using bioinformatics analysis, we explored and identified key genes as the predicted targets in the crosstalk among KEAP1 oncogenic signatures of KRAS mutation that were linked with development, metastasis, and poor prognosis in CRC. We further investigated the correlations between the clinical characteristics and expressions of prognostic genes among the KRAS and KEAP1‐related key hub genes in CRC, which as predicted targets to find the potential herbal ingredients in HERB database, further to verify the active herbal ingredients could regulate the key targets that related to KEAP1‐NFE2L2 in KRAS mutant CRC. The newest TCGA data reveals that the mutation of KRAS is found in 44% of CRC patients. In total, 28 genes were identified as differentially expressed genes (DEGs), and the hub genes such as CDKN2A, SPP1, FOS, BCL2L11, and HPSE were verified. Results indicated that key genes (SOX9, SPP1) significant correlation with the target prediction of the active herbal ingredients by molecular docking analysis. Furthermore, KEAP1, NFE2L2, SOX9 expression were decreased significantly with the treatment of potential ingredients. Furthermore, cyclopamine could enhance the sensitivity of HCT116 cells, downregulated the expression of SPP1, and induced activation of KEAP1‐NFE2L2 pathway, which cell death are characteristic features of apoptosis, and enhanced anticancer effect. Therefore, KEAP1‐related genes might be important oncogenic signatures in KRAS mutant CRC cells and cyclopamine was identified as a potential ingredient and regulated the predict targets of SOX9 and SPP1, may be expand the efficacy and range of novel and effective therapeutics.

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.

Selective PI3Kδ inhibitor TYM-3-98 suppresses AKT/mTOR/SREBP1-mediated lipogenesis and promotes ferroptosis in KRAS-mutant colorectal cancer

Colorectal cancer (CRC) is one of the most common tumors of the digestive system worldwide. KRAS mutations limit the use of anti-EGFR antibodies in combination with chemotherapy for the treatment of CRC. Therefore, novel targeted therapies are needed to overcome the KRAS-induced oncogenesis. Recent evidence suggests that inhibition of PI3K led to ferroptosis, a nonapoptotic cell death closely related to KRAS-mutant cells. Here, we showed that a selective PI3Kδ inhibitor TYM-3–98 can suppress the AKT/mTOR signaling and activate the ferroptosis pathway in KRAS-mutant CRC cells in a concentration-dependent manner. This was evidenced by the lipid peroxidation, iron accumulation, and depletion of GSH. Moreover, the overexpression of the sterol regulatory element-binding protein 1 (SREBP1), a downstream transcription factor regulating lipid metabolism, conferred CRC cells greater resistance to ferroptosis induced by TYM-3–98. In addition, the effect of TYM-3–98 was confirmed in a xenograft mouse model, which demonstrated significant tumor suppression without obvious hepatoxicity or renal toxicity. Taken together, our work demonstrated that the induction of ferroptosis contributed to the PI3Kδ inhibitor-induced cell death via the suppression of AKT/mTOR/SREBP1-mediated lipogenesis, thus displaying a promising therapeutic effect of TYM-3–98 in CRC treatment.

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.

PDP1 promotes KRAS mutant colorectal cancer progression by serving as a scaffold for BRAF and MEK1

The oncogenic role of KRAS in colorectal cancer (CRC) progression is well-established. Despite this, identifying effective therapeutic targets for KRAS-mutated CRC remains a significant challenge. This study identifies pyruvate dehydrogenase phosphatase catalytic subunit 1 (PDP1) as a previously unrecognized yet crucial regulator in the progression of KRAS mutant CRC. A substantial upregulation of PDP1 expression is observed in KRAS mutant CRC cells and tissues compared to wild-type KRAS samples, which correlates with poorer prognosis. Functional experiments elucidate that PDP1 accelerates the malignance of KRAS mutant CRC cells, both in vitro and in vivo. Mechanistically, PDP1 acts as a scaffold, enhancing BRAF and MEK1 interaction and activating the MAPK signaling, thereby promoting CRC progression. Additionally, transcription factor KLF5 is identified as the key regulator for PDP1 upregulation in KRAS mutant CRC. Crucially, targeting PDP1 combined with MAPK inhibitors exhibits an obvious inhibitory effect on KRAS mutant CRC. Overall, PDP1 is underscored as a vital oncogenic driver and promising therapeutic target for KRAS mutant CRC.

Andrographolide sensitizes KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the EGFR/AKT and PDGFRβ/AKT signaling pathways

BackgroundCetuximab, an inhibitor targeting EGFR, is widely applied in clinical management of colorectal cancer (CRC). Nevertheless, drug resistance induced by KRAS-mutations limits cetuximab's anti-cancer effectiveness. Furthermore, the persistent activation of EGFR-independent AKT is another significant factor in cetuximab resistance. Nevertheless, the mechanism that EGFR-independent AKT drives cetuximab resistance remains unclear. Thus, highlighting the need to optimize therapies to overcome cetuximab resistance and also to explore the underlying mechanism. PurposeThis work aimed to investigate whether and how andrographolide enhance the therapeutic efficacy of cetuximab in KRAS-mutant CRC cells by modulating AKT. MethodsThe viabilities of CRC cell lines were analyzed by CCK-8. The intracellular proteins phosphorylation levels were investigated by Human Phospho-kinase Antibody Array analysis. Knockdown and transfection of PDGFRβ were used to evaluate the role of andrographolide on PDGFRβ. The western blotting was used to investigate Wnt/β-catenin pathways, PI3K/AKT, and EMT in KRAS-mutant CRC cells. The animal models including subcutaneous tumor and lung metastasis were performed to assess tumor response to therapy in vivo. ResultsAndrographolide was demonstrated to decrease the expression of PI3K and AKT through targeting PDGFRβ and EGFR, and it enhanced cetuximab effect on KRAS-mutant CRC cells by this mechanism. Meanwhile, andrographolide helped cetuximab to inhibit Wnt/β-catenin, CRC cell migration and reduced Vimentin expression, while increasing that of E-cadherin. Lastly, co-treatment with cetuximab and andrographolide reduced the growth of KRAS-mutant tumors and pulmonary metastases in vivo. ConclusionsOur findings suggest that andrographolide can overcome the KRAS-mutant CRC cells' resistance to cetuximab through inhibiting the EGFR/PI3K/AKT and PDGFRβ /AKT signaling pathways. This research provided a possible theory that andrographolide sensitizes KRAS-mutant tumor to EGFR TKI.

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.

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.

35P Enhancing efficacy of the MEK inhibitor trametinib in KRAS-mutated colorectal cancer cells

35P Enhancing efficacy of the MEK inhibitor trametinib in KRAS-mutated colorectal cancer cells

PDE4DIP contributes to colorectal cancer growth and chemoresistance through modulation of the NF1/RAS signaling axis

Phosphodiesterase 4D interacting protein (PDE4DIP) is a centrosome/Golgi protein associated with cyclic nucleotide phosphodiesterases. PDE4DIP is commonly mutated in human cancers, and its alteration in mice leads to a predisposition to intestinal cancer. However, the biological function of PDE4DIP in human cancer remains obscure. Here, we report for the first time the oncogenic role of PDE4DIP in colorectal cancer (CRC) growth and adaptive MEK inhibitor (MEKi) resistance. We show that the expression of PDE4DIP is upregulated in CRC tissues and associated with the clinical characteristics and poor prognosis of CRC patients. Knockdown of PDE4DIP impairs the growth of KRAS-mutant CRC cells by inhibiting the core RAS signaling pathway. PDE4DIP plays an essential role in the full activation of oncogenic RAS/ERK signaling by suppressing the expression of the RAS GTPase-activating protein (RasGAP) neurofibromin (NF1). Mechanistically, PDE4DIP promotes the recruitment of PLCγ/PKCε to the Golgi apparatus, leading to constitutive activation of PKCε, which triggers the degradation of NF1. Upregulation of PDE4DIP results in adaptive MEKi resistance in KRAS-mutant CRC by reactivating the RAS/ERK pathway. Our work reveals a novel functional link between PDE4DIP and NF1/RAS signal transduction and suggests that targeting PDE4DIP is a promising therapeutic strategy for KRAS-mutant CRC.

Osthole inhibits malignant phenotypes and induces ferroptosis in KRAS-mutant colorectal cancer cells via suppressing AMPK/Akt signaling.

Ferroptosis is a form of cell death driven by iron-dependent lipid peroxidation. Intriguingly, KRAS-mutant cancers are particularly vulnerable to ferroptosis. Osthole is a natural coumarin extracted from Cnidium spp. and other Apiaceous plants. In the present study, we explored the antitumor potential of osthole in KRAS-mutant colorectal cancer (CRC) cells. Cell viability assay, EdU incorporation assay, flow cytometry, tumor xenograft model, western blot, immunochemistry staining, immunofluorescence, transcriptome RNA sequencing and quantitative reverse transcription-PCR were performed to evaluate the influence of osthole treatment on KRAS-mutant CRC cells. We found that osthole treatment suppressed proliferation and tumor growth of KRAS-mutant CRC cell lines HCT116 and SW480. Moreover, osthole treatment increased ROS production and induced ferroptosis. Osthole treatment also promoted autophagy, but inhibition of autophagy by ATG7 knockdown or 3-MA showed no influence on osthole-induced ferroptosis. In comparison, osthole increased lysosomal activation, and co-treatment with lysosome inhibitor Baf-A1 attenuated osthole-induced ferroptosis. Besides, osthole treatment reduced the phosphorylation of AMPK, Akt and mTOR in HCT116 and SW480 cells, while restored AMPK signaling by AMPK agonist AICAR partially abrogated ferroptosis induced by osthole treatment. Finally, co-treatment with osthole increased the cytotoxicity of cetuximab in KRAS-mutant CRC cells in vitro and in vivo. Our results suggested that the natural product osthole exerted its anticancer effects in KRAS-mutant CRC cells via inducing ferroptosis, and this was partially through inhibiting AMPK/Akt/mTOR signaling. Our results may expand our current knowledge for the use of osthole as an anticancer agent.

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

Statins enhances antitumor effect of oxaliplatin in KRAS-mutated colorectal cancer cells and inhibits oxaliplatin-induced neuropathy

BackgroundKRAS mutations are fraught with the progression of colorectal cancer and resistance to chemotherapy. There are pathways such as extracellular regulated protein kinase 1/2 (ERK1/2) and Akt downstream and farnesylation and geranylgeranylation upstream that are activated upon mutated KRAS. Previous studies have shown that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, are effective to treat KRAS mutated colorectal cancer cells. Increased doses of oxaliplatin (L-OHP), a well-known alkylating chemotherapeutic drug, causes side effects such as peripheral neuropathy due to ERK1/2 activation in spinal cords. Hence, we examined the combinatorial therapeutic efficacy of statins and L-OHP to reduce colorectal cancer cell growth and abrogate neuropathy in mice.MethodsCell survival and confirmed apoptosis was assessed using WST-8 assay and Annexin V detection kit. Detection of phosphorylated and total proteins was analyzed the western blotting. Combined effect of simvastatin and L-OHP was examined the allograft mouse model and L-OHP-induced neuropathy was assessed using cold plate and von Frey filament test.ResultsIn this study, we examined the effect of combining statins with L-OHP on induction of cell death in colorectal cancer cell lines and improvement of L-OHP-induced neuropathy in vivo. We demonstrated that combined administration with statins and L-OHP significantly induced apoptosis and elevated the sensitivity of KRAS-mutated colorectal cancer cells to L-OHP. In addition, simvastatin suppressed KRAS prenylation, thereby enhancing antitumor effect of L-OHP through downregulation of survivin, XIAP, Bcl-xL, and Bcl-2, and upregulation of p53 and PUMA via inhibition of nuclear factor of κB (NF-κB) and Akt activation, and induction of c-Jun N-terminal kinase (JNK) activation in KRAS-mutated colorectal cancer cells. Moreover, simvastatin enhanced the antitumor effects of L-OHP and suppressed L-OHP-induced neuropathy via ERK1/2 activation in vivo.ConclusionTherefore, statins may be therapeutically useful as adjuvants to L-OHP in KRAS-mutated colorectal cancer and may also be useful in the treatment of L-OHP-induced neuropathy.

Abstract LB043: Pharmacological inhibition of ubiquitination sensitizes to DNA damaging agents by preventing homologous recombination repair

Abstract Therapies inducing DNA damage have been the backbone of cancer treatment for decades, but they often cause significant adverse effects reducing quality of life. Recently, inhibition of poly-ADP-ribose polymerase (PARP) has been shown to target tumor cells with deficient DNA homologous recombination (HR) double-strand break repair by converting single-strand DNA breaks into toxic double-strand breaks that cannot be resolved. This approach has been advantageous as healthy cells with functional HR repair are less effective, resulting in a more tolerable treatment. However, most cancers will not respond to PARP inhibition. Here, using a genome-wide CRISPR knockout functional genomics screen we identified that depletion of ubiquitination enzymes induced sensitivity to PARP inhibition in HR proficient ovarian cancer cells, as well as KRAS-mutant colorectal cancer cells which are generally resistant to PARP and other DNA damaging agents. Validation studies across multiple cancer lineages using cell line and organoid models confirmed that either silencing or pharmacological inhibition of ubiquitin-activating enzyme E1 (UBA1), which is required for ubiquitination, sensitized to PARP inhibition and other DNA damaging therapies. In vivo testing of combination PARP and UBA1 inhibition was well tolerated, and significantly extended survival of patient-derived xenografts compared to either therapy alone. Mechanistically, we found that UBA1 inhibition not only blocked HR repair to sensitize to PARP inhibition, but it also induced global increases in PARylation, which was subsequently inhibited by PARP inhibition. Taken together, these results demonstrate that inhibition of UBA1 sensitizes to PARP via multiple pathways, and the combination is well tolerated making it an ideal strategy to expand the therapeutic benefit of PARP inhibition. Citation Format: Sharad Awasthi, S. Stephen Yi, Daniel J. McGrail, Nidhi Sahni. Pharmacological inhibition of ubiquitination sensitizes to DNA damaging agents by preventing homologous recombination repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB043.

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.

LncRNA SPINT1-AS1/miR-433-3p/E2F3 positive feedback loop promotes the KRAS-mutant colorectal cancer cell proliferation, migration and invasion.

Colorectal cancer (CRC) features high prevalence and mortality. Long non-coding RNAs (lncRNAs) exert nonnegligible roles in human cancer development. Nevertheless, the functions of most lncRNAs still remain unexplored. We currently focused on detecting the influence of SPINT1 antisense RNA 1 (SPINT1-AS1) on CRC development and investigating into the potential regulatory mechanism. RT-qPCR analysis first confirmed that SPINT1-AS1 exhibited high expression in KRAS-mutant (KRASMUT) CRC cells. Through series of functional experiments, we observed that knockdown of SPINT1-AS1 weakened KRASMUT CRC cell proliferation, migration and invasion. Afterwards, the implementation of mechanism assays help to verify that SPINT1-AS1 sequestered microRNA-433-3p (miR-433-3p) to regulate the expression of E2F transcription factor 3 (E2F3). Besides, E2F3 was validated to activate the transcription of SPINT1-AS1 in turn. Rescue experiments confirmed the functional influence of SPINT1-AS1/miR-433-3p/E2F3 on CRC cells. In summary, the molecular axis of SPINT1-AS1/miR-433-3p/E2F3 forms a positive loop which might become a potential biomarker in CRC.

Platycodin D sensitizes KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the PI3K/Akt signaling pathway.

Cetuximab is a monoclonal antibody against epidermal growth factor receptor that blocks downstream signaling pathways of receptor tyrosine kinases, including Ras/Raf/MAPK and PI3K/Akt, thereby inhibiting tumor cell proliferation and inducing cancer cell apoptosis. Owing to KRAS mutations, the effectiveness of cetuximab is usually limited by intrinsic drug resistance. Continuous activation of the PI3K/Akt signaling pathway is another reason for cetuximab resistance. Platycodin-D, a bioactive compound isolated from the Chinese herb Platycodon grandiflorum, regulates Akt in different trends based on tissue types. To investigate whether platycodin-D can sensitize KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the PI3K/Akt signaling pathway, HCT116 and LoVo cells were treated with cetuximab and platycodin-D. LY294002 and SC79 were used to regulate Akt to further evaluate whether platycodin-D sensitizes cells to cetuximab by inhibiting Akt. Our results confirmed that platycodin-D increased the cytotoxic effects of cetuximab, including inhibition of growth, migration, and invasion, via downregulation of PI3K and Akt phosphorylation in HCT116 and LoVo cells both in vitro and in vivo. Given these data, platycodin-D may sensitize KRAS-mutant colorectal cancer cells to cetuximab via inhibition of the PI3K/Akt signaling pathway.

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.

Abstract 1056: Determining efficacy of combining the MEK inhibitor trametinib with vincristine identified by unbiased high throughput screening in RAS-mutated colorectal cancer cells

Abstract Background: Colorectal cancer (CRC) is a heterogeneous disease with various driver genetic mutations. Metastatic colorectal cancer (mCRC) is the second leading cause of cancer related deaths in the US. Greater than 50% of patients with mCRC harbor mutations in the oncogenic drivers KRAS or NRAS. While direct targeting of specific mutations in RAS is in the early stages of development in patients with mCRC, targeting most mutations in RAS is technically challenging. Hence, prior efforts have been focused on targeting MEK, a downstream mediator of RAS. Unfortunately, several MEK inhibitors that have entered clinical trials have failed to exhibit clinical efficacy as a monotherapy or in combination with select agents in patients with mCRC. Thus, identifying combination therapies with unbiased screening methods has the potential to improve the efficacy of MEK targeting in patients with mCRC. Aims: We aimed to perform unbiased high throughput screening (HTS) to identify drugs that, when combined with the MEK inhibitor trametinib, would enhance its efficacy. Methods: We performed unbiased HTS with KRAS mutated CRC cells grown in 3D using the MEK inhibitor trametinib as the base drug, and two different “clinically ready” compound libraries; 1) the NCI oncology set V, and, 2) a custom clinical compound set composed of drugs either approved by the FDA or in clinical trials. Using the Bliss model of synergy, vincristine was identified to be synergistic with trametinib. Effects of combining trametinib with vincristine were analyzed in vitro by 1) cell growth assays, and 2) measuring changes in cell signaling and apoptosis mediators by RPPA and western blotting. The efficacy of this combination was also examined in vivo using KRAS mutated patient derived xenografts (PDXs). Results: HTS studies demonstrated that combining trametinib with vincristine was synergistic in 3D CRC cell culture. This combination was further validated by MTT and colony formation assays. Analyses of markers for apoptosis by RPPA and western blotting demonstrated that the combination increases cell death in multiple CRC cell lines when compared to single agents. Furthermore, combining trametinib with vincristine led to significant inhibition in tumor growth when compared to the monotherapies using RAS-mutated PDXs in vivo. The combination was well tolerated by mice in vivo with minor decreases in body weight. Conclusions: Our unbiased HTS along with in vitro and in vivo validation studies demonstrated that combining trametinib with vincristine can enhance the efficacy of MEK targeted therapy in RAS mutated CRC cells. Furthermore, our in vivo studies were performed using drug doses that reflect clinically achievable doses in humans and thus serve as basis for future clinical studies to determine the efficacy of this drug combination in patients with RAS mutated mCRC. Citation Format: Susmita Ghosh, Fan Fan, Jason Roszik, Reid Powell, Yong Park, Clifford Stephan, Lee M. Ellis, Rajat Bhattacharya. Determining efficacy of combining the MEK inhibitor trametinib with vincristine identified by unbiased high throughput screening in RAS-mutated colorectal cancer cells [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 1056.