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Study of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to the lack of isogenic models. To address this, we used healthy primary breast luminal epithelial cells of different genetic ancestry and their hTERT-immortalized counterparts to identify transcriptomic changes associated with immortalization. Elevated expression of TONSL (Tonsoku-like, DNA repair protein) was identified as one of the earliest events during immortalization. TONSL, which is located on chromosome 8q24.3, was found to be amplified in approximately 20% of breast cancers. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity, but overexpression was insufficient for neoplastic transformation. However, TONSL-immortalized primary cells overexpressing defined oncogenes generated estrogen receptor–positive adenocarcinomas in mice. Analysis of a breast tumor microarray with approximately 600 tumors revealed poor overall and progression-free survival of patients with TONSL-overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors, including NF-κB and limited access to the tumor-suppressor p53. TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in the homologous recombination (HR) and Fanconi anemia pathways. Consistent with these results, TONSL-overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was essential for growth of TONSL-amplified breast cancer cell lines in vivo, and these cells were sensitive to TONSL–FACT complex inhibitor CBL0137. Together, these findings identify TONSL as a regulator of epithelial cell immortalization to facilitate cancer initiation and as a target for breast cancer therapy. Significance: The chr.8q24.3 amplicon-resident gene TONSL is upregulated during the initial steps of tumorigenesis to support neoplastic transformation by increasing DNA repair and represents a potential therapeutic target for treating breast cancer.

Background: Heterozygous hotspot mutations in the RNA splicing factor SF3B1, occur in 3% of unselected breast cancers and are associated with oestrogen receptor (ER+) breast cancer (BC) where they are enriched in metastatic disease and are associated with a poor clinical outcome. SF3B1 mutations drive distinct signatures of alternative splicing through cryptic 3’ splice site selection leading to global transcriptomic and proteomic changes. The functional consequences of the mis-splicing events and resultant genetic vulnerabilities are poorly understood and precision medicine approaches that exploit these characteristics are not clinically available (Table 1). Methods: To understand the role of SF3B1 mutations in ER+ BC, we generated a series of SF3B1 mutant (SF3B1MUT) isogenic cell lines which were characterised using RNA-sequencing and high content mass-spectrometry proteomic profiling. SF3B1 interactome analysis was also performed using immunoprecipitation of SF3B1 followed by mass-spectrometry. The molecular consequences of aberrant splicing were investigated using a targeted screening approach of 280 genes predicted to be alternatively spliced in SF3B1MUT BC, while high-throughput drug screens were used to identify novel therapeutic options for patients with SF3B1MUT breast cancer using isogenic cells. Hits were validated in vitro and in vivo using cell line and patient derived xenografts. Results: Transcriptomic and proteomic profiling of SF3B1MUT cells identified global alternative 3’ splice site selection and subsequent proteomic changes induced by the mutations. Investigation of the SF3B1K700E interactome identified an enrichment of SF3B1K700E binding with ER, aberrant splicing of ER target genes, global rewiring of ER chromatin binding and resistance to endocrine therapy. Silencing of the aberrantly spliced candidate genes PPIH, TRIM37, HIGD1A, BRD9, and PHKG2 significantly enhanced the growth of the SF3B1 mutant cells, suggestive of a dose dependent tumour suppressive effect. Through synthetic-lethal drug screens we found that SF3B1MUT cells are selectively sensitive to PARP inhibitors. SF3B1MUT cells display a defective response to PARPi induced replication stress. Mechanistically, this occurs via defective ATR signalling in SF3B1MUT cells, which upon PARPi exposure leads to increased replication origin firing and loss of pChk1 (S317) induction. The resultant replication stress leads to failure to resolve DNA replication intermediates via the endonuclease MUS81 and cell cycle stalling at the G2/M checkpoint. These defects can be further targeted by ATM, CDK7 or FACT inhibition, when used in combination with PARPi treatment. This SF3B1MUT selective PARPi sensitivity is preserved across multiple cell lines and patient derived tumour models. In vivo, PARPi produce profound anti-tumour effects in multiple SF3B1MUT cancer models and eliminate distant metastases. Conclusions: Our integrative analysis reveals mechanistic insight into the role of SF3B1 mutations in endocrine therapy response in ER+ breast cancers, where altered SF3B1 induces ER-transcriptional re-programming. We further identified a robust synthetic-lethal relationship of mutant SF3B1 with PARP inhibition that is caused by a defective response to PARPi induced replication stress. Furthermore, we identified several potential selective combination strategies together with PARPi that are selective for SF3B1MUT cells. Together, these data provide the pre-clinical and mechanistic rationale for assessing already-approved PARPi in a biomarker-defined subset of advanced ER+ BC. Table 1. Identified potential therapies for SF3B1 mutant cancers from this study and the literature Citation Format: Phil Bland, Harry Saville, Abigail Read, Patty Wai, Gareth Muirhead, Lucinda Curnow, Jadwiga Nieminuszczy, Nivedita Ravindran, Marie John, Somaieh Hedayat, Holly Barker, James Wright, Lu Yu, Ioanna Mavrommati, Barrie Peck, Mark Allen, Patrycja Gazinska, Helen Pemberton, Aditi Gulati, Sarah Nash, Farzana Noor, Naomi Guppy, Ioannis Roxanis, Samantha Barlow, Helen Kalirai, Sarah Coupland, Ronan Broderick, Samar Alsafadi, Alexandre Houy, Marc-Henri Stern, Stephen Pettit, Jyoti Choudhary, Syed Haider, Wojciech Niedzwiedz, Christopher Lord, Rachael Natrajan. Mutations in the RNA Splicing Factor SF3B1 drive endocrine therapy resistance and confer a targetable replication stress response defect through PARP inhibition. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-05.

Background: A large fraction of human DCIS (>50%) may not need the multimodality treatment options currently offered to all patients. More importantly, while we may be overtreating many, we cannot identify those most at risk for invasion/metastasis. Revealing the cellular and molecular mechanisms by which some DCIS remain indolent while others advance to invasive and metastatic breast cancers is currently a clinical unmet need. Methods: To address this gap, we developed the Mouse-INtraDuctal (MIND) model, by which patient-derived (PDX) DCIS epithelial cells are injected intraductally and allowed to progress naturally in mice. Single cell RNA-sequencing (scRNA-seq) was utilized to profile the DCIS epithelial and stroma cells in progressors vs. non-progressors. To distinguish between stromal (diploid) cells and tumor (aneuploid) cells, we calculated Copy Number Aberration (CNA) profiles from RNA using CopyKAT. Cell-type specific differential gene expression analysis of DCIS epithelial cells and microenvironment cell types in progressors and non-progressors was performed. We also predicted putative ligand:receptor interactions between the tumor cells and cell types in the microenvironment by CellPhoneDB. Results: Among 37 PDX DCIS MIND models followed for a median of 9 months, 20 (54%) grafted into 95 glands, showed in vivo invasive progression (progressed) while 17 (46%), injected into 107 glands, remained non-invasive (non-progressed). ScRNA-seq was performed on 13 DCIS samples including 10 progressors and 3 non-progressors. Aneuploid cells were further analyzed to identify deferentially expressed genes that were upregulated in progressors compared to non-progressors (log2 fold=1, FDR p< 0.05).. Notable genes included NEAT1, EIF4EBP1, SCGB2A2, TFF1 and TFF3 that were upregulated in the progressors. NEAT1, the core structural component of the paraspeckles, is frequently overexpressed in human cancers and its expression is correlated with worse survival in cancer patients. NEAT1 drives tumor progression by regulating genes involved in cellular growth, migration, invasion, metastasis, EMT, stemness, radio- and chemoresistance, supporting its role as a potential biomarker and therapeutic target. TFF1/TFF3 mRNAs show increased expression in metastatic breast cancers. EIF4EBP1 is located on chrom 8p11-p12 which is frequently amplified in breast cancer and is associated with poor clinical prognosis. Further analysis using Cancer Hallmarks identified mitotic spindle, interferon signaling, DNA repair, oxidative phosphorylation and P53 pathway among the top signatures that were upregulated in the progressors. CellPhoneDB identified expression of several receptor/ligand interactions including CD74/MIF involved in epithelial/stromal and stromal/stromal cross talks that may play a role in DCIS invasive progression. Conclusions: Future studies will validate our findings using patient DCIS samples with known long-term outcome and in vivo MIND models to further refine risk associated biomarkers for invasion/metastasis and to identify more effective treatments. Citation Format: Aditi Rastogi, Fariba Behbod, Nicholas Navin, Jerome Lin, Linheng Li, Hua Li, Andrew K. Godwin, Alastair M. Thompson, Timothy Fields, Yan Hong. Epithelial/stromal cross talks that induce malignant transition of human ductal carcinoma in situ. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-08.

Background: In contrast to the IMpassion130 trial evaluating atezo + nab-pac [Schmid, NEJM 2018], the randomized phase 3 IMpassion131 trial (NCT03125902) did not demonstrate significantly improved progression-free survival (PFS; primary endpoint) and showed no improvement in overall survival (OS; secondary endpoint) with the addition of atezo to pac as first-line therapy for mTNBC in either the PD-L1+ or the intention-to-treat (ITT) population [Miles, Ann Oncol 2021]. In IMpassion130, enhanced PFS and OS improvement with atezo + nab-pac were seen in the basal-like immune-activated (BLIA) subtype, whereas potential resistance to atezo + nab-pac was observed in the luminal androgen receptor (LAR) subtype [Emens, ASCO 2021]. LAR may be more prevalent in Asian populations [Ding, Oncotarget 2019], which represented one-third of patients enrolled in IMpassion131 and could have influenced the overall result. PFS outcomes were numerically worse in Asian vs non-Asian subgroups in IMpassion131 [Miles, Ann Oncol 2021]. To investigate potential reasons for observing different effects in IMpassion130 and IMpassion131, we explored the prevalence and impact on clinical outcomes of TNBC molecular subtypes and race in IMpassion131. Patients and Methods: Patients with mTNBC (no prior systemic therapy or ≥12 months since [neo]adjuvant chemotherapy) were randomized 2:1 to atezolizumab 840 mg or placebo (days 1, 15), both with paclitaxel 90 mg/m2 (days 1, 8, 15), every 28 days until disease progression or unacceptable toxicity, stratified by tumor PD-L1 status, prior taxane, liver metastases, and geographic region. Molecular subtypes according to Burstein classification were determined by RNA sequencing (Illumina TruSeq RNA Access) of baseline tumor samples. Prevalence of Burstein molecular subtypes [Burstein, Clin Cancer Res 2015] was assessed in Asian (of whom 69% were enrolled in China) and non-Asian populations; clinical outcomes were assessed according to molecular subtype and race using Cox proportional hazards analysis. All analyses were performed using the final data cut-off (Sep 4, 2020; median follow-up duration: 14.4 months). Results: The biomarker-evaluable population (BEP; n=471) was representative of the ITT population (n=651) with respect to baseline characteristics and PFS hazard ratio (HR; 0.75 [95% CI 0.61-0.92] vs 0.81 [95% CI 0.68-0.96], respectively). Distribution of molecular subtypes in the BEP was: 30% BLIA, 41% basal-like immune suppressed (BLIS), 24% LAR, 5% mesenchymal (MES), similar to IMpassion130. Among the BLIA samples, 82% were PD-L1+ and 18% PD-L1-; corresponding percentages were 41% vs 59% for BLIS, 31% vs 69% for LAR, and 32% vs 68% for MES. Compared with non-Asian patients, the Asian subgroup (n=117) included more LAR (31% vs 22%) and fewer MES (1% vs 6%) samples; this was particularly pronounced in the cohort enrolled in China (n=79; 37% LAR, 0% MES). PFS was improved with atezo + pac in the BLIA subtype (HR 0.66, 95% CI 0.45-0.97). None of the Burstein subgroups derived OS benefit from atezo + pac. Findings were similar irrespective of PD-L1 status. Direction of effect for PFS and OS favored the placebo + pac arm in the LAR Asian subgroup (n=30). Conclusion: In these exploratory analyses, the distribution of molecular subtypes and enhanced effect of atezo + pac in the BLIA subtype are consistent with findings from IMpassion130. The lack of improved efficacy with the combination of atezo + pac in the IMpassion131 trial cannot be explained by overrepresentation of a Burstein subtype less sensitive to atezo in the trial population. Citation Format: Fabrice André, Regula Deurloo, Aditi Qamra, David Cameron, Joseph Gligorov, Andreas Schneeweiss, Carlos Barrios, Binghe Xu, Ching-Wei Chang, Luciana Molinero, Shilpen Patel, Andrea Liptrot, Leilani Morales, David Miles, Joyce O’Shaughnessy. Activity of atezolizumab (atezo) plus paclitaxel (pac) in metastatic triple-negative breast cancer (mTNBC) according to Burstein molecular subtype: Analysis of the IMpassion131 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD10-05.

Background: Inheritance of BRCA1 and BRCA2 mutations is associated with increased risk of breast and ovarian cancers. Previous studies with low-throughput flow cytometry-based assays suggested elevated number of luminal progenitor cells in the breast tissues of BRCA1 mutation carriers compared to breast tissues of non-carriers. However, breast epithelial cell-specific transcriptome differences between BRCA1, BRCA2 mutation carriers, and non-carriers and how these differences alter susceptibility to transformation are yet to be elucidated. Methods: We generated a single cell transcriptome atlas of breast tissues from BRCA1 (six samples, 17,220 cells), BRCA2 (four samples, 25,046 cells) mutation carriers and non-carriers (11 samples, >50,000 cells). Using previously described markers, epithelial cells were sub-clustered into basal, luminal progenitor, and mature luminal cells. Genes differentially expressed in epithelial cells of BRCA1 and BRCA2 mutation carriers compared to those in non-carrier donors were subjected to Ingenuity Pathway Analysis to determine signaling pathways uniquely active in BRCA1 and BRCA2 mutant epithelial cells. Breast epithelial cells derived from three donor types were immortalized using hTERT and then transformed with PIK3CAH1047R mutant or H-RasG12V ± SV40-T/t antigens, and tumorigenicity was determined in vivo. Results: BRCA1 but not BRCA2 mutations altered the ratio between basal, luminal progenitor and mature luminal cells in breast tissues compared to breast tissues in non-carriers. A unique cluster of cells within luminal progenitors was underrepresented in case of BRCA2 mutation carriers compared to non-carriers or BRCA1 mutation carriers. BRCA1 or BRCA2 mutations specifically altered transcriptomes which are an integral part of mTOR and MYCN signaling, and the translational machinery. Signaling pathway alterations in epithelial cells unique to BRCA1 mutations included YAP1, BRD4, SMARCA4, and TGFβ1 signaling. BRCA2 mutations were associated with upregulation of IL-6, FOXO3, and TNFSF11 signaling. Breast epithelial cells from BRCA2 mutation carriers but not BRCA1 mutation carriers or non-carriers modified to overexpress hTERT + PIK3CAH1047R generated tumors in NSG mice. These tumors displayed large cystic structures with basilar epithelial cells lining the rim of cysts with focal areas of cellular hyperplasia and neoplastic cells that extended into the lumen. However, BRCA1/2 mutation status did not influence tumorigenicity by hTERT+ H-RASG12V +SV40-T/t antigens. Conclusions: Our studies provide a high resolution transcriptome atlas of breast epithelial cells of BRCA1 and BRCA2 mutation carriers, which also reveal potentially targetable signaling networks uniquely deregulated in these cells. BRCA2 mutations are associated with distinct susceptibility to PIK3CA mutation-driven transformation. Since PIK3CA mutations are observed in clinically normal breast tissues, screening for such mutations in BRCA2 mutation carriers may help to detect pre-neoplastic or early stage breast cancer. Citation Format: Harikrishna Nakshatri, Poornima Bhat-Nakshatri, Duojiao Chen, Katie Chen, Henry Mang, Christopher A Herodotou, Aditi S Khatpe, Patrick C McGuire, Xiaoling Xuei, Yunlong Liu, George Sandusky, Anna Maria Storniolo. Single cell transcriptomic analysis reveals the effects of BRCA1 and BRCA2 mutations on distinct signaling networks and cancer susceptibility [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-07-09.

Background: Despite recent advances in the treatment of AML, most approaches are rarely curative and most patients (pts) succumb to relapsed disease. The effectiveness of stem cell transplant and associated graft vs. leukemia effect implies an important role for immune-based therapy in producing long lasting remissions. Traditional approaches using immunotherapy have failed to establish a suitable surface target or treatment paradigm that is effective in myeloid malignancies. Hu8F4 is a humanized T-cell receptor-like monoclonal antibody that binds to the conformational epitope of PR1 bound to HLA-A2, which is highly, differentially expressed on the surface of AML compared to normal progenitors. Methods: We conducted a first in human, phase I dose escalation trial of Hu8F4 in pts with myeloid malignances. Pts with R/R AML, MDS, CMML, and myeloid blast phase of CML with adequate organ function and PS ≤ 2 were eligible. Pts were treated on 7 escalating dose levels, ranging from 0.01 mg/kg to 10 mg/kg IV on D1 & 15. Initial dose levels required 1 pt per dose (0.01, 0.03, 0.1, 0.3, 1), followed by 3 pts per dose (3, 10). Results: 10 pts with R/R AML have been enrolled, with a median age of 65 years (range, 23-77), including 6 females (60%). Pts had received a median of 4 (1-4) prior therapies; 5 pts (50%) had a PS of 2. At enrollment, the median WBC was 1.9 (0.1 - 18.4), median BM blasts were 32% (8 - 76); 9 (90%) pts had complex karyotype and 3 (30%) had a TP53 mutation. All pts had > 98% surface expression of PR1 on the myeloid blasts. Hu8F4 Cmax ranged up to 160,000 ng/mL with t1/2 of 48 hours and clearance of 2.61 hr*ng/mL at the highest dose. Weak anti-drug antibodies were observed after week 4 in 2 of 3 pts treated at 3 mg/kg. With a median follow up of 3.5 months (1.1 - 9.9), pts have received a median of 1 (1-4) cycle of therapy. Two pts had decline in BM blasts and 4 had stable disease. Routine peripheral blood testing revealed sharp decline in peripheral blasts immediately after infusion of Hu8F4 on D1 and 15 with associated elevation in serum LDH in some pts and a rise in normal granulocytes, consistent with on-tumor effects. The pharmacokinetic parameters and transient blast reduction indicated a possible sink effect mediated by high levels of circulating blasts. SAEs documented on study were mostly disease-related and included infections, cytopenias, hemoptysis, pneumonia, and GI bleeding. Treatment related AEs were temporally related to the infusion included hypotension (Grade 2: N=2), rigors (Grade 2: N=2; Grade 1: N=1). All infusion reactions were observed at dose levels of 3 and 10 mg/kg, but were transient, and managed with steroids and antihistamines. All pts proceeded with their next dose without further issues. No cytokine release syndrome or neurologic toxicity was observed. Correlative studies support antibody dependent cellular cytotoxicity and phagocytosis as important mechanisms of anit-AML activity. Conclusion: Hu8F4 was well tolerated with no dose-limiting toxicities observed at the maximum planned dose. On-target peripheral blast reduction temporally related to infusion suggests biological activity. Real-time pharmacokinetic data on study indicate a possible sink effect that may be overcome by a more frequent dosing strategy. Citation Format: Tapan M. Kadia, Hagop Kantarjian, Gheath Alatrash, Anna Sergeeva, Hong He, Lisa St. John, Priya Koppikar, Celine Kerros, Abhishek Maiti, Courtney Dinardo, Elias Jabbour, Serge Vesrstovsek, Naveen Pemmaraju, Nitin Jain, Ghayas Issa, Guillermo Montalban-Bravo, Aditi Shastri, Daniel Couriel, Rhona Pinsoy, Sapna Parshottam, Richard Champlin, Jorge Cortes, Jeffrey Molldrem. Phase I study of the T-cell receptor-like antibody Hu8F4 in patients with advanced hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT101.

Papillomaviruses are species specific, non-enveloped, double-stranded DNA viruses that have an ~8kb circular genome enclosed by a 55 nm icosahedral capsid. Human papillomaviruses (HPV) are associated with both benign and malignant epithelial lesions. High-risk HPV subtypes are the causative agents of cervical and the majority of oropharyngeal squamous cell carcinomas. Persistent HPV infection is required for HPV-associated tumorigenesis; however, cellular factors needed to support the viral persistence and HPV genes expression are not completely understood. Several transgenic mouse models, developed in last decades, helped tremendously to study HPV-induced carcinogenesis. However, the absence of HPV non-coding upstream regulatory region and late coding region, as well as targeted early genes expression driven by an artificial promoter - features that are shared by all existent HPV transgenic mice - limit our ability to investigate HPV-associated malignant transformation and the role of host factors in this process. Therefore, we generated HPV16 transgenic mouse by using CRISPR/Cas9-mediated genome editing to insert the full-length HPV16 genome into the Rosa26 locus. Two founder mice, with single HPV copy and with tandem HPV integration, were used to establish mouse colonies. Interestingly, regardless of HPV copy number, ~30% of the HPV knock-in mice exhibited early death during the first 2 weeks after birth, while survived pups were significantly smaller than the same age wild-type mice, and had severe hair loss. Intriguingly, the growth retardant and hair loss phenotype in the HPV16 knock-in mice disappeared in ~4 weeks, most likely indicating that viral genes compromise specific stages of mouse development. We evaluated the expression of HPV genes during development, in different mouse organs, and in established fibroblasts and epithelial cell cultures. Our newly developed HPV16 knock-in mouse model provides viral gene expression from natural viral promoters offering an essential basis to explore the role of cellular factors in HPV-driven pathologies. Citation Format: Xue Li, Travis Schrank, Aditi Kothari, Hina Rehmani, Jason Tasoulas, Sulgi Kim, Wendell Yarbrough, Natalia Isaeva. Novel mouse model to study HPV associated pathologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB350.

Colorectal cancer (CRC) is one of the most life-threatening gastrointestinal cancers, with around 1.9 million new cases and 935,000 deaths in the year 2020 worldwide. It accounts for 12% and 8% of all estimated new cases of cancer and 11% and 9% of all cancer deaths in Hispanic men and women, respectively. Long-term cumulative exposure to environmental factors such as reactive oxygen species (ROS) has been implicated to cause molecular damage and DNA modifications that are critical for CRC pathogenesis through stress-survival pathway genes. However, limited number of studies related to the role of these pathways have not been conducted for the Hispanic population. The identification and validation of new ethnicity-specific transcriptomic markers within the stress-survival pathways is important for improving treatment, prognosis, and detection strategies. In this study, we have explored the role of stress-survival pathway genes in Hispanic and Non-Hispanic White (NHW) CRC tissues. In one of the previously published studies from our lab we used microarray and RNA-seq datasets obtained from the gene expression omnibus (GEO), the cancer genome atlas (TCGA), and the oncomine databases to identify 28 genes associated with CRC. These genes were screened for transcript level expressions in six CRC cell lines and a normal colon cell line, and in cDNA arrays containing the tumor (n=40) from different CRC stages, and control (n=8) samples by qRT-PCR. The protein level expressions were evaluated by immunohistochemistry (IHC) in CRC tissue microarrays (TMAs) containing different stages of tumor (n = 108) and control (n = 12) tissues. All 28 genes were also analyzed for their transcript-level expressions in Hispanic (n=10) and NHW (n=10) tumors and corresponding non-tumor adjacent (NATs; n=3) tissues. The stress-survival pathway genes associated with cell cycle regulation such as CHEK1, MCM10, PDCD2L, CCNB1, CDK1 and CDK4, and an oxidative stress marker PRDX4 were upregulated at both transcript and protein levels when compared to its normal counterparts. Additionally, the genes CHEK1, MCM10, BCL2L1, CSE1L, ESPL1, GLA, GPX2, RRM2B, SH3GLB1, TNFRSF12A, TRAF5 and TRIB3 were observed to be upregulated whereas GPX1, NOXA, NQO1, SLC7A11, BCL2L12, CCNB1, CDK1, CDK4, FOXM1, PDCD2L and SOD2 were found to be downregulated in Hispanic tumor tissues when compared to NHWs. Only CDK1, CHEK1, FOXM1 and NQO1 were seen to be differentially expressed in the Hispanic and NHW NATs; however, their expression patterns were different from that observed in the respective tumor tissues. Overall, our current findings evaluate the expression of stress-survival pathway genes across different CRC cell lines, stages, and Hispanic and NHW tissues. The genes identified to be differentially expressed in Hispanic tissues may be used as potential biomarkers or as therapeutic targets specific to the Hispanic population in future. Citation Format: Urbashi Basnet, Aditi Kulkarni, Frances A. Rangel, Abhijeet R. Patil, Sourav Roy. Differential expression of stress-survival pathway genes related to Hispanic colorectal cancer disparities. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5232.

Incidence of HPV associated head and neck squamous cell carcinoma (HNSCC) is on the rise, displaying a significantly favorable prognosis and overall better survival as compared to HPV negative HNSCC. Intense radiotherapy is the primary medical care leading to treatment associated morbidity in patients, thereby generating a need for de-intensification strategies. Biomarkers that can identify patients with less aggressive tumors as candidates for de-escalation therapy would revolutionize treatment. We applied an unbiased approach (weighted gene correlation network analysis (WGCNA)) that allows detection of autocorrelated gene sets on 3 independent cohorts of HPV+ HNSCC and created 22 consensus transcriptional modules by selecting genes that grouped together in WGCNA analyses from all 3 cohorts. Interestingly, only 1 module intrinsically divided HPV+ HNSCC into 2 subtypes, displaying different mutational profiles, mutational signatures, HPV gene expression patterns, HPV integration status, and patient survival. Gene set enrichment analysis revealed that this module was enriched in NF-kB genes and strongly associated with NF-κB signaling, confirming our prior findings that defined HPV+ HNSCC subtypes by presence or absence of NF-kB regulators, TRAF3 or CYLD defects and by the NF-κB activity classifier. Our studies show that NF- κB -driven intrinsic tumor characteristics contribute to increased sensitivity of NF- κB active HPV+ head and neck tumors to radiation, providing patients survival benefits. Indeed, TRAF3 or CYLD deletion activated NF-kB and dramatically increased radiation sensitivity of HPV+ head and neck cancer cells. In line with our RNA seq analysis, we found that activation of NF-κB via TRAF3/CYLD deletion significantly correlated with marked downregulation of NRF2 activity and reduced nuclear localization in in HPV+ HNSCC cells. Interestingly, TRAF3 CRISPR KO cells had lower NRF2 protein levels that were restored by MG132 treatment, indicating an involvement of KEAP1/CUL3 mediated proteasomal degradation of NRF2. In summary, our data unveils a unique relation between NF-κB pathway and radiosensitivity in HPV+ HNSCC. The distinct biological characteristics that separate patients into two subclasses associated with favorable prognosis could serve as a biomarker for personalized therapy. Citation Format: Aditi Kothari, Travis Parke Schrank, Wendell Gray Yarbrough, Natalia Isaeva. NF-κB and NRF2 signaling pathways affect prognosis in HPV-associated head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6164.

Skeletal muscle dysfunction due to the effects of cancer secretome is observed in multiple cancer types and extreme dysfunction is manifested as cachexia. Major preclinical studies on cancer-associated muscle defects utilized mouse models or mouse C2C12 mouse myoblast cell line for in vitro studies. Because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required to fully comprehend the effects of cancer secretome on skeletal muscle. Here, we report a simple method to establish skeletal muscle stem cell line (hMuSC), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with hMuSCs to myotube transition. Cancer cell line derived factors accelerated stem to myotube differentiation with accompanying changes including an increase in PAX7+/MyoD+ myogenic progenitor cells. Among the pathways activated by cancer-derived factors include inflammatory pathway involving CXCL8 (also called IL-8), glucocorticoid receptor (GR) pathway, and wound healing pathway. Furthermore, cancer-derived factors significantly altered splicing machinery in hMuSCs. Additionally, AKT and p53 pathways that function in metabolic/survival pathways of the skeletal muscle were adversely affected when hMuSCs were exposed to cancer cell-derived factors. Cancer-derived factors increased the expression levels of previously known cachexia-associated genes such as MT-2, ZIP14, and PDK4. Thus, the model system not only recapitulates results of previous mouse studies but also provides much needed human model system that can easily be adapted for large scale studies to explore the epigenomic changes during hMuSC differentiation and to screen for drugs that restore skeletal muscle function in various diseases. Citation Format: Ruizhong Wang, Brijesh Kumar, Poornima Bhat-Nakshatri, Aditi Sanjay Khatpe, Michael P. Murphy, Kristen E. Wanczyk, Emma H. Doud, Amber L. Mosley, Yunlong Liu, Duojiao Chen, Ed Simpson, Hongyu Gao, Harikrishna Nakshatri. A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3666.

Background: In recent years, a relationship between the tumor microenvironment (TME) and patient response to targeted cancer immunotherapy has been suggested. We applied machine-learning algorithms on H&E stained tissue to study the TME in metastatic non-small cell lung cancer (NSCLC) patients. Our goal was to identify digital pathology (DP) features associated with outcome under combination treatment or monotherapy with atezolizumab (atezo), an anti-PD-L1 therapy, and relate those features to other data modalities. We analyzed patient data from two phase 3 clinical trials, OAK (docetaxel versus atezo in 2L+ NSCLC) and IMpower150 (bevacizumab, carboplatin, and paclitaxel (BCP) versus BCP+atezo (ABCP) in advanced 1L non-squamous NSCLC). Methods: As part of our effort to build a DP-based tumor-immune microenvironment atlas, digitized H&E images were registered onto the PathAI research platform. Over 200K annotations from 90 pathologists were used to train convolutional neural networks (CNNs) that classify slide-level human-interpretable features (HIFs) of cells and tissue structures from images and deployed on images from OAK and IMpower150. HIFs and PD-L1 status were associated with outcome in all samples in each arm in OAK and results were validated in IMpower150, using Cox proportional hazard models. Bulk RNAseq was run using samples extracted from the same area as the H&E slide. Results: We identified a composite feature capturing the ratio of immune cells to fibroblasts in the stroma predictive of both overall survival (OS) (HR=0.74 p=0.0046) and progression-free survival (PFS) (HR=0.87 p=0.14). While patients primarily benefit from atezo if they are PD-L1 high, we found that even PD-L1 negative patients benefited from atezo when enriched for this feature (22C3 PD-L1 assay: OS HR=0.59 p=0.015, PFS HR=0.8 p=0.25; SP142 PD-L1 assay: OS HR=0.74 p=0.12, PFS HR=0.88 p=0.45). We thus recognized a DP feature that was predictive for positive outcome with atezo treatment, independent of PD-L1 levels. This association was then validated in IMpower150 comparing ABCP to BCP, both overall (OS HR=0.69 p=0.012) and in PD-L1 negative patients (SP263 assay OS HR=0.56 p=0.034). Integrating with RNAseq, patients enriched for this DP feature showed similar enrichment for B and T gene signatures and depletion in CAF-related gene signatures, thus showing the harmonization of TME between different data modalities. Conclusions: Using a deep learning-based assay for quantifying pathology features of the TME from H&E images in two NSCLC trials, we identified a novel biomarker predictive of outcome to PD-L1 targeting therapy, even in PD-L1 low & negative patients. Importantly, our work shows how different data modalities (DP, gene expression) can be integrated to further our understanding of the TME. Citation Format: Aditi Qamra, Minu K. Srivastava, Eloisa Fuentes, Ben Trotter, Raymond Biju, Guillaume Chhor, James Cowan, Steven Gendreau, Webster Lincoln, Lisa McGinnis, Luciana Molinero, Namrata S. Patil, Amber Schedlbauer, Katja Schulze, Adam Stanford-Moore, Laura Chambre, Ilan Wapinski, David S. Shames, Hartmut Koeppen, Stephanie Hennek, Jane Fridlyand, Jennifer M. Giltnane, Assaf Amitai. Digital pathology based prognostic & predictive biomarkers in metastatic non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5705.

Background: Immune-modulating therapies have limited efficacy in colorectal cancers (CRC). This resistance to treatment is attributed to low neoantigen expression, and the inability of T cells to kill HLA-I negative tumor cells, especially cancer stem cells (CSCs). Advantageously, NK cells can preferentially kill CSCs via stress ligand recognition. We have previously shown that glycosaminoglycans (GAGs) of a specific chain length, and synthetic mimetics thereof, selectively inhibit colon CSCs. In the current study, we evaluated the immunomodulatory role of natural GAGs and synthetic GAG mimetics (SGMs) in eliciting Natural Killer effector functions that regulate the anti-tumor immune response. Methods: 8-12 wk old C57bl/6 mice were treated with SGM or natural GAG (Fucoidan, heparin hexasaccharide (HS06)) and vehicle for 2 wks. Splenocytes were expanded in IL-2-containing media with or without respective GAG/SGMs, and subjected to co-culture with syngeneic MC38 colon cancer cells. The effects of each treatment on immune cell profile and anti-cancer properties were evaluated using a) multiplex flow cytometry Cytek® immune profiling assay and b) MTT assay. In vitro, MACS-enriched human peripheral blood NK cells were stimulated ex vivo with GAG/NSGMs and analyzed for activation using flow cytometry and cytotoxicity towards primary human colon cancer-derived spheroids. PDL-1 expression and CSC marker expression in GAG/SGM-treated MC38 spheroids was analyzed using western blotting and qPCR. Results: Unbiased multiplex flow cytometry revealed a robust activation of NK cells, evidenced by increased CD335 (NKp46) expression in SGM or Fucoidan-treated mice. SGM pulsed splenocytes also showed increased cytotoxicity toward MC38 cells compared to vehicle controls ex vivo. Furthermore, our preliminary findings showed that human peripheral blood NK cells (CD45+CD3-CD56+) showed activation (increased CD69 expression) with both GAGs and SGMs but showed increased proliferation (Ki67+) upon ex vivo stimulation with GAG oligosaccharide HS06 and not polymeric GAG. Furthermore, GAG treatment significantly enhanced activated autologous NK cell-induced apoptosis in primary human tumor-derived spheroids. Mechanistically, a significant reduction in Pdl-1 protein expression was observed in MC38 spheroids concurrent with the downregulation of various CSCs maker genes, e.g., Oct4, Sox2, and c-my when treated with various SGMs in vitro. Conclusions: The work highlights the role of novel SGMs in the activation of NK cells to target CSCs and it has major implications for development of novel immunotherapy strategies in CRC. Citation Format: Pavani Pingali, Jackson Faulx, Aditi Nandi, Umesh R. Desai, Bhaumik B. Patel. Role of natural killer (NK) cells in glycoasminoglycan-mediated anti-cancer immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1808.

Background: We have previously identified miR-1307-5p is exclusively expressed in OSCC tumors and is significantly upregulated in chemo-resistant patients. In this study, we investigate the mechanism of miR-1307-5p in conferring therapeutic refractoriness and demonstrate reversal of cisplatin induced chemoresistance. This study provides a potentially new therapeutic target for treating recurrent oral carcinomas. Methods: Expression of miR-1307-5p was validated in OSCC patients (n=86), OECM1 cell line and cisplatin resistant CD44+ subpopulation. miR-1307-5p was knocked down in OECM1 and CD44+ cells and its effect on cell proliferation, apoptosis, cell cycle, migration, invasion and sphere forming abilities was measured. We examined the transcriptome of these patients (n=30) and combined this data with The Cancer Genome Atlas (TCGA) dataset for target prediction (TargetScan) and network analysis (Ingenuity Pathway Analysis). The hub genes were considered to be potential targets of miR-1307-5p and were validated by luciferase assay and Western Blotting. Results: Sequencing analysis revealed exclusive expression of miR-1307-5p in the OSCC patient cohort with significant upregulation in chemo-resistant cases (FC:4.82 ± 2.38, p-value:0.01). miR-1307-5p upregulation separated the chemoresistant and remission cohorts (p0.05). Combinatorial treatment of cisplatin and miR-1307-5p inhibitor reduced the proliferation rate of CD44+ cells by 66.23% in comparison to standalone treatment of the inhibitor (p:0.034). Thus, indicating role of miR-1307-5p in reversal of chemo-resistance. Gene target identification revealed miR-1307-5p could promote therapeutic refractoriness by suppressing the expression of EHF, which regulates cancer stem cell renewal by targeting Sox-2, Oct-4 and Nanog. Dual-luciferase gene reporter assay revealed direct suppression of EHF by miR-1307-5p. Further, knockdown of miR-1307-5p increased endogenous EHF mRNA and protein expression in cancer cells. Conclusion: miR1307-5p downregulates EHF which is an inhibitor of many stemness genes like Sox-2, Oct-4 and Nanog. Knockdown of miR-1307-5p reverses Cisplatin resistance by upregulating EHF thus suppressing the emergence of cancer stem cells in OSCC. Citation Format: Aditi Patel, Shanaya Patel, Shreya Lotia, Vivek Tanavde. miR-1307-5p reverses cisplatin resistance in oral squamous cell carcinomas (OSCC) by targeting EHF, a modulator of stemness genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1743.

Clinical sequencing of tumor samples is now a component of routine cancer care used to identify predictive biomarkers of drug response, refine patient cancer diagnoses, assess heritable cancer risk, or inform patient prognosis. Most genomic alterations are accurately annotated with tools such as the Variant Effect Predictor (VEP) that infer the effects of these alterations on the mRNA and protein by following basic rules of transcription, mRNA post-transcriptional processing, and translation. However, some genomic variants are not as easily captured by these rules, which can cause inappropriate or unclear annotation of the protein effect. For example, a variant that alters an existing splice site or creates a new one can activate or inactivate the protein. Similarly, specific mutations in the non-coding promoter region of a gene may de-regulate gene expression (e.g., TERT promoter mutations). In some instances, these so-called variants with unexpected effects (VUE) may have therapeutic implications. For example, while ~2-3% of GIST tumors harbor KIT exon 11 deletions that extend into the non-coding intron between exons 10 and 11 and are shown to cause in-frame deletions, they are typically misclassified as inactivating splice site mutations, precluding patients with tumors harboring these mutations from receiving standard care imatinib. Thus, while many VUEs are functionally characterized and documented in the literature, there are no resources to systematically identify, curate and store these events such that they can be correctly annotated in routine clinical sequencing leading to suboptimal treatment decisions. To address this unmet clinical need, we built a novel bioinformatic resource, the Repository for Variants with Unexpected Effects (reVUE - cancerrevue.org). The reVUE resource consists of (1) an intuitive website listing curated VUEs with their observed effects as demonstrated by functional characterization in peer-reviewed literature and (2) an application programming interface (API) for programmatic annotation of variants. We built reVUE as a crowd-sourced resource to enable contributions of VUEs to the repository from the clinical and scientific communities. Both the software and data are openly available. Our goal is to enable clinical bioinformatic pipelines to accurately annotate all DNA mutations, including those with unanticipated protein coding effects, some of which can have therapeutic implications. Citation Format: Ino de Bruijn, Aditi Gopalan, Xiang Li, Hongxin Zhang, Onur Sumer, Thomas Y. Cong, Tona Gonzalez, Madelaine Rangel, Nikolaus Schultz, Debyani Chakravarty. reVUE: repository for variants with unexpected effects. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6578.

Colorectal cancer (CRC) is one of the most life-threatening gastrointestinal cancers with around 1.9 million new cases and 935,000 deaths in the year 2020 worldwide. With limited access to health care, Hispanics tend to present with later-stage CRC when compared to NHWs, which likely accounts for their inferior survival after CRC diagnosis. Given the rising incidence of CRC among 20-39-year-old Hispanic individuals and the rapid growth in this segment of the population, this is a significant problem to study. As tumorigenesis is contingent upon oxidative stress, reactive oxygen species (ROS) may be one of the important progenitors in carcinogenesis. Here, we have hypothesized that ethnicity-based variations in the stress survival response axis and other related pathways may influence the capacity to suppress ROS, repair DNA damage, and regulate cell death signaling pathways in Hispanic CRC patients. To decipher the role of stress-survival pathway genes in Hispanic CRC disparities, we performed a meta-analysis of microarray and RNA-seq datasets obtained from the GEO, Oncomine, and TCGA and identified 28 stress-survival pathway-associated genes in the CRC datasets. These genes were screened for transcript-level expression by qRT-PCR in one normal colon and six CRC cell lines. cDNA arrays containing normal, and Stage I-IV human CRC samples were used to determine the transcript level expressions of the target genes in different stages of CRC from human tissues. Protein level expressions were evaluated by immunohistochemistry in CRC tissue microarrays containing different stages of tumor and normal human CRC tissues. These genes were also analyzed for their transcript level expressions in Hispanic (n=10) and NHW (n=10) tissues. Twenty-three out of the 28 genes were found to be differentially expressed in Hispanic CRC tissues when compared to NHW CRC tissues. Genes such as CHEK1 and MCM10 were found to be upregulated, whereas BCL2L12, CCNB1, CDK1, CDK4, FOXM1, and PDCD2L were seen to be down-regulated. Transcriptomic analysis (RNA-seq) revealed that 213 and 363 genes, respectively, were differentially expressed in NHW and Hispanic CRC tissues compared to the normal adjacent tissues. The genes involved in cytochrome P450 and PPAR pathways were found to be enriched in the Hispanic cohort. Cytokine IL17 signaling and chemical carcinogenesis pathways were enriched in Hispanic males, whereas PI3K-Akt pathway and chemokine signaling pathways were enriched in Hispanic females as compared to their NHW counterparts. The PPAR and IL17 signaling were seen to be enriched in early-stage (I/II) Hispanic CRC tissues, however, TGF-b and coagulation cascades were enriched in the later stages (III/IV) of Hispanic CRC tissues as compared to NHWs. The genes identified to be differentially regulated in Hispanic tissues and the pathways in which they are involved in should lead to potential biomarkers or therapeutic targets specific to the Hispanic population. Citation Format: Sourav Roy, Aditi Kulkarni, Urbashi Basnet, Soumya Nair. Genes and pathways related to Hispanic colorectal cancer disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1905.

Colorectal cancer (CRC) was reported as the third most diagnosed cancer worldwide with approximately 1.93 million new cases reported in 2020. Within the U.S., the incidence and death rates differ by ethnicity. Among Hispanics, CRC accounted for 12% and 8% of all estimated new cases of cancer and 11% and 9% of all cancer deaths in men and women, respectively, in 2018. Though CRC is preventable, and the overall survival rate is approximately 90% when detected at early stages, only about 39% of CRC patients are diagnosed in the early stages, mostly because screening rates in the US are low, especially among Hispanics. Access to health care for Hispanics is limited when compared to non-Hispanic Whites (NHWs); as a result, they tend to present with later-stage CRC, which likely accounts for their inferior survival after CRC. Essential to improving treatment, prognosis, and detection strategies is the identification and validation of new ethnicity-specific transcriptomic profiles. Here, we have conducted RNA sequencing from Hispanic and NHW CRC (n=10 each) and normal adjacent tissues (NATs; n=3 each), to identify the differentially expressed genes (DEGs) in these two populations. The Illumina sequencing reads were mapped against the reference human genome, and the read counts were normalized using the median of ratios method using DESeq2 software. The transcriptomic analysis revealed that as compared to the respective NATs, 7222 and 7873 genes were observed to be differentially expressed in NHW and Hispanic CRC tissues. Among these, 213 and 363 genes were unique to the NHW and Hispanic CRC cohorts, respectively. Within the Hispanic cohort, gender-based analyses revealed that 504 and 266 genes were differentially expressed in the female and male donors (n=5 each), respectively, as compared to the NHW counterparts. Further, 271 and 475 genes were found to be differentially expressed in early (stage I/II; n=5) and late (stage III/IV; n=3) stages of Hispanic CRC tissues, respectively, as compared to the NHW counterparts. Pathway enrichment analysis of the unique DEGs from the Hispanic and NHW cohorts was conducted using Ingenuity Pathway Analysis software. The genes involved in cysteine biosynthesis, ferroptosis, and GABA receptor signaling pathways were seen to be enriched in the NHW cohort, whereas the genes involved in glucocorticoid receptor (GR) signaling, bile, and androgen biosynthesis pathways were enriched in the Hispanic cohort. Previous studies have implicated, GR signaling to promote tumor heterogeneity and invasion in CRC via CDK1, or bile acids to regulate numerous genes including p53, CDK1, cyclooxygenase 2, IL8, and various miRNAs leading to CRC metastasis. As each pathway likely regulates specific cellular behaviors, the DEGs identified from this study will constitute potential ethnicity-specific biomarkers or attractive targets for the development of novel therapeutic interventions. Citation Format: Soumya Nair, Aditi Kulkarni, Sourav Roy. Transcriptomic profiling of Hispanic colorectal cancer disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1933.

Comprehensive CtDNA and CTC profiling of treatment naïve early-stage head and neck cancer patients reveals early signatures of disease progressionBackground: We performed comprehensive ctDNA analysis on early-stage HNC patients in a pilot study to determine the mutational landscape in HNC patients with a known tobacco history. Methods: We analysed ctDNA of 18 early-stage HNC patients for genomic landscapes using the illumina NextSeq 2000 NGS. A custom-designed OncoIndex gene panel was used for the hybrid capture target-enrichment of critical cancer genes. Panel was designed to detect cancer targeting exonic sequence of 600 genes reporting SNVs and indels along fusions and copy number amplification. The gene panel detected genome-wide signatures including bTMB, MSI (microsatellite instability), HRD (homologous recombination deficiency) prediction and calculate cfDNA tumor fraction. Results: 80 % patients showed presence of at least one CTC in peripheral blood, possibly indicating the progressive disease at the time of presentation. Comprehensive genomic profile obtained from plasma cfDNA of early-stage HNC cancer patients predominantly had low bTMB and MSI Scores (99 % patients). However, HRD and LOH matrix was high for 60 % patients indicating highly dysregulated DNA repair activities. Concurring to these observations, 98 % patients had mutations in key tumor suppressor and DNA damage response (DDR) genes possibly resulting in their loss of function. Besides DNA damage pathway, 60% patients harboured alterations in RTK genes including FGFR, EGFR and PDEGFR family and 32% patients showed activating mutations in Erk1 and its upstream regulators. MSH2 was the most prominently mutated gene (37%) followed by FGFR (32%). Surprisingly, unlike HPV positive advanced HNC cases, TP53 mutations were not detected in any patient, though alterations in TS genes were most prevalent in the study population. 51% alterations resulted into truncated proteins possibly impairing their functions, while 42% alterations were point mutations, 6 % were frameshift and 1 % indels. Presence of mutations in BRAF, PDGFR, FGFR and KIT genes suggested for the potential therapy resistance. Tumor fraction representing ctDNA showed elevated range from 20 % to 45 % with a corresponding ploidy between 2 to 4. Conclusions: Comprehensive ctDNA profile showed major gene alterations in TS and DDR response pathway genes besides mutations in proliferative signaling members. TP53 mutation was not detected, although critical tumor suppressor and DDR genes were predominantly mutated, suggesting for a unique mutation pattern associated with early-stage HNC due to tobacco etiology. Our results suggest that comprehensive ctDNA analysis along CTC profiling can predict the disease progression beforehand and may offer new treatment options to early-stage HNC patients. Citation Format: Gowhar Shafi, Atul Bharde, Fauzul Moubeen, Kanchan Hariramani, Alain D’Souza, Trupti Kad, Bhagwat Jadhav, Sangita Prajapati, Aditi Rani, Madhura Basavalingegowda, Mohan Uttarwar, Gourishankar Aland, Sreeja Jayant, Aravindan Vasudevan, Pankaj Chaturvedi, Jayant Khandare. Comprehensive circulating tumor DNA and CTC profiling of treatment naïve early-stage head and neck cancer patients reveals early signature of disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3379.

Introduction: Colorectal cancer (CRC) is the third most common cancer worldwide, and is associated with high morbidity and mortality. Approximately 50% of all CRC patients develop metastases. Tumor microenvironment (TME), including extracellular matrix (ECM), plays an important role in tumor progression and metastasis in CRC, indicating an important role of agents targeting ECM in the treatment of CRC. ECM acts as niche to support cancer cells. In this study, the role of metformin as a therapeutic agent in modulating ECM components of CRC has been studied. Methods: HCT116 (aggressive, poorly differentiated) and HT 29 (less aggressive, moderately differentiated), human CRC cell lines were treated with different doses of metformin (0.1, 2.5 and 5 mM) for 14 days in vitro and subsequently microarray analysis was done to identify differentially expressed genes. To observe the role of metformin in modulating the ECM, ECM-receptor interaction pathway was given primary focus among the list of pathways identified by DAVID (Database for Annotation, Visualization and Integrated Discovery) bioinformatics resources. The seven differentially expressed genes of ECM-receptor interaction pathway (elucidated by microarray) were -COL3A1, COL4A2, COL5A2, VTN, ITGA6, TNXB and LAMC3. These genes were validated using RT-qPCR in both the cell lines. Protein expression was analyzed by Western blotting. Immunohistochemistry (IHC) was done in different histopathological grades of CRC samples to evaluate the expression and localization of ECM proteins. Results: The gene expression of COL4A2, COL5A2, ITGA6, VTN and TNXB by RT-qPCR was decreased with metformin treatment and these results were consistent with microarray data. However, discrepancy was observed in the gene expression of COL3A1 and LAMC3 between microarray and RT-qPCR. Expression of COL3A1, VTN was found to be increased whereas a decrease in expression of ITGA6 was observed in different histopathological grades of CRC samples with IHC. The expression of PI3K and AKT proteins were decreased in HCT116 cells with metformin treatment justifying its anticancer properties. Conclusions: Anticancer role of metformin via mTOR and PI3K-AKT signaling has been studied before, but this study identifies the novel ECM proteins in CRC that can be modulated by metformin. The results from this study indicates the promising role of metformin in CRC as it is associated with very less side effects and can influence various aspects of cancer biology. Citation Format: Amar Preet Kaur, Aditi Bhattacharya, Muzaffer Ahmed Bhat, Aakriti Bansal, Sudip Sen. Evaluating the effect of metformin on components of extracellular matrix in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1269.

Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent malignant neoplasm of the pancreas, responsible for over 90% of pancreatic cancer cases. Despite recent therapeutic advances, the five-year overall survival rate for PDAC is only 11% and therapies remain limited. It is now known that deficiency in DNA damage repair (DDR) renders PDAC cells vulnerable to certain DNA damage agents, for example, poly (ADP-ribose) polymerase inhibitors (PARPi). Most of the DDR genes mutated in PDAC are crucial for the proper functioning of the homologous recombination repair (HRR) pathway, and loss-of-function alterations in DDR genes sensitize cells to DNA-damaging agents. Since this treatment strategy is currently limited to 5-10% of PDAC cases with known mutations in the DDR genes, it underscores a dire need to understand new therapeutic avenues and expand the PDAC patient population that could benefit from this therapeutic approach. We have recently published that BARD1 (BRCA1- Associated- Ring- Domain- 1), an obligate binding partner of BRCA1, is upregulated in PDAC cells exposed to PARPi/platinums. We found that transient inhibition of BARD1 rendered PDAC cells extremely sensitive to olaparib (PARPi)/oxaliplatin (platinum), and enhanced DNA damage. Based on these results, we created two genetic models of BARD1 inhibition and utilized these model systems to investigate if loss of BARD1 chemo-sensitizes PDAC cells to other PARPis and DNA damage agents. Doxycycline inducible shRNA and BARD1 CRISPR KO models were created to inhibit gene expression of BARD1 in PDAC cells. We first characterized these model systems to confirm the loss of BARD1 protein and mRNA expression by western blot and qRT-PCR analyses. Using colony formation and Pico green cell survival assays, we found that inhibiting BARD1 resulted in slower growth of pancreatic cancer cells in vitro. Moreover, expression of several cell cycle genes from G2-M phase was significantly decreased (p=0.05). These studies were recapitulated in a mouse xenograft subcutaneous model, where BARD1 KO and WT PDAC cells were injected subcutaneously and tumor growth was analyzed over 25-30 days. Kaplan-Meier curves were calculated and plotted using Log-rank test. We found that inhibiting BARD1 significantly (p=0.026) delayed formation of PDAC tumors in vivo. Next, we analyzed the efficacies of various DNA damaging agents when BARD1 was inhibited. Silencing or CRISPR KO of BARD1 enhanced efficacy of mitomycin C, irinotecan, as well as other PARPis, which are currently in clinical trials (rucaparib, veliparib) in vitro and lowered their IC50s. These results indicate that targeting BARD1 has the potential to benefit patients in terms of novel or improved treatments. Our ongoing and future studies will expand on these findings to understand BARD1-mediated pathway and evaluate sensitivity of multiple DNA damage agents by screening a library of > 250 compounds. Citation Format: Sohum Patel, Anoohya Arkala, Avinoam Nevler, Carolyn Coats, Charles J. Yeo, Aditi Jain. Targeting BARD1 slows PDAC growth in vitro and in vivo and sensitizes cells to DNA damaging agents. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6102.

Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase is present in AML cells in 25% of patients, causing constitutive activation and aberrant signaling. FLT3-ITD+ AML patients have a high relapse rate following chemotherapy and short disease-free and overall survival. Incorporation of FLT3 inhibitors (FLT3is) into therapy for FLT3-ITD+ AML has improved outcomes, but FLT3i efficacy is limited by onset of resistance, which occurs by diverse mechanisms. The tumor suppressor protein phosphatase 2A (PP2A) is inactivated in FLT3-ITD+ AML, and we previously showed that PP2A-activating drugs (PADs), including DT-061 and FTY720, enhance FLT3i efficacy through enhanced proteasomal degradation of c-Myc and Pim-1, mediated by activation of the serine/threonine kinase GSK-3β (Mol Cancer Ther 20:676, 2021). Here we sought to determine whether concurrent treatment with PADs overcomes resistance to FLT3is. We studied MOLM-14 human AML cells, with heterozygous FLT3-ITD, and FLT3i-resistant MOLM-14 cells with D835Y tyrosine kinase domain and F691L gatekeeper FLT3 mutations and M14(R)701 MOLM-14 cells (from Dr. Donald Small, Johns Hopkins) with a G12D NRAS activating mutation. IC50s, determined by WST-1 cytotoxicity assays, of Type 1 FLT3i gilteritinib in MOLM-14, D835Y, F691L and M14(R)701 cells were 9.2, 18.6, 56 and 57.5 nM, and the Type II FLT3i quizartinib, 2.8, 211, 659 and >4,000 nM. Both DT-061 and FTY720 sensitized MOLM-14 D835Y cells to quizartinib (IC50s Citation Format: Moaath Mustafa Ali, Aditi Chatterjee, Jonelle K. Lee, Mario Scarpa, Maria R. Baer. Effects of PP2A-activating drugs on FLT3 inhibitor resistance mediated by diverse mechanisms in acute myeloid leukemia with FLT3-ITD. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5013.