Tumor Suppressor Function Research Articles

Knockout of Brca1-interacting factor Ola1 in female mice induces tumors with estrogen suppressible centrosome amplification

Obg-like ATPase 1 (OLA1) is a binding protein of Breast cancer gene 1 (BRCA1), germline pathogenic variants of which cause hereditary breast cancer. Cancer-associated variants of BRCA1 and OLA1 are deficient in the regulation of centrosome number. Although OLA1 might function as a tumor suppressor, the relevance of OLA1 deficiency to carcinogenesis is unclear. Here, we generated Ola1 knockout mice. Aged female Ola1+/− mice developed lymphoproliferative diseases, including malignant lymphoma. The lymphoma tissues had low expression of Ola1 and an increase in the number of cells with centrosome amplification. Interestingly, the proportion of cells with centrosome amplification in normal spleen from Ola1+/− mice was higher in male mice than in female mice. In human cells, estrogen stimulation attenuated centrosome amplification induced by OLA1 knockdown. Previous reports indicate that prominent centrosome amplification causes cell death but does not promote tumorigenesis. Thus, in the current study, the mild centrosome amplification observed under estrogen stimulation in Ola1+/− female mice is likely more tumorigenic than the prominent centrosome amplification observed in Ola1+/− male mice. Our findings provide a possible sex-dependent mechanism of the tumor suppressor function of OLA1.

Abstract 6864: Tumor suppressor function of the ubiquitin ligase Siah1a in melanoma is mediated by myeloid derived suppressor cells

Abstract Growing evidence supports the important role of myeloid-derived suppressor cells (MDSC) in melanoma progression and response to therapy. MDSC contributes to a pro-tumorigenic phenotype, yet the mechanistic basis for the immune suppressor properties acquired by MDSC remains largely unknown. Mammals have two homologous Siah genes, Siah1 (Siah1a and Siah1b in mice) and Siah2. In the context of melanoma, the ubiquitin ligases Siah1a and Siah2 have melanoma-intrinsic and -extrinsic (immune) functions. Correspondingly, ablation of Siah1a/2 in melanoma or its microenvironment attenuates melanoma development in a number of mouse melanoma models. Given that number of macrophage clusters infiltrated into melanoma when grown in Siah2 KO mice, we set to assess macrophage function in melanoma. Selective ablation of Siah1a or Siah2 in macrophages, using Lyz2Cre, revealed that Siah1a but not Siah2 elicits a tumor suppressor function in melanoma. Inoculation of mouse melanoma Yummer1.7 cells (Braf mutated and Pten deleted) in mice lacking Siah1a in macrophages, resulted in bigger melanoma tumors compared to melanoma grown in either WT or mice in which macrophages were ablated of Siah2. FACS analysis of melanoma tumors grown in mice harboring Siah1a mutant macrophages revealed a significant increase in MDSC, which coincided with a decrease in CD4+ and CD8+ cells, compared with WT mice. These observations suggest that a lack of Siah1a in macrophages leads to an expansion of MDSC that affects surrounding immune cells, including T cells. RNA-seq analysis of MDSC that were differentiated in vitro from bone marrow cells of WT or Siah1a ablated macrophage mice revealed increased expression of genes involved in proliferation and alternative macrophage activation in MDSC lacking Siah1a when compared to the WT genotype. Mapping mechanisms underlying Siah1a tumor suppressor function is expected to reveal novel regulatory cues in melanoma control by the MDSC population while mapping novel means for stratifications of melanoma to therapy, and possibly new means for therapy of this tumor type. Citation Format: Marzia Scortegagna, Yuanning Du, Yongmei Feng, Ze'ev Ronai. Tumor suppressor function of the ubiquitin ligase Siah1a in melanoma is mediated by myeloid derived suppressor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6864.

Abstract 1706: Investigating the interaction between ETS family members and mutant p53

Abstract Cancer cases are on the rise globally requiring a deeper understanding of the disease and identification of novel therapeutic targets. Mutations in genes often result in tumor formation. TP53 encodes p53, a tumor suppressive transcription factor, and is mutated in nearly 50% of all cancer cases. Once mutated, p53 loses its tumor suppressive function while simultaneously gaining oncogenic function. p53 mutations disrupt direct p53 binding to DNA and promotes p53 binding to regulatory regions of oncogenes via protein-protein interactions with other transcription factors. One of these interacting partners is ETS2, a member of the ETS transcription factor family. ETS binding sites are present in 50% of all mutant p53 occupied regulatory elements. Other ETS family members have also been linked to mutant p53 but these interactions have either been deemed weak (ETS1) or have yet to be identified as direct (ERG). It is my aim to determine the scope of interaction between the ETS family and mutant p53 and to elucidate whether this interaction is required for the observed oncogenic phenotypes of ETS proteins and mutant p53.To determine which ETS proteins interact with mutant p53 purified ETS proteins were used for affinity pull-down of purified mutant p53. The entire ETS family interacted with mutant p53 to some degree, and several ETS proteins had stronger interactions than ETS2. Truncation studies were used to determine interaction domains. One interacting region was the conserved DNA binding domain, which may explain interaction across the entire family. ERG and ETS2, both strong mutant p53 interactors, had two interaction interfaces. I hypothesize that the second interaction interface defines strong interactors. To determine if interacting ETS are responsible for targeting mutant p53 to the genome, chromatin immunoprecipitation sequencing measured mutant p53 binding in the presence or absence of different ETS factors. These data indicate that each of the conditions resulted in different p53 binding patterns and that there is a requirement for ETS in mutant p53 binding. Lastly, I wanted to determine if there was a correlation between p53 mutation and expression of strong mutant p53 interactors in certain cancer types. Analysis of TCGA samples showed that in ovarian cancers with p53 mutations, ETS factors that are strong mutant p53 interactors were upregulated in a mutually exclusive pattern, while weak interactors tended to be downregulated, or unchanged. Similar trends were observed among some other cancer types. My studies have shown that several ETS proteins interact with mutant p53 in vitro, recruit p53 to the genome, and that this interaction pattern correlates with expression in mutant p53 driven cancers. Additionally, studies are ongoing to determine phenotypes related to this interaction. Ultimately, if ETS/mutant p53 interactions are deemed important for oncogenesis, these will be attractive targets for drug development. Citation Format: Stephanie Metcalf, Peter Hollenhorst. Investigating the interaction between ETS family members and mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1706.

Abstract 4641: Preclinical evaluation of the novel curcumin Analogue AKT-100 demonstrates therapeutic effectiveness in p53-mutated cancer cells

Abstract Among the myriad of molecular alterations associated with cancer, loss of the normal tumor suppressive functions of p53, the “guardian of the genome,” is the most consistent. Thus, the possibility of targeting oncogenic, mutant p53 proteins has long been a therapeutic goal. However, that aspiration has not been fully realized, leaving patients vulnerable to aggressive disease that is resistant to chemotherapy and to targeted agents such as PARP inhibitors and immunotherapy owing to the loss of wild type p53 functionality and the gain of new oncogenic functions when mutant p53 is expressed. An effective therapeutic strategy is urgently needed for p53 mutant cancers, and our team is developing novel analogues of curcumin with the potential to convert mutant p53 to a wild type conformation and restore its tumor suppressive functions. In this study, we investigated the effects of AKT-100, which was developed from the parent molecule and curcumin analogue HO-3867. Effects were compared to another drug believed to reactivate p53 functionality, APR-246, using CyQUANT IC50 cell proliferation assays and bulk and single cell RNA sequencing. Structural analyses are underway investigating the specific sites of interaction of AKT-100 with various mutant forms of p53. Our findings indicate that, first, AKT-100 exhibits impressive cell killing in multiple ovarian and serous endometrial cancer cells at concentrations ranging from 100-300 nM and is synergistic with the PARP inhibitor olaparib. Second, RNA sequencing demonstrates the reactivation of wild type p53 genes associated with normal cell cycle regulation (induction of CDKN1A encoding p21 and GADD45A), apoptosis (induction of PMAIP1 encoding Noxa and DR5 encoding Death Receptor 5), and the inhibition of DNA replication and multiple alternative mechanisms of DNA repair employed by malignant cells. Third, RNA sequencing indicates that AKT-100 acts through both p53-dependent and independent pathways to inhibit cancer cell proliferation. Gene expression is impacted in ways that portend a therapeutic effect as well as highlight potential resistance pathways (e.g. significant induction of PLK5 encoding polo like kinase 5) that can be further targeted to create synthetic lethal drug combinations with AKT-100 chosen specifically to target the various mutant forms of p53. Taken together, these results identify novel curcumin analogues in early development with the potential to improve therapy for p53 mutant cancers. Through this strategy it is also possible to identify and block resistance pathways that are activated by different forms of mutant p53 in response to AKT-100 and to create synthetic lethal precision drug combinations. Citation Format: Kimberly K. Leslie, Jamie L. Padilla, Geneva L. Williams, Hua-Ying Fan, Cristian G. Bologa, Eric R. Prossnitz, Jun-Yong Choe, Lane E. Smith, Alexander Goss, Robert J. Lake, Avinash D. Sahu. Preclinical evaluation of the novel curcumin Analogue AKT-100 demonstrates therapeutic effectiveness in p53-mutated cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4641.

Abstract 4536: MTAP loss alters the epigenetic landscape and demonstrates superior therapeutic sensitivity to concomitant PRMT5 and PARP inhibition in cholangiocarcinoma

Abstract 5’-methylthioadenosine phosphorylase (MTAP), is deleted in 15% of all cancers (estimated 100,000 patients per year in the United States), including 12-15% of cholangiocarcinoma (CCA) cases. MTAP deficiency accumulates 5’-methylthioadenosine (MTA) that subsequently binds protein arginine methyl transferase 5 (PRMT5) and inhibits its activity. This renders selective targeting of MTAP null tumors with agents (MRTX1719, AMG193, TNG462, TNG908) targeting MTA bound PRMT5 (PRMT5:MTA), while sparing surrounding normal MTAP wild-type (WT) tissue. Early signs of clinical activity was seen with MRTX1719, including objective responses in patients with MTAP loss tumors, including biliary tract cancer from the phase I/II study (NCT05245500). Despite these advances, prolonged targeted therapy poses the risk of development of acquired resistance demanding the need for better therapeutic strategies. To address this pressing need, we co-treated CCA cell lines with MRTX1719 and PARP inhibitor olaparib. We hypothesize that since PRMT5 is a critical regulator of splicing and DNA damage pathways, inhibition of PRMT5 disrupts these downstream processes creating an additional vulnerability to DNA damage inducing agents such as PARP inhibitors. Our preliminary data combining MRTX1719 with olaparib in three MTAP loss CCA cell lines (RBE, YSCCC, TFK1) show synergy (combination index; CI<1), and significant reduction in cell viability as well as colony formation ability when compared to monotherapy in vitro. To further gain mechanistic insights into the biology of MTAP loss, we stably expressed MTAP in MTAP null CCA cell line, RBE and demonstrated that MTAP loss drives tumorigenesis in vivo highlighting its tumor suppressor function in CCA. Mass spectrometry-based profiling of histone modifications from the isogenic RBE cell lines showed heightened methylation and reduced acetylation marks in the absence of MTAP. We also noted substantial differences in the distribution of splicing events, with increased intron retention, 3’ alternative splicing and decreased exon skipping as a result of MTAP loss. Similar differences in proportion of splicing events were also observed in MTAP loss CCA patient derived xenografts as compared to WT models. Collectively, these data implicate MTAP to be a crucial player in modulation of the chromatin and splicing events that may drive therapeutic response to PRMT5 inhibition. Citation Format: Pooja A. Shah, Ajay Kumar Saw, William Padron, Ming Zhao, Argun Akcakanat, Kurt Evans, Funda Meric-Bernstam, Milind Javle, Jordi Rodon, Kunal Rai. MTAP loss alters the epigenetic landscape and demonstrates superior therapeutic sensitivity to concomitant PRMT5 and PARP inhibition in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4536.

Abstract 640: In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations

Abstract p53 tumor suppressor mutations result in loss of function of p53 in approximately 50% of all human cancers.TP53 missense, nonsense mutations and p53 deletion are present in about 50%, 12% and 5% of all p53 driven cancers, respectively. To date there is no effective treatment for rescuing p53 function in cancers using a pan-p53 approach. The use of ADGN-531 is a new potent strategy combining mRNA with a tumor selective peptide nanocarrier to rescue of p53 tumor suppressor function. ADGN-531 contains proprietary p53-mRNA complexed with proprietary short amphipathic peptides in the form of nanoparticles. ADGN-531 was evaluated on 20 different cancer cell lines harboring various p53 mutations. In-vivo efficacy of IV-administered ADGN-531 nanoparticles was evaluated on colorectal SW403 (p53 nonsense/KRASG12V), osteosarcoma SaOs2 (p53 null) and NSCLC NCI-H358 (p53 null/KRASG12C) mouse xenografts at a dose of 0.25-1.0 mg/kg weekly. ADGN-531/sotorasib synergy was evaluated in vitro on NCI-H358 (p53 null/KRASG12C) and Mia-PACA (p53R48W/KRASG12C) cells and in vivo on NCI-H358 mouse xenograft. ADGN-531 markedly inhibits the growth of a large panel of cancer cells harboring p53-missense or nonsense mutations or p53-deletion, by rescuing p53 transcriptional activity and subsequent expression of its target proteins. ADGN-531 induces cell cycle arrest in G1 due to p21 upregulation and apoptosis following BAX and PUMA activation. IV-administration of ADGN-531 resulted in dose responsive tumor growth inhibition in SaOs2, SW403 and H358 xenografts independent of KRAS status. We demonstrated a strong synergy in a NSCLC H358 xenograft (tumor regression 80%) when restoring P53 function with ADGN-531 (0.5mg/kg, weekly) along with inhibition of KRASG12C mutant using sotorasib (10mg/kg, daily). ADGN-531 potentiated the effect of sotorasib on sotorasib resistant cells harboring high level of KRASG12C in permanent active state. ADGN-531 treatments are well tolerated, no sign of clinical toxicity was detected after single or repeated administrations. ADGN-531 is effective in rescuing p53 function both in vitro and in vivo across a wide range of p53 alterations. Our study provides a proof-of-concept that restoration of tumor suppressor function by ADGN-531 could be used as a single agent therapy in P53 altered tumors independent of other driver mutations e.g., KRAS, or with other therapies for potent combinatorial cancer treatment. Citation Format: Gilles Divita, Audrey Grunenberger, Elodie Czuba, Veronique Josserand, Melanie Guidetti, Neil Desai. In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 640.

Abstract 3170: Epigenetic identification and functional characterization of a novel RING finger protein tumor suppressor in nasopharyngeal carcinoma and esophageal carcinoma

Abstract RING finger proteins play crucial roles in normal physiology and pathophysiology as important regulators of ubiquitin-dependent proteasomal protein degradation. Many RING finger proteins have also been implicated in tumorigenesis either as oncogenes or tumor suppressors. Through cancer CpG methylome studies, we identified RING finger protein 150 (RNF150) as a methylated target gene in nasopharyngeal carcinoma (NPC) and esophageal carcinoma (ESCC). RNF150 was found to be ubiquitously expressed in human normal tissues but frequently downregulated in NPC and ESCC cells due to its promoter methylation. Its downregulation is associated with poor survival in patients with head and neck cancer and esophageal cancer. We also found that RNF150 is a functional tumor suppressor by inhibiting tumor cell growth and inducing cell cycle arrest in NPC and ESCC cells. Furthermore, ectopic expression of RNF150 inhibited NPC and ESCC cell migration/invasion by suppressing epithelial-mesenchymal transition (EMT) through upregulation of epithelial marker E-cadherin and downregulation of mesenchymal markers (vimentin, N-cadherin, Snail, and Slug). RNF150 is located in the cytoplasm and represses several oncogenic signaling pathways including the Wnt/β-catenin and NF-kB signaling pathways. Liquid chromatography-mass spectrometry (LC-MS) was performed to elucidate RNF150 binding proteins. KEGG enrichment analysis showed that glycolysis/gluconeogenesis, pyruvate metabolism, central carbon metabolism in cancer, and AMPK signaling were the top 4 pathways with significant enrichment, suggesting potential targeting of metabolic molecules by RNF150. Thus, our results provide insights into a new ring finger protein member, RNF150, as a functional tumor suppressor for NPC and ESCC. Our study also suggests a potential epigenetic biomarker for the early dection of NPC and ESCC. Citation Format: Xiaoxue Chai, Feifei Luo, Shujie Bao, Liuzhen Meng, Anthony T. Chan, Brigette B. MA, Qian Tao, li LiLi. Epigenetic identification and functional characterization of a novel RING finger protein tumor suppressor in nasopharyngeal carcinoma and esophageal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3170.

Abstract 3025: Germline p53 R181 variants and DNA binding cooperativity in tumorigenesis

Abstract TP53 is the most frequently mutated gene in cancer, and its encoded protein p53 has many tumor-suppressive functions. There are several different classes of mutant p53 acquired in human cancer and inherited in cancer-prone families with Li-Fraumeni Syndrome, where individuals have an 80-90% increased risk of cancer. These include structural mutations that are generally (e.g R175H) or locally (e.g Y220C) misfolded, DNA contact mutations (e.g R273H); and oligomerization mutations (A347D). Cooperativity mutations affect binding of the p53 tetramer to DNA via disruption of a salt bridge formed by the negatively charged glutamic acid (E) 180 residue of one p53 monomer and the positively charged arginine (R) 181 residue of another. p53 “cooperativity” mutations at the R181 residue (R181H and R181C) have been increasingly identified in cancer-prone families undergoing genetic testing; however, the mechanism by which these variants disrupt p53 tumor suppression in humans is not understood. We show that the purified DNA binding domains of p53 variants R181H and R181C bind less cooperatively to the p53 binding site in the CDKN1A (p21) promoter, despite retaining wild-type levels of structural stability. RNA-sequencing of CRISPR knock-in colorectal and breast cancer cell lines shows reduced ability of R181H and R181C to transactivate a curated set of ~300 known p53 target genes. Upon treatment with p53 activating molecule Nutlin-3a, the R181-mutant cells fail to cell cycle arrest in the G1 phase and maintain high proliferative rates. Interestingly, we observe some residual apoptotic activity in R181H and R181C mutant cells treated with DNA-damaging agent 5-fluorouracil, despite losing the transactivation of proapoptotic p53 targets; this suggests that these mutants retain the p53 transcription-independent mechanism of apoptosis which is observed in other p53 variants such as P47S and A347D. These studies will define the pathogenicity of R181 variants and guide therapeutic intervention for patients that harbor these p53 mutations. Citation Format: Renyta Moses, Ryan Hausler, Gregory Kelly, Alexandra Indeglia, Sven Miller, John Karanicolas, Maureen Murphy, Kara Maxwell. Germline p53 R181 variants and DNA binding cooperativity in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3025.

Abstract 1996: Novel mechanism of temozolomide resistance in glioblastoma

Abstract Intrinsic expression of PD-1 has been discovered in different types of tumurs, exhibiting either oncogenic or tumor-suppressing function. In glioblastoma (GBM), intrinsic PD-1 has been found to play a role in promoting the proliferation and self-renewal of brain tumor-initiating cells through nuclear factor κB without involving PD-L1 ligation, with unknown factors activating this signalling pathway. Meanwhile, TMZ resistance significantly contributes to treatment failure and an extremely poor prognosis for GBM. This study investigated whether intrinsic PD-1 is also expressed in differentiated human GBM cell lines and its role in GBM proliferation and temozolomide (TMZ) resistance. In vitro studies demonstrated the presence of PD-1 in both U87 and U251 TMZ-sensitive and -resistant cell lines, with a higher expression on TMZ-resistant cell lines. PDCD1 knockdown led to decreased GBM progression, including cell proliferation, colony formation and cell migration, in TMZ-resistant U87 and U251 cell lines using cell viability assays. It also resensitizes these cell lines to TMZ. Overexpression of PDCD1 in the PDCD1-knockdown cell lines increases their proliferation and restores their resistance to TMZ. Treatment of U87 and U251 TMZ-resistant cell lines with TMZ upregulated PD-1 expression. An orthotopic xenograft mouse model is further deployed by injecting previously cultured U87 and U251 cell lines into the mouse brains. A smaller tumour size is observed in the model with PDCD1 knockdown model than the one without PDCD1 knockdown when treated with TMZ. This study reveals that intrinsic PD-1 expression contributes to TMZ resistance in GBM and TMZ may be a factor activating the PD-L1-independent PD-1 signalling. PD-1 and its downstream signalling pathway, such as SHP-2 and IKK, may be potential therapeutic targets for GBM patients with TMZ resistance. Co-administration of TMZ and agents inhibiting the intrinsic PD-1 signalling pathway can potentially produce synergistic therapeutic effects. The higher expression of PD-1 on TMZ-resistant GBM than on TMZ-sensitive GBM also suggests that the intrinsic PD-1 level can be a predictor of TMZ outcome. Citation Format: Yuet Yi Charmaine Hung, Mei Yee Karrie Kiang, Ka Kit Gilberto Leung. Novel mechanism of temozolomide resistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1996.

Abstract 3027: p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas

Abstract High-Grade Serous Ovarian Cancer (HGSOC) is the most common and aggressive gynecological malignancy. It is typically diagnosed in late stages due to lack of diagnostic tests and current therapies available are not efficacious when the tumor is advanced. HGSOC is characterized by ubiquitous loss of functional p53 tumor suppressor protein function, largely due to point mutations that arise very early in carcinogenesis. P53 mutations are detected in pre-neoplastic cells of the fallopian tube mucosa, called p53 signature, and in early precursor lesions called serous tubal intraepithelial carcinoma (STIC). Some mutations promote the misfolding and aggregation of p53, transforming the protein into a cancer-promoting oncogene. Here, we hypothesize that the transition from folded, soluble to aggregated mutant p53 underlies the transformation from a pre-neoplastic lesion (p53 signature, STILs), to STICs and eventually HGSOC. We first set to determine the proportion of cells carrying soluble vs misfolded mutant p53 in fallopian tube tissues collected from n=10 patients undergoing salpingo-oophorectomy. We performed conformation-sensitive staining and quantification of folded and unfolded p53 cases and demonstrated that p53 misfolding, a mandatory precursor to aggregation, is present in STICs and HGSOCs, but notably absent from p53 signatures and surrounding healthy tissue. To better characterize the role of aggregated p53 in the progression of HGSOC, we took advantage of primary human fallopian tube secretory epithelial cells (FTSEC) that mimic early and more advanced stages of carcinogenesis. We utilize the FTSEC cells line FT282, immortalized by introducing hTERT and the aggregation-prone mutant TP53R175H that is found in p53 signature lesions and STILs, as well as the FT282-CCNE1, derived from FT282 cell line with overexpression of CCNE1, representing a more malignant phenotype.We used these models to investigate p53 aggregation, characterize the mutant p53 interactome and test the efficacy of ReACp53, a p53 aggregation-targeting peptide we developed. Our results demonstrated that p53 aggregation is more severe in the FT282-CCNE1 line, with a higher susceptibility to ReACp53 compared to the FT282 cells. Overall, our results demonstrated that aggregation of mutant p53 is a structural defect that can distinguish between pre-malignant and malignant HGSOC lesions and can potentially be targeted, offering a potential window for early intervention in halting ovarian cancer progression. Citation Format: Sara Sartini, Lexi Omholt, Neda A. Moatamed, Alice Soragni. p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3027.

Abstract 5506: HOST-Factor: A comprehensive platform for single-cell highplex immunofluorescence staining, digital imaging, spatial mapping, and quantitative analysis of the tumor microenvironment

Abstract Solid tumor complexity extends beyond the genetic and functional landscapes of heterogenous cancer cells, encompassing the tumor microenvironment (TME). Elucidating the TME’s complexity requires a comprehensive assessment of its cellular composition, functional states, and spatial distributions. We developed the Harmonic Output of Stromal Traits (HOST) to identify TME cells, and the HOST-Factor to quantify their functional states. The HOST-Factor is a numerical value that reflects the relative contribution of cancer-associated fibroblasts (CAFs) to tumor-suppressive or tumor-promoting functions. Our workflow combines automated cycling highplex immunofluorescent microscopy with artificial intelligence (AI)-guided image analysis. This generates HOST-Factor values for each identified TME cell within selected regions of interest, providing spatial distribution data. The TME signature encompasses 15 immune cells and 14 CAF antibody-detected biomarkers. We applied our workflow to ten human pancreatic cancer specimens, generating OME-TIFF output images. This cancer model was used due to its significant TME makeup. The 29 highplex AI-based digital image analysis was conducted using the Phenoplex™ workflow from Visiopharm. The workflow included deep-learning-based tissue morphologic and cellular segmentations, cellular phenotyping, and integration of spatial/location data. Biomarker subsets were visualized, and a user-trained algorithm was used for tissue segmentation. Nuclear segmentation was done using a pre-trained algorithm on a DAPI-labeled DNA channel. Cellular phenotyping was performed using thresholds based on visual assessment and confirmation of positivity. Spatial neighborhood plots and metrics, heatmaps and partitioned t-SNE plots were generated for the dataset for downstream analysis. Importantly, the workflow's visualization templates, pre-trained nuclear/cytoplasm segmentation tools, and neighborhood plots and metrics, are reusable and fully customizable for new datasets. Using HOST-Factor values, we successfully classified cancer and TME cells, along with their functional states, and spatial distributions. This AI-based computational approach and user-friendly workflow provides a simple and effective way to obtain single-cell information from multiplex immunofluorescence images, making spatial phenotyping of several cell populations in tissues more accessible to researchers, providing a fully amendable means for future clinical translation. Citation Format: Janusz Franco-Barraza, Fabian Schneider, Rasmus Norré-Sørensen, Rasmus Ahrenkiel-Lyngby, Caneta Brown, Dan Winkowski, James Robert Mansfield, Edna Cukierman. HOST-Factor: A comprehensive platform for single-cell highplex immunofluorescence staining, digital imaging, spatial mapping, and quantitative analysis of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5506.

Abstract 2509: Abundant glucocorticoid receptor (GR) expression in human tumors: A tissue microarray study on more more than 18,000 tumors

Abstract The glucocorticoid receptor (GR, or GCR) is a nuclear receptor protein for cortisol and other glucocorticoids and regulates the transcription of thousands of genes involved in metabolism, development, stress, and inflammatory response. In cancer, both an oncogenic and a tumor suppressive function of GR has been proposed. To comprehensively evaluate GR expression in normal and tumorous tissues, a tissue microarray containing 18,570 samples from 147 different tumor types and subtypes and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry (IHC). GR positivity was found in 76.3% of 14,349 interpretable cancers, including 18.5% with weak, 19.6% with moderate, and 38.3% with strong positivity. GR positivity of at least a fraction of tumors was found in all 147 analyzed tumor types and at least one strongly GR positive tumor was found in 136 tumor types. 77 of our 147 tumor entities showed GR positivity of variable intensity in all analyzed cases. 43 further tumor entities showed GR positivity in 80-99.9% of cases. 21 tumor entities had a GR positivity rate between 50 and 80%, including urothelial carcinoma as well as gastric and esophageal adenocarcinoma. Only 6 tumor types had less than 50% GR positive. These included adenomas (21.7-32.5%), adenocarcinomas (17%) and neuroendocrine carcinomas (45.5%) of the colorectum. Reduced GR staining was significantly associated with adverse histopathological and clinical features in several tumor types. Low GR expression was linked to grade progression in pTa (p<0.0001) and to nodal metastasis in pT2-4 urothelial carcinoma of the urinary bladder (p=0.0051), advanced pT stage (p=0.0006) and estrogen receptor positivity (p=0.0126) in breast carcinomas of no special type (NST), as well as to high grade (p≤0.0076), advanced pT stage (p<0.0001), distant metastasis (p=0.0081), and poor overall survival (p=0.002) in clear cell renal cell carcinoma. GR expression was unrelated to clinic-pathological parameters in gastric, pancreatic, and colorectal adenocarcinoma as well as in serous high-grade carcinoma of the ovary. In summary, our data demonstrate frequent GR expression across all cancer types. Significant associations between reduced GR expression and unfavorable tumor features in some but not all tumor types suggest that the functional importance of GR regulated genes for cancer progression depends on the cell of tumor origin. Citation Format: Christina Tsourlakis, Frederike Wegener, Katharina Möller, Martina Kluth, Claudia Hube-Magg, Christian Bernreuther, Guido Sauter, Andreas H. Marx, Ronald Simon, Till Krech, Christoph Fraune, Natalia Gorbokon, Maximilian Lennartz, Sarah Minner, Florian Viehweger. Abundant glucocorticoid receptor (GR) expression in human tumors: A tissue microarray study on more more than 18,000 tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2509.

Abstract 5550: Crosstalk of estrogen receptor Β with tumor microenvironment signaling explains tumor suppressor actions in TNBC

Abstract Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer lacking molecular targets, possessing one of the poorest prognoses of all breast cancers. Recent studies using breast tumor samples show an association between the expression of estrogen receptor β (ERβ) and improved survival of patients with TNBC. ERβ - whose function largely opposes that of oncogenic ERα - is involved in the regulation of many tumor repressive pathways and has lately been observed to partake in immune modulation, thus presenting a promising target for TNBC. Discovering selective ERβ agonists is critical to understanding the potential of activating the receptor as a therapy approach for TNBC. It enables us to elicit estrogen signaling response specific to the tumor suppressive function of the β receptor subtype. We have set out a screening process to test and identify ligands that display the greatest activation and specificity for ERβ as determined by reporter assays and changes in downstream target gene expression. We selected to study in vitro and in vivo the compounds with optimal biological activity to delineate tumor-intrinsic and -extrinsic effects and validate the mechanism of action employing human and mouse cell models that reflect the heterogeneity of the disease. We propose that activation of ERβ decreases the proliferative and metastatic ability of breast cancer cells by altering cellular pathways and crosstalk with immune cells. The results of these studies help lay the groundwork for further research on the clinical potential of ERβ especially in the realm of immunotherapy and treatment of TNBC. Citation Format: Kristina Diana A. Zambo, Harika Nagandla, Kim Cuong Cap, Aireana Phillips, Fotis Nikolos, Wei Qian, Jianying Zhou, Jenny Chang, Christoforos Thomas. Crosstalk of estrogen receptor Β with tumor microenvironment signaling explains tumor suppressor actions in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5550.

Abstract 5628: Aberrant 3'UTR processing regulates a tumor suppressor network in AML

Abstract Differentiation blockade is a hallmark of acute myeloid leukemia (AML). While inactivating mutations in tumor suppressor genes (TSGs) have been well-studied, non-genetic mechanisms that impair TSGs to promote myeloid differentiation and influence drug response remain poorly understood. In this study, we performed genome-wide loss-of-function CRISPR/Cas9 screens to identify suppressors of myeloid differentiation. These screens converged on the zinc finger transcriptional repressor, ZBTB7A whose ablation restricted myeloid maturation and promoted a dysregulated inflammatory state to protect leukemic cells from undergoing differentiation. Moreover, genetic deletion of ZBTB7A or its paralog, ZBTB7B impeded leukemia differentiation and conferred resistance to differentiation-inducing agents. Mechanistically, we found that leukemia cells hijack 3’UTR processes to reduce mRNA expression of the tumor suppressor gene paralogs ZBTB7A and ZBTB7B. These findings provide a molecular framework of altered 3’UTR regulation that orchestrate a tumor suppressive function and promote resistance to AML differentiation-based therapies. Citation Format: Eric Wang, Cuijuan Han, Alexander Calderon, Abimbo Lawal, Sakthi Harini Rajendran, Sadik Karma, Nidhi Hariramani, Iannis Aifantis, Aristotelis Tsirigos. Aberrant 3'UTR processing regulates a tumor suppressor network in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5628.

Abstract 2635: An epigenetic regulation targeting inhibitory receptors and attenuates CD8 T cell exhaustion in breast cancer

Abstract T cell exhaustion is a state of dysfunctional T cell response characterized by prolonged antigen exposure and diminished effector functions, a significant cause of tumor tolerance, and poor response to immune checkpoint blockade (ICB) therapy. T cell exhaustion is a highly dynamic process involved with a network of genetic and epigenetic regulation that program the T cell functions. Epigenetic regulation by microRNAs targets multiple genes involved in T cell function and is expected to be a major contributor to the functional programming of tumor-associated T cells. However, the underlying causal mechanisms remain to be determined. Here, we show that the microRNA miR-379-5p is a critical epigenetic regulator for promoting T cell function and suppressing exhaustion induced by chronic stimulation. The microRNA is identified by integrating bioinformatic interrogation of the miRNA databases cross-referencing with the TCGA database for genes involved in T cell functioning and their correlation with clinical outcomes, coupled with molecular profiling of miRNAs in experimental T exhaustion. Notably, the expression levels of miR-379-5p in tumor tissues, which encompass cancer cells and the surrounding stromal components, including immune cells, are closely associated with favorable prognosis of various cancer types. Further molecular and cellular characterization of miR-379-5p entailing cell culture, animal models, and patient-derived tumor organoids shows that it exerts suppressive effects by targeting specific sequences in the 3’ untranslated region (UTR) of critical immune checkpoint genes in T cells, thereby enhances the effector functions of cytotoxic T cells for tumor clearance. Ectopic expression of miR-379-5p shifts the evolution of primary T cells toward memory-like effector cells while silencing miR-379-5p induces exhaustion and abrogates the cytotoxic activity of primary T cells. We further demonstrate that transcriptional downregulation of miR-379-5p in T cells by a nuclear receptor transcription factor which is responsible for initiating the exhausting process impairs the tumor suppression function of T cells. Our studies indicate that miR-379-5p can function as a tumor suppressor by protecting T cells from exhaustion and bears the potential for developing novel strategies to improve the response to ICB therapy in breast cancer. Citation Format: You-Zhe Lin, Wan-Rong Wu, Feng-Chi Chung, Yi-Chun Shen, Chuan-Chun Lee, Hong-Wei Li, Guang-Jan Lin, Wei-Chung Chengx, Shao-Chun Wang. An epigenetic regulation targeting inhibitory receptors and attenuates CD8 T cell exhaustion in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2635.

Abstract 634: Tuba-seq and CRISPR/Cas9 analysis of TBX2 subfamily genes: Exploring contextual tumor suppression in lung adenocarcinoma

Abstract Lung adenocarcinoma (LUAD) is a multifaceted and genetically diverse cancer, shaped by a myriad of oncogenic and tumor suppressive events. Through recent clinical and in vitro investigations, we have discovered that the TBX2 subfamily of genes, encompassing TBX2, TBX3, TBX4, and TBX5, are linked to LUAD progression. Specifically, we have observed marked downregulation of their expression in clinical LUAD specimens, and reduced growth in LUAD cell lines when the subfamily is re-activated- hinting at a potential tumor suppressive function. Paradoxically, the role of TBX2 subfamily genes in other cancer types remains enigmatic, with varying reports of both oncogenic and tumor suppressive effects. In this study, we harnessed the power of Tumor Barcoding with Ultradeep Barcode Sequencing technology (Tuba-seq) and CRISPR/Cas9-mediated genome editing to systematically investigate the effects of knocking-out Tbx genes and associated partners EGR1, CHD2, A20 and ATF3 in LUAD preclinical models. We investigated their effects in both normal lung epithelium and Ras-driven lung tumors via in situ gene editing and assessed tumor burden via histology and targeted DNA sequencing of tumor barcodes. TBX2 subfamily gene losses appear to have a profound effect on tumor initiation and early growth, which somewhat attenuates with progression. In Ras-driven tumors, we found that these genes are moderately tumor suppressive when measuring mean growth rate, although these effects (sans Egr1 loss) are more pronounced at 6 weeks of growth than at 20 weeks. Strikingly, however, we observed an unprecedented increase in total tumor burden when deleting these genes in genomically-normal (Kras w.t.) cells, including 4-20+ fold increase in Tbx2, Tbx3, Tbx4, and Tnfaip3-deficient cell lineages 20-week following transduction. Increases in total lineage burden of this magnitude are typically only seen for frequently-deleted hallmark tumor suppressor genes such as Tp53, Stk11, or Pten. This increased burden, however, seems to primarily affect early-stage progression, as no appreciable increases in mean lineage size relative to w.t. lineages are observed after 40 weeks of growth. Our study reveals the context-specific functions of the TBX2 subfamily while introducing the first in vivo model for TBX2-driven LUADs. This model facilitates the development of targeted therapeutic strategies and encourages accurate tumor modeling across various progression stages and diverse genetic backgrounds. Citation Format: Athar Khalil, Jaff Maltas, Mira Rahm, Zachary Faber, Madeline Bedrock, Xiangzhen Wei, Bindi Patel, Christopher McFarland. Tuba-seq and CRISPR/Cas9 analysis of TBX2 subfamily genes: Exploring contextual tumor suppression in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 634.

Abstract 1262: Comparative analysis of TP53 alleles in pancreatic ductal adenocarcinoma

Abstract The transcription factor p53 is a tumor suppressor capable of inducing cell cycle arrest, senescence, and apoptosis. Both truncating loss-of-function mutations and point mutations of the TP53 gene (mouse gene Trp53), which encodes for the p53 protein, are frequently observed across human cancers. While point mutations in p53 result in loss of canonical tumor suppressor function, recent studies suggest that these mutated proteins acquire pro-tumorigenic gain-of-function properties. For example, Trp53R172H mutations (the murine homolog of TP53R175H, the most common human TP53 variant) enhance metastatic spread relative to deletion mutations in mouse models of advanced pancreatic ductal adenocarcinoma (PDAC). To further explore the differential functions of p53 point and deletion mutants in cancer progression, we performed a rigorous comparative analysis of Trp53 alleles in vivo using Mosaic Analysis with Double Markers (MADM) in mice. MADM uses stochastic mitotic recombination to induce two genotypically distinct daughter cells - created at 1-to-1 ratios - expressing different Trp53 variants dependent on the genotype of the mouse (+/+ vs. -/-, R172H/R172H vs. +/+, R172H/R172H vs. -/-) and simultaneously label them with unique genetically encoded fluorescent markers (TdTomato vs. GFP). We integrated MADM into a faithful Kras-driven model of pancreatic tumorigenesis and used fluorescence microscopy to trace subclonal populations with various p53 alleles within the same mouse. By analyzing the ratio of TdTomato+ to GFP+ cells, we studied the functional differences between alleles in driving pancreatic cancer at various stages of progression. We found that Trp53−/− cells exhibited greater tumor initiation of preinvasive pancreatic intraepithelial neoplasia (PanINs) and early PanIN cell expansion compared to Trp53+/+ cells. Surprisingly, Trp53R172H/R172H cells were comparable in number to Trp53+/+ cells in early PanINs but were outcompeted by Trp53−/− cells.In contrast, both Trp53R172H/R172H and Trp53−/− cells were capable of facilitating the transition to advanced PDAC with comparable survival times. These phenotypic differences imply that p53R172H retains tumor suppressive properties in early pancreatic tumorigenesis, but similar to the loss of p53, it is incapable of constraining progression to advanced disease, arguing that p53R172H is a separation of function mutation in vivo. Ongoing studies examining the molecular differences between Trp53 alleles by fluorescence-activated cell sorting and RNA-sequencing will shed further light onto the underlying mechanisms that explain these phenotypic differences. Citation Format: Andy Tang, Sherry Agabiti, Hannah Chung, Mandar Deepak Muzumdar. Comparative analysis of TP53 alleles in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1262.

Abstract 5682: Micro RNA level changes in prostate cancer cells treated with MDMX and MDM2 inhibitors

Abstract INTRODUCTION: Prostate Cancer (PCa) is the most common non-cutaneous cancer and the second leading cause of cancer death among men in the United States. Most of the PCa cases are diagnosed in men between the ages of 65-74. Due to the recent push for frequent prostate screenings, these cancers are being detected early and treated much faster, leading to a 5-year survival of a localized or regional PCa to >99%. However, the 5-year survival of PCa with distant metastasis is significantly reduced to 32%, especially with involvement of osteoblastic bone lesions, lung lesions, and brain lesions. Therefore, there is a clinical need to identify prognostic markers and new targets for developing therapies and effectively treating advanced PCa. Previous studies have shown that levels of MDMX and MDM2, oncogenic inhibitors of p53 tumor suppressor protein, are significantly elevated in PCa cells. Inhibition of MDM2 and MDMX using specific inhibitors has been shown to elevate the levels of p21, p27, and p53 in LNCaP (Lymph Node Carcinoma of the Prostate) cells leading to cell death. Recent studies have demonstrated that micro RNAs (miRNAs) play an important role in supporting the tumor suppressor function of p53 through regulating the balance between p53 and MDM2. In this regard, several miRNAs were found to be significantly altered to modulate the level and intracellular mechanisms of p53 and MDM2 in various cancers. OBJECTIVES: To determine the relative levels of miRNA31, miRNA-34a and miRNA-128 in LNCaP cells that were treated with NSC-207895 (MDMX inhibitor), SJ-172550 (MDMX inhibitor), and RG-7388 (MDM2 inhibitor). METHODS: LNCaP cells were treated with NSC-207895 (20 uM), SJ-172550 (20 uM) or RG-7388 (2 uM) for 24 hrs. After completing the treatments, the miRNAs were extracted from the LNCaP cells using the miRNeasy Tissue/Cells Advanced Kit from Qiagen. We analyzed the levels of miRNA-31, miRNA-34a, and miRNA-128 in the treated samples using qRT-PCR analysis method and compared the levels to the control. During the qRT-PCR analysis the cDNA was amplified using Meridian Biosciences qPCR Master Mix and specific primers for miRNAs from Origene were used according to the manufacturer's protocol. RESULTS: Our experimental results indicate that inhibition of MDMX using NSC-207895 and SJ-172550 can elevate the levels of p21, p27, and p53 leading to cell death. Determination of cell death markers in the past revealed induction of necroptosis following drug treatments. Our current results indicate measurable changes in the levels of miRNA-31 and miRNA-34a following inhibition of MDMX using SJ-172550. We are in the process of determining the possible link between the changes in the levels of miRNAs and cell death. ACKNOWLEDGEMENT: This research was supported by the Royal Dames of Cancer Research, Ft. Lauderdale, Florida. Citation Format: Rojin Fatirkhorani, Ahmed Saleh Alsarrani, Shyam Sundar Jaganathan, Umamaheswari Natarajan, Appu Rathinavelu. Micro RNA level changes in prostate cancer cells treated with MDMX and MDM2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5682.

Abstract 2751: Functional assessment of STK11 C-terminal domain variants

Abstract STK11 mutations in KRAS-driven lung adenocarcinomas (LUADs) are associated with aggressive tumor phenotypes characterized by increased risk of metastasis and lower overall and progression free survival. STK11 is a serine-threonine kinase that is comprised of an N-terminal domain containing a nuclear localization signal, a kinase domain, and a C-terminal domain (CTD). In LUAD, STK11 loss of function impacts many aspects of coordinated cell motility and promotes alterations characteristic of metastasis including cell polarity and adhesion. Both kinase activity and subcellular localization are important for STK11 tumor suppressor function. Our objective was to develop a reliable method to identify pathogenic STK11 mutations. Using archival data from the University of Vermont Medical Center, we identified 28 STK11 missense variants that had not yet been functionally characterized. We evaluated the functional impact of these variants on STK11 kinase activity through detection of autophosphorylation using an in-vitro kinase assay, as well as their ability to induce p53 activity through a luciferase reporter. Of the variants studied, 5 were CTD mutations. These variants retained kinase activity and the ability to bind STK11’s partners STRAD-alpha and MO25 in co-immunoprecipitation assays. Although the CTD of STK11 has no catalytic activity, it is thought to be integral for translocation to the cytoplasm where it can colocalize with actin and the cell membrane. STK11 dependent regulation of directional migration requires both proper localization to the actin cytoskeleton and kinase activity. Moreover, it has been reported that the C-terminal polybasic motif of STK11 (aa403-426), consisting of 3 Lysine and 5 Arginine residues is key for its localization and activation of AMPK at the plasma membrane. Therefore, when ascertaining functional status of STK11 in vivo, it is important to consider kinase independent STK11 functions. In LUAD cell lines, we show through immunofluorescent microscopy that expression of a truncated construct encoding STK11 with a CTD deletion results in nuclear sequestration. Additionally, point mutants in 3 of the polybasic motif residues (R409W, K416E and K423E) retain kinase activity and STRAD-alpha binding, but also lead to nuclear sequestration - highlighting the importance of a multipronged approach when assessing STK11 function. Future studies will assess the ability of these C-terminal domain mutants to phosphorylate cytoplasmic substrates, and their impact on cell migration and invasion. Additionally, we will utilize the TurboID proximity ligation system to assess the effect of C-terminal domain point mutations on binding partner accessibility. Citation Format: Gopika Nandagopal, Sean Lenahan, Hannah Ross, Hailey Sarausky, David Seward, Paula Deming. Functional assessment of STK11 C-terminal domain variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2751.

Abstract 7237: Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy

Abstract BRCA1 and BRCA-2 proteins are involved in the repair of DNA damage such as double-strand breaks during S and G2 phases, by intervening in homologous recombination (HR) steps. Breast cancers arising from BRCA-1 mutation carriers (10% of BC) and from epigenetically silenced BRCA-1 (30% of sporadic BC) are associated with a lack of expression and/or function of BRCA-1, which induces genomic instability. Herein, we describe a new potent strategy combining mRNAs with a tumor selective peptide nanocarrier, to selectively rescue BRCA-1 function as potential therapeutic approach in cancers. Proprietary BRCA-1 derived mRNA were complexed with proprietary short amphipathic peptides and the resulting nanoparticles were evaluated on several breast cancer cells, harboring different BRCA-1 alterations. Sensitivity to Veliparib (PARPi) following the nanoparticle treatments were evaluated on PARPI resistant and PARPi sensitive cells. The recruitment of RAD51 to sites of double stranded DNA break was quantified by immunofluorescence. In-vivo efficacy of varying sequences of BRCA-1 mRNA-peptide nanoparticles (2.0 mg/kg, weekly, IV; ADGN-901, -902, -903) and veliparib (50mg/kg, daily, oral) were evaluated on breast cancer (HCC1937-BRCA-1 deficient, PARPi resistant) mouse xenografts. We showed that ADGN-901 inhibits the proliferation of BRCA-1 deficient cell lines from 20 to 60% depending on the type of BRCA-1 alterations. ADGN-902, specifically reduces the proliferation of cells expressing N-terminal truncated BRCA-1, by 50 to 60% and ADGN-903 inhibits proliferation >60% of all cells harboring defects in BRCA-1. Interestingly, ADGN-901 and ADGN-903 were able to resensitize the PARPi resistant cells to veliparib inhibition. We demonstrated that ADGN-903 restores BRCA-1 function in homologous recombination. ADGN-903 prevents RAD51 recruitment to the damage site by reducing RAD51 protein level as well as formation of RAD51 foci following irradiation. IV administration of ADGN-901 and ADGN-903 (2.0 mg/kg) had a strong inhibitory effect on HCC-1937 tumor growth while ADGN-902 and veliparib were inactive. ADGN-901 reduced tumor growth by 95% and ADGN-903 abolished tumor growth with strong tumor regressions. ADGN-901 and ADGN-903, were also evaluated on large tumors of about 500-600 mm3 and were highly effective in preventing further progression and achieving shrinkage of large tumors. ADGN-901 and ADGN-903 treatments are well tolerated, without inducing clinical toxicity. BRCA-1 mRNA-peptide nanoparticles are effective in rescuing BRCA-1 function and inducing tumor regressions in BRCA-1 altered breast cancer cells and tumors. Our study provides a proof-of-concept that restoration of wild type BRCA-1 expression could be a potent therapeutic strategy for BRCA-1 altered tumors whether or not they are PARPi resistant. Citation Format: Gilles Divita, Audrey Grunenberger, Léa Cabrera, Nathalie Durany, Neil Desai. Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7237.