Colon Tumor Research Articles

Abstract 4755: Extracellular GRP78 inhibition reverses drug and immune resistance in cancer cells

Abstract CBT300 was created from Creative BioTherapeutics’ discovery that drug resistant recurrent cancer cells up-regulate a survival pathway that results in the expression of extracellular Glucose-Regulated Protein 78 (ecGRP78) in the tumor microenvironment (TME). This TME ecGRP78 binds to cancer and immune cell surfaces, which induces a cascade of events to increase drug resistance, immune suppression, and cancer stem cell (CSC) formation. CBT300 targets surface bound ecGRP78 that has been found on breast, lung, ovarian, prostate, melanoma, multiple myeloma, colon, pediatric and adult brain tumors. We can now show that inhibition of ecGRP78 can a) induce apoptosis of drug resistant tumor cells, b) eliminate drug and immune resistance showing synergistic effects with chemotherapy and immunotherapy, c) decrease the amount of chemotherapy about 90% in combination with CBT300. Recent publications show that ecGRP78 is found on many types of tumor cell surfaces but not on normal cell surfaces. In fact, cell surface bound ecGRP78 is important for many aspects of cancer development, including cell survival, proliferation, chemoresistance, angiogenesis, metastasis formation, immune suppression, and stem cell formation. Recently, it has been shown that increased ecGRP78 expression in metastatic breast cancer, glioblastoma and multiple myeloma patients was significantly associated with later stage, increased distant metastasis, increased aggressiveness, shorter disease-free survival, and decreased overall survival. In studies to help understand how ecGRP78 causes tumor progression and drug and immune resistance, we discovered a novel ecGRP78 binding transmembrane protein on TNBC, and brain cancer cells called Receptor Tyrosine Kinase Orphan Receptor-1 (ROR1). Using the GRP78 binding domain from ROR1 and a human Fc domain, we created a biologic fusion protein that is a potent and specific ecGRP78 inhibitor called CBT300. We now show that CBT300’s elimination of csGRP78 destabilizes and removes oncofetal proteins ROR1, and Cripto, and checkpoint protein PD-L1 from tumor cell surfaces resulting in reversal of chemoresistance, reduction in immune suppression, inhibition of stem cell phenotype and increased tumor cell apoptosis. Our results with CBT300 demonstrate proof of concept data in vitro and in vivo for our novel ecGRP78 inhibitor on several drug resistant cancers demonstrating the potential to provide a major advance in the treatment of drug resistant cancers either alone or in combination with lower doses of chemotherapy. Exploiting a novel mechanism of action with a non-toxic, efficacious and cost effective biologic therapy that has shown increased survival in recurrent cancers will bring new hope for these patients. Citation Format: Donald J. Davidson, Jorryn Zelek, Elizabeth Stolarik, Mariah Thigpen, Anita Davidson, DPT. Extracellular GRP78 inhibition reverses drug and immune resistance in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4755.

Abstract 1425: Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status

Abstract Background: Epigenetic silencing of MHC-I, and poor T cell infiltration and function in the tumor microenvironment (TME) are prevalent in solid malignancies and associate with lack of response to immune checkpoint blockade (ICB). Novel combination therapies may overcome these barriers, thereby providing clinical benefit across solid tumors. STAR0602 is a first-in-class bifunctional therapeutic molecule, that simultaneously engages a nonclonal mode of T cell receptor (TCR) activation with costimulation to promote activation and expansion of αβ T cell subsets expressing distinct variable β (Vβ) TCR chains. Here, we examined the tumor-suppressive ability and mode of action of a murine surrogate of STAR0602 (mSTAR1302) in combination with the class I HDAC inhibitor Entinostat, an epigenetic modulator that enhances tumor immune cell recognition, in multiple tumor models, including ICB-refractory. Methods: mSTAR1302 and/or Entinostat were administered to mice bearing ICB-responsive breast EMT6 wild-type (wt) and ICB-refractory EMT6 β2-microglobulin (B2m)-deleted and CT26 (Kras G12Dmut colon) tumors. Anti-tumor efficacy, survival, and protective memory were monitored. Immune cell depletion studies were conducted. Comprehensive proteomic and immune profiling of tumor, tumor-draining lymph node (tdLN), and spleen cell populations was conducted. Antigen-specific IFNg T cell responses were evaluated by ELISpot. Results: Combination therapy demonstrated significant enhancement in tumor suppression and prolonged survival in CT26 (MHC-I+), EMT6 wt (MHC-I+) and EMT6 β2m-deleted (MHC-Inull) tumors relative to single agent therapy, as well as enhanced tumor-specific protective memory responses. Tumor suppression was independent of CD4+ T cell immunity and associated with sustained activation and expansion of Vβ13 CD8+ T cells. Conclusions: Ongoing mechanistic studies indicate combination therapy to elicit complimentary mechanisms of tumor suppression beyond CD8+ T cells. Ongoing proteomic and single-cell transcriptomic investigation of MHC-I competent and null tumors will provide a deeper insight into the mode of action of this combination therapy. Collectively, these data support the use of STAR0602 in combination with Entinostat for the treatment of solid malignancies, including for patients harboring ICB-refractory tumors devoid of MHC class I. Citation Format: Katherine E. Lothstein, Asma S. Khelifa, Masaya Miyamoto, Christine M. Minnar, Lisa Poppe, Nicholas Roller, Andrew Bayliffe, Ke Liu, Jacques Moisan, Madan Katragadda, Zhen Su, Jeffrey Schlom, Sofia R. Gameiro. Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status [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 1425.

Abstract 2660: CXCL9/10 is critical for immunotherapeutic efficacy by recruiting and activating T lymphocytes in tumor microenvironment

Abstract Background: Tumor immunotherapies targeting PD-(L)1 exhibit anti-tumor efficacy in only about 20% of patients with a variety of cancers. Literature has demonstrated that “hot tumor” which contains high T lymphocytes in tumor microenvironment exhibits a better response to immunotherapies than “cold tumor”. Therefore, we hypothesize that T lymphocyte-recruited factors play the roles determining the immunotherapeutic efficacy, including CXCL9, 10, and 11, which are located in human chromosome 4 with consistent co-expression and protein structure. Aims: The aim of this study is to investigate whether CXCL9/10 is critical in determining the anti-PD-(L)1 immunotherapeutic efficacy. Methods: GEPIA, cBioPortal, and Kaplan-Meier Plotter were used to investigate the gene expression, co-expression, and immunotherapeutic association for CD274 (PD-L1), ICAM1, and CXCL9/10 in lung, colon, and liver cancers. qPCR and flow cytometry were used to detect the gene expression in tumor cells and T lymphocytes, particularly comparing the differences between LL/2 and CT26 cells whereas LL/2 is immunotherapy-resistant and CT26 is immunotherapy-sensitive. CD8+ T cell transwell assay and qPCR were used to measure the function of CXCL10 on CD8+ T cell migration and activation, whereas IFNγ and IL-2 as the activation markers. Results: We found that CXCL9/10 overexpressed in a variety of tumors, including lung, colon, and liver tumors with a correlation with PD-L1. However, PD-L1 was not overexpressed in most tumors. High PD-L1 and CXCL10 are basically associated with better survival rates in tumor patients receiving immunotherapies. Correlation analysis revealed that IRF-1 and STAT1 were correlated with PD-L1 and CXCL10 expression in lung cancer. Since literature has indicated that LL/2-derived syngeneic tumor exhibits immunotherapy-resistant and CT26 exhibits immunotherapy-sensitive, we further compared the gene expression between them. We found that IFNA, IFNG, CXCL10, and PD-L1 all expressed higher levels in CT26 cells than LL/2. Moreover, we found that T lymphocytes specifically expressed high CXCR3, a receptor of CXCL9/10, including CD4+ T and CD8+ T cells by flow cytometry. As expected, CD8+ T cells can be recruited in a transwell assay by IFNα and CXCL9. In addition, we found that IFNα induced CXCL10 expression in monocytes and CXCL10 increased activation markers IFNγ and IL-2 in CD4+ T and CD8+ T cells. Conclusions: This study revealed that CXCL9/10 expression is correlated with PD-L1 in tumors, resulting in better immunotherapeutic efficacy. The evidence demonstrated that CT26 intrinsically exhibited higher IFNs and CXCL9/10 compared to LL/2. Therefore, CXCL9/10 potentially recruits and activates CD8+ T cells to tumor microenvironment, transferring “cold tumor” to “hot tumor”. This study indicates that CXCL9/10 is critical for immunotherapeutic efficacy. Citation Format: Chun-Chia Cheng, Jungshan Chang, Zong-Lin Sie, Ai-Sheng Ho, Cheng-Liang Peng, Chun-Chao Chang. CXCL9/10 is critical for immunotherapeutic efficacy by recruiting and activating T lymphocytes in tumor microenvironment [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 2660.

Abstract 4058: Nanoparticle delivery of interleukin-2 induces anti-tumor immune response and therapeutic efficacy in mouse tumor models

Abstract Immune checkpoint inhibitors (ICIs) have demonstrated robust responses in some solid tumor types. However, the low response rate, which is associated with lack of pre-existing tumor-infiltrating lymphocytes (TILs), remains a significant challenge. Interleukin-2 (IL-2) can effectively expand TILs and has been FDA-approved for metastatic renal cancer and melanoma. However, IL-2 has several drawbacks—including short circulation half-life, dose-limiting toxicities, and counterproductive activation of regulatory T cells (Tregs)—resulting in less than 10% complete response rate. Numerous efforts have been made to enhance IL-2 formulations, but none have yet received FDA approval. In this study, we utilized our proprietary nanoparticle platform (Pdx-NP™) to improve IL-2 formulation. Pdx-NP™ can load IL-2 with near-complete binding, resulting in a 160-nm nanoconstruct (IL2-NP), suitable for both local and intravenous administrations. To evaluate the efficacy, we utilized bilateral melanoma (B16F10) and colon (MC38) tumor models, in which the local tumor was treated intratumorally for 3 doses and the distal tumor was left untreated to assess the systemic anti-tumor immune response. In the B16F10 model, IL2-NP treatment demonstrated favorable immune profiles in local and distal tumors and the draining lymph nodes, such as increased CD8+ T cell proliferation, CD8+ T cells/Treg ratio, and recruitment of dendritic cells and macrophages, compared to saline or free IL-2 counterparts. These positive immune responses translated into significant growth reduction of IL2-NP treated tumors and improvement in survival of the mice compared to free IL-2. In the MC38 tumor model, the IL2-NP treatment resulted in complete regression of both local and distal tumors in 43% of the mice. When combined with ICIs (αPD-1 and αCTLA-4), the treatment achieved a cure rate of 100%, whereas ICIs alone yielded only a 29% cure rate. In conclusion, IL2-NP induces robust proliferation and activation of TILs and is efficacious both as a monotherapy and in combination with ICIs. This is due to the ability of Pdx-NP™ to protect and retain therapeutics within tumors, with an 8-fold increase in tumor accumulation over the free drug counterpart. Our ongoing efforts are focused on the targeted delivery of IL-2 to tumors upon systemic administration, using αPD-L1 antibody-conjugated Pdx-NP™. This antibody-conjugated nanoparticle has an elimination half-life (t1/2) of about 25 hrs in non-human primates. Paired with αPD-L1 for tumor homing, this nanoconstruct is hypothesized to enhance IL-2 accumulation in tumors compared to free IL-2, which has t1/2 of only 1.5 hrs in human. The current clinical landscape of cancer immunotherapy focuses on combinational approaches, and our versatile Pdx-NP™ platform, which is capable of co-delivering different classes of therapeutics, is well suited for this endeavor. Citation Format: Ruijie Wang, Alyssa Wallstrum, Natalie White, Pramod Kumar, Tongchatra Watcharawittayakul, Noah A. Crumrine, Cole Baker, Jeremy Saito, Moataz Reda, Worapol Ngamcherdtrakul, Wassana Yantasee. Nanoparticle delivery of interleukin-2 induces anti-tumor immune response and therapeutic efficacy in mouse tumor 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 4058.

Abstract 4356: An isoform-resolution transcriptomic atlas of colorectal cancer from long-read single-cell sequencing

Abstract Colorectal cancer (CRC), a complex and molecularly heterogenous disease that lacks robust predictive markers and targeted therapies, is the second leading cause of cancer death worldwide. In recent years, technological advances in short-read single-cell RNA sequencing (scRNA-seq) have been instrumental in deciphering tumor cell heterogeneities. However, most studies have focused on gene-level expression quantification without considering alterations in transcript structures arising from widespread alternative end processing and/or splicing which are frequently dysregulated in cancer. Here, we combined short- and long-read scRNA-seq of CRC patient samples to build the first isoform-resolution CRC transcriptomic atlas. We identified 394 dysregulated transcript structures in tumor epithelial cells, including 299 derived from the coupling of multiple alternative splicing events. Additionally, we characterized genes and isoforms associated with different colon epithelial lineages and tumor cell subpopulations exhibiting varying levels of stemness and differentiation, as well as their distinct prognostic implications. Finally, we built an algorithm by integrating mass spectrometry data and novel peptides derived from predicted open reading frames of recurrent tumor-specific transcripts to curate a panel of recurring neoepitopes. Overall, this study unveils the transcriptomic landscape of CRC and may drive the identification of neoantigens based on full-length transcriptomic atlases to aid future development of more universal neoantigen-based cancer vaccines. Citation Format: Zhongxiao Li, Bin Zhang, Jia Jia Chan, Hossein Tabatabaeian, Qing Yun Tong, Xiao Hong Chew, Xiaonan Fan, Charlene Chan, Patrick Driguez, Faith Cheong, Shi Wang, Bei En Siew, Ian Jse-Wei Tan, Kai-Yin Lee, Bettina Lieske, Wai-Kit Cheong, Dennis Kappei, Ker-Kan Tan, Xin Gao, Yvonne Tay. An isoform-resolution transcriptomic atlas of colorectal cancer from long-read single-cell sequencing [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 4356.

Abstract 5108: Immunomodulatory and anti-tumor effect of radiation and induced telomere damage to treat pediatric high-grade gliomas

Abstract Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine glioma (DIPG) are among the most treatment refractory cancers in children. Despite multimodal therapy, prognosis is dismal with survival of <1 year for DIPG and a 5-year overall survival <10% for other pHGGs. Radiotherapy remains the only standard of care for DIPG that extends the survival by few months. In recent years, immunotherapy is emerging as a potential alternative treatment option. However, several factors have hindered the use of immunotherapy for pHGGs including “cold” immune environment with lack of immune cells infiltration, low tumor mutational burden, and lack of neo-antigen expression that prevents tumor recognition by the immune system. Therefore, novel therapies that activate the immune system while evading the tumor immunosuppression are highly needed. Here, we aim to demonstrate the immunomodulatory anti-tumor effects of THIO (6-thio-2’deoxyguanosine), a nucleoside analog and a telomerase-dependent telomere targeting agent, in DIPG mouse models. Our preclinical in vitro and in vivo data have demonstrated the efficacy of THIO in inducing telomeric damage, G2/M cell cycle arrest, and apoptosis in high-risk medulloblastoma and DIPG. Importantly, we have demonstrated that THIO crosses the blood-brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. THIO was shown to induce anti-tumor immunity through micronuclei formation leading to cGAS-STING pathway mediated activation of the innate and adaptive immune response in colon, lung, and hepatocellular carcinoma tumor models. Our preliminary data demonstrate the THIO-dependent induction of micronuclei formation co-localizing with cGAS and leading to STAT1 activation in DIPG cell lines. We have also shown that THIO synergistically sensitizes DIPG cells to ionizing radiation (IR), significantly decreasing cell proliferation. We are currently testing the combination of THIO+IR in an orthotopic mouse model for DIPG. We will present results on the potential of THIO and IR treatments to stimulate anti-tumor immunity through the activation of STING pathway in a syngeneic mouse model of DIPG. These preclinical studies will support the potential use of THIO and IR to treat children with high-risk pediatric brain tumors. Citation Format: Umaru Banlanjo, Shiva Senthil Kumar, Sergei Gryaznov, Rachid Drissi. Immunomodulatory and anti-tumor effect of radiation and induced telomere damage to treat pediatric high-grade gliomas [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 5108.

Abstract 2698: TCF-1 is a critical regulator of intraepithelial lymphocytes in colorectal carcinoma

Abstract Tissue-resident immune cells reside within the gastrointestinal tract known as intraepithelial lymphocytes (T-IELs), including αβ and γδ T-IELs. This unique population of T cells constantly survey and are critical in maintaining the gastrointestinal epithelium. We show that T-IELs in various regions of the gastrointestinal tract have distinct features. T-IELs in the small intestine exhibit high expression of cytotoxic molecules important for cancer defense while colon T-IELs are distinctively regulated by the transcription factor, TCF-7/TCF-1, which suppresses their effector and cytotoxic properties, including reduced expression of granzymes, and their abundance is dependent on the microbiome. Targeted deletion of TCF-1 in γδ T-IELs using pre-clinical mouse models resulted in a distinct effector profile and reduced colon tumor burden. Furthermore, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers compared with normal healthy colon, strongly correlating with an enhanced γδ T-IEL effector phenotype and improved patient survival. Further investigation aims to elucidate diverse mechanisms by which TCF-1 controls tissue-specific anti-tumor immunity and uncover the important factors controlling TCF-1 expression. Our study underscores the necessity to consider tumor and organ microenvironment-specific factors in optimizing immunotherapy, advancing our understanding of T cell function in the colon to pave the way for innovative CRC treatments. Citation Format: Marina H. Yakou, Sonia Ghilas, Kelly Tran, Yang Liao, Shoukat Afshar-Sterle, Anita Kumari, Kevin Schmid, Christine Dijkstra, Chantelle Inguanti, Simone Ostrouska, Jordan Wilcox, Maxine Smith, Pavitha Parathan, Amr Allam, Hai-Hui Xue, Gabrielle T. Belz, John M. Mariadason, Andreas Behren, Grant R. Drummond, Roland Ruscher, David S. Williams, Bhupinder Pal, Wei Shi, Matthias Ernst, Dinesh Raghu, Lisa A. Mielke. TCF-1 is a critical regulator of intraepithelial lymphocytes in colorectal carcinoma [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 2698.

Abstract 3439: Factors affecting the performance of existing epigenetic clocks to predict chronological age in colorectal tumor and normal tissues

Abstract Epigenetic age (EA) may serve as a risk stratification biomarker for early-onset colorectal cancer (EOCRC, <50 years old), which has been rising substantially in recent decades. We utilized publicly available data in a descriptive analysis to compare epigenetic clocks in colorectal tissue and to investigate differences between chronological age (CA) and biological age, as measured by EA, in relation to demographic and clinical features. We analyzed DNA methylation (DNAm) array data on 144 participants (96 CRC cases, 48 healthy controls) from the Colonomics Study (CLX; Barcelona, Spain) and 331 CRC cases (51 EOCRC, 316 average-onset (≥50) colorectal cancer (AOCRC)) from The Cancer Genome Atlas (TCGA) as well as corresponding demographic and clinical variables. Among CLX participants, the 96 CRC cases provided colon tumor and paired normal mucosa samples, while the 48 controls provided healthy colon mucosa samples. TCGA included primary tumor samples from 331 cases with colon or rectal adenocarcinoma (TCGA-COADREAD). The 2013 Horvath clock estimated EA based on CA, while epiTOC, estimated the relative stem cell division rate from tissue biospecimens. We examined the correlations between CA and EA stratified by case/control status, sample type, sex, and age of onset (TCGA only). Tissue from CLX controls demonstrated a strong, positive correlation (r>0.9) between CA and EA for both the 2013 Horvath and epiTOC clocks. Using the Horvath clock, CA and EA correlations in normal mucosa and tumor tissue samples from CLX varied, with r = 0.74 and r = 0.41, respectively, and were weaker using the epiTOC clock, with r = 0.26 (normal) and r = 0.10 (tumor). When CLX cases were stratified by sex, the CA and EA (Horvath) correlations among female cases were r = 0.80 (normal) and r = -0.12 (tumor), while the correlations for males were stronger at r = 0.58 (normal) and r = 0.31 (tumor). This is in comparison to the epiTOC clock with females showing correlations of r = -0.02 (normal) and r = 0.41 (tumor), while males had r = 0.06 (normal) and r = 0.11 (tumor). In TCGA, correlations for EOCRC were r = -0.20 (Horvath) and r = -0.08 (epiTOC) and for AOCRC were r = 0.29 and 0.20, respectively. By sex, correlations for females with EOCRC for Horvath and epiTOC were r = -0.09 and r = -0.07, respectively, while correlations for males with EOCRC were r = -0.02 and r = -0.08, respectively. For TCGA cases with AOCRC, the correlations between CA and EA were r = 0.35 and r = 0.22 for females, and r = 0.26 and r = 0.20 for males, respectively for Horvath and epiTOC. This study revealed substantial variability in the ability of existing epigenetic clocks to accurately predict chronological age, with tissue type and sex strongly influencing the resulting correlations. Our findings highlight the importance of considering these factors when choosing an epigenetic clock. Citation Format: Christopher L. Benson, Ferran Moratalla-Navarro, Anna Diez-Villanueva, Matthew A. Devall, Victor Moreno, Stephanie L. Schmit, Fredrick R. Schumacher. Factors affecting the performance of existing epigenetic clocks to predict chronological age in colorectal tumor and normal tissues [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 3439.

Abstract 99: Versican regulates T cell abundance within the colorectal cancer microenvironment

Abstract Background: Clinical benefit from immunotherapy is seen in a minority of patients with metastatic colorectal cancer (CRC). Identifying how the tumor microenvironment (TME) influences immune infiltration and treatment response is an unmet need. Versican (VCAN), a chondroitin sulfate proteoglycan, common in the CRC TME has immunoregulatory properties and can be cleaved releasing an immunostimulatory product, versikine (Vkine). Here we evaluate the impact of the abundance of VCAN and Vkine on CD8+ T cell infiltration in CRC. Methods: Samples include 306 primary colon tumors, an additional 176 primary CRCs with matched liver metastases (175 samples), and 328 normal samples (238 colon and 90 liver). Samples were stained via immunohistochemistry (IHC) for VCAN, Vkine, and CD8. CD8+ tumor infiltrating lymphocytes (TILs) were counted per high powered field (HPF, 400X magnification) and presented as averages. Intensity of VCAN and Vkine expression were scored from 0-3+. Samples designated a 0/1 were considered low accumulation and samples scored as a 2/3+ were designated high. Samples scored low for VCAN but high for Vkine were designated VCAN proteolytic predominant (VPP) and all other combinations considered VCAN proteolytic weak (VPW). To create an in vitro model, the mouse MC38 cell line was CRISPR engineered to over-express VCAN. Conditioned media was then concentrated and added to a collagen matrix in a transwell with activated T cells. The number of cells and their subtype that migrated through the matrix were evaluated with flow cytometry. Results: VCAN accumulation was highest in cancerous stroma with 58% of cancers being VCAN high and only 14% of adjacent normal tissues (p<0.001). Vkine accumulation was similar between groups, with 67% of tumors and 54% of adjacent normal samples scored as Vkine high. The VPP phenotype was least common in tumor tissues with only 27% of tumor samples designated VPP versus 43% of adjacent normal samples (p<0.001). VCAN high tumors had significantly fewer TILs with 4 TILs/HPF vs 13 in the VCAN low tumors (p<0.001). High levels of Vkine correlated with greater TIL abundance (Vkine high: 10 TILs/HPF, Vkine low: 5 TILs/HPF; p=0.02). Versican proteolysis had the strongest correlation with TIL abundance in cancers (VPP: 18 TILs/HPF, VPW: 5 TILS/HPF; p<0.001). In vitro, the addition of VCAN containing media into transwell matrices reduced both CD4+ and CD8+ T cell migration through the matrix (Control: 4277 CD8+ T cells, VCAN: 2603 CD8+ T cells; p<0.001 and Control: 7770 CD4+ T cells, VCAN: 5340 CD4+ T cells; p=0.007). Conclusions: VCAN accumulation in human CRCs is common and correlates with reduced TIL abundance, whereas VCAN proteolysis correlates with increased TILs. In vitro, VCAN demonstrated the ability to reduce T cell migration. Future studies will explore the mechanism by which VCAN abundance leads to decreased T cell infiltration and the importance of this as an immunotherapy biomarker. Citation Format: Sean Kraus, Philip Emmerich, Mitch Hayes, Kristina Matkowskyj, Wei Zhang, Cheri Pasch, Fotis Asimakopoulos, Dustin A. Deming. Versican regulates T cell abundance within the colorectal cancer microenvironment [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 99.

Abstract 4039: Enhanced anti-cancer efficacy of the AhR Inhibitor DA-4505 in combination with anti-PD-1 treatment: Attenuation of lung metastasis and elimination of tumor

Abstract Background: The Aryl hydrocarbon receptor (AhR) is one of the most predominant regulators of cancer metabolism. AhR plays a crucial role in inhibiting the activation of immune cells and promoting the growth of tumor cells. Here, we propose that a best-in-class AhR inhibitor, DA-4505, improves anti-tumor efficacy in combination with anti-PD-1. Methods: To explore the anti-tumor effects, DA-4505 was administered at a daily dose of 10 mg/kg either alone or in combination with anti-PD-1 (10 mg/kg) in a syngeneic mouse model. Tumor volume, survival rates, and metastasis were measured, and immune profiles were evaluated using mIHC, flow cytometry, and scRNAseq. Results: The synergistic anti-tumor effects of the DA-4505 and aPD-1 combination therapy were observed across four mouse tumor models (CT26, 4T1, LLC, TC1). The combination group demonstrated a significant improvement in anti-tumor efficacy compared to the monotherapy groups. Despite the moderate response of aPD-1 in the CT26 colon tumor model, the combination group displayed heightened anti-tumor effects through increase in CD8 T cell activity (P<0.05). In the LLC and TC1 lung cancer models, the combination therapy demonstrated improved anti-tumor effects and survival rates (P<0.001). Notably, the LLC model exhibited a significant increase in M1-type macrophages, while the TC1 model showed a significant augmentation in functional markers of CD3+ T cells within the combination group (P<0.05). In both models, there was a significant reduction in the ratio of immunosuppressive M-MDSC and PMN-MDSC in the combination group. Immune depletion assays elucidated that DA-4505 mediated anti-tumor effects by increase in CD8+ T cells, NK cells, and macrophages in the CT26 model. In contrast, the LLC model exhibited the contribution of macrophages and CD4+ T cells to anti-tumor effects in the combination group (P<0.05). To assess the impact on metastasis inhibition, an orthotopic model of 4T1 mouse tumor was established, revealing a significantly increased anti-tumor effects and survival rate in the combination group, accompanied by a reduced lung metastasis ratio. H&E staining confirmed diminished migration of tumor tissue to adjacent areas. scRNAseq analysis of CT26 tumor samples indicated consistent changes at the gene expression level, reflecting a decrease in metastasis-related MMP and VEGF genes in the combination group and an increasing trend in cytotoxicity-related genes following DA-4505 treatment. Conclusion: The AhR inhibitor, DA-4505, has demonstrated enhanced anti-tumor efficacy when administered in combination with anti-PD-1. Furthermore, it not only promoted tumor elimination but also demonstrated significant efficacy in inhibiting metastasis. This study highlights the potential of combining AhR inhibitors with immunotherapies for clinical treatment. Citation Format: Dong Kwon Kim, Sujeong Baek, Seung Min Yang, Young Taek Kim, Joon Yeon Hwang, Seul Lee, Seong-san Kang, Mi hyun Kim, Heekyung Han, Kwangmin Na, Chai Young Lee, Yu Jin Han, Taedong Han, Hyounmie Doh, Jongho Cho, Dajeong Kim, Daewon Cha, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Mi Ran Yun, Jii Bum Lee, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. Enhanced anti-cancer efficacy of the AhR Inhibitor DA-4505 in combination with anti-PD-1 treatment: Attenuation of lung metastasis and elimination of tumor [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 4039.

Abstract 693: The effect of butyrophilin 1A1 blockade in combination with radiation treatment in tumor models

Abstract Expression of radiation-induced immune escape molecules, including PD-L1, contributes to the efficacy of cancer immunotherapy. We used an unbiased in vivo gene knockdown screen in naïve T-cells and introduced them into irradiated host tumors to identify novel immunosuppressive genes activated during radiation. From the screen, we found that butyrophilin 1A1 (BTN1A1) knockdown causes T-cell expansion in irradiated tumors. This points to the potential immune checkpoint role of BTN1A1. We validated that BTN1A1 expression is inducible in activated T-cells and that blocking BTN1A1 contributes to T-cell activation. We tested an anti-mouse BTN1A1 antibody (STC109) in mouse colon tumor models (CT26 tumors), we observed partial anti-tumor efficacy using single agent STC109, which was enhanced by the combination of STC109 with radiation. We also got similar result in in mouse lung tumor models (LLC tumors) using anti-mouse BTN1A1 antibody, and the combination treatment showed an improved suppression of tumor growth and lung metastasis compared to single agent. Moreover, our data shown that knockout of BTN1A1 in 4T1 tumor models shown dramatic defect of tumor growth, and the BTN1A1 ko tumors are more sensitive to radiotherapy. These findings indicate the role of BTN1A1 in tumor immune response from radiation stress, and that anti-BTN1A1 has strong potential to enhance efficacy of radiotherapy for cancer treatment. Citation Format: Nan Li, Chen Braun, Young-Seung Kim, Andrew Park, Stephen S Yoo, Steven H. Lin. The effect of butyrophilin 1A1 blockade in combination with radiation treatment in tumor 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 693.

Abstract 700: Impact of heterogeneity for mismatch repair activity on colon tumor development and therapeutic response

Abstract Background: Colorectal cancers (CRC) that are deficient for DNA mismatch repair (dMMR) proteins have high levels of genetic instability and consequently high mutational burden, creating numerous neoantigens that can elicit an immune response. Therefore, dMMR CRC are the best candidates for immune checkpoint inhibition (ICI) compared to proficient DNA mismatch repair (pMMR) CRC. Early clinical trials revealed that only 30-55% metastatic dMMR CRC responded to ICI. Recent studies utilizing allograft models have demonstrated that tumor immune landscape can be primed with radiopharmaceutical therapy (RPT). Although early results have been quite promising, allograft models often fail to accurately predict efficacy for many reasons, but most often are due to a lack of heterogeneity. Methods: To better understand how heterogenous MMR expression impacts tumor development and response to ICI alone or in combination with RPT, we developed a new mouse model that develops three tumor types: (1) homotypic dMMR tumors that express green fluorescent protein, (2) homotypic pMMR tumors that express red fluorescent protein, and (3) heterotypic tumors with a mixture of dMMR and pMMR cells. Surveillance bright field and fluorescent colonoscopies allow us to assess treatment response by measuring tumor size and proportion of dMMR and pMMR tumor cells in real-time. These outcomes are verified through ex vivo imaging and histological analysis. Results: Tumor response to anti-PD-L1 ICI was dependent on MMR status. Homotypic dMMR tumors exhibited the strongest response: significantly smaller in treated mice than controls, exhibited areas of vascular congestion and increased CD8+ cytotoxic T-cells infiltration. Response of heterotypic MMR tumors varied depending on percentage of dMMR cells; tumors with a high percentage of dMMR cells remained small, whereas tumors with a low percentage of dMMR had an outgrowth of pMMR cells. Treated and untreated homotypic pMMR tumors were indistinguishable. To enhance the response of all three tumor types, mice were treated with dual ICI treatment, anti-PD-L1 and anti-CTLA-4, or RPT. Animals administered the tri-therapy (dual ICI + RPT) survived significantly longer with fewer tumors than animals treated with either dual ICI or RPT alone. Conclusions: Our innovative mouse model allows for the highly detailed characterization of tumor response to therapies. Simplistic allograft models may not adequately account for the dynamic effects that neighboring cell death may have on radio-resistant cells, on tumor vascular perfusion and oxygenation, or on tumor immune infiltration and adaptive immune recognition of remaining cells. We begin to fill these critical gaps in knowledge and therefore the results from our studies will help guide the translation and integrated use of ICIs and RPTs in clinic. Citation Format: Santina M. Snow, Dawn Albrecht, Paul A. Clark, Caroline P. Kerr, Joseph J. Grudzinski, Justin J. Jeffery, Hansel Comas Rojas, Reinier Hernandez, Kristina A. Matkowskyj, Jamey P. Weichert, Zachary S. Morris, Richard B. Halberg. Impact of heterogeneity for mismatch repair activity on colon tumor development and therapeutic response [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 700.

Abstract 6623: Ala59 mutants of KRAS cooperate with Nf1 loss to enhance colon tumorigenesis

Abstract Understanding the role of KRAS allele heterogeneity in tumor development, and its influence on therapeutic efficacy, is critical for the improvement of cancer treatment. The Kirsten rat sarcoma (KRAS) gene is the most frequently mutated oncogene in colorectal cancer (CRC) and patients with CRC tumors positive for KRAS mutations have poorer outcomes and limited treatment options. Extensive work over the past four decades has shown that common oncogenic alleles of KRAS are associated with hyperactivation of the MAPK signaling pathway (i.e. RAF/MEK/ERK), and it is for this reason that patients with KRAS mutant tumors are excluded from targeted therapies against EGFR. Recently, we characterized two alleles of KRAS, KRASA59T and KRASA59E, that paradoxically promote KRAS activation but are impaired in their ability to promote BRAF dimerization (Johnson et al. Mol Cell. 2022. PMID: 35202574). Consistent with these biochemical functions, KRASA59T and KRASA59E are enriched in CRC tumors that already have genetic alterations in genes that regulate MAPK signaling (e.g. EGFR, BRAF, NRAS, NF1). Further, unlike other described oncogenic alleles of KRAS, clinical data suggest that these KRASA59T and KRASA59E may not confer intrinsic resistance to anti-EGFR therapies at all. While KRASA59T and KRASA59E may not promote BRAF dimers, here we show that KRASA59T and KRASA59E does promote dimerization between ARAF and CRAF, and using an unpublished conditional mouse mode of K-Ras (i.e. LSL-K-RasA59E) we tested (1) whether Ala59 alleles are oncogenic or (2) require cooperation with additional MAPK pathway activating mutations, and (3) whether Ala59 alleles can promote intrinsic resistance to EGFR inhibition. Here, we show that endogenous K-RasA59E can disturb colon homeostasis and decrease mouse survival in an autochthonous colon tumor model, demonstrating that A59E alone is an oncogenic allele of KRAS in the colon. Breeding of LSL-K-RasA59E mice with mice harboring a conditional knockout of Nf1 (i.e. Nf1fl/fl), a GTPase activating protein (GAP) that promotes activation of wildtype RAS, show that combined expression of K-RasA59E and wildtype RAS activation cooperatively perturb colon homeostasis and decrease mouse survival. Thus, while KRASA59E may not require a second genetic alteration to activate the MAPK signaling pathway, it can cooperate with these alterations to increase the severity of disease. Finally, using mouse colon derived organoids, we show that endogenous K-RasA59E neither promotes EGF independence of organoid growth nor confers on these organoids a resistance to EGFR inhibition. Thus, our data support a unique role for RAF dimer function in in KRAS mutant tumors of the colon in both development and sensitivity to drugs, as well as argue for the inclusion of KRASA59T and KRASA59E bearing CRC tumors in anti-EGFR therapies. Citation Format: Christian William Johnson, Elizabeth M. Terrell, Josh Cook, Bing Shui, Eve O'Donoghue, Shannon Hull, Moon Hee Yang, Shikha Sheth, Rona Yaeger, Scott Kopetz, Deborah K. Morrison, Kevin Haigis. Ala59 mutants of KRAS cooperate with Nf1 loss to enhance colon tumorigenesis [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 6623.

Abstract 762: The combination of EPA and naproxen suppresses colon tumorigenesis via altering the TME, and promoting epithelial maturation in cancerous crypts

Abstract Introduction: Colorectal cancer (CRC) is a leading cause of cancer-associated mortality. Our recent study in Pirc rats revealed that the combination of eicosapentaenoic acid and sodium naproxen exhibited additive tumor protection. While the results from this preclinical study were impressive, showing tumor suppression in excess of 95%, potential mechanisms underlying this tumor protection remain unclear. The purpose of this study was to determine the transcriptomic changes resulting in the tumor suppression afforded by the combination of EPA and naproxen supplementation in the diet. Methods: Six week-old Pirc rats were fed either a control AIN-93G diet, or AIN-93G diet supplemented with 2% EPA combined with 200 ppm naproxen, for a total of 20 weeks. Global transcriptomic analysis was performed using RNA sequencing (RNA-Seq) of colon tumors and tumor-adjacent normal tissue from 26-week-old Pirc rats. 100-bp paired-end sequencing was performed on an Illumina NovaSeq to a depth of 25 million reads per sample. Reads were trimmed with Trimmomatic-v0.39, and reads were mapped to the mRatBN7.2.105 rat reference genome using HISAT2. Differentially expressed genes were calculated using DESeq2, and gene set enrichment analysis (GSEA) were performed using Rstudio. Results: DESeq2 identified 809 differentially expressed genes (DEGs, 2-fold change > 1, FDR < 0.05) between large tumors from AIN-93G fed rats versus suppressed tumors from rats fed the combination of EPA and naproxen (490 up-regulated, 319 down-regulated). GSEA analysis revealed activation of inflammatory pathways including IL-17 signaling, JAK-STAT signaling, complement activation, cytokine signaling, ribosome biogenesis, and cell cycle were enriched in untreated control. In contrast, suppressed tumors from animals supplemented with EPA and naproxen were enriched in pathways including negative regulation of Wnt signaling, cell fate specification, ion channel complexes, and nutrient absorption. Furthermore, suppressed tumors shared significant GSEA pathway overlap with normal tissue, leading us to believe the combination of EPA and naproxen may be preventing tumorigenesis in part by maintaining cells in a differentiated state. Conclusion and Future directions: Inflammation plays a key role in tumorigenesis and promoting cancer stem cell niches in tumors. According to our analysis, the combination of EPA and naproxen attenuated cancer-associated inflammation and reduced proliferation in part by augmenting tumor differentiation. Future studies will utilize single-nuclei RNA-Seq (snRNA-Seq) to determine at a single cell resolution the transcriptomic changes of epithelial and stromal populations within the TME. This work was supported by National Cancer Institute task order 75N91019F00132. Citation Format: Ryan Beach, Michael Martinez, Vignesh Gunasekharan, Daniel W. Rosenberg. The combination of EPA and naproxen suppresses colon tumorigenesis via altering the TME, and promoting epithelial maturation in cancerous crypts [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 762.

Abstract 7349: Ligand-receptor signaling analysis and tumor-immune microenvironment deconvolution reveal differential adipose-tumor crosstalk in lean and obese colon cancer patients

Abstract Obesity is a well-defined risk factor for colon cancer (CC). In the current study, we investigated the paracrine influences between tumors and adjacent adipose tissue to understand the interactions between adipose-tumor using transcriptomics data. Stage I-III newly diagnosed CC patients (n = 130) as part of the ColoCare Study at the Huntsman Cancer Institute, University of Utah, and University of Heidelberg, Germany were included in the current study. Fresh-frozen colon tumor tissues and adjacent adipose tissues naïve to neoadjuvant treatment were collected during surgery. RNA sequencing was done using the NovaSeq X. Patients were classified as having a BMI <30 kg∙m−2 (lean) or BMI ≥30 kg∙m−2 (obese). Tumor-adipose signaling was investigated separately for lean versus obese patients. Differentially expressed genes (DEGs, FDRq<0.05 and |log2FC|>2) between paired tumor-adipose samples were identified through DESeq2, respectively for lean and obese participants. DEGs were filtered to obtain exclusively up-regulated genes for 1) tumor tissue in the obese 2) tumor tissue in the lean 3) adipose tissue in the obese 4) adipose tissue in the lean. Ligand-receptor pairs for adipose-adipose signaling (A-A), tumor-tumor signaling (T-T), adipose-tumor signaling (A-T), and tumor-adipose signaling (T-A) in tissues from lean versus obese patients were determined by mapping the exclusively up-regulated genes in the FANTOM 5 database. Single sample Gene Set Enrichment Analysis (ssGSEA) was performed to score each sample based on signatures of interest, i.e. TIMEx immune cell types. The associations of signatures between tumor-adipose were assessed by evaluating Spearman’s correlation coefficients on the enrichment scores of each signature for paired tumor-adipose samples. From RNA-seq, there were 430 protein-coding DEGs (144 up-regulated in the tumor, 286 up-regulated in the adipose) identified exclusively in the obese and; 704 protein-coding DEGs (421 up-regulated in the tumor, 283 up-regulated in the adipose) identified exclusively in the lean patients. In the obese, we observed ligand-receptor-mediated T-T signaling through FGF5-FGFR3 and PYY-NPY4R; T-A signaling through GCG-GCGR; A-A signaling through ICAM1-IL2RA. In the lean, we found ligand-receptor-mediated T-T signaling through ADAM2-ITGA6 and LAMB3-ITGA6; T-A signaling through DLL3-NOTCH2; A-A signaling through DLK1-NOTCH2. In addition, ssGSEA analysis showed different association patterns for the lean versus obese regarding the TIMEx immune cell types. Our results suggest differential cross-talk between adipose and tumor tissue in lean vs. obese patients could influence the progression of CC. The next steps are to validate these interactions by using in vivo preclinical models to interrogate novel therapeutic targets for reducing the impact of obesity on CC. Citation Format: Tengda Lin, Victoria M. Bandera, Elaine M. Glenny, Caroline Himbert, Jennifer Ose, Christy Warby, Olena Aksonova, Alessandro Carpanese, Chris Stubben, David Nix, Kenneth M. Boucher, Peter Schirmacher, Ildiko Strehli, Jolanta Jedrzkiewicz, Courtney L. Scaife, Bartley Pickron, Alexander Brobeil, Martin Schneider, Christoph Kahlert, Erin M. Siegel, Adetunji T. Toriola, David Shibata, Christopher I. Li, Jane C. Figueiredo, Jatin Roper, Biljana Gigic, Stephen D. Hursting, Cornelia M. Ulrich, Aik Choon Tan. Ligand-receptor signaling analysis and tumor-immune microenvironment deconvolution reveal differential adipose-tumor crosstalk in lean and obese colon cancer patients [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 7349.

Abstract 4281: BMP signaling impaired telocytes-Foxl1+disrupt the extracellular matrix network enabling the development of colitis-associated cancer

Abstract The relationship between colon stem cells (SCs), its surrounding niche microenvironment like the extracellular matrix (ECM) and the mesenchymal niche cells is complex and dynamic. ECM proteins are not only a scaffolding system for SCs, but their biophysical properties also contribute to localizing signals and creating gradients leading to the SCs homeostasis. Foxl1+-Telocytes (TCFoxL1+) are mesenchymal niche cells implicated in cell-cell communication and the production of BMP ligands. Upon chronic inflammation, mice lacking BMP signaling in TCFoxL1+ (BmpR1aΔFoxL1+), developed colonic tumors with microenvironment exhibiting abnormal TCFoxL1+ structure, increased BMP expression and myofibroblastic-like cancer-associated fibroblasts (myCAF) subpopulation. Yet, how the TCFoxL1+ influences SCs niche ECM proteins during tumorigenesis is less understood. We aimed to investigate how the loss of BMP signaling in TCFoxL1+ affects ECM network impacting the niche microenvironment promoting the development of CAC. DSS-based chronic inflammatory challenge in mutant and control mice, quantitative-MS-based strategy was performed to determine the proteome of the transformed regions solely in epithelial-mesenchymal tissue following colon deconstruction. Expression of the proteins of interest were validated by Western Blots or immunostainings. Quantitative analysis of collagen network was assessed using phyton pipeline U-net in Sirius red-stained colon analyzed under polarized light. Differential significant expression of 798 proteins, with 342 upregulated and 456 downregulated, were found in the tumors of mutant mice compared to controls. Mfap5 involved in CAF activation was the highest upregulated protein. We found upregulated, laminin subunits α4 and β2, decorin, lumican as well as transgelin (myCAF marker), which are proteins known for contributing to the stiffening of the ECM during colorectal cancer. Adamdec1 which prevents aberrant ECM accumulation and thrombospodin-1, an ECM glycoprotein whose low levels have been related to tumor growth, were both found to be downregulated. Data extracted from Python's U-Net pipeline demonstrated that tumors, in mutant mice, exhibited higher frequencies of straighter collagen fibers compared to wavier fibers in controls. Collagen fiber distribution was shown to present an even angle distribution in controls whereas BmpR1aΔFoxL1+ mice presented a preferential orientation. These results suggest that defective TCFoxL1+ commits the surrounding niche microenvironment, especially its ECM, toward the development and progression of CAC. Citation Format: Vilcy Reyes Nicolas, Alain B. Alfonso, Jennifer Raisch, François M. Boisvert, Marc-Antoine Lauzon, Nathalie Perreault. BMP signaling impaired telocytes-Foxl1+disrupt the extracellular matrix network enabling the development of colitis-associated 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 4281.

Abstract 4360: Integrative analysis of multiplex interphase FISH copy number with whole exome sequencing identifies tumor evolution in Stage II colon cancer

Abstract Colon cancer ranks as the second leading cause of cancer-related death worldwide. Although standard therapies are highly effective in stage II lymph-node negative colon cancers, about 10-15% of these patients show disease relapse within the next 5-year period after intended curative surgery. Extensive prior research has highlighted the role of intratumor heterogeneity, arising from genomic instability, as a leading contributor to metastasis in colon cancer. In the current study, we pioneered the integration of Multiplex Interphase FISH (miFISH) and Whole Exome Sequencing (WES) data aiming to depict intratumor heterogeneity and delineate the landscape of genetic aberrations that underlie the early metastatic potential in stage II colon cancer patients. To this aim, we have showcased the genome-wide copy-number alterations and single nucleotide mutations derived from WES analysis of samples obtained from primary stage II colon tumors and their patient-matched liver metastases of nine patients using multi-region sampling. miFISH was employed for the concurrent quantification of copy-numbers for nine genes relevant to colon cancer and a centromeric control probe in intact tumor nuclei derived from archival patient material allowing us to establish ploidy baselines and gain and loss patterns for the genes analyzed. Our study was revealing of several copy number alterations, such as gains of chromosomes 1q, 7, 8q, 13q and 20, and losses affecting chromosomes 17p and 18, which were confirmed by miFISH. Based on our SNV data, most frequently mutated genes were APC, TP53, KRAS. We were able to show several mutations in genes belonging to specific pathways most commonly altered in colorectal cancer, such as APC, TCF7L2, AXIN2, FBXW7 in WNT signaling, SMAD4 in TGF-ß signaling, PIK3CA and PTEN in PI3K signaling and KRAS in RTK-RAS signaling pathways. In nearly all cases, subclones that led to metastasis were already present in the primary cancer, indicating a direct progression from the primary to metastatic disease. However, the divergence from the primary tumor to metastasis enabled us to uncover several potential genetic aberrations present in the metastasis but not observed in the primary tumor. Moreover, in one of our cases, despite harboring APC and TP53 mutations in both the primary and metastatic tumor, unique mutations for these genes along with several distinct mutations in the metastasis were identified. These observations suggest that the metastasis might not be related to the primary tumor analyzed and may have arisen from an independent lesion. In conclusion, applying miFISH and WES to decipher the mutational and copy number landscape in metastatic stage II colon cancer provided comprehensive insights into the complex and heterogenous nature of primary colon cancer and the clonal evolution leading to subsequent liver metastases. Citation Format: Sanam Yaghoubi, Kerstin Heselmeyer-Haddad, Ivan Archilla, Carolina Parra, Darawalee Wangsa, Giancarlo Castellano, Jack Zhu, Diba Yaghoubi, Sara Lahoz, Veronica Pablo-Fontecha3, Daniela Hirsch, Wei-Dong Chen, Thomas Ried, Miriam Cuatrecasas, Paul S. Meltzer, Jordi Camps. Integrative analysis of multiplex interphase FISH copy number with whole exome sequencing identifies tumor evolution in Stage II colon 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 4360.

Abstract 7299: Preventing CRC progression in FAP rat model using a combinational targeting of TRAIL signaling and inflammation

Abstract Colorectal Cancer (CRC) is among the leading causes of mortality globally, with high incidence rates in developed countries including the US. While FAP patients are prone to develop CRC in their lifetime, recent rising trends among young adults is also a major concern. Hence, preventing CRC would aid in reducing cancer mortality, especially in high-risk FAP patients for which there are no FDA approved preventive agents. Here, we evaluated TRAIL-inducing, orally active small molecule ONC201, alone and in combination with the NSAID naproxen (NAP) for CRC prevention in the PIRC rat model representing FAP cohort. Male PIRC rats were bred inhouse and randomized by age into placebo and intervention groups (N=15). Starting at 6 weeks of age, experimental groups were administered either ONC201 alone by gavage [25mg/kg; 2X/week or 50mg/kg; 1X/week]; NAP in AIN-76A diet [200 ppm; continuously or 1 week ON/OFF]; or a combination of both agents, while placebo group received vehicle only. After 26 weeks of treatment, rats were euthanized and intestines were evaluated for tumor incidence and multiplicity. H&E stained colonic tumor sections were histologically classified as hyperplastic polyps, adenomas (AD), and adenocarcinomas (ADCA). Gross tumor data indicated significant reduction in colonic polyps in both the NAP alone and NAP+ONC201 groups without any overt-toxicites. Histological analyses also demonstrated strong suppression of AD progression to ADCA as well as a significant decrease in the number of ADs and ADCAs (p<0.05) in the treatment groups compared to the placebo. ONC201 regimens modestly reduced AD (19%-27% less, p<0.05) but showed strong inhibition of ADCA (45%-59% less, p<0.0001). A significant reduction of AD and ADCA was seen in NAP continuous (54% & 87% less respectively; p<0.0001) and intermittent (34% & 68% less respectively; p<0.0001) dosing regimens. As anticipated, the NAP continuous treatment had better efficacy than the intermittent dosing regimen. Notably, the combination treatment showed >90% (p<0.0001) inhibition of ADCA and 40%-57% (p<0.0001) inhibition of ADs. Importantly, ONC201+ NAP treatment also resulted in an additional 45% - 70% reduction of ADCA (p<0.0001) when compared to the NAP alone treatment. Thus, our data showed that the combination groups with NAP intermittent dosing demonstrated ~90% inhibition of ADCA, suggesting the synergistic effect of the two agents in preventing CRC progression. Molecular analysis of the rat tumors and additional analyses in in-vitro tumoroid models suggested induction of TRAIL (TRAIL, DR5, FADD), increased apoptosis (Caspases), and a decrease in proliferation markers (PCNA, Cyclin D1) and pro-inflammatory cytokines (IL1b, IL13 etc.). Our data suggests NAP/ONC201 combo has no toxicities, warranting further investigation of CRC interception in high-risk FAP patients (Funding by NCI-PREVENT 75N91019D00020-75N91020F00004). Citation Format: Venkateshwar Madka, Gopal Pathuri, Karthikkumar Venkatachalam, Surya P. Singh, Anil Singh, Nicole Stratton, Stanley Lightfoot, Shizuko Sei, Mark S. Miller, Chinthalapally V. Rao. Preventing CRC progression in FAP rat model using a combinational targeting of TRAIL signaling and inflammation [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 7299.

Abstract 2496: Molecular characterization of stage III colon cancer patients with recurrence after adjuvant chemotherapy

Abstract Introduction: Patients with stage III colon cancer are routinely treated with resection followed by adjuvant chemotherapy (ACT) with a fluoropyrimidine and oxaliplatin. This one-size-fits-all approach does not account for heterogeneity in tumor biology and fails to cure ~30% of patients, highlighting the urgency for biomarkers to better understand and predict resistance to ACT or to offer alternative targeted treatment. Aim: To identify molecular characteristics predictive of resistance to standard ACT in stage III colon cancer. Methods: Patients with resected stage III colon cancer who received ACT (93% CAPOX, 7% CAP) were selected from the Prospective Dutch Colorectal Cancer cohort (PLCRC). RNA exome capture sequencing was performed on resected FFPE tumor tissue to compare differential gene expression, hallmark pathway enrichment, immune cell deconvolution and consensus molecular subtypes (CMS) between patients with versus without recurrence after surgery and ACT. Postsurgery circulating tumor DNA (ctDNA) detected in PLCRC-PROVENC3 (separate abstract) was used to select a subgroup with minimal residual disease (MRD) postsurgery, as sensitivity analysis of patients with recurrence despite ACT (chemoresistant) versus curation by ACT (chemosensitive). Results: For 264 out of 278 patients (95%), tumor-derived RNA sequencing libraries were of sufficient quality. 67 patients (25%) with recurrence (median after 13 months [IQR 7-23]) were compared to 178 patients with at least 2 year recurrence-free follow-up (median 42 months [IQR 30-53]). A total of 320 genes were differentially expressed between patients with versus without recurrence. Genesets defining epithelial-mesenchymal transition (p = 0.002) and angiogenesis (p < 0.001) were enriched in the primary tumor of patients with recurrence, in part explained by the estimated larger fraction of stroma (Wilcoxon effect size r 0.18, p < 0.001) and cancer-associated fibroblasts (CAFs) (r 0.19, p = 0.003). In contrast, the fraction of CD4+ T cells was lower (r 0.14, p = 0.001). Consistently, CMS4 was found more frequently in tumors from patients with versus without recurrence (57% versus 34%, p < 0.001). Of the subgroup with ctDNA-based MRD postsurgery (26 out of 171 assessed patients), 19 patients (73%) with recurrence (median after 12 months [IQR 7-17]) were compared to 7 patients without recurrence (minimum follow-up 32 months). This sensitivity analysis showed similar trends in pathways, immune cells and CMS as the main analysis, with a more pronounced effect size for CAFs (r 0.27). Conclusion: Stage III colon tumors of patients who experience recurrence after resection and ACT are enriched for stroma and CAFs, while displaying less CD4+ T cells. The observed trends are sustained in the patients with MRD postsurgery. These findings may help to better predict resistance to standard ACT and elucidate targets for alternative personalized treatment. Citation Format: Ingrid Franken, Steven Ketelaars, Carmen Rubio-Alarcón, Sietske van Nassau, Suzanna Schraa, Gerrit Meijer, Mark Sausen, Miriam Koopman, Geraldine Vink, Sanne Abeln, Remond Fijneman, Jeanine Roodhart, on behalf of the PLCRC-MEDOCC group. Molecular characterization of stage III colon cancer patients with recurrence after adjuvant chemotherapy [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 2496.

Abstract 5635: TPM4 overexpression promotes colon epithelial cell tumorigenesis in vitro and correlates with human colon tumor progression in vivo

Abstract Because colon cancer still exerts a heavy burden on human health, we are interested in identifying very early events in colon epithelial cell tumorigenesis in order to develop strategies to treat and possibly prevent colon carcinogenesis. We hypothesize that aberrant expression of genes involved in crypt-luminal colon epithelial cell migration, needed for proliferative, undifferentiated stem cells at the crypt bottom to mature into growth arrested, mature effector cells near the lumen, may disrupt normal differentiation and initiate events that can trigger the transition from a normal to premalignant state in colon epithelial cells. Forced upregulation of tropomyosin 4 (TPM4), an actin-binding protein that exerts many effects on contractility and migration, prevents certain aspects of Caco2 maturation induced by contact inhibition of growth, including increased expression of the digestive enzyme sucrose isomaltase and glutathione S-transferase alpha 1. Furthermore, TPM4.2-overexpressing cells exhibit numerous morphological abnormalities. Compared to untransfected Caco2 cells and empty-vector stable transfectant controls, TPM4.2-overexpressing Caco2 cell clones elaborate aberrant thin membrane protrusions and expanded membrane ruffles. Particularly striking are defective maintenance of dome structures at middle (day 14) and late (day 21) time points of induced differentiation in TPM4.2-overeexpressing clones. We further hypothesize that TPM4 is overexpressed in human colon tumors, particularly at early stages of tumorigenesis. In order to test this hypothesis, we examined TPM4 RNA expression in datasets obtained from human colon tissue at premalignant as well as tumorigenic stages. TPM4 trends toward overexpression in premalignant, relative to normal, human colon tissue (uninvolved mucosa from patients with familial adenomatous polyposis, or FAP) and is significantly overexpressed in adenocarcinoma tissue relative to normal controls. Interestingly, TPM4 overexpression may be more pronounced at earlier, rather than later, stages of adenocarcinoma. Our results suggest that aberrant TPM4 overexpression in colon epithelial cells can promote adoption of a tumorigenic state. Certain aspects of Caco2 cell maturation are disrupted by forced TPM4 overexpression during induced differentiation, and TPM4 overexpression correlates with early stages of colon tumorigenesis. Misregulation of TPM4 gene expression may therefore represent a critical molecular event that can block normal differentiation and induce early events in colon epithelial cell tumorigenesis. TPM4 expression level may therefore prove to be a useful biomarker that can, along with other diagnostics, help to determine tumorigenic progression in human colon tissue and to develop novel strategies to prevent colon cancer and treat it at early stages. Citation Format: Giulio Ferrero, Barbara Pardini, Alessio Naccarati, Michael Papetti. TPM4 overexpression promotes colon epithelial cell tumorigenesis in vitro and correlates with human colon tumor progression in vivo [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 5635.