Preclinical Models Of Colorectal Cancer Research Articles

ATG16L1 in myeloid cells limits colorectal tumor growth in ApcMin/+ mice infected with colibactin-producing Escherichia coli via decreasing inflammasome activation

ABSTRACT Escherichia coli strains producing the genotoxin colibactin, designated as CoPEC (colibactin-producing E. coli), have emerged as an important player in the etiology of colorectal cancer (CRC). Here, we investigated the role of macroautophagy/autophagy in myeloid cells, an important component of the tumor microenvironment, in the tumorigenesis of a susceptible mouse model infected with CoPEC. For that, a preclinical mouse model of CRC, the Apc Min/+ mice, with Atg16l1 deficiency specifically in myeloid cells (Apc Min/+ /Atg16l1[∆MC]) and the corresponding control mice (Apc Min/+ ), were infected with a clinical CoPEC strain 11G5 or its isogenic mutant 11G5∆clbQ that does not produce colibactin. We showed that myeloid cell-specific Atg16l1 deficiency led to an increase in the volume of colonic tumors in Apc Min/+ mice under infection with 11G5, but not with 11G5∆clbQ. This was accompanied by increased colonocyte proliferation, enhanced inflammasome activation and IL1B/IL-1β secretion, increased neutrophil number and decreased total T cell and cytotoxic CD8+ T cell numbers in the colonic mucosa and tumors. In bone marrow-derived macrophages (BMDMs), compared to uninfected and 11G5∆clbQ-infected conditions, 11G5 infection increased inflammasome activation and IL1B secretion, and this was further enhanced by autophagy deficiency. These data indicate that ATG16L1 in myeloid cells was necessary to inhibit colonic tumor growth in CoPEC-infected Apc Min/+ mice via inhibiting colibactin-induced inflammasome activation and modulating immune cell response in the tumor microenvironment. Abbreviation: AOM, azoxymethane; APC, APC regulator of WNT signaling pathway; ATG, autophagy related; Atg16l1[∆MC] mice, mice deficient for Atg16l1 specifically in myeloid cells; CASP1, caspase 1; BMDM, bone marrow-derived macrophage; CFU, colony-forming unit; CoPEC, colibactin-producing Escherichia coli; CRC, colorectal cancer; CXCL1/KC, C-X-C motif chemokine ligand 1; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; MC, myeloid cell; MOI, multiplicity of infection; PBS, phosphate-buffered saline; pks, polyketide synthase; qRT-PCR, quantitative real-time reverse-transcription polymerase chain reaction; siRNA, small interfering RNA; TME, tumor microenvironment; TNF/TNF-α, tumor necrosis factor.

A phase 1, first-in-human, open-label study evaluating the safety, tolerability, pharmacokinetics, and efficacy of TT125-802 in patients with advanced solid tumors.

TPS3171 Background: TT125-802 is a selective, potent, orally bioavailable small molecule inhibitor of the bromodomain of CBP and p300, two highly homologues lysine acetyltransferases. CBP/p300 act as transcriptional co-activators with multiple domains and functions, including well-documented roles in cell cycle regulation and several critical tumorigenic pathways (1-3). TT125-802 has shown mono-therapy efficacy in preclinical models of castration-resistant prostate cancer, KRAS-mutated colorectal and non-small cell lung cancer (4,5). Furthermore, TT125-802 is capable to interfere with transcriptional resistance mechanisms limiting targeted therapies (6) which will be explored clinically upon determination of a safe, well-tolerated, and biologically active single-agent dose. Methods: This study aims to establish the safety, tolerability, and pharmacokinetic profile of TT125-802. The maximum tolerated dose/recommended dose for expansion (MTD/RDE) of repeat daily dosing for TT125-802 monotherapy will be determined in subjects with advanced solid tumors in a sequential escalating cohorts design, (1 cycle = 21 days) using the Bayesian Logistics Regression Model (BLRM) with sentinel patients (2-day window for first patient enrolled in each cohort). Cohorts will consist of 3 to 6 subjects who will be followed over a dose-limiting toxicity (DLT) period of at least 21 days. TT125-802 will be administered orally once daily; additional schedules may be evaluated. Dose escalation is planned to follow two-fold increments. It may be adapted upon dose level revision and guided by pharmacokinetic analysis. Additional subjects (up to 6) may be enrolled in at least two monotherapy cohorts deemed safe and tolerable to optimize the recommended phase 2 dose (RP2D). Up to a total of 20 additional subjects may be enrolled. Translational/pharmacodynamic endpoints: Exploratory endpoints will provide proof-of-principle and early biomarker data for CBP/p300 inhibition. These analyses will include: 1. Single-cell gene expression evaluation of peripheral blood mononuclear cells (PBMCs) at multiple time points, 2. anagen hair bulb bulk RNAseq analysis, and 3. paired tumor biopsies will be analyzed with single-cell RNA sequencing. Together, these investigations are expected to support optimal dose determination. Additionally, circulating tumor DNA might be evaluated to define molecular responses. Outlook: The recommended phase 2 dose will be used further to explore the efficacy of TT125-802 as monotherapy. TT125-802 will be evaluated with targeted therapies (for example, KRASG12C/D, BRAF, EGFR, etc., inhibitors) to exploit the blocking of transcriptional escape mechanisms driven by CBP/p300. 1. Iyer, 2007. 2. Dutta, 2016. 3. Attar, 2017. 4. AACR2023 posters #6268. 5. AACR poster #3907. 6. AACR2023 poster #3907. Clinical trial information: 2022-500849-24-00 .

Review of Prodrug and Nanodelivery Strategies to Improve the Treatment of Colorectal Cancer with Fluoropyrimidine Drugs.

Fluoropyrimidine (FP) drugs are central components of combination chemotherapy regimens for the treatment of colorectal cancer (CRC). FP-based chemotherapy has improved survival outcomes over the last several decades with much of the therapeutic benefit derived from the optimization of dose and delivery. To provide further advances in therapeutic efficacy, next-generation prodrugs and nanodelivery systems for FPs are being developed. This review focuses on recent innovative nanodelivery approaches for FP drugs that display therapeutic promise. We summarize established, clinically useful FP prodrug strategies, including capecitabine, which exploit tumor-specific enzyme expression for optimal anticancer activity. We then describe the use of FP DNA-based polymers (e.g., CF10) for the delivery of activated FP nucleotides as a nanodelivery approach with proven activity in pre-clinical models and with clinical potential. Multiple nanodelivery systems for FP delivery show promise in CRC pre-clinical models and we review advances in albumin-mediated FP delivery, the development of mesoporous silica nanoparticles, emulsion-based nanoparticles, metal nanoparticles, hydrogel-based delivery, and liposomes and lipid nanoparticles that display particular promise for therapeutic development. Nanodelivery of FPs is anticipated to impact CRC treatment in the coming years and to improve survival for cancer patients.

An Anti-VEGF-B Antibody Reduces Abnormal Tumor Vasculature and Enhances the Effects of Chemotherapy

The vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key regulators of blood vessel formation, including in tumors, where their deregulated function can promote the production of aberrant, leaky blood vessels, supporting tumor development. Here we investigated the VEGFR1 ligand VEGF-B, which we demonstrate to be expressed in tumor cells and in tumor stroma and vasculature across a range of tumor types. We examined the anti-VEGF-B-specific monoclonal antibody 2H10 in preclinical xenograft models of breast and colorectal cancer, in comparison with the anti-VEGF-A antibody bevacizumab. Similar to bevacizumab, 2H10 therapy was associated with changes in tumor blood vessels and intra-tumoral diffusion consistent with normalization of the tumor vasculature. Accordingly, treatment resulted in partial inhibition of tumor growth, and significantly improved the response to chemotherapy. Our studies indicate the importance of VEGF-B in tumor growth, and the potential of specific anti-VEGF-B treatment to inhibit tumor development, alone or in combination with established chemotherapies.

Focused Ultrasound as Targeted Therapy for Colorectal Cancer: A Comprehensive Review

Traditional cancer treatments have not significantly improved the survival rates for individuals with colorectal cancer. As a result, there is a dire need to explore novel treatment modalities that can target cancer-specific niches, transform cold colorectal tumors into hot ones, and disrupt the tumor niche. Therapeutic focused ultrasound, recognized for its capacity to induce thermal and mechanical impacts on tissue, can potentially eliminate cancer cells and elicit the body’s anticancer reaction by disrupting the tumor microenvironment. This article provides an overview of recent developments in employing therapeutic focused ultrasound (TFUS) to enhance the body’s natural defenses against colorectal cancers. It also discusses studies examining the utility of TFUS in treating colorectal cancer patients and recent research indicating its potential to stimulate the body’s anticancer response in various in vitro and in vivo colorectal cancer models. Furthermore, it explores the therapeutic effects of TFUS on the immune system in colorectal cancers. This article also highlights the safety and effectiveness of TFUS in managing colorectal cancer, providing relief from pain, and potentially improving survival rates. Given the indications that TFUS may bolster the body’s immune response and augment the impacts of TFUS therapy in clinical and preclinical colorectal cancer models, it has the potential to emerge as a pivotal tool in clinical settings.

Promising Anticancer Prodrugs Based on Pt(IV) Complexes with Bis-organosilane Ligands in Axial Positions.

We report two novel prodrug Pt(IV) complexes with bis-organosilane ligands in axial positions: cis-dichloro(diamine)-trans-[3-(triethoxysilyl)propylcarbamate]platinum(IV) (Pt(IV)-biSi-1) and cis-dichloro(diisopropylamine)-trans-[3-(triethoxysilyl) propyl carbamate]platinum(IV) (Pt(IV)-biSi-2). Pt(IV)-biSi-2 demonstrated enhanced in vitro cytotoxicity against colon cancer cells (HCT 116 and HT-29) compared with cisplatin and Pt(IV)-biSi-1. Notably, Pt(IV)-biSi-2 exhibited higher cytotoxicity toward cancer cells and lower toxicity on nontumorigenic intestinal cells (HIEC6). In preclinical mouse models of colorectal cancer, Pt(IV)-biSi-2 outperformed cisplatin in reducing tumor growth at lower concentrations, with reduced side effects. Mechanistically, Pt(IV)-biSi-2 induced permanent DNA damage independent of p53 levels. DNA damage such as double-strand breaks marked by histone gH2Ax was permanent after treatment with Pt(IV)-biSi-2, in contrast to cisplatin's transient effects. Pt(IV)-biSi-2's faster reduction to Pt(II) species upon exposure to biological reductants supports its superior biological response. These findings unveil a novel strategy for designing Pt(IV) anticancer prodrugs with enhanced activity and specificity, offering therapeutic opportunities beyond conventional Pt drugs.

Abstract 1658: CFT1946, a potent, selective BRAF V600X mutant-specific degrader demonstrates superior activity as a single agent to clinically approved BRAF inhibitors and standard of care combinations in preclinical models of BRAF V600X melanoma, CRC, NSCLC, and brain metastasis

Abstract Activating mutations in BRAF at residue V600 (typically V600E) lead to dysregulation of the MAPK pathway and occur in approximately 8% of all human cancers including 60% of melanoma, up to 12% of colorectal cancer (CRC), and 4% of NSCLC. Currently approved BRAF inhibitors (BRAFi) are selective for BRAF V600X mutant proteins and are typically used in combination with MEK inhibitors (MEKi) in melanoma and NSCLC, or anti-EGFR antibodies such as cetuximab in CRC. However, their activity is limited by primary or acquired resistance often mediated by RAF dimer-inducing mechanisms. Furthermore, progression of BRAF V600X melanoma after BRAFi/MEKi treatment frequently involves brain metastasis, and currently approved BRAFi have relatively poor brain penetration. CFT1946 is a potent, orally bioavailable, cereblon-based BiDACTM degrader that selectively degrades BRAF V600X mutant protein and is currently under investigation in a Phase I clinical trial. CFT1946 has the potential to overcome limitations of clinically approved BRAFi, as degradation of BRAF V600X should abrogate RAF dimer-driven resistance and paradoxical activation. Indeed, we have previously demonstrated that CFT1946 is efficacious in an A375 BRAF V600E/NRAS Q61K xenograft model of BRAFi resistant melanoma. Here we substantially expand our preclinical characterization of CFT1946 across multiple models of BRAF V600X-driven cancers including BRAF V600X-driven CRC and NSCLC, additional BRAFi-resistant melanoma models, and a brain metastatic melanoma model. Single agent CFT1946 outperformed the SOC encorafenib + cetuximab combination in a panel of BRAF-V600X CRC xenograft models. Pathway analysis in BRAF V600X CRC models revealed that CFT1946 suppresses EGFR-mediated MAPK pathway reactivation, a mechanism known to diminish the impact of approved BRAF inhibitors in this indication. CFT1946 also demonstrated regression in a BRAF-V600X NSCLC PDX model where the SOC dabrafenib + trametinib showed only modest tumor growth inhibition. Consistent with our previous results in the A375 melanoma model, single agent CFT1946 showed superior activity versus the SOC dabrafenib + trametinib combination in all additional melanoma models tested, with complete regression achieved using a CFT1946 + trametinib combination in a PDX model bearing a BRAF-V600E kinase duplication that showed minimal response to dabrafenib + trametinib. Finally, using an A375 intracranial model, treatment with CFT1946 gave robust, dose dependent efficacy and survival advantage over encorafenib. The promising activity of CFT1946 in a broad range of BRAF V600X preclinical models supports its ongoing clinical investigation in BRAF V600 mutant solid tumors (NCT05668585). Citation Format: Bridget Kreger, Jacob R. Stephenson, Mathew E. Sowa, Samantha A. Perino, Laura L. Poling, Eunju Hurh, Michael J. Thomenius, Yanke Liang, Stewart L. Fisher, Roy M. Pollock. CFT1946, a potent, selective BRAF V600X mutant-specific degrader demonstrates superior activity as a single agent to clinically approved BRAF inhibitors and standard of care combinations in preclinical models of BRAF V600X melanoma, CRC, NSCLC, and brain metastasis [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 1658.

Abstract 672: Small molecule NSC59984 stimulates mitochondria-dependent ferroptosis and overcomes integrated stress response pro-survival signaling in pre-clinical pancreatic and colorectal cancer models

Abstract Pancreatic and colorectal cancer are leading causes of cancer deaths worldwide. Resistance to current therapeutics is a significant challenge in treating these cancers. Ferroptosis, a non-apoptotic iron-dependent form of cell death characterized by overwhelming lipid peroxidation, has emerged as a potential strategy to overcome drug resistance. Our lab previously identified the small-molecule NSC59984 as a p53 pathway restoring compound that induces reactive oxygen species, requires p73, and targets mutant p53 for ubiquitin-mediated proteolysis involving MDM2 and HSP90. In pre-clinical models of pancreatic and colorectal cancer, we now show that the small-molecule NSC59984 induces lipid peroxidation and causes reactive oxygen species-dependent apoptosis as monotherapy. However, when combined with ferroptosis inducers or cystine deprivation, NSC59984 potently induces ferroptosis in a mitochondrial complex III-dependent manner. Using CRISPR/Cas9 in pancreatic cancer cells, we further show that HRI, an EIF2-alpha kinase responsible for the cellular response to mitochondrial stress, induces the integrated stress response (ISR) upon treatment with NSC59984 or ferroptosis inducers. The ISR serves as a mechanism for resistance to cell death induced by NSC59984 or ferroptosis inducers, causing upregulation of key anti-oxidative stress and anti-ferroptosis proteins, including the cystine importer SLC7A11 and the GPX4-stabilizing protein HSPA5/BIP/GRP78. The combination therapy overcomes the ISR to induce potent cell death. Our work demonstrates the importance of the interplay between mitochondria and the ISR during ferroptosis induction in pancreatic and colorectal cancer cells. Citation Format: Praveen R. Srinivasan, Arielle J. De La Cruz, Maximilian Pinho-Schwermann, Andrew George, William J. MacDonald, Shengliang Zhang, Wafik S. El-Deiry. Small molecule NSC59984 stimulates mitochondria-dependent ferroptosis and overcomes integrated stress response pro-survival signaling in pre-clinical pancreatic and colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 672.

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

Abstract KRASG12C inhibitors have achieved impressive results in the clinic. However, intrinsic or acquired resistance to therapy invariably develops leading to disease progression in most treated patients. Emerging pre-clinical and clinical evidence points to multiple mechanisms being responsible for resistance to KRASG12C inhibitors, including receptor tyrosine kinase (RTK) activation, KRASG12C amplification, activation of alternative signaling pathways including PI3K, mTOR, YAP/TAZ, acquisition of KRAS mutations that would preclude compound binding (e.g. R68S, Y96C/D, H95D/Q/R), conversion of the G12C codon mutation, and additional activating KRAS mutations (G13D, Q61H). In the clinic, many of the described mutations were detected at low frequency, some patients displayed multiple mutations and in 40% of patients no potential driving mutations were identified. It is therefore likely that other non-genetic alterations are also involved in resistance to KRASG12Ci monotherapy. Here, we used preclinical models of KRASG12C non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) to study intrinsic and acquired resistance to KRASG12C inhibitors. Increased expression of the RAS superfamily gene MRAS was observed in most KRASG12C-naïve tumor cells upon treatment with a KRASG12C inhibitors. In line with this, we observed an increase of MRAS-GTP levels in cells treated with KRASG12Ci in vitro. Interestingly, the increase in active MRAS did not lead to concomitant upregulation of MAPK signaling. Long term treatment with adagrasib was used for generating in vitro and in vivo KRASG12C models with acquired resistance to KRASG12Ci. A strong increase in MRAS expression was observed under these conditions, that surpassed the levels seen with short term treatment in naïve models. This data suggests that activation of MRAS-SHOC2-PP1C complex may play a role in compensating for the KRASG12C-driven signaling in both the intrinsic and acquired resistance setting. Knockdown of SHOC2 to baseline levels resulted in re-sensitization to KRASG12Ci monotherapy and to the effects of combinations in adagrasib-resistant NCI-H358 NSCLC cells. Pathway suppression was deepened by combining adagrasib with either a SOS1i (e.g. BI-3406) or SHP2i (e.g. TNO155) resulting in enhanced anti-tumor effects observed in the KRASG12Ci-resistant models. Our findings underscore the role of MRAS in acquired resistance in KRASG12Ci and the potential to overcome this with vertical pathway combinations, such as SOS1i or SHP2i. Citation Format: Venu Thatikonda, Kaja Kostyrko, Sabine Jurado, Simone Lieb, Melanie Hinkel, Donat Alpar, Oliver Bergner, Astrid Jeschko, Mark Pearson, Marco H. Hofmann. MRAS the forgotten member of the GTPase superfamily plays a role in KRASG12Ci resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1926.

Abstract 1869: The AI-powered KisoSeek™ platform identifies functional anti-TROP2 heavy-chain antibodies with potent anti-cancer activity

Abstract Functional heavy-chain antibodies (HCAbs) are promising therapeutic agents for targeted cancer therapy. Since they do not have a light chain, HCAbs have a small antigen-binding domain that can unlock new functionalities by reaching epitopes that are inaccessible to classical antibodies. HCAbs can be obtained by immunizing camelids, but this approach is expensive, time-consuming, and results in a limited antibody repertoire. Previously, we engineered the KisoMouse™- a transgenic mouse with multi-camelid species VHH/VH genes and the complete alpaca D and J gene segments integrated into the mouse IgH locus, along with a functional loss of the heavy chain CH1 domain to eliminate light chain pairing. KisoMouse™ are healthy and generate a robust serum HCAb response against a diverse range of immunogens. Trophoblast cell surface antigen-2 (TROP2) is a membrane protein overexpressed in numerous solid tumor types such as breast, colon, and lung cancers. TROP2 is associated with tumor aggressiveness and decreased survival and has emerged as an attractive therapeutic target. An anti-TROP2 antibody-drug conjugate has been approved for breast and bladder cancer, but challenges related to payload toxicity and resistance highlight the need for alternative therapeutics. We had previously identified an anti-TROP2 HCAb with high potency in in vivo preclinical models of human breast and colorectal cancer. Conventional antibody discovery approaches are resource-intensive and can fail to identify rare antibodies. To overcome such limitations, we developed the KisoSeek™ platform, a proprietary AI-powered toolkit that categorizes antibodies in an immune response based on their structural and biophysical features. With KisoSeek™, we analyzed the entire antibody repertoire generated from KisoMouse™ immunized with TROP2 and rapidly identified HCAbs with similar features as the high potency reference antibody described above. We selected three additional antibodies based on their in vitro TROP2 affinity, competitiveness for the reference antibody’s epitope, and yield in CHO cells. These candidates showed comparable potent efficacy in inducing regression in human breast and colorectal xenograft models, akin to the original reference antibody. In summary, the KisoSeek™ platform identified novel anti-TROP2 HCAbs out of the KisoMouse™ antibody repertoire that were similar to our original reference antibody. This demonstrates how KisoMouse™ and KisoSeek™ are two powerful complementary platforms for novel clinical antibody discovery. By categorizing the entire antibody repertoire and identifying antibodies with similar characteristics as known functional antibodies, KisoSeek™ has the potential to leverage existing discovery efforts to uncover many more rare yet potent therapeutic antibodies - the proverbial needles in a haystack. Citation Format: Sophie E. Cousineau, Amit Subedi, Richard Wargachuk, Juliana The, Xiaowei Wang, Wenyang Hou, Amogh Nair, Wansu Qiu, Alex Zhou, Tiffany Cheng, Morteza Babaie, Benyamin Gojogh, Marinieve Montero, Hiba Zahreddine, Elijus Undzys, Lucy Lai, Gordon Ngan, Luis Da Cruz, David Young. The AI-powered KisoSeek™ platform identifies functional anti-TROP2 heavy-chain antibodies with potent anti-cancer activity [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 1869.

Abstract 5909: Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer

Abstract Objectives: Given the intricate relationship between inflammation and ovarian cancer (OC) development and progression, anti-inflammatory drugs may have potential in the prevention and treatment of OC. Sulindac exhibits potent anti-inflammatory effects through inhibition of COX1/COX2 pathways and promising anti-tumor activity in pre-clinical models of colorectal and endometrial cancer. Thus, we investigated the efficacy of sulindac as an anti-tumorigenic agent in OC pre-clinical cell and mouse models. Methods: The human OC cell lines, MES and OVCAR5, and the K18-gT121+/-;p53fl/fl;Brca1fl/fl (KpB) mouse model of high-grade serous OC were used. Cellular proliferation was assessed by MTT and colony count assays. Apoptosis was assessed using cleaved caspase 3, 8, and 9 assays. To measure cellular stress, production of reactive oxygen species was measured by DCFH-DA assay, and the JC1 assay was used to assess changes in mitochondrial membrane potential. Cellular adhesion was evaluated by laminin assay, and cellular migration was determined by wound healing assay. Western immunoblotting was used to assess the effects of sulindac on downstream targets related to cellular stress, apoptosis, cellular invasion potential, cell cycle control, and inflammation. The KpB mice were treated with sulindac (7.5 mg/kg, oral gavage, daily) or placebo for four weeks. Results: Sulindac inhibited cellular proliferation in a dose-dependent manner in the MES and OVCAR5 cells, with IC50s of 75.3 and 75.69 μM, respectively. Sulindac (100 μM) reduced colony formation by 81% in the MES cells and by 49% in the OVCAR5 cells (p<0.01). Treatment of MES and OVCAR5 cells with sulindac increased ROS production and decreased mitochondrial membrane potential, in a dose-dependent manner (p<0.01). Sulindac induced activity of cleaved caspase 3, 8, and 9, in MES and OVCAR5 cells (p<0.01). In addition, sulindac (100 μM) effectively decreased cellular adhesion by 31% in MES cells and by 23% in OVCAR5 cells (p<0.01). These results were confirmed by Western immunoblotting demonstrating that sulindac downregulated the anti-apoptotic proteins Bcl-xl and Mcl-1 and upregulated the pro-apoptotic proteins Bax and cellular stress proteins Bip, ATF-4, and PDI, in both cell lines. Sulindac downregulated expression of several epithelial-mesenchymal transition proteins in both cell lines, including beta-catenin, snail, and slug. Sulindac also downregulated expression of cell cycle proteins CDK4, CDK5, and Cyclin D1 and inflammatory protein COX-2 in both cell lines. After 4 wks of treatment, sulindac significantly inhibited tumor growth by 71% (1.89 g vs 0.54 g, p<0.01) in KpB mice. Conclusions: Our results find that sulindac exhibited anti-tumorigenic effects in OC cell lines and the KpB OC mouse model. Given these promising pre-clinical results, further studies on the repurposing of sulindac for the prevention and treatment of OC is warranted. Citation Format: Nikita Sinha, Shuning Chen, Jennifer Haag, Xiaochang Shen, Boer Deng, Ziyi Zhao, Chunxiao Zhou, Victoria Bae-Jump. Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian 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 5909.

Abstract 3241: Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated colorectal cancer

Abstract Background: BRAFV600E in colorectal cancer (CRC) defines a unique subgroup of patients with poor prognosis and scarcely benefit from BRAF inhibitor-based treatment. Interestingly, BRAFV600E tumors demonstrated profound CpG island methylator phenotype (CIMP), suggesting extensive epigenetic dysregulation. Thus, we investigated epigenomic regulations in this subset of tumors with treatment-induced DNA demethylation. Methods: We treated six cell lines and two patient-derived xenograft (PDX) models of BRAFV600E CRC with vehicle, 5-azacitidine (DNMTi), vemurafenib (BRAFi), and combination treatment. PDX tumors treated with either vehicle or 5-azacitidine were collected on day 21 and sequenced for DNA methylation (Infinium HumanMethylation 450k BeadChip array), histone marks (ChIP-seq), chromatin accessibility (ATAC-seq), and gene expression (RNA-seq). Comprehensive post-translational modifications (PTMs) in histones with DNMTi or BRAFi treatment were assessed by mass spectrometry (MS). The combinational effect of 5-azacitidine with EZH2 or RNF2 inhibitors (Polycomb repressive complex activity (PRC) inhibition) was assessed through cell viability assays and colony formation assays in BRAFV600E CRC cell lines. Results: Analysis of TCGA COAD/READ tumors demonstrated a higher DNA methylation profile in BRAFV600E tumors than wild-type tumors even among the CIMP population (FDR < 1e-4). The combination of DNMTi with BRAFi showed selective additivity in vitro; however, did not improve efficacies in PDXs. The 5-azacitidine treatment induced profound demethylation in PDXs compared to control samples, predominantly attributed to promoter regions (48%). However, DNA demethylation failed to reactivate classic CIMP markers or tumor suppressor genes in CRC (e.g., CDKN2A, MGMT). Interestingly, demethylated genomic regions gained H3K27 trimethylation, repressive histone mark, in azacitidine-treated samples compared to control (9,431 vs 5,147 peaks respectively). We validated the elevation of H3K27 trimethylation as well as additional histone PTMs following DNMTi in MS-based histone analysis. Finally, combining a demethylating agent with EZH2i or RNF2i, which either deplete H3K27 trimethylation or block the PRC activity respectively, has demonstrated additive growth inhibitory effects in BRAFV600E CRC cell lines (p <0.05). Conclusions: This study unveiled intriguing aspects of demethylation agent treatment, which led to the compensatory engagement of other epigenomic markers, particularly repressive histone marks, resulting in limited efficacies to azacitidine treatment in preclinical BRAFV600E CRC models. This finding suggests the existence of adaptive interactions between epigenetic modifiers and potential clinical strategies in combinational epigenetic therapeutics. Citation Format: Hey Min Lee, Ajay Kumar Saw, Van Karlyle Morris, Anand Kamal Singh, Stefania Napolitano, Alexey Sorokin, Preeti Kanikarla Marie, Oluwadara Coker, Oscar Eduardo Villarreal, Nazanin Esmaeili Anvar, Kunal Rai, Scott Kopetz. Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated 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 3241.

Targeting two radiation-induced immunosuppressive pathways to improve the efficacy of normofractionated radiation therapy in a preclinical colorectal cancer model

Purpose We have previously demonstrated in a murine colorectal cancer model that normofractionated RT (normoRT: 18 × 2 Gy) induced MDSC infiltration and PD-L1 expression, while hypofractionated RT (hypoRT: 3 × 8 Gy) induced Treg. Here, we wanted to assess whether the association of normoRT with treatments that target two radiation-induced immunosuppressive pathways (MDSC and PD-L1) could improve tumor control. Materials and methods Subcutaneous tumors were induced using colon tumor cells (CT26) in immunocompetent mice (BALB/c) and were treated with RT alone (18 × 2 Gy or 3 × 8 Gy), or concomitantly with 5-Fluorouracil (5FU) (10 mg/kg) to deplete MDSC, and/or anti-PD-L1 (10 mg/kg). We assessed the impact of these combinations on tumor growth and immune cells infiltration by flow cytometry. In addition, we performed tumor rechallenge experiments and IFN-γ ELISpots to study the long-term memory response. Results Even though tumor growth was significantly delayed in the RT + 5FU compared to 5FU and untreated groups (p < .05), there was no significant difference between RT + 5FU (CRT) and RT alone. The rate of MDSC increased significantly 1 week after the end of normoRT (8.09% ± 1.03%, p < .05) and decreased with the addition of 5FU (3.39% ± 0.69%, p < .05). PD-L1 expressing tumor cells were increased after treatment. Adding anti-PD-L1 significantly delayed tumor growth, achieved the highest complete response rate, and induced a long-lasting protective specific anti-tumor immunity. Conclusions These results tend to demonstrate the interest of inhibiting two radiation-induced immunosuppressive mechanisms. In patients, the combination of normoRT with 5FU is already the standard of care in locally advanced rectal cancer. Adding an anti-PD-L1 to this treatment could show promising results.

BCL-XL inhibitors enhance the apoptotic efficacy of BRAF inhibitors in BRAFV600E colorectal cancer

Metastatic BRAFV600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAFV600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAFV600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAFV600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-XL, and that combining encorafenib with a BCL-XL inhibitor significantly enhances apoptosis in BRAFV600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-XL inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-XL inhibition, we also examined the effect of combining encorafenib with the BCL-XL -targeting PROTAC DT2216, and the novel BCL-2/BCL-XL inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAFV600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-XL inhibition significantly enhances apoptosis in pre-clinical models of BRAFV600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients.

Near-Infrared In Vivo Imaging of Claudin-1 Expression by Orthotopically Implanted Patient-Derived Colonic Adenoma Organoids

Background: Claudin-1 becomes overexpressed during the transformation of normal colonic mucosa to colorectal cancer (CRC). Methods: Patient-derived organoids expressed clinically relevant target levels and genetic heterogeneity, and were established from human adenoma and normal colons. Colonoids were implanted orthotopically in the colons of immunocompromised mice. This pre-clinical model of CRC provides an intact microenvironment and representative vasculature. Colonoid growth was monitored using white light endoscopy. A peptide specific for claudin-1 was fluorescently labeled for intravenous administration. NIR fluorescence images were collected using endoscopy and endomicroscopy. Results: NIR fluorescence images collected using wide-field endoscopy showed a significantly greater target-to-background (T/B) ratio for adenoma versus normal (1.89 ± 0.35 and 1.26 ± 0.06) colonoids at 1 h post-injection. These results were confirmed by optical sections collected using endomicroscopy. Optical sections were collected in vivo with sub-cellular resolution in vertical and horizontal planes. Greater claudin-1 expression by individual epithelial cells in adenomatous versus normal crypts was visualized. A human-specific cytokeratin stain ex vivo verified the presence of human tissues implanted adjacent to normal mouse colonic mucosa. Conclusions: Increased claudin-1 expression was observed from adenoma versus normal colonoids in vivo using imaging with wide field endoscopy and endomicrosopy.

Influenza virus infection enhances tumour-specific CD8+ T-cell immunity, facilitating tumour control.

Influenza A virus (IAV) can cause severe respiratory infection leading to significant global morbidity and mortality through seasonal epidemics. Likewise, the constantly increasing number of cancer diseases is a growing problem. Nevertheless, the understanding of the mutual interactions of the immune responses between cancer and infection is still very vague. Therefore, it is important to understand the immunological cross talk between cancer and IAV infection. In several preclinical mouse models of cancer, including melanoma and colorectal cancer, we observed that IAV infection in the lung significantly decreased the tumour burden. Concomitantly, tumour-specific CD8+ T-cells are strongly activated upon infection, both in the tumour tissue and in the lung. CD8+ T-cell depletion during infection reverses the reduced tumour growth. Interestingly, IAV infection orchestrated the migration of tumour-specific CD8+ T-cells from the tumour into the infected lung. Blocking the migration of CD8+ T-cells prevented the anti-tumoural effect. Thus, our findings show that viral respiratory infection has significant impact on the anti-tumour CD8+ T-cell response, which will significantly improve our understanding of the immunological cross talk between cancer and infection.

RET is a sex-biased regulator of intestinal tumorigenesis

Ret is implicated in colorectal cancer (CRC) as both a proto-oncogene and a tumor suppressor. We asked whether RET signaling regulates tumorigenesis in an Apc-deficient preclinical model of CRC. We observed a sex-biased phenotype: ApcMin/+Ret+/- females had significantly greater tumor burden than ApcMin/+Ret+/- males, a phenomenon not seen in ApcMin/+ mice, which had equal distributions by sex. Dysfunctional RET signaling was associated with gene expression changes in diverse tumor signaling pathways in tumors and normal-appearing colon. Sex-biased gene expression differences mirroring tumor phenotypes were seen in 26 genes, including the Apc tumor suppressor gene. Ret and Tlr4 expression were significantly correlated in tumor samples from female but not male ApcMin/+Ret+/- mice. Antibiotics resulted in reduction of tumor burden, inverting the sex-biased phenotype such that microbiota-depleted ApcMin/+Ret+/- males had significantly more tumors than female littermates. Reconstitution of the microbiome rescued the sex-biased phenotype. Our findings suggest that RET represents a sexually dimorphic microbiome-mediated “switch” for regulation of tumorigenesis.

Orthotopic MC-38 Allograft as a Robust Preclinical Model of Colorectal Carcinoma.

Colorectal cancer (CRC) is a significant global health concern, requiring effective preclinical models for studying its development and testing therapies. Mouse models have been used, including spontaneous tumors, carcinogen exposure, and tumor cell implantation as xenografts or at orthotopic sites. Here, we describe an orthotopic preclinical model of CRC, which provides a valuable tool for studying tumor growth and the tumor microenvironment, offering a more accurate representation of human CRC compared to xenograft models.

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Vasopressin Analog [V4Q5]dDAVP Exerts Cooperative Anticancer Effects in Combination With Low-Dose 5-Fluorouracil on Aggressive Colorectal Cancer Models.

Colorectal cancer (CRC) is a leading cause of cancer-associated mortality worldwide. Despite being an essential component of systemic chemotherapy for advanced CRC, 5-fluorouracil (5-FU) clinical use has severe limitations, such as high toxicity, low selectivity and drug resistance. [V4Q5]dDAVP (1-deamino-4-valine-5-glutamine-8-D-arginine vasopressin) is a peptide vasopressin analog and a selective agonist of the arginine vasopressin type 2 membrane receptor (AVPR2), expressed in microvascular and tumor tissue. This synthetic compound has well-proven antitumor and antimetastatic activity in different tumor types, including metastatic CRC. The objective of this work was to assess the potential combinational benefits in preclinical CRC models after [V4Q5]dDAVP addition to 5-FU. Effects on cellular viability, cell cycle progression, apoptosis and molecular mechanisms associated to [V4Q5]dDAVP treatment in combination with 5-FU were evaluated in murine CT-26 and human COLO-205 cell lines. In vivo, impact of dual therapy was explored on CRC tumor growth and metastatic spread. In CRC cells, [V4Q5]dDAVP (1 µM) addition to sub-IC50 5-FU concentrations resulted in the enhancement of cytostatic effects induced by chemotherapy. Reduction of cell viability after combined treatment was associated with cell cycle arrest in the G0/G1 phase, induction of apoptosis and increased gene expression of the cyclin-dependent kinase inhibitor p21 (CDKN1A) and the tumor suppressor p53 (TP53) in malignant cells, as assessed by flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. In vivo, intravenous administration of [V4Q5]dDAVP (0.3 µg/kg) in combination with safe low doses of 5-FU (50 or 80 mg/kg for CT-26 or COLO-205 tumor models, respectively) effectively abrogated CRC growth, reducing aggressiveness of primary lesions and increasing survival of tumor-bearing mice. In addition, concomitant administration of [V4Q5]dDAVP and 5-FU inhibited pulmonary metastasis formation by CT-26 cells in immunocompetent mice, especially reducing macrometastatic disease. [V4Q5]dDAVP seems to enhance the efficacy of 5-FU-based chemotherapy in CRC by modulating tumor progression, as well as metastatic dissemination, suggesting its potential role as a safe and cost-effective co-adjuvant agent for the management of advanced CRC.