Cancer Pathways Research Articles

Abstract C021: Tumor-associated microbiota suppresses anti-tumor immunity by driving cDC1 dysfunction in lung cancer

Abstract Type 1 conventional dendritic cells (cDC1s) are critical for initiating and sustaining tumor-reactive CD8 T cells; lack of functional cDC1s is a key driver for failed tumor immunosurveillance and immunotherapy. However, what factors cause the cDC1 insufficiency in the tumor microenvironment (TME) remain poorly understood. Commensal microbiota has emerged as a key regulator of TME and the efficacy of cancer immunotherapies. Nonetheless, most studies have focused on the intestinal microbiome; it remains underexplored whether and how the local microbiota within tumors influences the anti-cancer immunity. Using the autochthonous Kras LSL-G12D/+ ; p53 flox/flox (KP) mouse model of lung cancer, we found an increased local bacterial burden and altered bacterial composition associated with lung tumors. Importantly, this local microbiota suppresses the anti-tumor CD8 T cell response both at baseline and in response to ICB therapies. Specifically, single-cell RNA-seq analysis revealed that the microbiota shapes the functional subsets of tumor-reactive CD8 T cells. Bacterial depletion promotes the accumulation of stem-like Tpex cells (precursor of exhausted T cells) in the TME, which serve as a reservoir of effector CD8 T cells and mediate an effective response upon ICB treatment. To distinguish the function of the local microbiota within TME from the distal gut microbiota, we developed a new method of aerosolized antibiotic treatment, which selectively ablates the intra-tumoral lung microbiota while preserving the intact gut microbiota. Using this approach, we demonstrated that removal of the intra-tumoral lung microbiota markedly improves the tumor-reactive T cell response and sensitizing “cold” lung tumors to ICB therapies. Mechanistically, we identified cDC1s as a central player in microbiota-mediated suppression of anti-tumor immunity. Local microbiota diminishes cDC1 quality and quantity in the lung TME, resulting in impaired T cell priming and Tpex maintenance. At the molecular level, our data revealed that bacterial products derived from the local microbiota induce type I interferon expression in tumor-associated myeloid cells and enhance GM-CSF production from cancer cells: chronic type I interferon and GM-CSF signaling acts cooperatively to reduce the functional cDC1 population. Combining in vitro cDC1 assays, competitive bone marrow chimera experiments and cDC1-specific knockout models, we establish the causal relationships between the local microbiota, IFN-I and GM-CSF signaling in cDC1s, and the tumor response to ICB. Altogether, our study uncovered a dominant role of tumor-associated microbiota in inhibiting cDC1s and suppressing anti-tumor immunity through type I IFN and GM-CSF dependent pathways in lung cancer. Our findings not only identify the intra-tumoral microbiota as a critical therapeutic target for lung cancer treatment, but also provide new insights into the tissue-specific, context-dependent regulatory mechanisms of cDC1s in the TME. Citation Format: Qiang Dong, Chengcheng Jin. Tumor-associated microbiota suppresses anti-tumor immunity by driving cDC1 dysfunction in lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C021.

Oxidative stress drives endometrial fibrosis via TGF-β1/MAPK signaling pathway in breast cancer.

Breast cancer patients have high serum reactive oxygen species (ROS) levels, which exert toxicity on the ovary. However, it is still unclear whether tumor-derived ROS play a role in endometrial development and function in breast cancer. Breast cancer patients and healthy controls were recruited and endometrial thickness was measured by transvaginal ultrasound (TVUS). Xenograft tumors of the breast cancer cell line MDA-MB-231 in a female BALB/c nude mice model were established, and the therapeutic mechanism of vitamin C (VC) was investigated on uterine pathology invivo and the contribution of co-culture of breast cancer cell and endometrial epithelial cell on this process was examined invitro. Median thickness in endometria was lower in breast cancer patients and tumor-bearing mice compared to controls. A gene signature of uteri in tumor-bearing mice demonstrated differential expression of genes (DEGs) regulating extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT), and activation of TGF-β and MAPK signaling pathways. In addition, ROS, EMT- and ECM-related protein levels were enhanced in uteri in tumor-bearing mice, as well as in Ishikawa cells which were co-cultured with MDA-MB-231 cells compared to controls. Supplementation with VC reduced endometrial damage, inhibited the EMT process and collagen deposition, and maintained better histologic architecture of uteri in tumor-bearing mice via inactivation of the TGF-β1/p38MAPK pathway. In women with breast cancer oxidative stress in the endometrium results in a fibrotic response as a consequence of EMT. VC could alleviate endometrial fibrosis via TGF-β1/p38MAPK pathway and provide new predictive and therapeutic targets for fertility preservation in younger breast cancer patients.

Targeting regulated cell death pathways in cancers for effective treatment: a comprehensive review

Cancer is a complex disease characterized by specific “mission-critical” events that drive the uncontrolled growth and spread of tumor cells and their offspring. These events are essential for the advancement of the disease. One of the main contributors to these events is dysregulation of cell death pathways—such as apoptosis, necroptosis, ferroptosis, autophagy, pyroptosis, cuproptosis, parthanatos and—allows cancer cells to avoid programmed cell death and continue proliferating unabated. The different cell death pathways in cancers provide useful targets for cancer treatment. This review examines recent progresses in the preclinical and clinical development of targeting dysregulated cell death pathways for cancer treatment. To develop effective cancer therapies, it is essential to identify and target these mission-critical events that prevent tumor cells from timely death. By precisely targeting these crucial events, researchers can develop therapies with maximum impact and minimal side effects. A comprehensive understanding of the molecular and cellular mechanisms underlying these regulated cell death pathways will further the development of highly effective and personalized cancer treatments.

Expression of HECTD2 predicts peritoneal metastasis of gastric cancer and reconstructs immune microenvironment.

Peritoneal metastasis (PM) is a common metastasis site and death cause of gastric cancer, which is a complex biological process, but there is currently a lack of effective prediction and treatment targets. In this study, we first analyzed the differential gene expression of gastric cancer patients with or without peritoneal metastasis, and identified the HECT domain E3 ubiquitin protein ligase 2 (HECTD2) as the core gene of PM in gastric cancer. The current study shows that the role of HECTD2 in tumor is contradictory. In this study, our results show that the low expression of HECTD2 indicates that the survival rate of overall survival (OS), progression-free survival (PFS), disease-specific survival (DSS), and disease-free survival (DFS) are better, and can be used as an important component of prognostic indicators. In addition, through pathway enrichment analysis, we found that HECTD2 was mainly involved in metastasis related pathways such as extracellular matrix remodeling and cell adhesion in gastric cancer, and high expression of HECTD2 could activate epithelial-mesenchymal transition (EMT) metastasis related pathways in gastric cancer. In regulating the metastasis of gastric cancer cells, HECTD2 can also change the surrounding microenvironment, induce the enrichment of interstitial components and build an immune microenvironment conducive to tumor progression, while patients with low expression of HECTD2 may be more likely to benefit from immunotherapy. In conclusion, HECTD2 may be a novel biomarker for the diagnosis and prognosis of peritoneal metastasis of gastric cancer, providing basis for the mechanism of peritoneal metastasis of cancer and clinical medication.

Abstract PR009: mascRNA directs nutrient-insensitive activation of the LARS-mTOR pathway in breast cancer

Abstract Cellular growth and metabolism are tightly regulated by nutrient availability, yet cancer cells frequently lose these controls, enabling unchecked proliferation even in the absence of sufficient nutrients. While non-coding RNAs are known to regulate metabolic enzyme expression, their direct role in nutrient-sensing pathways in cancer remains unclear. MALAT1-associated small cytoplasmic RNA (mascRNA) is a 61-nucleotide small RNA with a tRNA-like structure, cleaved from the 3’ end of the long non-coding RNA MALAT1. mascRNA has been shown to promote hepatocellular carcinoma proliferation and enhance protein translation in HEK293 cells, though its underlying molecular mechanism is not fully understood. We assessed mascRNA levels in over 70 human breast cancer patient samples using BaseScope assays and found that mascRNA is detectable in the cytoplasm of tumor cells, but not in normal mammary tissues or stromal cells adjacent to tumors. However, the levels of mascRNA varied significantly among patient samples, irrespective of molecular subtypes or tumor stages, suggesting that mascRNA is associated with other aspects of tumor biology. Functional studies in breast cancer cell lines revealed that mascRNA overexpression promotes cell proliferation, invasion, and metabolic pathways linked to ATP generation. Specifically, mascRNA increases basal glycolysis and mitochondrial oxidative phosphorylation and significantly enhances mitochondrial spare capacity, particularly in triple-negative breast cancer (TNBC) cell lines. We also found that mascRNA overexpression activates both mTORC1 and AMPK, indicating a deviation from the normal negative feedback loop between these two pathways. To dissect the molecular mechanism, we performed mascRNA pull-down assays and mass-spectrometric analyses. We identified RNA splicing factors, tRNA-modifying enzymes, and several aminoacyl-tRNA synthetases as mascRNA-interacting proteins. Among these, leucyl-tRNA synthetase (LARS), a component of the multi-tRNA synthetase complex, is known for its non-canonical role in activating mTORC1 at the lysosomal membrane. We found that, in MDA-MB-231 TNBC cells, mascRNA overexpression led to increased LARS localization to the lysosomal membrane where it can activate mTORC1. Interestingly, while control MDA-MB-231 cells responded to glucose and leucine availability to regulate the lysosomal localization of LARS and mTOR, mascRNA- overexpressing cells constitutively maintained LARS and mTOR localization to the lysosome, as well as mTORC1 phosphorylation, regardless of nutrient status. In summary, our data demonstrate that mascRNA aberrantly activates the LARS-mTORC1 axis in breast cancer cells independent of nutrient levels, likely through direct interaction with LARS, promoting its localization to the lysosome. This suggests that mascRNA enables breast cancer cells, and possibly other cancer types, to sustain uncontrolled growth even in nutrient-deprived conditions. Elevated mascRNA levels may thus represent a highly efficient metabolic adaptation of cancer cells. Citation Format: Bohye Park, Joshua K Stone, Steve Lim, Shuko Harada, Erin Ahn. mascRNA directs nutrient-insensitive activation of the LARS-mTOR pathway in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR009.

Abstract A025: RNA-Based Therapeutics to target the p53 pathway in high-grade serous ovarian cancer: TAp63-Regulated Oncogenic LncRNAs (TROLLs) as novel therapeutic targets in cancer

Abstract High-grade serous ovarian cancer (HGSOC) is the most common and malignant type of ovarian cancer with a high frequency of p53 mutation and hyperactivation of the AKT pathway. Even though roughly 85% of patients achieve remission after initial surgical debulking and chemotherapy, four out of five HGSOC patients will experience disease recurrence, resulting in a 5-year survival rate of 43%. We have identified and characterized two TAp63 Regulated Oncogenic Long non-coding RNAs (TROLL-2 and TROLL-3) whose transcription is induced by mutant p53 and high expression level correlate with a more aggressive ovarian cancer grade and phosphorylation of AKT. In-depth analysis demonstrated that TROLL-2 and TROLL-3 directly activate AKT in tumor cells, thus linking p53 alterations to AKT activity. The use of AKT inhibitors in the clinic is limited because of frequent adverse effects, likely due to the constitutive expression of AKT in all cells. TROLL-2 and TROLL-3 expression is limited to tumor cells and provides a more specific target for decreasing AKT activation, potentially minimizing off-target effects. We have optimized reduction of TROLL-2 and TROLL-3 expression using antisense oligonucleotides (ASOs) in an ex vivo perfusion platform for 3D culture of microtumors from primary patient derived tumors. Samples show maximum reduction of proliferation (EdU) and phosphorylation of AKT when TROLL-2 and TROLL-3 ASOs are used concurrently. We also performed phospho-proteomics and single cell RNAseq to compare individual ASO-mediated knockdown of each lncRNA to combined targeted ASO treatments. We have developed primary patient derived xenograft models using ovarian tumors orthotopically implanted into NSG mice generating a relevant preclinical model to determine the efficacy of ASO treatment. To reduce the rapid degradation and clearance of ASOs in circulation, ASOs were encapsulated into lipid nanoparticles and metal-organic frameworks (MOFs) and we are currently testing these in vivo. Since AKT activation is associated with increased staging and resistance to platinum and targeted therapies in ovarian cancer patients, our data provides preclinical rationale for the implementation of ASOs and the use of new delivery methods as a relevant translational therapy and novel method to exploit the specificity of TROLL-2 and TROLL-3 expression in HGSOC. Citation Format: Ashley Lui, Marco Napoli, Jaden R Baldwin, Christina L Carr, Angie Rivera, Duy Nguyen, W. Gregory Sawyer, Michael R Dunne, Erin George, Omar K Farha, Michelle Teplensky, Elsa R Flores. RNA-Based Therapeutics to target the p53 pathway in high-grade serous ovarian cancer: TAp63-Regulated Oncogenic LncRNAs (TROLLs) as novel therapeutic targets in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A025.

Abstract B011: RNA-based therapeutics to target the p53 pathway in cancer

Abstract Mutations in the TP53 gene and activation of the PI3K/AKT pathway are the two most frequent genetic alterations across multiple human tumors. We discovered a crucial mechanism of crosstalk between these two events consisting of two oncogenic lncRNAs, TROLL-2 and TROLL-3, whose expression is induced by mutant p53. We demonstrated that these lncRNAs drive tumor and metastasis formation in several orthotopic models, including breast cancer, lung adenocarcinoma, and melanoma. The investigation of these mouse models and a pan-cancer analysis of human cancers showed that these lncRNAs are markers of tumor progression and lead to the phosphorylation and subsequent activation of AKT by promoting the cytoplasmic localization of the scaffold protein WDR26. Notably, we have now proven that TROLL-2 and TROLL-3 are also involved in the response to cancer therapies. Indeed, the downregulation of these lncRNAs via siRNAs bypasses the AKT-driven resistance to chemotherapeutics (e.g. Adriamycin, docetaxel, and paclitaxel) in triple-negative breast cancer cells, to inhibitors of mutant KRAS (e.g. sotorasib) and EGFR (e.g. erlotinib) in lung adenocarcinoma cells, and to inhibitors of PARP (e.g. niraparib) in ovarian cancer cells. To target these lncRNAs in a clinically relevant manner, we designed LNA GapmeRs, also known as antisense oligonucleotides (ASOs), to downregulate these two lncRNAs directly. The efficacy of the ASOs was confirmed in ex vivo specimens of tumors and metastases of lung adenocarcinoma and ovarian cancer patients. These clinical specimens were also analyzed via single-cell RNA sequencing and phospho-proteomics to unveil the common vs. unique downstream effects of targeting each lncRNA. When administered together, the ASOs targeting TROLL-2 and TROLL-3 effectively decreased the levels of these lncRNAs, which in turn caused WDR26 sequestration in the nucleus, thus preventing the activation of the AKT pathway and the proliferation of these cancer specimens. Furthermore, we confirmed that the antitumor activities of these ASOs were also maintained in vivo, as proven by the decreased tumor proliferation observed in breast cancer xenograft models. Finally, to improve the delivery of these ASOs in vivo, the usage of non-viral systems, including lipid-based and metallic-based nanoparticles, is being tested in both ex vivo and in vivo preclinical models. Overall, our results unveiled TROLL-2 and TROLL-3 as suitable targets to counteract tumor growth and promote the efficacy of chemotherapeutics and targeted therapies in ex vivo and in vivo cancer models. Citation Format: Marco Napoli, Ashley K Lu, Avani A Deshpande, Jaden R Baldwin, Christina L Carr, Michael R Dunne, Omar K Farha, Michelle H Teplensky, Elsa R Flores. RNA-based therapeutics to target the p53 pathway in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B011.

Abstract I001: Long noncoding RNAs at the intersection of cancer pathways

Abstract There are thousands of long noncoding RNAs (lncRNAs) in mammalian genomes. Yet, their functional significance in health and disease states remains poorly understood. We investigate the intersection of lncRNAs and cancer biology. On the one hand, we dissect the molecular and cellular mechanisms by which lncRNAs modulate cancer pathways. On the other hand, we develop mouse models to interrogate the roles of lncRNAs in tumorigenesis at the organismal level. Our work has uncovered insights into the transcriptional regulatory roles of lncRNAs during the cellular response to genotoxic and oncogenic stress. In parallel, we have elucidated roles of lncRNAs as drivers of tumor progression and metastatic dissemination. The combined functional and mechanistic studies have revealed the importance of lncRNAs in cancer and suggest therapeutic approaches to target lncRNAs or their downstream effectors in cancer. Citation Format: Nadya Dimitrova. Long noncoding RNAs at the intersection of cancer pathways [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I001.

Therapeutic potential of β-hydroxybutyrate in the management of pancreatic neoplasms: exploring novel diagnostic and treatment strategies.

Pancreatic neoplasm, a highly aggressive and often fatal cancer, poses challenges due to late detection and nonspecific symptoms. Therefore, both early diagnosis and appropriate therapeutic approaches are necessary to augment the condition of these patients. Cancer cells undergo metabolic deregulation, which enables their proliferation, survival, and invasion. As a result, it is crucial to focus on the metabolic pathways in prevalent cancers and explore treatment strategies that target these pathways to control tumor growth effectively. This is particularly relevant in cancers like pancreatic cancer, which undergo numerous metabolic alterations. The ketogenic regimen, characterized by low carbohydrate and protein contents and high-fat sources, does not involve caloric restriction. This allows for the induction of ketogenesis and an increase in ketone bodies, while insulin and glucose levels remain low even after meals. This unique metabolic state may influence the tumor microenvironment. Given the lack of unanimous agreement on the precise role and mechanism of the ketogenic diet, this review aims to clarify the diagnostic value and accuracy of ketone bodies in various types of pancreatic tumors and explore the potential anti-cancer effects of the ketogenic diet when used alone or in conjunction with chemotherapy, also to determine the potential of the ketogenic diet to be used as adjuvant therapy. The outcomes of this study are instrumental in enhancing our understanding of the benefits and drawbacks associated with employing this diet for the management and diagnosis of pancreatic cancer.

HPB SO17 - A rare case of Goblet cell adenocarcinoma of appendix presenting as primary ampullary tumour

Abstract Background Goblet cell adenocarcinoma (GCA), formerly known as goblet cell carcinoid,is a rare appendiceal tumour with amphicrine differentiation that has distinct morphologic and clinical features compared to carcinomas seen elsewhere in the gastrointestinal tract. The tumour histologically resembles both carcinoids and adenocarcinomas. It is found in 0.3–0.9 % of appendectomy specimens; hence, ampullary GCAs are exceptionally uncommon. Method A 57-year-old Caucasian male presented with a two-month history of weight loss, pruritus, and dark urine. His blood tests revealed deranged liver enzymes. His past medical history was unremarkable. Abdominal CT identified a double duct sign, no detectable mass. Endoscopic ultrasound (EUS) revealed a dilated biliary system terminating in a slightly enlarged ampulla (15mm) without any obvious mass lesions. Endoscopic retrograde cholangiopancreatography (ERCP) was performed showing a distal common bile duct (CBD) stricture. Cytology samples were negative for malignancy, and stents were placed in the CBD and pancreatic duct (PD). Pancreaticoduodenectomy was advised given the high risk of malignancy. Results Histopathology report showed clusters of goblet-like mucinous cells in an infiltrative pattern, in addition to classic neuroendocrine tumor morphology, reminiscent of goblet cell adenocarcinoma of the appendix, staged as pT3pN1M0. This is consistent with primary ampullary GCA given the normal appendix on preoprative CT imaging. Conclusion To our knowledge, this is the fourth case of ampullary GCA report in medical literature. There is no consensus on adjunct chemotherapy regimen for systemic treatment. Chicago consensus group (2018) recommends following the colorectal cancer pathway for goblet cell adenocarcinoma. In our experience, given the anatomical location of the tumour and the normal appendix, FOLFIRINOX was offered in line with ampullary adenocarcinoma cancer pathway.

Associations of radiotherapy receipt with lung cancer risk by histological subtype among breast cancer survivors in the United States.

Radiotherapy for breast cancer has been associated with an increased risk of secondary malignancies, including primary lung cancer. Whether this association varies by histological subtype of lung cancer remains unknown. Based on the data from 12 Surveillance, Epidemiology, and End Results registries, we examined the association between radiotherapy receipt and the risk of subtype-specific subsequent primary lung cancer (SPLC) among female first primary breast cancer cases diagnosed between ages 20 and 84 from 1992 to 2020. More than half (53%) of the 550,007 breast cancer survivors identified had undergone radiotherapy as part of their initial breast cancer treatment. Over an average follow-up of 9.7 years, 8014 survivors developed SPLCs. For small-cell carcinoma, the standardized incidence ratio (SIR) compared with the general population was higher for survivors who received radiotherapy (SIR = 1.15, 95% confidence interval [CI] = 1.06-1.25) but similar for those who did not receive radiotherapy (SIR = 1.00, 95% CI = 0.91-1.09), with the difference in SIRs being statistically significant (p = .003). Similar associations were found for squamous cell carcinoma (SIRyes = 1.16, 95% CI = 1.08-1.24 vs. SIRno/unknown = 1.06, 95% CI = 0.98-1.15; p = .07). The increased risks were confined to ipsilateral SPLC, with the greatest SIRs for small-cell carcinoma occurring 5-10 years since breast cancer diagnosis (SIR = 1.83, 95% CI = 1.53-2.19) and for squamous cell carcinoma with a latency of 10 years or more (SIR = 1.64, 95% CI = 1.42-1.88). In contrast, the risk of developing adenocarcinoma did not vary by radiotherapy receipt (SIRyes = 1.23, 95% CI = 1.18-1.28 vs. SIRno/unknown = 1.17, 95% CI = 1.12-1.22; p = .18), indicating additional risk factors in play. The findings suggest a distinct carcinogenic pathway of radiation-induced lung cancer across histological subtypes and may inform risk-stratified surveillance guidelines for SPLC.

Abstract PR012: A novel plasma-based epigenomic assay for comprehensive profiling of cancerous and inflammatory disease states

Abstract Background: We developed a method for comprehensive epigenomic profiling from 1 ml of patient (pt) plasma, targeting histone modifications and DNA methylation. This approach can be applied to plasma from pts with cancer and inflammatory conditions, allowing us to infer expression levels of diagnostic markers and drug targets, measure activity of therapeutically targetable transcription factors, and detect non-genomic mechanisms of resistance. By examining the transcriptional status of therapeutic gene targets and pathways, our method addresses the need for new approaches in liquid biopsy (LBx) assays, providing clinically actionable information that enhances the understanding of cancer and inflammatory biology and therapeutic response. Methods: We developed an innovative multimodal assay capable of revealing genome-wide transcriptional activity of promoters and enhancers from chromatin, as well as surveying the DNA methylome. Utilizing this assay on plasma samples from pts with 5 non- cancer conditions and 15 different tumor types, we generated comprehensive genome-wide transcriptional profiles across 21 conditions. Results: We identified transcriptional activation in cancer pt plasma of cancer-associated biomarkers (e.g., HER2, DLL3) in a tumor-specific manner and in autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus/lupus nephritis. Epigenomic profiling simultaneously inferred the expression of multiple pertinent ADC and radio-immune targets (e.g., MET, HER2, HER3, TROP2, B7H4 in breast cancer and PSMA in prostate cancer), and SLFN11, a biomarker for sensitivity to these agents. The multimodal platform assesses transcriptional regulation genome-wide, enabling analysis of select genes as well as related pathways. For example, a multimodal classifier robustly predicted HER2 status across multiple HER2-expressing tumors. HER2 classification of breast cancer pts by epigenomic LBx was concordant with standard tissue-based IHC for 64/72 (89%) breast cancer samples (AUC 0.9). Accurate classification was achieved for 11/14 (79%) gastroesophageal adenocarcinoma and 4/4 (100%) ovarian cancer pts. A multi-modal predictive model trained on FOLH1 plasma epigenomic regulatory signals demonstrated significant accuracy in predicting quantitative readouts from PSMA PET scans (total SUVmean) in metastatic castration resistance prostate cancer pts and was associated with response to pluvicto. Epigenomic signatures also delineated activation of the endocrine signaling pathway in breast cancer and multiple mechanisms of resistance to androgen receptor signaling inhibition in prostate cancer, including reactivation of the androgen receptor, glucocorticoid signaling bypass, and histological transformation to a neuroendocrine subtype. Conclusions: This comprehensive epigenomic LBx assay offers a platform for simultaneous genome-wide transcriptional assessment of disease and therapy-relevant genes and pathways, marking a paradigm shift in our ability to assess the underlying transcriptional states of cancer and inflammatory diseases. Citation Format: Travis Clark, Anthony D'Ippolito, Aparna Gorthi, Jonathon Beagan, Jamey Guess, Amy Donahue, Mandy Greene, Alyssa Lau, Fernando Ramirez, Kristian Cibulskis, Jenna Wurster, Michael Coyne, Mary McGillicuddy, Baovy Tran, Tyrone Tamakloe, Hathairat Sawaengsri, Corrie Painter, Juliann Chmielecki, Geoff Otto, Matthew Eaton, Carl Barrett. A novel plasma-based epigenomic assay for comprehensive profiling of cancerous and inflammatory disease states [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR012.

The 2-week wait pathway for suspected head and neck cancers in patients with throat and voice symptoms: referral patterns, common clinical practice and diagnostic efficacy of NICE guidelines.

The 2-week wait (2ww) referral pathway has been introduced into UK clinical practice to increase the early detection of cancer and improve survivals. The efficiency of this system for head and neck (H&N) cancers has been questioned over the years because of evidence of low pick-up rates. H&N cancers present with a wide variety of non-specific symptoms, particularly throat and voice symptoms. These symptoms need to be accurately interpreted together with risk factors if they are to be addressed adequately and overload of cancer facilities avoided. One of the most common outcomes of H&N 2ww referrals is laryngopharyngeal reflux (LPR), a common condition that could be diagnosed and managed in the primary care setting with a prescription of proton pump inhibitors (PPI) trials. We retrospectively analysed a cohort of consecutive patients referred on the 2ww pathway for throat and voice symptoms at University College London Hospital H&N cancer clinic during two months in 2019. A total of 101 patients (43.6% men, mean age 53.3 years) were included. Throat and voice symptoms were described as intermittent in 52.5% and non-lateralised in 88.1%. Diagnosis of LPR was made in 59.4% of the referrals. A PPI trial was prescribed by general practitioners (GPs) in only 7.9% of cases. The cancer pick-up rate in our cohort is 2.9%. An improved awareness of the symptoms of LPR could guide GPs to prescribe trials of PPIs in low-risk patients before rushing into a referral on a cancer pathway. This would improve the 2ww process on many levels, reduce the burden on the National Health Service and avoid patients' psychological distress.

Abstract 4137833: The plasma cell-free transcriptome non-invasively profiles the immune response and myocardial damage in immune checkpoint inhibitor-induced myocarditis.

Introduction: Immune checkpoint inhibitor-induced myocarditis (ICI-m) is a severe complication of cancer immunotherapy, marked by cardiac inflammation. Despite its high mortality rate, diagnosing ICI-m remains challenging due to the lack of sensitive and non-invasive diagnostic modalities. Plasma cell-free mRNA (cf-mRNA) has recently been highlighted as a promising tool for non-invasive tissue profiling. Hypothesis: The plasma cf-mRNA profile contains disease-specific immune and tissue transcriptomic signatures of ICI-m that can be leveraged for disease-specific precision diagnostics. Aims: To establish and validate a novel cf-mRNA bench and bioinformatic pipeline for noninvasive profiling of the immune and tissue landscape of patients with ICI-m. Methods: We isolated, DNase-treated, and sequenced bulk cfRNA from frozen plasma of patients with ICI-treated cancer patients with no autoimmunity (group A, n=5), extracardiac autoimmunity (group B, n=7), and ICI-m with or without extracardiac autoimmunity (group C, n=10). Publicly available healthy control cfRNA was downloaded (n=30) as external controls. We used cell type-specific gene panels curated from single-cell RNA-sequencing datasets of circulating immune cells and cardiac cells for cellular deconvolution of the cf-mRNA. Signature scores of the sum of the counts of each cell type-specific gene were used to compare cell type contributions between patient groups. Results: Cancer and inflammatory gene pathways were significantly enriched in the cf-mRNA of all ICI-treated cancer patients. Cardiac pathways and conduction genes were significantly enriched in group C vs. A and B patients. Signature scores of Temra CD8+ T cells and cardiomyocytes, and gene expression of CCL5 and MYH6 were elevated in group C patients. Our custom ICI-m classifier containing 3 cardiac- and 3 immune cell-specific genes differentiated group C from A (AUC 0.903, 95% CI 0.858-0.903) and B patients (AUC 0.983, 95% CI 0.970-0.983) effectively, outperforming a diagnostic classifier obtained from unsupervised feature selection from all differentially expressed genes. Conclusions: Using our novel plasma cf-mRNA platform, we developed a diagnostic classifier that captures the disease-specific immune and tissue transcriptomic signatures of ICI-m, highlighting the advantages of this disease-specific approach over conventional biomarker discovery, and demonstrating the broader implications of the platform for the field of precision diagnostics.

CSIG-07. JAK1/STAT1/3 COOPERATES WITH THE CIC-FUSIONS TO DRIVE CIC-REARRANGED SARCOMAS

Abstract CIC-rearranged sarcoma (CRS) is an rare disease driven by a specific fusion protein involving the CIC gene. Occurrence in the brain is 3% in all CRS patients. The native CIC protein is a transcriptional repressor of the (RTK)/Ras/ERK signaling pathway, which is one of the most tumorigenic pathways in cancer. The most common rearrangement is with the double homeobox 4 (DUX4) transcription factor (CIC-DUX4), and others, such as CIC-NUTM1 fusions, have been identified in a subset of pediatric primitive neuroectodermal tumors. However, the molecular mechanisms by which CIC-fusions drive CRS remain unknown. Preliminary data shows that CIC-DUX4/NUTM1 fusions activate JAK and its downstream effector STAT1/3. We hypothesize that the JAK/STAT1/3 signal transduction pathway cooperates with CIC-fusions to induce the expression of oncogenic transcription factors ETV1/4/5 and drive these sarcomas. We show high levels of JAK1/STAT1/3 activation in patient-derived CRS cell lines (NCC-SCC-89/C) as compared to other sarcoma cell lines without the fusion. JAK1 inhibition using Solicitinib and Ruxolitinib reduced phosphorylation of STAT1/3 as well as protein and mRNA expression of ETV1/4/5, promoter activity of ETV5, cell proliferation and tumorgenicity. Interestingly DUX4 is involved in histone acetylation and STAT1 has been shown to activate p300/CBP complex which lead us to evaluating the role of STAT1 in the gene activation CRS. We elucidate mechanistically that the fusion proteins increase binding of STAT1/3 at the promoters of ETV 1/4/5. Importantly, JAK1 inhibition effectively reduces histone acetylation induced by CIC-fusions at the promoters of ETV1/4/5. To evaluate the pre-clinical effect of targeting the JAK1/STAT1/3 pathway, A CRS cell line (NCC-SCC-89/C) were grafted into NSG mice. Ruxolitinib treatment significantly reduced tumor volume, STAT activation, and ETV1/4/5. In conclusion, we show that the JAK1/STAT1/3 pathway plays a critical role in cooperating with CIC-fusions to drive CRS, providing insight into potential therapeutic avenues for these aggressive tumors.

DeePathNet: a transformer-based deep learning model integrating multi-omic data with cancer pathways.

Multi-omic data analysis incorporating machine learning has the potential to significantly improve cancer diagnosis and prognosis. Traditional machine learning methods are usually limited to omic measurements, omitting existing domain knowledge, such as the biological networks that link molecular entities in various omic data types. Here we develop a Transformer-based explainable deep learning model, DeePathNet, which integrates cancer-specific pathway information into multi-omic data analysis. Using a variety of big datasets, including ProCan-DepMapSanger, CCLE, and TCGA, we demonstrate and validate that DeePathNet outperforms traditional methods for predicting drug response and classifying cancer type and subtype. Combining biomedical knowledge and state-of-the-art deep learning methods, DeePathNet enables biomarker discovery at the pathway level, maximizing the power of data-driven approaches to cancer research. DeePathNet is available on GitHub at https://github.com/CMRI-ProCan/DeePathNet.

Gene expression-based modeling of overall survival in Black or African American patients with lung adenocarcinoma

IntroductionLung cancer is a leading cause of cancer-related deaths worldwide. Black/African American (B/AA) populations, in particular, exhibit the highest incidence and mortality rates of lung adenocarcinoma (LUAD) in the United States.MethodsThis study aims to explore gene expression patterns linked to LUAD in B/AA and case-matched white patients, with the goal of developing predictive models for prognosis. Leveraging RNA sequencing data from The Cancer Genome Atlas (TCGA) database, genes and pathways associated with overall survival (OS) were identified.ResultsThe OS-associated genes in B/AA patients were distinct from those in white patients, showing predominant enrichment in immune-related pathways. Furthermore, mRNA co-expression network analysis revealed that OS-associated genes in B/AA patients had higher levels of interaction with various pathways, including those related to immunity, cell-ECM interaction, and specific intracellular signaling pathways. Notably, a potential B/AA-specific biomarker, C9orf64, demonstrated significant correlations with genes involved in immune response. Unsupervised machine learning algorithms stratified B/AA patients into groups with distinct survival outcomes, while supervised algorithms demonstrated a higher accuracy in predicting survival for B/AA LUAD patients compared to white patients.DiscussionIn total, this study explored OS-associated genes and pathways specific for B/AA LUAD patients. Further validation and clinical application of these findings are warranted to address disparities and improve outcomes in diverse patient populations.

TMIC-46. TRANSCRIPTIONAL HETEROGENEITY AND MECHANISTIC PATHWAYS OF RECURRENT GLIOBLASTOMA: INSIGHTS FROM A PRE-CLINICAL RECURRENT TUMOR MODEL

Abstract Despite aggressive therapy combining surgical resection, radiation and chemotherapy, glioblastoma (GBM) almost always recurs. Currently, there is no standard treatment for recurrent GBM, and patient inclusion in clinical trials is based on the genetic and molecular profile of the primary tumor even though the effects of chemoradiation on the transcriptional subtype in matched primary and recurrent GBM remain largely understudied. Paralleling the GLASS consortium, we sought to perform a longitudinal assessment of GBM recurrence after therapy based on molecular subtype with the hypothesis that recurrent tumors exhibit a distinct transcriptional landscape different from treatment-naive tumors. To address this, we utilized primary tumor-derived glioma stem cells (GSCs) representing classical and proneural subtypes. We then undertook in vivo intracranial implantation studies and performed standard concomitant treatment that mimics the Stupp protocol. Upon bulk RNAseq data analyses of untreated (n=21) and recurrent tumors (n=17) we identified that while the molecular subtype of recurrent tumors does not change post therapy, their transcriptional profile is more heterogenous compared to untreated tumors for both subtypes. Hierarchical cluster analyses revealed distinct sub-clusters of recurrent tumors. Common mechanisms of recurrence include upregulation of angiogenesis pathways and PI3k/Akt/mTOR signaling pathways. Interestingly, recurrent tumors exhibit upregulation of genes involved in cellular metabolism and membrane transport/ potential. The source of heterogeneity in recurrent tumors in both subtypes appears to be due to the diversity in expression of gene sets involved in hallmark of cancer pathways such as cell cycle, Notch and Wnt signaling pathways and heterogeneity in oncogenic signatures and cell states resembling fetal or neural cells. Further research into the recurrent tumor microenvironment by spatial multi-omics approaches using this pre-clinical recurrent GBM model will elucidate the linkage between cellular as well as molecular heterogeneity and enable identification of targets that can help drive therapeutic decisions for recurrent GBM patients.

Elevated ITGA3 expression serves as a novel prognostic biomarker and regulates tumor progression in cervical cancer

Patients with advanced and recurrent cervical cancer often lack satisfactory treatment outcomes. Thus, it is necessary to seek reliable biomarkers that provide the ability to identify the disease at an early stage and predict the patient prognosis, providing new strategies for the treatment of cervical cancer. The sequencing data of ITGA3 were retrieved from public datasets. Immune infiltration and sensitivity of potential immunotherapy and chemotherapy have been analyzed between two subgroups. Functional analysis was applied to excavate the related pathways of ITGA3 in cervical cancer. Furthermore, the impact of ITGA3 in tumor progression has been verified in vitro. The results revealed that the level of ITGA3 was upregulated in cervical cancer, and was positively correlated with worse prognosis. The tumor microenvironment of patients in the high-risk group was immunosuppressed. Patients in high-risk group may not benefit from immunotherapy, but be may be sensitive to several chemotherapy drugs. Notably, the angiogenesis, epithelial mesenchymal transition, and PI3K pathway were increased in high-risk group. Collectively, ITGA3 is a marker of poor prognosis and promotes tumor progression by regulating PI3K/AKT pathway in cervical cancer. Our results provide new insights for potential molecular targeted therapy and prognostic prediction of cervical cancer.

Epoxymicheliolide Reduces Radiation-Induced Senescence and Extracellular Matrix Formation by Disrupting NF-κB and TGF-β/SMAD Pathways in Lung Cancer.

Lung cancer is a major cause of cancer-related mortality, and radiotherapy is often limited by tumor resistance and side effects. This study explores whether epoxymicheliolide (ECL), a compound from feverfew, can enhance radiotherapy efficacy in lung cancer. We tested ECL on A549 and PC-9 lung cancer cell lines to evaluate its effect on x-ray irradiation. We measured apoptosis, NF-κB pathway inhibition, TGF-β secretion reduction, and epithelial-mesenchymal transition suppression. Invivo, C57BL/6 mice with lung tumors received ECL and radiotherapy. ECL enhanced the antiproliferative effects of x-ray irradiation, induced apoptosis in senescent cells, inhibited the NF-κB pathway, reduced TGF-β levels, and suppressed epithelial-mesenchymal transition. ECL also inhibited tumor growth and improved survival in mice. ECL is a promising adjunct to radiotherapy for lung cancer, improving treatment outcomes by targeting multiple tumor progression mechanisms. It offers potential for enhanced management of lung cancer.