Therapeutic Approach For Colorectal Cancer Research Articles

Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer.

Bromodomain and extraterminal domain (BET) family proteins are epigenetic master regulators of gene expression via recognition of acetylated histones and recruitment of transcription factors and co-activators to chromatin. Hence, BET family proteins have emerged as promising therapeutic targets in cancer. In this study, we examined the functional role of bromodomain containing 3 (BRD3), a BET family protein, in colorectal cancer (CRC). Invitro and vivo analyses using BRD3-knockdown or BRD3-overexpressing CRC cells showed that BRD3 suppressed tumor growth and cell cycle G1/S transition and induced p21 expression. Clinical analysis of CRC datasets from our hospital or The Cancer Genome Atlas revealed that BET family genes, including BRD3, were overexpressed in tumor tissues. In immunohistochemical analyses, BRD3 was observed mainly in the nucleus of CRC cells. According to single-cell RNA sequencing in untreated CRC tissues, BRD3 was highly expressed in malignant epithelial cells, and cell cycle checkpoint-related pathways were enriched in the epithelial cells with high BRD3 expression. Spatial transcriptomic and single-cell RNA sequencing analyses of CRC tissues showed that BRD3 expression was positively associated with high p21 expression. Furthermore, overexpression of BRD3 combined with knockdown of, a driver gene in the BRD family, showed strong inhibition of CRC cells invitro. In conclusion, we demonstrated a novel tumor suppressive role of BRD3 that inhibits tumor growth by cell cycle inhibition in part via induction of p21 expression. BRD3 activation might be a novel therapeutic approach for CRC.

ATF3-CBS signaling axis coordinates ferroptosis and tumorigenesis in colorectal cancer

The induction of ferroptosis is promising for cancer therapy. However, the mechanisms enabling cancer cells to evade ferroptosis, particularly in low-cystine environments, remain elusive. Our study delves into the intricate regulatory mechanisms of Activating transcription factor 3 (ATF3) on Cystathionine β-synthase (CBS) under cystine deprivation stress, conferring resistance to ferroptosis in colorectal cancer (CRC) cells. Additionally, our findings establish a positively correlation between this signaling axis and CRC progression, suggesting its potential as a therapeutic target. Mechanistically, ATF3 positively regulates CBS to resist ferroptosis under cystine deprivation stress. In contrast, the suppression of CBS sensitizes CRC cells to ferroptosis through targeting the mitochondrial tricarboxylic acid (TCA) cycle. Notably, our study highlights that the ATF3-CBS signaling axis enhances ferroptosis-based CRC cancer therapy. Collectively, the findings reveal that the ATF3-CBS signaling axis is the primary feedback pathway in ferroptosis, and blocking this axis could be a potential therapeutic approach for colorectal cancer.

Artificial Intelligence Applications in the Treatment of Colorectal Cancer: A Narrative Review.

Colorectal cancer is the third most prevalent cancer worldwide, and its treatment has been a demanding clinical problem. Beyond traditional surgical therapy and chemotherapy, newly revealed molecular mechanisms diversify therapeutic approaches for colorectal cancer. However, the selection of personalized treatment among multiple treatment options has become another challenge in the era of precision medicine. Artificial intelligence has recently been increasingly investigated in the treatment of colorectal cancer. This narrative review mainly discusses the applications of artificial intelligence in the treatment of colorectal cancer patients. A comprehensive literature search was conducted in MEDLINE, EMBASE, and Web of Science to identify relevant papers, resulting in 49 articles being included. The results showed that, based on different categories of data, artificial intelligence can predict treatment outcomes and essential guidance information of traditional and novel therapies, thus enabling individualized treatment strategy selection for colorectal cancer patients. Some frequently implemented machine learning algorithms and deep learning frameworks have also been employed for long-term prognosis prediction in patients with colorectal cancer. Overall, artificial intelligence shows encouraging results in treatment strategy selection and prognosis evaluation for colorectal cancer patients.

Downregulation of γ-Catenin by miR-195-5p Inhibits Colon Cancer Progression, Regulating Desmosome Function.

Desmosomes are essential structures for ensuring tissue functions, and their deregulation is involved in the development of colorectal cancer (CRC). JUP (γ-catenin) is a desmosome adhesion component that also acts as a signaling hub, suggesting its potential involvement in CRC progression. In this context, we recently demonstrated that miR-195-5p regulated JUP and desmosome cadherins expression. In addition, miR-195-5p gain of function indirectly modulated the expression of key effectors of the Wnt pathway involved in JUP-dependent signaling. Here, our purpose was to demonstrate the aberrant expression of miR-195-5p and JUP in CRC patients and to functionally characterize the role of miR-195-5p in the regulation of desmosome function. First, we showed that miR-195-5p was downregulated in CRC tumors compared to adjacent normal tissue. Then, we demonstrated that JUP expression was significantly increased in CRC tissues compared to adjacent normal tissues. The effects of miR-195-5p on CRC progression were assessed using in vitro transient transfection experiments and in vivo miRNA administration. Increased miR-195-5p in colonic epithelial cells strongly inhibits cell proliferation, viability, and invasion via JUP. In vivo gain of function of miR-195-5p reduced the numbers and sizes of tumors and significantly ameliorated the histopathological changes typical of CRC. In conclusion, our findings indicate a potential pharmacological target based on miR-195-5p replacement as a new therapeutic approach in CRC.

Discovery of New Fusobacterium nucleatum Inhibitors to Attenuate Migratory Capability of Colon Cancer Cells by the Drug Repositioning Strategy.

Recent studies revealed that intestinal microbiota played important roles in colorectal cancer (CRC) carcinogenesis. Particularly, Fusobacterium nucleatum was confirmed to promote the proliferation and metastasis of CRC. Therefore, targeting F. nucleatum may be a potential preventive and therapeutic approach for CRC. Herein, 2,272 off-patent drugs were screened inhibitory activity against F. nucleatum. Among the hits, nitisinone was identified as a promising anti-F. nucleatum lead compound. Further optimization of nitisinone led to the discovery of more potent derivatives. Particularly, compounds 19q and 22c showed potent anti-F. nucleatum activity (MIC50 = 1 and 2 μg/mL, respectively) with low cytotoxicity. Among them, compound 19q effectively attenuated the migratory ability of MC-38 cells induced by F. nucleatum. Preliminary mechanism studies suggested that nitisinone and its derivatives might act by downregulating nitroreductase and tryptophanase. Thus, the development of small molecule F. nucleatum inhibitors represents an effective strategy to treat CRC.

Cholesterol in colorectal cancer: an essential but tumorigenic precursor?

Colorectal cancer (CRC) is one of the most lethal human malignancies, and with the growth of societies and lifestyle changes, the rate of people suffering from it increases yearly. Important factors such as genetics, family history, nutrition, lifestyle, smoking, and alcohol can play a significant role in increasing susceptibility to this cancer. On the other hand, the metabolism of several macromolecules is also involved in the fate of tumors and immune cells. The evidence discloses that cholesterol and its metabolism can play a role in the pathogenesis of several cancers because there appears to be an association between cholesterol levels and CRC, and cholesterol-lowering drugs may reduce the risk. Furthermore, changes or mutations of some involved genes in cholesterol metabolism, such as CYP7A1 as well as signaling pathways, such as mitogen-activated protein kinase (MAPK), can play a role in CRC pathogenesis. This review summarized and discussed the role of cholesterol in the pathogenesis of CRC as well as available cholesterol-related therapeutic approaches in CRC.

Probiotics as a therapeutic approach in colorectal cancer.

Colorectal cancer (CRC) is a growing concern worldwide. In recent decades, the incidence of CRC has increased, and this has been attributed to changes in lifestyle. The lack of physical activity, smoking habits, and a diet high in red meat and fat and low in fiber are important aspects of these deleterious changes in lifestyle. The increase in the incidence of CRC has impelled researchers to investigate methods for preventing and treating CRC with greater efficacy and fewer complications. Probiotics are an attractive and potentially promising therapeutic approach. They have been evaluated by a large number of preclinical and clinical studies in recent years, and it has been found that they can play a role in the prevention, treatment, and management of complications of CRC. This review provides a concise summary of the mechanisms of action of probiotics. Furthermore, it focuses on the results of clinical and preclinical studies that evaluated probiotics' effects on CRC management. It also discusses the effects of different strains of probiotics and their combination in CRC treatment.

Nanoformulations of Plant-Derived Compounds as Emerging Therapeutic Approach for Colorectal Cancer.

Colorectal cancer (CRC) represents one of the most daunting health problems accompanied by progressive undesirable socio-economic effects. Phytochemicals, bioactive ingredients majorly found in plants, have gained momentum for their potential against CRC occurrence and regression. However, these phytoconstituents are not exempt from biopharmaceutical drawbacks; therefore, novel strategies, especially nanotechnology, are exploited to surmount the aforementioned bottlenecks. The current paper aims to comprehensively review the phytochemical-based nanoformulations and their mechanisms in the setting of CRC. Electronic databases including Scopus, PubMed, and Web of Science were searched with the keywords "colon cancer" or "colorectal cancer", and "plant", "phytochemical", "extract", or "herb", and "nano", "nanoformulation", "Nanoencapsulation", "nanoparticle", "nanostructure", or "nanoliposome", until January 2021. Of the 1230 research hits, only 69 articles were consequently analyzed. The results indicated nanoformulations of several secondary plant metabolites such as berberine, camptothecin, colchicine, apigenin, chrysin, fisetin, quercetin, curcumin, gallic acid, resveratrol, and ursolic acid have profound effects in a broad range of preclinical models of CRC. A wide variety of nanoformulations have been utilized to deliver these phytochemicals, such as nanocomposite, nanocolloids, and mesoporous silica nanoparticles, which have consequently decreased tumor angiogenesis and mitochondrial membrane potential, increased radical scavenging activity, induced cell cycle arrest at different phases of the cancer cell cycle, and induction of apoptosis process via decreased anti-apoptotic proteins (BRAF, CD44, and Bcl-2) and increased in pro-apoptotic ones (Bax, Fas, caspase 3,8, and 9), as well as modulated biopharmaceutical properties. Chitosan and PEG and their derivatives are among the polymers exploited in the phytochemicals' nanoformulations. To conclude, nanoformulated forms of natural ingredients depicted outstanding anti-CRC activity that could hold promise for help in treating CRC. However, well-designed clinical trials are needed to build up a whole picture of the health profits of nanoformulation of natural products in CRC management.

MicroRNAs and colorectal cancer: clinical potential and regulatory networks.

Colorectal cancer (CRC) is a serious global health concern, with a high incidence and mortality rate. Although there have been advancements in the early detection and treatment of CRC, therapy resistance is common. MicroRNAs (miRNAs), a type of small non-coding RNA that regulates gene expression, are key players in the initiation and progression of CRC. Recently, there has been growing attention to the complex interplay of miRNAs in cancer development. miRNAs are powerful RNA molecules that regulate gene expression and have been implicated in various physiological and pathological processes, including carcinogenesis. By identifying current challenges and limitations of treatment strategies and suggesting future research directions, this review aims to contribute to ongoing efforts to enhance CRC diagnosis and treatment. It also provides a comprehensive overview of the role miRNAs play in CRC carcinogenesis and explores the potential of miRNA-based therapies as a treatment option. Importantly, this review highlights the exciting potential of targeted modulation of miRNA function as a therapeutic approach for CRC.

Cetuximab Enhances the Efficacy of MRTX1133, a Novel KRASG12D Inhibitor, in Colorectal Cancer Treatment.

Kirsten Rat Sarcoma viral oncogene homolog (KRAS) has remained undruggable for decades. KRAS has predominantly been used to evaluate the applicability of anti-Epidermal Growth Factor Receptor (EGFR) antibody drugs. However, various KRAS inhibitors have recently emerged. Unfortunately, KRAS inhibitors have not been effective against colorectal cancer. Therefore, this study aimed to determine the effects of MRTX1133, a novel KRASG12D inhibitor, in combination with an anti-EGFR antibody, cetuximab, on signal transduction and cell proliferation. The KRASG12D-mutated LS513 and KRAS wild-type CACO-2 human colon cancer cell lines were utilized. The KRASG12D mutation was stably transduced into the CACO-2 cells using a retrovirus. We evaluated the effects of the drugs using the CCK-8 assay and assessed the activity of proteins related to the MAPK pathway using western blotting. We demonstrated that the administration of MRTX1133, a novel KRASG12D inhibitor, to KRASG12D-mutated colorectal cancer cells led to feedback activation of the ERK pathway via EGFR activation, inducing drug resistance. Intriguingly, when MRTX1133 was used in combination with cetuximab, KRASG12D-mutant colorectal cancer growth was effectively inhibited, both in vitro and in vivo. The combination of MRTX1133 and cetuximab serves as a potential and promising therapeutic approach for colorectal cancer with KRASG12D mutation. KRASG12D is a frequent genetic mutation not only in colorectal cancer, but also in pancreatic and lung cancer, and the results of this study open new avenues for potential treatment of many cancer patients.

Polyphenol-Based Nanoparticles: A Promising Frontier for Enhanced Colorectal Cancer Treatment.

Colorectal cancer (CRC) poses a significant challenge in healthcare, necessitating the exploration of novel therapeutic strategies. Natural compounds such as polyphenols with inherent anticancer properties have gained attention as potential therapeutic agents. This review highlights the need for novel therapeutic approaches in CRC, followed by a discussion on the synthesis of polyphenols-based nanoparticles. Various synthesis techniques, including dynamic covalent bonding, non-covalent bonding, polymerization, chemical conjugation, reduction, and metal-polyphenol networks, are explored. The mechanisms of action of these nanoparticles, encompassing passive and active targeting mechanisms, are also discussed. The review further examines the intrinsic anticancer activity of polyphenols and their enhancement through nano-based delivery systems. This section explores the natural anticancer properties of polyphenols and investigates different nano-based delivery systems, such as micelles, nanogels, liposomes, nanoemulsions, gold nanoparticles, mesoporous silica nanoparticles, and metal-organic frameworks. The review concludes by emphasizing the potential of nanoparticle-based strategies utilizing polyphenols for CRC treatment and highlights the need for future research to optimize their efficacy and safety. Overall, this review provides valuable insights into the synthesis, mechanisms of action, intrinsic anticancer activity, and enhancement of polyphenols-based nanoparticles for CRC treatment.

Targeted hyperactivation of AKT through inhibition of ectopic expressed SHIP1 induces cell death in colon carcinoma cells and derived metastases

Current therapeutic approaches for colorectal cancer (CRC) focus on the suppression of oncogenic kinase signaling. Here, we test the hypothesis that targeted hyperactivation of the PI3K/AKT-signaling may lead to trigger CRC cell death. Recently we found that hematopoietic SHIP1 is ectopically expressed in CRC cells. Here we show that SHIP1 is more strongly expressed in metastatic cells than in the primary cancer cells, which allows for an increase in AKT signaling in metastatic cells, giving them an advantage from an evolutionary point of view. Mechanistically, the increased SHIP1 expression reduces the activation of the PI3K/ AKT signaling to a value that is below the threshold that leads to cell death. This mechanism gives the cell a selection advantage. We show that genetic hyperactivation of PI3K/AKT-signaling or blocking the activity of the inhibitory phosphatase SHIP1, induces acute cell death in CRC cells, because of excessive accumulation of reactive oxygen species. Our results demonstrate that CRC cells critically depend on mechanisms to fine-tune PI3K/AKT activity and show SHIP1 inhibition as an unexpectedly promising concept for CRC therapy.

Current and emerging therapeutic approaches for colorectal cancer: A comprehensive review

Current and emerging therapeutic approaches for colorectal cancer: A comprehensive review

Abstract 2581: Fatty acid synthase regulates expression of Notum in colorectal cancer

Abstract Background: Upregulation of lipid synthesis has been associated with poor clinical outcomes in colorectal cancer (CRC). Fatty acid synthase (FASN) synthesizes 16-carbon fatty acid palmitate which can be utilized for post-translational modifications of various proteins. Notum, a palmitoleoyl-protein carboxylesterase, is involved in the negative regulation of the Wnt signaling via its role in de-palmitoylation of Wnt ligands and has been identified as a marker for poor prognosis in CRC. However, the crosstalk between FASN and Notum has not been reported. Our preliminary data suggest that FASN regulates β-catenin signaling and Notum expression. Therefore, the purpose of this study is to elucidate (I) the mechanisms of how FASN regulates expression of Notum and (II) the contribution of the FASN/Notum axis to CRC. Methods: Tumor and normal intestinal organoids were established from transgenic mice models, ApcMin and Apc/VillinCre-ERT2, with inducible hetero- and homozygous deletion of FASN. The effect of genetic deletion and pharmacological inhibition of FASN on organoid growth and viability was assessed by 4-hydroxytamoxifen (4-OHT) and TVB-3664 (a FASN inhibitor) treatments, respectively. LIVE/DEAD™ Viability/Cytotoxicity Cell Viability Kit and Cell Titer-Glo® 3D Cell Viability Assays were used for quantitative analysis of growth and viability. HCT116, NTC and FASN shRNA, and SW480, control and FASN overexpression, cells were used for analysis. Results: ERT2-mediated deletion of Apc leads to upregulation of FASN and Notum expression in mouse intestinal tissues and organoids. RNA-seq analysis of adenomas from Apc/VillinCre mice showed that hetero- and homozygous germline deletion of FASN is associated with a significant decrease in the number of adenomas, expression of Notum and CRC stem cell markers. Using qRT-PCR and Western blot, we confirmed that FASN downregulation is associated with a decrease in active β-catenin, Notum, and stem cell markers. Consistently with Apc/VillinCre model, downregulation of FASN results in a decrease in bud formation in Apc/VillinCre-ERT2 organoids, and viability and size in ApcMin organoids. Furthermore, overexpression of FASN increases the levels of active and total β-catenin, Notum, and stem cell markers expression in SW480 cells. In contrast, shRNA-mediated deletion of FASN decreases expression of Notum in HCT116 cells. Conclusion: Downregulation of FASN leads to a decrease in expression of Notum and is associated with morphological changes and a significant decrease in viability in organoid models. Conversely, FASN overexpression upregulates Notum expression suggesting a potential cross-talk between de novo lipid synthesis and Notum. Delineating the role of FASN regulation of stemness via altered β-catenin signaling and expression of Notum and other stem cell markers will provide the rationale for targeting the FASN/Notum axis as a preventative or early-stage therapeutic approach in CRC. Citation Format: Courtney Olivia Kelson, James Drury, Daheng He, Chi Wang, Yekaterina Zaytseva. Fatty acid synthase regulates expression of Notum in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2581.

Evaluation of CDK9 Inhibition by Dinaciclib in Combination with Apoptosis Modulating izTRAIL for the Treatment of Colorectal Cancer.

Treatment options for colorectal cancer (CRC), especially in advanced stages are still insufficient. There, the discovery of Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was a bright spot. However, most cancers show resistance toward apoptotic signals. Cyclin-dependent kinase 9 (CDK9) plays a crucial role in cell cycle progression in most tissues. We recently demonstrated the role of CDK9 in mediating TRAIL resistance. In this work, we investigated the role of CDK9 in colorectal cancer. Immunohistochemical analysis of CDK9 expression in cancer and normal tissues of CRC specimens was performed. The effect of selective CDK9 inhibition in combination with TRAIL on CRC cells was analyzed via cell viability, colony formation, and induction of apoptosis by flow cytometry. The mechanism of action was conducted via western blotting. We now have confirmed overexpression of CDK9 in cancer tissues, with low expression associated with poorer survival in a subset of CRC patients. In-vitro, CDK9 inhibition could strongly promote TRAIL-induced cell death in TRAIL-resistant CRC cells. Mechanistically, CDK9 inhibition induced apoptosis by downregulation of antiapoptotic proteins, myeloid leukemia cell differentiation protein 1 (Mcl-1) and FLICE-inhibitory protein (c-FLIP). Overall, we identified CDK9 as a prognostic marker and combined CDK9 inhibition and TRAIL as a novel and promising therapeutic approaches for colorectal cancer.

Roles of anoikis in colorectal cancer therapy and the assessment of anoikis-regulatory molecules as therapeutic targets.

Colorectal cancer (CRC) is a deadly malignancy and therapeutic approaches for CRC are evolving every day. Anoikis is a key mechanism for programmed cell death of cancer cells that undergo anchorage-independent growth at a different matrix than the one which is expected. Yet, anoikis is a less studied mechanism of cell death in comparison to other mechanisms such as apoptosis. Relating to this, resistance to anoikis among cancer cells remains critical for improved metastasis and survival in a new environment evading anoikis. Since CRC cells have the ability to metastasize from proximal sites to secondary organs such as liver and promote cancer in those distant sites, a clear knowledge of the mechanisms essential for anchorage-independent growth and subsequent metastasis is necessary to counteract CRC progression and spread. Therefore, the identification of novel drug candidates and studying the roles of anoikis in assisting CRC therapy using such drugs can prevent anchorage-independent cancer cell growth. Additionally, the identification of novel biomarkers or therapeutic targets seems essential for implementing superior therapy, impeding relapse among malignant cells and improving the survival rate of clinical patients. As there are no reviews published on this topic till date, anoikis as a mechanism of cell death and its therapeutic roles in CRC are discussed in this review. In addition, several molecules were identified as therapeutic targets for CRC.

RGD-Coated Polymer Nanoworms for Enriching Cancer Stem Cells

Cancer stem cells (CSCs) are primarily responsible for tumour drug resistance and metastasis; thus, targeting CSCs can be a promising approach to stop cancer recurrence. However, CSCs are small in numbers and readily differentiate into matured cancer cells, making the study of their biological features, including therapeutic targets, difficult. The use of three-dimensional (3D) culture systems to enrich CSCs has some limitations, including low sphere forming efficiency, enzymatic digestion that may damage surface proteins, and more importantly no means to sustain the stem properties. A responsive 3D polymer extracellular matrix (ECM) system coated with RGD was used to enrich CSCs, sustain stemness and avoid enzymatic dissociation. RGD was used as a targeting motif and a ligand to bind integrin receptors. We found that the system was able to increase sphere forming efficiency, promote the growth of spheric cells, and maintain stemness-associated properties compared to the current 3D culture. We showed that continuous culture for three generations of colon tumour spheroid led to the stem marker CD24 gradually increasing. Furthermore, the new system could enhance the cancer cell sphere forming ability for the difficult triple negative breast cancer cells, MBA-MD-231. The key stem gene expression for colon cancer also increased with the new system. Further studies indicated that the concentration of RGD, especially at high doses, could inhibit stemness. Taken together, our data demonstrate that our RGD-based ECM system can facilitate the enrichment of CSCs and now allow for the investigation of new therapeutic approaches for colorectal cancer or other cancers.

Targeting Lysosomes in Colorectal Cancer: Exploring the Anticancer Activity of a New Benzo[a]phenoxazine Derivative

Colorectal cancer (CRC) has been ranked as one of the cancer types with a higher incidence and one of the most mortal. There are limited therapies available for CRC, which urges the finding of intracellular targets and the discovery of new drugs for innovative therapeutic approaches. In addition to the limited number of effective anticancer agents approved for use in humans, CRC resistance and secondary effects stemming from classical chemotherapy remain a major clinical problem, reinforcing the need for the development of novel drugs. In the recent years, the phenoxazines derivatives, Nile Blue analogues, have been shown to possess anticancer activity, which has created interest in exploring the potential of these compounds as anticancer drugs. In this context, we have synthetized and evaluated the anticancer activity of different benzo[a]phenoxazine derivatives for CRC therapy. Our results revealed that one particular compound, BaP1, displayed promising anticancer activity against CRC cells. We found that BaP1 is selective for CRC cells and reduces cell proliferation, cell survival, and cell migration. We observed that the compound is associated with reactive oxygen species (ROS) generation, accumulates in the lysosomes, and leads to lysosomal membrane permeabilization, cytosolic acidification, and apoptotic cell death. In vivo results using a chicken embryo choriollantoic membrane (CAM) assay showed that BaP1 inhibits tumor growth, angiogenesis, and tumor proliferation. These observations highlight that BaP1 as a very interesting agent to disturb and counteract the important roles of lysosomes in cancer and suggests BaP1 as a promising candidate to be exploited as new anticancer lysosomal-targeted agent, which uses lysosome membrane permeabilization (LMP) as a therapeutic approach in CRC.

Insights on Ferroptosis and Colorectal Cancer: Progress and Updates.

Patients with advanced-stage or treatment-resistant colorectal cancer (CRC) benefit less from traditional therapies; hence, new therapeutic strategies may help improve the treatment response and prognosis of these patients. Ferroptosis is an iron-dependent type of regulated cell death characterized by the accumulation of lipid reactive oxygen species (ROS), distinct from other types of regulated cell death. CRC cells, especially those with drug-resistant properties, are characterized by high iron levels and ROS. This indicates that the induction of ferroptosis in these cells may become a new therapeutic approach for CRC, particularly for eradicating CRC resistant to traditional therapies. Recent studies have demonstrated the mechanisms and pathways that trigger or inhibit ferroptosis in CRC, and many regulatory molecules and pathways have been identified. Here, we review the current research progress on the mechanism of ferroptosis, new molecules that mediate ferroptosis, including coding and non-coding RNA; novel inducers and inhibitors of ferroptosis, which are mainly small-molecule compounds; and newly designed nanoparticles that increase the sensitivity of cells to ferroptosis. Finally, the gene signatures and clusters that have predictive value on CRC are summarized.

Abstract PR009: RNF43 G659fs is an oncogenic and immune-modulating colorectal cancer mutation and sensitizes tumor cells to PI3K/mTOR inhibition

Abstract Introduction: The RNF43 p.G659fs mutation occurs with a frequency of approximately 8% in colorectal cancer (CRC) and is enriched among microsatellite-instability high tumors. However, despite RNF43 being implicated as a Wnt-signaling tumor suppressor, the function of the RNF43 p.G659fs mutation remains undescribed, with no specific therapies directed against it. We hypothesized that large-scale drug screening coupled with functional validation and testing in CRC pre-clinical models would reveal the functional role and selective vulnerabilities of this alteration, leading to new therapeutic approaches in CRC. Methods: We targeted the RNF43 659 site with CRISPR-Cas9 to generate isogenic RNF43659mut clones of CRC cell lines. We leveraged the Broad Institute’s drug repurposing library of over 5,300 compounds to identify selective hits (z<-3 vs DMSO, >5% selective killing in mutant vs WT) in RNF43659mut isogenic cells. These hits were validated in secondary screens and top drugs were tested in patient-derived organoid and xenograft models of the RNF43 p.G659fs mutation. We assessed the functional role of the mutation using cell viability and colony formation assays, quantitative mass spectrometry proteomics, immunoprecipitation and ubiquitination experiments. The immunomodulatory role of RNF43659mut was assessed through bulk and single-cell RNA Seq analysis of mutant cell lines and patient-derived CRC specimens. Results: Using edited cell line models, we demonstrated that RNF43 p.G659fs has a Wnt-independent oncogenic role in CRC. We performed a comprehensive drug screen in isogenic RNF43659mut HT29 CRC cells with a repurposing library of 5,363 compounds and discovered that RNF43659mut is selectively targeted by PI3K/mTOR inhibitors (p = 1.34 x 10-5, chi-square test). Secondary screens in LS513 RNF43659mut-edited and HCT116 RNF43G659fs-overexpressing cells confirmed these results and we further showed that alpelisib and PF-04691502 (a dual PI3K/mTOR inhibitor) selectively killed patient-derived organoids with the RNF43 p.G659Vfs mutation and reduced tumor growth in RNF43659mut CDX mice (p=0.0005, Student’s t-test). RNF43 p.G659fs increased PI3K/AKT signaling in vitro and in vivo through binding, ubiquitinating and degrading p85 and this was reversed by PI3K/mTOR inhibition, RNF43 knockout or RSPO stimulation. Additionally, in pre-clinical models and single-cell studies of human CRCs (>350,000 single cells), we found that RNF43659mut modulates interferon activity and NK cells in the CRC tumor microenvironment. Conclusions: Through functional and pharmacologic studies in pre-clinical models and human CRCs, we described that RNF43 p.G659fs activates PI3K/AKT signaling and modulates the CRC immune microenvironment. We showed that these effects are reversed with PI3K/mTOR inhibitors and that this class of drugs selectively targets RNF43659mut CRCs. Our results have important therapeutic implications for patients with CRC. Citation Format: Marios Giannakis, Lishan Fang, Dane Ford-Roshon, Max Russo, Casey O'Brien, Xiaozhe Xiong, Carino Gurjao, Maximilien Grandclaudon, Srivatsan Raghavan, Steven M. Corsello, Steven A. Carr, Namrata Udeshi, James Berstler, Ewa Sicinska, Kimmie Ng. RNF43 G659fs is an oncogenic and immune-modulating colorectal cancer mutation and sensitizes tumor cells to PI3K/mTOR inhibition [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR009.