Tumor Phenotype Research Articles

Abstract 4367: PTEN/AKT signal promotes cholangiocyte fate in liver tumorigenesis by inducing SOX9 overexpression and crosstalk with Notch activation

Abstract Cholangiocarcinoma (CCA) is the second most dominant primary liver malignancy next to hepatocellular carcinoma (HCC), and among the most mortal among human cancers. The PI3K/AKT signaling pathway was considered a permissive signal for the development of CCA. To explore how the PI3K/AKT signal contributes to CCA development, we deleted Pten, the lipid phosphatase that negatively regulates the PI3K/AKT signal in the liver. The Lv-PTEN (PtenloxP/loxP; Alb-Cre+) mice developed a mixed HCC and CCA tumor phenotype with all mice developing CCA by 12-month age. Treatment of Lv-PTEN mice with DDC at 3 months to obstruct the bile duct leads to an earlier CCA oncogenesis. Deletion of Akt2 in the Lv-PTEN mice (Lv-DM, Akt2loxP/loxP; PtenloxP/loxP; Alb-Cre+) significantly attenuates tumor development. Compared with the Lv-PTEN mice, loss of AKT2 in Lv-DM mice robustly and significantly reduced the expression of SOX9, a cholangiocyte gene. This data suggests that the AKT signal may permit a cholangiocyte fate by inducing SOX9 expression. Supporting the role of PTEN/AKT in permitting a cholangiocyte fate, SOX9 expression is also induced in the livers of mice where Pten deletion is targeted to the hepatocytes via injection of AAV8-TGB-Cre (Hp-PTEN, PtenloxP/loxP; R26RYFP; AAV8-TBG-Cre). Similar to the Lv-PTEN mice, treatment with DDC induced early onset of CCA development in the Hp-PTEN mice. We next explore Notch signal for its crosstalk with PTEN loss that permits CCA development. Notch signal is robustly induced in the tumors of the Lv-PTEN mice and induced with DDC treatment in both Lv-PTEN and Hp-PTEN livers. We showed that exposure to Jag1 ligand-coated extracellular matrix or expression of NICD also induced the expression of SOX9 while DAPT time-dependently attenuated the expression of SOX9. In the Hp-Pten mice treated with DDC, inhibiting the Notch pathway with DAPT attenuated ductal reaction and led to downregulation of SOX9 in the Lv-PTEN livers, suggesting a positive regulatory role of Notch on SOX9. These data support that Notch activation regulates SOX9 and collaborates with PTEN loss to drive cholangiocyte fate and CCA development. Citation Format: Qi Tang, Jingyu Chen, Ni Zeng, Lina He, Shefali Chopra, Diala Alhousari, Phillip Nguyen, Guo Zhang, Bangyan L. Stiles. PTEN/AKT signal promotes cholangiocyte fate in liver tumorigenesis by inducing SOX9 overexpression and crosstalk with Notch activation [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 4367.

Abstract 3733: Gut microbiota-dependent bile acidtaurodeoxycholic acid, TDCAfacilitates anti-tumor immune responses by modulating cytotoxic CD8 T cell immunity

Abstract Gut microbiota are critical for effective immune checkpoint blockade therapy (ICT) for cancer. However, the underlying mechanisms by which gut microbiota and/or microbiota-derived metabolites augment antitumor response are largely unknown. Here, we utilized a preclinical melanoma (B16-F10) model and treated groups of mice (Jackson, C57BL/6J, female, 6-8 wks, n=14-26 per group) with seven distinct antibiotic treatments to induce unique gut microbiomes and metabolomes. Mice were then treated with ICT (anti-PD-1 and anti-CTLA-4), resulting in markedly heterogeneous tumor phenotypes (tumor volume and weight) as dictated by the specific antibiotic treatment. Using machine learning (e.g. random forest classifier), we identified metabolite candidates most strongly associated with ICT response (i.e. tumor volume and weight).One metabolite candidate, the bile acid taurodeoxycholic acid, TDCA, had previously been identified by my group as associated with anti-PD-1/anti-CTLA-4 response in human melanoma patients (PMID 28923537). Administration of TDCA alone is sufficient to overcome antibiotic-induced ICT hyporesponsiveness in mice. Utilizing both in vitro/ex vivo immune cell assays and in vivo preclinical models, we have validated that TDCA treatment directly boosted the antitumor cytotoxic CD8+ T cell responses (i.e. IFN-gamma production, anti-tumor cytotoxicity, tumor growth, etc.) without affecting innate immune responses. Bulk RNAseq of CD8 T cell analysis indicated that TDCA modulate CD8 T cell function through IFN pathway. Together, our findings suggest that the gut microbial metabolite TDCA could promote immunotherapy efficacy through regulation of CD8+ T cell immunity and has the potential to be utilized as a novel therapeutic agent to enhance ICT efficacy. Citation Format: Wenng li, Hesuiyuan Wang, Laura Coughlin, Nicole Poulides, Priscilla Del Valle, Jake Lichterman, Xiaowei Zhan, Chen Liao, Suzette N. Palmer, Xin Li, Andrew Y. Koh. Gut microbiota-dependent bile acidtaurodeoxycholic acid, TDCAfacilitates anti-tumor immune responses by modulating cytotoxic CD8 T cell immunity [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 3733.

Abstract 169: Prevalence, immune checkpoint expression and spatial interplay of intraepithelial lymphocytes, macrophages, and dendritic cells in 4740 human carcinomas

Abstract Background: Although there is raising evidence that the density and the level of immune checkpoint expression of immune cell subpopulations that are in direct contact to the tumor cells (intraepithelial) can predict response to immune checkpoint therapy and patient’s outcome, a comprehensive assessment of intraepithelial immune cell subpopulations and their spatial interplay is lacking. Material and methods: To comprehensively assess the density, level of immune checkpoint expression, and spatial interplay of 48 immune checkpoint expressing intraepithelial leukocyte subpopulations in 46 carcinoma entities, 4740 tumor samples in a tissue microarray format were stained with 21 antibodies using our BLEACH&STAIN mfIHC approach. A deep learning-based framework comprising 18 different convolutional neuronal networks (U-Net and DeepLabv3+) were used for image analysis. Results: The mean intraepithelial immune cell density of CD8+ cytotoxic T-cells, CD4+ T-helper cells, FOXP3+ Tregs, CD20+ B-cells, M1/M2 macrophages and CD11c+ dendritic cells varied markedly between tumor entities and individual tumors. For example, 88 (±90) cells/mm2 were found in tubular breast cancer, 661 (±729) cells/mm2 in colorectal cancer, and 1280 (±1334) to 2325 (±2131) cells/mm2 in squamous cell cancers from various origins. The abundance of T-cell subpopulations, M2 macrophages, and dendritic cells was highly concordant (range r=0.49 to 0.92) across all tumor entities, while the density of M1 macrophages was unrelated (r=0.1, p<0.001). Unsupervised hierarchical clustering across all tumor entities revealed a cluster A of 634 patients from almost all different tumor entities with an exceptionally high density of intraepithelial immune cells, thus a highly inflamed tumor microenvironment that was characterized by the strongest interaction between intraepithelial CD8+ T-cell and CD4+ T-helper cells, M2 macrophages, as well as dendritic cells (DC, p<0.002 each) along with the highest expression level of TIM3, PD-1, CLTA-4 and PD-L1. In contrast, a cluster C of 2245 patients with the lowest intraepithelial immune cell infiltration was characterized by a significantly increased M1 proportion (p<0.001) and an enriched proportion of regulatory T-cells (p<0.001). Across all analyzed tumor entities, the intraepithelial highly inflamed cluster A was significantly linked to low pT (p<0.001). Conclusion: The data from this study provide a comprehensive characterization of intraepithelial immune cells across 46 different human carcinomas and identify an inflamed phenotype defined by strong interactions of intraepithelial CD8+ cytotoxic T-cells, CD4+ T-cells, dendritic cells, and M2 macrophages along with highest levels of TIM3, PD-1, PD-L1, and CTLA-4 expression that is linked to a favorable tumor phenotype. Citation Format: Zhihao Huang, Tim Mandelkow, Jan H. Müller, Andreas H. Marx, Till Krech, Guido Sauter, Julia Ebner, Maximilian Lennartz, Ronald Simon, Martina Kluth, Claudia Hube-Magg, Sarah Minner, Elena Bady, Niclas C. Blessin. Prevalence, immune checkpoint expression and spatial interplay of intraepithelial lymphocytes, macrophages, and dendritic cells in 4740 human carcinomas [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 169.

Abstract 3231: Pharmacological inhibition of the m6A RNA reader, YTHDC1, as a novel approach to targeting biomolecular condensates in cancer

Abstract Epigenetic dysregulation is a hallmark of cancer, and tumors have evolved complex mechanisms to fine-tune oncogenic gene expression. In acute myeloid leukemia (AML), one such regulatory mechanism involves post transcriptional modification of RNAs. The most abundant post transcriptional modification is methylation of adenosine at the N6 position (m6A). This modification is catalyzed by the m6A “writer”, METTL3 and METTL14 complex, and is removed by “erasers” such as FTO and ALKBH5. m6A RNAs can regulate a variety of cellular processes which can impact tumor phenotypes, including RNA stability, splicing, and nuclear export; and METTL3 catalytic inhibitors are currently in clinical development for AML and other cancers (NCT05584111).The m6A “reader”, YTHDC1, has been recognized as a critical contributor to the proliferation and survival of AML cells. As the primary nuclear m6A reader, YTHDC1 localizes with m6A marked RNAs in phase-separated assemblies, termed biomolecular condensates, helping to maintain the stability of oncogenic transcripts such as MYC. Here, we have employed our proprietary microfluidics based screening platform (termed PhaseScanTM) to identify modulators of YTHDC1 biomolecular condensates. Evaluation of early screening hits helped identify that disruption of the interaction between YTHDC1 and m6A RNA is a functionally relevant mechanism of action (MoA) that results in dissolution of YTHDC1 condensates. Using computational and knowledge based approaches, we have identified other structurally distinct inhibitors that occupy the RNA binding pocket of YTHDC1 and disrupt its interaction with m6A RNA at nanomolar potencies. These compounds disrupt cellular YTHDC1 condensates, suppress oncogenic gene expression, and result in anti proliferative and pro-differentiation phenotypes in AML cell lines. Compounds are selective for YTHDC1 over other YTH family members and are also selective in dissolving YTHDC1 condensates over other nuclear and cytosolic condensate systems. Our optimized chemical matter has suitable ADME properties to support in vivo studies. Thus, targeting YTHDC1 biomolecular condensates represents a novel approach to the treatment of AML. Citation Format: Richard C. Centore, Mark Charles, Mansi Arora, Yiwen Chen, Matthew Watson, Charlotte Kelley, Magdalena Czekalska, Marius Rebmann, Mahmoud Ghandi, Jerome Cattin, Nagakumar Bharatham, Prathima Radhakrishnan, Alex Howarth, Gale Rudlaff, William E. Arter, Seema Qamar, Kadi Saar, Douglas Williamson, Andrew Seeber, Tuomas Knowles, Shilpi Arora. Pharmacological inhibition of the m6A RNA reader, YTHDC1, as a novel approach to targeting biomolecular condensates in 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 3231.

Abstract 6121: Racial differences in the intratumoral immune cell infiltrate in luminal breast cancer

Abstract Background: Black women have a higher rate of breast cancer mortality compared with their non-Hispanic White (NHW) counterparts, with an 80% higher likelihood of breast cancer death from axillary node-negative, ER-positive (a.k.a. luminal) tumors (Hoskins et al, JAMA Oncol 2021). Black women are 30% more likely to have ER-positive tumors with a high-risk gene expression profile, indicating a role for tumor biology in this disparity (Hoskins et al, JAMA Oncol 2023). The underlying biological mechanisms responsible are unknown. The aim of this study was to investigate the immune cell infiltrate in ER-positive breast tumors as a potential driver of disproportionately aggressive luminal tumor biology among Black women. Methods: Gene expression data from ER-positive tumors diagnosed in women identified as Black or NHW in The Cancer Genome Atlas (TCGA) were deconvoluted with the CIBERSORT tool to estimate the relative intratumoral proportion of 22 immune cell types within each tumor. Differential analysis of imputed immune cell fractions comparing race was conducted with the Mann-Whitney test. P-values were adjusted for multiple comparisons with the false discovery rate (FDR) method. Linear regression of previously reported ancestry estimates for breast cancer patients in TCGA (Huo, et al, JAMA Oncol 2017) was used to test for association between African ancestry and proportion of regulatory T cells (Tregs) in tumors from Black women. Results: CIBERSORT analysis of tumors found a significantly increased proportion of Tregs in tumors from Black (n=86) than NHW (n=534) patients, with log2 fold-change= 0.74 (FDR-corrected p-value= 0.01). Plasma cells, T follicular helper cells and M0 macrophages were increased in tumors from Black women with uncorrected p-values < 0.05, but racial differences in these cell types were not significant after FDR correction (p >0.1). Ancestry analysis among Black women indicated that African ancestry was not associated with the intratumoral proportion of Tregs (1 SD change in proportion African ancestry was associated with 0.06 standard deviation increase in Tregs, p > 0.3). Confirmation of findings with a racially diverse breast tumor TMA from an independent cohort is ongoing and results will be presented. Conclusions: Increased pro-tumorigenic Treg infiltrate in ER-positive breast tumors from Black women may contribute to a disproportionately aggressive tumor phenotype and racial survival disparity. The findings could have therapeutic implications and suggest that immune checkpoint inhibitors may have increased activity against luminal breast cancer in Black patients. Although this study did not show a statistically significant effect for genetic admixture within African Americans, larger studies are needed to distinguish the contribution of ancestry vs. social context, environmental, and behavioral factors as the driver of this racial difference in tumor biology. Citation Format: Neha Hippalgaonkar, George Chlipala, Dezheng Huo, Garth Rauscher, Naiche Adler, Jan Kitajewski, Kent Hoskins. Racial differences in the intratumoral immune cell infiltrate in luminal breast 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 6121.

Abstract 1458: Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness

Abstract Pancreatic ductal adenocarcinoma (PDAC) represents a very aggressive type of pancreatic cancer (9% survival rate at 5 years). PDX1 is an important transcription factor for embryonic development of the pancreas, endocrine lineage differentiation and the maintenance of mature beta cells. Notably, in PDAC patients, the expression of PDX1 is downregulated in tumor cells compared to adjacent non-tumoral tissue. Interestingly, increasing tumor PDX1 expression has been shown to enhance the overall survival rate in PDAC patients. Therefore, our aim was to analyze the role of PDX1 on tumor aggressiveness of PDAC cells. To induce PDX1 expression, PANC1 cells were treated with BRD7552 (a PDX1 inducer) for 9 days. Overexpression of PDX1 was confirmed by Western Blot analysis and no cytotoxic effects were observed by MTT or Trypan Blue exclusion assays on treated cells compared to control. Wound healing and transwell migration assays showed a significant reduction in the migration rate compared to the control group. A higher proportion of cells in G1 phase was observed in treated cells compared to control by propidium iodide staining followed by flow cytometry assay. Furthermore, the cell confluence assay showed a significant reduction in proliferation rate in treated cells compared to control, but no differences were observed on the proportion of KI67+ and PH3+ cells by immunostaining. To assess the in ovo effects of BRD7552, treated PANC1 cells were implanted onto the chorioallantoic membrane of chick embryos and tumor growth was measured at different stages observing a significant reduction in tumor growth in PANC1 cells compared to control between days 3 and 8 post implantation. No significant differences in morphology or color were observed between treated and control implanted cells. Hematoxylin-eosin staining showed reduced invasion of treated cells into the chorioallantoic membrane compared to control. In conclusion, the overexpression of PDX1 affects cell cycle, reduces proliferation rate and inhibits migratory potential in vitro. Moreover, in an in ovo model this overexpression diminished tumor growth and invasion in PANC1 cells. In conclusion, the overexpression of PDX1 induced by BRD7552 drives pancreatic ductal adenocarcinoma to a less aggressive tumoral phenotype. Citation Format: Jimena Mosna, Federico Garde, Marcelo Stinson, Candela Pastore, Pablo Sanchis, Abel Carcagno. Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness [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 1458.

Abstract 1587: Multimodal spatial transcriptomics uncover distinct tumor microenvironment states and cell-cell communication networks in molecular pancreatic cancer subtypes

Abstract The tumor microenvironment (TME) presents a complex ecosystem comprising a diversity of immune and stromal cell populations that influence tumor progression, drug delivery and therapy outcome. High levels of inter- and intra-tumor heterogeneity in TME state are driven by molecular tumor phenotypes. Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancer types, displays a high genetic heterogeneity along with an immunosuppressive TME. However, it remains largely elusive how distinct TME states are mechanistically influenced, and which molecular processes dictate the emergence of different modes of immunosuppression within the TME of molecular PDAC subtypes.Here, we performed a systematic analysis of functional associations between the major molecular PDAC subtypes, the classical and mesenchymal subtype, and their TME states. We describe the TME composition and complex cell-cell communication networks within PDAC subtypes using a multimodal integration of spatial transcriptomics (ST) with complementary approaches, such as scRNA-seq, MS-based Secretomics and multiplexed histocytometry. We generated ST data sets (Visium) of a tumor cohort derived from a Kras-driven PDAC mouse model. To analyze TME composition as well as spatial niches and cell type communities, we computationally enhanced the spot-resolution of ST datasets, followed by cell type deconvolution and cell-cell communication analysis to identify subtype-specific TME communities and communication networks.To this end, we generated a large resource of PDAC mouse models which represent molecular subtypes of the disease and mimic the heterogeneity of TME states found in human PDAC cohorts. This analysis revealed that a set of subtype-specific secreted factors shape the immunosuppressive PDAC TME via direct and indirect communication networks with immunosuppressive myeloid and T cells in molecular PDAC subtypes. Multimodal ST analysis delineated spatial subtype-specific TME communities as well as spatial communication patterns. We functionally investigate the tumor cell intrinsic regulation of the identified subtype-specific secreted factors, providing potential therapeutic vulnerabilities for more effective combinatorial subtype-specific therapies including immunotherapeutic approaches. Citation Format: Stefanie Baerthel, Chiara Falcomatà, Vanessa Goelling, Daniele Lucarelli, Rushin Gindra, Constantin Schmitt, Jonathan Swietlik, Felix Meissner, Marc Schmidt-Supprian, Dieter Saur. Multimodal spatial transcriptomics uncover distinct tumor microenvironment states and cell-cell communication networks in molecular pancreatic cancer subtypes [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 1587.

Abstract 6991: Transcriptomic profiling of human and mouse models highlights YAP pathway hyperactivation as the underlying mechanism of the biological and clinical aggressiveness of Numb-defective bladder cancer

Abstract In this study, we identify a previously uncharacterized role for the tumor suppressor, Numb, in bladder tumorigenesis, defining new predictive biomarkers and potentially actionable targets for the treatment of non-muscle-invasive bladder cancers. We conducted a comprehensive IHC analysis of Numb expression leveraging two retrospective longitudinal cohorts comprising 356 post-cystectomy muscle-invasive bladder cancer (MIBC) patients and 77 high-grade non-muscle-invasive (NMIBC) patients with transurethral resections. We found that Numb expression is frequently deregulated in the tumor compared to the adjacent normal urothelium, with NumbLow status correlating with poorer prognosis both in MIBC, associated with advanced stage at diagnosis, vascular invasion, and worse overall survival, and in NMIBC, where a NumbLow status associates with a higher rate of progression to life-threatening MIBC disease. In a transgenic mouse model with targeted deletion of the Numb gene in the basal layer of the bladder mucosa, we observed that loss of Numb per se is sufficient to drive malignant transformation of the normal urothelium and accelerates tumorigenesis in carcinogen-induced models. Integrative transcriptomic analysis of human and mouse Numb-deficient BC models revealed consistent dysregulation of the YAP1/TAZ1 inhibitory Hippo pathway, identifying YAP1 transcriptional hyperactivation as the most distinguishing trait downstream of NUMB loss. Through genetic and pharmacological functional inhibition studies in relevant mouse and human BC cell line-based models, we provide proof-of-evidence that interfering with YAP pathway activation at multiple upstream levels of the Hippo cascade reverts the aggressive Numb loss-associated tumor phenotypes, in particular growth and invasive/migratory potential, thereby highlighting the YAP pathway as a potential therapeutical vulnerability for the treatment of clinically aggressive Numb-deficient human BC. We also identified a gene signature of early BC depending on loss of Numb that we extensively validated for its potential as a predictor of NMIBC to MIBC progression, arguing for its clinical application as a biomarker for NIMBC patient stratification for targeted anti-YAP signaling treatment to prevent MIBC transition. In summary, our study identifies a previously uncharacterized role of Numb in bladder cancer progression, highlighting its potential as a predictive biomarker of NMIBC to MIBC transition and response to targeted therapies, thereby advancing a framework for precision oncology for NMIBC patients at high risk of progression to life-threatening MIBC disease. Citation Format: Francesco Antonio Tucci, Rosa Pennisi, Maria Grazia Filippone, Roberta Bonfanti, Francesco Romeo, Damiano Rigiracciolo, Stefano Freddi, Francesca Sanguedolce, Giuseppe Renne, Giancarlo Pruneri, Gianluca Vago, Daniela Tosoni, Salvatore Pece. Transcriptomic profiling of human and mouse models highlights YAP pathway hyperactivation as the underlying mechanism of the biological and clinical aggressiveness of Numb-defective bladder 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 6991.

Abstract 2472: Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse models

Abstract Background: ENPP1, a nucleotide pyrophosphatase and phosphodiesterase, has emerged as an important modulator of anti-tumor immune activation, as it regulates the concentrations of cGAMP and ATP in the TME. ENPP1 expression in tumors is common and correlates with a cold tumor phenotype. Its loss has been reported to suppress metastasis, restore immune cell infiltration into the tumor and potentiate the response to immune checkpoint blockade, while, conversely, the overexpression of ENPP1 promoted metastasis and rendered tumors resistant to checkpoint blockade. RBS2418 was designed as a potent and selective inhibitor of ENPP1 and is in clinical development. Methods: ENPP1 enzyme inhibition was measured in vitro using cGAMP and ATP substrates. Effects of pH and serum and selectivity against related enzymes were evaluated. In vivo efficacy was assessed in Hepa1-6, and GL261-luc syngeneic liver and glioblastoma mouse models. Compound exposures were determined in tumor and blood samples. Cures were confirmed by re-challenge of cured animals with the same cancer cells. Results: RBS2418 inhibited hydrolysis of cGAMP and ATP in vitro with similar potency (Ki =0.14 and 0.13 nM). Inhibition was also similar in human serum, consistent with low binding affinity to human serum albumin. In humans, oral dosing at doses of 100 mg could inhibit ENPP1 enzyme activity during the full dosing interval. Dosing of RBS2418 was performed in mice to achieve trough concentrations of RBS2418 in tumors and plasma exceeding ENPP1 EC90 levels for the complete dosing interval. In the Hepa1-6 model treatment with RBS2418 for either 2 or 10 days resulted in significant reduction of tumor growth and prolongation of survival as compared to vehicle control. There was no significant difference between the RBS2418 groups. Complete tumor regression was observed in 44% of treated mice across the two treatment groups. These mice were resistant to rechallenge with Hepa1-6 tumor cells. In the GL261-luc model treatment with RBS2418 showed significant reduction of tumor burden and prolongation of survival as compared to vehicle control. The mice that achieved complete tumor regression were resistant to rechallenge with GL261-luc tumor cells. Conclusions: RBS2418 inhibited ATP and cGAMP hydrolysis by ENPP1 with similar high potency. The pharmacology profile was optimized to achieve low serum protein binding and oral bioavailability in humans. RBS2418 treatment was well tolerated in mice under conditions where tumor and plasma levels exceeding ENPP1 serum EC90 levels were achieved. In the syngeneic mouse models Hepa1-6 and GL261-luc significant tumor growth reductions, complete tumor regression and cures were observed. Resistance to cancer cell rechallenge was consistent with treatment-induced anti-tumor immunity. Citation Format: Ningwu Huang, Junjun Cheng, Ron Hawley, Klaus Klumpp. Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse 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 2472.

Abstract 6885: Investigating a novel role of DCLK1 isoforms in colitis and colitis associated colon cancer

Abstract Chronic intestinal inflammation in patients diagnosed with inflammatory bowel disease (IBD) significantly increases their risk of colorectal cancer (CRC). Despite the rising global frequency of IBD, the precise mechanisms that regulate the transition from IBD to colon cancer are poorly understood. Doublecortin-like kinase 1 (DCLK1) is a protein with two major isoforms, DCLK1-L and DCLK1-S. Previous studies have shown that hypermethylation of the DCLK-1α promoter encoding DCLK1-L, particularly in colorectal cancer, allows switching to the DCLK1-S isoform that confers an invasive tumor phenotype. The DCLK1-L is a tuft cell marker that protects against colitis by sensing and eliminating gut pathogenic infection through various mechanisms. On the contrary, the role of DCLK1-S in colitis and colitis-associated cancer has not been thoroughly investigated. Studies from our lab have identified a marked elevation of DCLK1-S in tissue samples of IBD patients. We also recently discovered that DCLK1-S is predominantly expressed in Ly6G+; MHCII- neutrophils, which coincide with elevated levels of inflammation and tissue damage in the colon. To further understand the role of DCLK1 isoforms in driving the progression of colitis to colon cancer, we bred Dclk1fl/fl mice with MRP8-Cre-ires/GFP mice to generate Dclk1fl/fl;MRP8-Cre+/- that eliminates DCLK1-L upon tamoxifen injection resulting in sustained expression of DCLK1-S in the granulocytes especially neutrophils. When infected with Citrobacter rodentium (CR), MRP8;Dclk1−/− mice compared to MRP8;Dclk1+/− or WT mice developed more severe colitis. We have also discovered a novel interaction between DCLK1-S and MMP13 in an unbiased docking study that correlated with significant co-localization of DCLK1-S with MMP13 in the colons of CR-infected mice. Employing a knock-in mice over-expressing the DCLK1-S +19 under the CAG promoter, the DCLK1-MMP13 co-localization correlated with severe ulceration and colitis with loss of crypts and immune cell infiltration including Ly6G+ neutrophils. Studies are underway to see if the severity of colitis in MRP8;Dclk1−/− mice translates into enhanced tumorigenesis and to see if potential interaction between DCLK1-S and MMP13 could provide a plausible mechanism of colitis progression to colon cancer through ECM remodeling and tissue damage. Citation Format: Kafayat Aderonke Yusuf, Badal C. Roy, Shrikant Anant, Shahid Umar. Investigating a novel role of DCLK1 isoforms in colitis and colitis associated 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 6885.

Abstract 2820: The effect of Pten haploinsufficiency on genomic and transcriptomic profiles in combined genetically-engineered mouse models for colonic neoplasia based on Apc inactivation

Abstract Background and Aim: We have reported genetically-engineered mouse models for colonic neoplasia by using our novel CDX2P-Cre mice, in which Cre is restrictedly expressed in colonic epithelial cells. Although colonic tumors are generated in CDX2P-Cre;Apcflox/+ (CPC;Apc) mice with highly penetrant rate, greater number and deeper invasion of colonic tumors were observed in CDX2P-Cre;Apcflox/+;Ptenflox/+ (CPC;Apc+Pten), possibly resulting from haploinsufficiency of Pten gene. We have also confirmed shorter lifespan in CPC;Apc+Pten than that in CPC;Apc (AACR Annual Meeting 2018). Here, we test if these phenotypes come from one copy loss of Pten itself or secondary genetic and/or transcriptomic alterations induced by Pten loss by using whole genome sequence (WGS) and RNA sequence from macroscopically-dissected colonic tumors (bulk RNA-seq). Methods: WGS for five tumors and five livers as counterpart controls in each mouse model was performed by DNBSEQ platforms. Bulk RNA-seq was performed for three tumors in each model. Results: In the analysis using B-allele frequency, copy-neutral LOH occurred in chromosome 18 including Apc loci specifically in colonic neoplasia in both CPC;Apc and CPC;Apc+Pten mice. In contrast, LOH was not observed in chromosome 19 including Pten loci in CPC;Apc+Pten mice. We also confirmed remarkably decreased coverage of reads in reference mapping in Apc locus, and there was a slight decrease of coverage in Pten locus. There were no additional driver gene mutations in CPC;Apc or CPC;Apc+Pten in WGS. All these results suggested just one copy loss of Pten resulting in aggressive tumor phenotype in CPC;Apc+Pten mice. In RNA-seq and subsequent GSEA comparing colonic tumors in CPC;Apc+Pten with those in CPC;Apc revealed upregulation of some signal pathways including mTORC1. Supporting this observation, number of colon tumors, especially adenocarcinomas, were decreased by rapamycin administration to CPC;Apc+Pten. Similarly, rapamycin was more effective in tumorids from CPC;Apc+Pten those from CPC;Apc. Conclusion: WGS reveals one copy loss of Pten locus is the only genetic alteration in CPC;Apc+Pten when compared with CPC;Apc. One copy loss of Pten is sufficient to upregulate mTORC1 pathway in a CPC;Apc+Pten mouse model, resulting in promotion of tumor formation and invasion. Citation Format: Haruki Sada, Hiroaki Niitsu, Hikaru Nakahara, Naoya Sakamoto, Hirotaka Tashiro, Hideki Ohdan, Takao Hinoi. The effect of Pten haploinsufficiency on genomic and transcriptomic profiles in combined genetically-engineered mouse models for colonic neoplasia based on Apc inactivation [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 2820.

Aberrant non-canonical NF-κB signalling reprograms the epigenome landscape to drive oncogenic transcriptomes in multiple myeloma.

In multiple myeloma, abnormal plasma cells establish oncogenic niches within the bone marrow by engaging the NF-κB pathway to nurture their survival while they accumulate pro-proliferative mutations. Under these conditions, many cases eventually develop genetic abnormalities endowing them with constitutive NF-κB activation. Here, we find that sustained NF-κB/p52 levels resulting from such mutations favours the recruitment of enhancers beyond the normal B-cell repertoire. Furthermore, through targeted disruption of p52, we characterise how such enhancers are complicit in the formation of super-enhancers and the establishment of cis-regulatory interactions with myeloma dependencies during constitutive activation of p52. Finally, we functionally validate the pathological impact of these cis-regulatory modules on cell and tumour phenotypes using in vitro and in vivo models, confirming RGS1 as a p52-dependent myeloma driver. We conclude that the divergent epigenomic reprogramming enforced by aberrant non-canonical NF-κB signalling potentiates transcriptional programs beneficial for multiple myeloma progression.

“Patchiness” in mechanical stiffness across a tumor as an early-stage marker for malignancy

Mechanical phenotyping of tumors, either at an individual cell level or tumor cell population level is gaining traction as a diagnostic tool. However, the extent of diagnostic and prognostic information that can be gained through these measurements is still unclear. In this work, we focus on the heterogeneity in mechanical properties of cells obtained from a single source such as a tissue or tumor as a potential novel biomarker. We believe that this heterogeneity is a conventionally overlooked source of information in mechanical phenotyping data. We use mechanics-based in-silico models of cell-cell interactions and cell population dynamics within 3D environments to probe how heterogeneity in cell mechanics drives tissue and tumor dynamics. Our simulations show that the initial heterogeneity in the mechanical properties of individual cells and the arrangement of these heterogenous sub-populations within the environment can dictate overall cell population dynamics and cause a shift towards the growth of malignant cell phenotypes within healthy tissue environments. The overall heterogeneity in the cellular mechanotype and their spatial distributions is quantified by a “patchiness” index, which is the ratio of the global to local heterogeneity in cell populations. We observe that there exists a threshold value of the patchiness index beyond which an overall healthy population of cells will show a steady shift towards a more malignant phenotype. Based on these results, we propose that the “patchiness” of a tumor or tissue sample, can be an early indicator for malignant transformation and cancer occurrence in benign tumors or healthy tissues. Additionally, we suggest that tissue patchiness, measured either by biochemical or biophysical markers, can become an important metric in predicting tissue health and disease likelihood just as landscape patchiness is an important metric in ecology.

Circular RNA circNCOA3 promotes tumor progression and anti-PD-1 resistance in colorectal cancer.

Aim: Circular RNAs (circRNAs) have been found to be involved in tumor progression, but their role in colorectal cancer (CRC) immune escape remains to be elucidated. Methods: circRNAs differentially expressed in responsive and resistant CRC tissues to programmed cell death 1 (PD-1) antibody therapy were identified by microarray analysis. The clinical and pathological significance of circNCOA3 was validated in a separate cohort of CRC samples. The function of circNCOA3 was explored experimentally. RNA immunoprecipitation and luciferase activity assays were conducted to identify downstream targets of circNCOA3. Results: The circNCOA3 was markedly overexpressed in CRC samples resistant to PD-1 blockade. circNCOA3 expression was significantly correlated with adverse tumor phenotypes and poor outcomes in CRC patients. Knockdown of circNCOA3 expression markedly suppressed the proliferative and invasive capability of CRC cells. Moreover, knockdown of circNCOA3 increased the proportion of CD8+ T cells while decreasing the proportion of myeloid-derived suppressor cells (MDSCs). Knockdown of circNCOA3 inhibited tumor growth and increased the sensitivity to PD-1 antibody treatment in mouse tumor models. Further studies revealed that circNCOA3 acted as a competing endogenous RNA (ceRNA) for miR-203a-3p.1 to influence the level of CXCL1. Conclusion: Our findings indicate that circNCOA3 might be useful as a potential biomarker to predict the efficacy and prognosis of CRC patients treated with anti-PD-1 therapy.

Artemisia argyi extracts overcome lapatinib resistance via enhancing TMPRSS2 activation in HER2-positive breast cancer.

Breast cancer stands as the predominant malignancy and primary cause of cancer-related mortality among females globally. Approximately 25% of breast cancers exhibit HER2 overexpression, imparting a more aggressive tumor phenotype and correlating with poor prognoses. Patients with metastatic breast cancer receiving HER2 tyrosine kinase inhibitors (HER2 TKIs), such as Lapatinib, develop acquired resistance within a year, posing a critical challenge in managing this disease. Here, we explore the potential of Artemisia argyi, a Chinese herbal medicine known for its anti-cancer properties, in mitigating HER2 TKI resistance in breast cancer. Analysis of the Cancer Genome Atlas (TCGA) revealed diminished expression of transmembrane serine protease 2 (TMPRSS2), a subfamily of membrane proteolytic enzymes, in breast cancer patients, correlating with unfavorable outcomes. Intriguingly, lapatinib-responsive patients exhibited higher TMPRSS2 expression. Our study unveiled that the compounds from Artemisia argyi, eriodictyol, and umbelliferone could inhibit the growth of lapatinib-resistant HER2-positive breast cancer cells. Mechanistically, they suppressed HER2 kinase activation by enhancing TMPRSS2 activity. Our findings propose TMPRSS2 as a critical determinant in lapatinib sensitivity, and Artemisia argyi emerges as a potential agent to overcome lapatinib via activating TMPRSS2 in HER2-positive breast cancer. This study not only unravels the molecular mechanisms driving cell death in HER2-positive breast cancer cells induced by Artemisia argyi but also lays the groundwork for developing novel inhibitors to enhance therapy outcomes.

Targeting SMAD-Dependent Signaling: Considerations in Epithelial and Mesenchymal Solid Tumors.

SMADs are the canonical intracellular effector proteins of the TGF-β (transforming growth factor-β). SMADs translocate from plasma membrane receptors to the nucleus regulated by many SMAD-interacting proteins through phosphorylation and other post-translational modifications that govern their nucleocytoplasmic shuttling and subsequent transcriptional activity. The signaling pathway of TGF-β/SMAD exhibits both tumor-suppressing and tumor-promoting phenotypes in epithelial-derived solid tumors. Collectively, the pleiotropic nature of TGF-β/SMAD signaling presents significant challenges for the development of effective cancer therapies. Here, we review preclinical studies that evaluate the efficacy of inhibitors targeting major SMAD-regulating and/or -interacting proteins, particularly enzymes that may play important roles in epithelial or mesenchymal compartments within solid tumors.

Modulating tumoral exosomes and fibroblast phenotype using nanoliposomes augments cancer immunotherapy.

Cancer cells program fibroblasts into cancer associated fibroblasts (CAFs) in a two-step manner. First, cancer cells secrete exosomes to program quiescent fibroblasts into activated CAFs. Second, cancer cells maintain the CAF phenotype via activation of signal transduction pathways. We rationalized that inhibiting this two-step process can normalize CAFs into quiescent fibroblasts and augment the efficacy of immunotherapy. We show that cancer cell-targeted nanoliposomes that inhibit sequential steps of exosome biogenesis and release from lung cancer cells block the differentiation of lung fibroblasts into CAFs. In parallel, we demonstrate that CAF-targeted nanoliposomes that block two distinct nodes in fibroblast growth factor receptor (FGFR)-Wnt/β-catenin signaling pathway can reverse activate CAFs into quiescent fibroblasts. Co-administration of both nanoliposomes significantly improves the infiltration of cytotoxic T cells and enhances the antitumor efficacy of αPD-L1 in immunocompetent lung cancer-bearing mice. Simultaneously blocking the tumoral exosome-mediated activation of fibroblasts and FGFR-Wnt/β-catenin signaling constitutes a promising approach to augment immunotherapy.

Cold Tumour Phenotype Explained Through Whole Genome Sequencing in Clinical Nasopharyngeal Cancer: A Preliminary Study.

Nasopharyngeal cancer (NPC) is a complex cancer due to its unique genomic features and association with the Epstein-Barr virus (EBV). Despite therapeutic advancements, NPC prognosis remains poor, necessitating a deeper understanding of its genomics. Here, we present a comprehensive whole genome sequencing (WGS) view of NPC genomics and its correlation with the phenotype. This study involved WGS of a clinical NPC biopsy specimen. Sequencing was carried out using a long read sequencer from Oxford Nanopore. Analysis of the variants involved correlation with the phenotype of NPC. A loss of genes within chromosome 6 from copy number variation (CNV) was found. The lost genes included HLA-A, HLA-B, and HLA-C, which work in the antigen presentation process. This loss of the major histocompatibility complex (MHC) apparatus resulted in the tumour's ability to evade immune recognition. The tumour exhibited an immunologically "cold" phenotype, with mild tumour-infiltrating lymphocytes, supporting the possible etiology of loss of antigen presentation capability. Furthermore, the driver mutation PIK3CA gene was identified along with various other gene variants affecting numerous signaling pathways. Comprehensive WGS was able to detect various mutations and genomic losses, which could explain tumour progression and immune evasion ability. Furthermore, the study identified the loss of other genes related to cancer and immune pathways, emphasizing the complexity of NPC genomics. In conclusion, this study underscores the significance of MHC class I gene loss and its probable correlation with the cold tumour phenotype observed in NPC.

Integrated machine learning-driven disulfidptosis profiling: CYFIP1 and EMILIN1 as therapeutic nodes in neuroblastoma

BackgroundNeuroblastoma (NB), a prevalent pediatric solid tumor, presents formidable challenges due to its high malignancy and intricate pathogenesis. The role of disulfidptosis, a novel form of programmed cell death, remains poorly understood in the context of NB.MethodsGaussian mixture model (GMM)-identified disulfidptosis-related molecular subtypes in NB, differential gene analysis, survival analysis, and gene set variation analysis were conducted subsequently. Weighted gene co-expression network analysis (WGCNA) selected modular genes most relevant to the disulfidptosis core pathways. Integration of machine learning approaches revealed the combination of the Least absolute shrinkage and selection operator (LASSO) and Random Survival Forest (RSF) provided optimal dimensionality reduction of the modular genes. The resulting model was validated, and a nomogram assessed disulfidptosis characteristics in NB. Core genes were filtered and subjected to tumor phenotype and disulfidptosis-related experiments.ResultsGMM clustering revealed three distinct subtypes with diverse prognoses, showing significant variations in glucose metabolism, cytoskeletal structure, and tumor-related pathways. WGCNA highlighted the red module of genes highly correlated with disulfide isomerase activity, cytoskeleton formation, and glucose metabolism. The LASSO and RSF combination yielded the most accurate and stable prognostic model, with a significantly worse prognosis for high-scoring patients. Cytological experiments targeting core genes (CYFIP1, EMILIN1) revealed decreased cell proliferation, migration, invasion abilities, and evident cytoskeletal deformation upon core gene knockdown.ConclusionsThis study showcases the utility of disulfidptosis-related gene scores for predicting prognosis and molecular subtypes of NB. The identified core genes, CYFIP1 and EMILIN1, hold promise as potential therapeutic targets and diagnostic markers for NB.

Identification and validation of an immunotherapeutic signature for colon cancer based on the regulatory patterns of ferroptosis and their association with the tumor microenvironment

The integrated landscape of ferroptosis regulatory patterns and their association with colon microenvironment have been demonstrated in recent studies. However, the ferroptosis-related immunotherapeutic signature for colon cancer (CC) remains unclear. We comprehensively evaluated 1623 CC samples, identified patterns of ferroptosis modification based on ferroptosis-associated genes, and systematically correlated these patterns with tumor microenvironment (TME) cell infiltration characteristics. In addition, the ferroptosis-regulated gene score (FRG-score) was constructed to quantify the pattern of ferroptosis alterations in individual tumors. Three distinct patterns of ferroptosis modification were identified, including antioxidant defense, iron toxicity, and lipid peroxidation. The characteristics of TME cell infiltration under these three patterns were highly consistent with the three immune phenotypes of tumors, including immune-inflamed, immune-excluded and immune-desert phenotypes. We also demonstrated that evaluation of ferroptosis regulatory patterns within individual tumors can predict tumor inflammatory status, tumor subtype, TME stromal activity, genetic variation, and clinical outcome. Immunotherapy cohorts confirmed that patients with low FRG-scores showed remarkable therapeutic and clinical benefits. Furthermore, the hub gene apolipoprotein L6 (APOL6), a drug-sensitive target associated with cancer cell ferroptosis, was identified through our proposed novel key gene screening process and validated in CC cell lines and scRNA-seq.