Mutant p53R172H Research Articles

Abstract C064: Mapping cellular plasticity through the lens of FRA1 during Kras G12D mediated pancreatic acinar to ductal metaplasia

Abstract Introduction: Acinar cells have been proposed as a cell-of-origin for pancreatic ductal adenocarcinoma (PDAC) after undergoing acinar to ductal metaplasia (ADM). ADM can be triggered by pancreatitis causing acinar cells to de-differentiate to a ductal-like state. We identify FRA1 (gene name Fosl1) as the most active transcription factor during Kras G12D acute pancreatitis-mediated injury. Herein, we explore the role of the FRA1 in the onset of ADM and neoplastic transformation. Methods: We generated tamoxifen (TAM)-inducible Ptf1aCre ERT ;Kras G12D ; Rosa26 YFP/YFP (TAM-KCY Fosl1 WT ) mice and bred in the Fosl1 fl/fl allele to create TAM- KCY Fosl1 KO mice and subjected them to caerulin (CCK analog) induced acute pancreatitis. First, we performed a time course of hematoxylin and eosin (H&E) images from mice either with Fosl1 WT or Fosl1 KO after induction of acute pancreatitis to understand the functional role of FRA1 during acinar de-differentiation. Next, after snap-freezing pancreata at the Day 5 post-caerulein timepoint and extracting nuclei, we sorted YFP+ nuclei for snATAC sequencing and bulk-RNA sequencing. We developed a pseudotime model and gene-regulatory network (GRN) to understand the dynamic landscape as cells transitioned from normal acinar to late ADM. Finally, to determine the functional role of in tumorigenesis, we bred an inducible mutant p53 R172H gain- of-function allele into TAM-KCY Fosl1 WT/KO mice to create TAM-KPCY Fosl1 WT/KO mice. These mice were subjected to the same tamoxifen and acute pancreatitis protocol as TAM-KCY Fosl1 WT/KO mice and monitored for tumor growth and overall survival. Results: A time course of H&E images demonstrated that FRA1 depletion slowed progression to maximum ADM in the first 5 days after caerulein treatment. We additionally observed a dramatic reduction in the amount of PanIN lesions formed between TAM-KCY Fosl1 WT and TAM-KCY Fosl KO mice, which occurred on day 7 in this caerulein model. Pseudotime analysis proposed a time point when FRA1 becomes active to prime ADM cells for recovery from inflammation and simultaneously undergo PanIN transformation. Furthermore, GRN analysis validated the differentially active transcription factors identified by pseudotime analysis of snATAC-seq. To elaborate on further timepoints, we observed a delay in the progression of TAM-KCY Fosl KO PanIN lesions at later Day 21, 8-week and 12-week timepoints. In the survival analysis, TAM-KPCY Fosl1 WT mice had a median survival time of 25.86 weeks, while TAM-KPCY Fosl1 KO mice survived for almost twice as long with a median survival time of 40.57 weeks. Histologically, the percentage of Ki-67- positive cells in TAM-KPCY Fosl1 WT tumors was significantly higher than in TAM-KPCY Fosl1 KO tumors, suggesting the loss of FRA1 reduce tumor aggressiveness. Conclusions: Our findings reveal that FRA1 is a critical mediator of ADM progression to PanIN and PDAC. Fosl1 genetic depletion can alter the kinetics of ADM formation and lead to a decrease in PanIN and eventual malignant transformation. Citation Format: Alina Li, Noriyuki Nishiwaki, Kensuke Sugiura, Kensuke Suzuki, Constanza Nicole Tapia Contreras, Dorsay Sadeghian, Anirban Maitra, Jason R Pitarresi, Rohit Chandwani, Peter A Sims, Anil K Rustgi. Mapping cellular plasticity through the lens of FRA1 during Kras G12D mediated pancreatic acinar to ductal metaplasia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C064.

Proteasome inhibition paradoxically degrades gain-of-function mutant p53 R273H in NSCLC and could have therapeutic implications.

Lung cancer is the leading cause of cancer mortality. Despite therapeutic advances in recent years, new treatment strategies are needed to improve outcomes of lung cancer patients. Mutant p53 is prevalent in lung cancers and drives several hallmarks of cancer through a gain-of-function oncogenic program, and often predicts a poorer prognosis. The oncogenicity of mutant p53 is related to its stability and accumulation in cells by evading degradation by the proteasome. Therefore, destabilization of mutant p53 has been sought as a therapeutic strategy, but so far without clinical success. In this study, we report that proteasome inhibition results in degradation of mutant p53 in non-small cell lung cancer (NSCLC) cell lines bearing the R273H mutant protein and show evidence that this was mediated by hsp70. NSCLC cell lines with the mutant R273H allele demonstrated increased susceptibility and apoptosis to proteasome inhibitors. These data suggest that proteasome inhibitors could have therapeutic implications in some subsets of TP53 mutated NSCLC.

Abstract 1270: p53-R172H mutation confers gain-of-function properties and promotes metastasis in squamous cell carcinoma

Abstract INTRODUCTION: Metastasis is associated with more than 90% of cancer-related mortality, and thus, there is a compelling need for innovative therapeutic breakthroughs. TP53 mutations are present in 60-70% of human cancers, especially in squamous cell cancers. For example, TP53 mutations are detected in up to 80% of esophageal squamous cell carcinomas (ESCCs). Accumulating evidence suggests that certain missense mutant forms of p53 such as R175 (R172 in mouse), R273 and R282 can acquire neomorphic pro-oncogenic activities that are referred to as gain-of-function (GOF). To elucidate novel mutant p53-dependent mechanisms in promoting metastasis, we conducted RNA-Seq, p53 ChIP-Seq and H3K27ac CUT&RUN-Seq on primary and metastatic tumor cells harvested from our mouse model of ESCC harboring Trp53R172H/−, Trp53−/− and Trp53+/+. Herein, we have identified p53-R172H gene targets in metastatic ESCC. One of the dominant effectors of p53-R172H-mediated metastasis was Colony stimulating factor-1 (Csf-1) signaling through its cognate receptor (Csf-1r), which we recently published in Cancer Discovery (PMID: 37676642). Overall, this study aims to investigate the GOF properties and mediators of mutant p53 in promoting ESCC metastasis. RESULTS/DISCUSSION: Using ex vivo cultures and in vivo tail-vein injections, we demonstrated increased colonization and metastatic capabilities of ESCC cells harboring p53-R172H in comparison to the tumor cells with null and wild-type p53. p53-R172H occupies more genes in the metastatic ESCCs than in the primary tumors that is independent of p53 expression levels. We identified 72 unique targets of p53-R172H with altered gene occupancy and expression that occurs during the transition from primary tumor to metastasis. Such targets in metastatic ESCC are exemplified by Csf-1 and Birc5 (encoding anti-apoptosis protein Survivin) that are also enriched with H3K27ac in their promoters. They are upregulated during metastasis compared to the primary tumors dependent upon p53 mutation status, which is reinforced by TCGA data and ESCC patient-derived tissue microarrays (TMAs). Interestingly, other frequently detected “hotspot” p53 mutations including R282W and R273C, which we introduced into the ESCC cells using base editing promote differential pro-tumorigenic activities. Indeed, the analysis of SCC datasets in TCGA reveals that specific p53 mutations are associated with altered overall survival, as well as differences in the enriched pathways. CONCLUSION: We have demonstrated GOF properties and mediators of p53-R172H in promoting ESCC metastasis that may be applicable to other SCCs. We are further expanding our analyses to additional “hotspot” p53 mutations to dissect the mechanisms of distinct p53 mutations in fostering ESCC metastasis, which can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Katherine Cunningham, Raul Navaridas Fernandez de Bobadilla, Karen J. Dunbar, Kensuke Sugiura, Noriyuki Nishiwaki, Saul Carcamo, Lois Resnick-Silverman, Dan Hasson, Andres J. Klein-Szanto, Alison M. Taylor, James J. Manfredi, Carol Prives, Anil K. Rustgi. p53-R172H mutation confers gain-of-function properties and promotes metastasis in squamous cell 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 1270.

Abstract B018: MYC is sufficient to generate mid-life high-grade serous ovarian and serous endometrial carcinomas in a BRCA wild type p53-R270H mouse model

Abstract Genetically engineered mouse models (GEMM) have fundamentally changed how cancer etiology, early detection, and treatment can be understood. Mouse models have been developed that support the fallopian tube as the origin of some ovarian cancers. However, serous endometrial cancer is a much rarer gynecologic cancer with fewer models available. The genetics of serous endometrial cancer are very similar that of high-grade serous ovarian cancer. The most common mutation found in both high-grade serous ovarian cancer and serous uterine cancer is p53 and in humans, MYC is amongst the most amplified genes in both tyeps of cancer. We developed a GEMM of high-grade serous ovarian cancer by using a fallopian tube epithelium specific promoter Ovgp1 to express MYC and dominant negative mutant p53-R270H. Female mice developed lethal cancer at an average of 15.1 months. Histopathological examination of mice revealed HGSOC characteristics including nuclear p53 and nuclear MYC in clusters of cells within the fallopian tube epithelium and ovarian surface epithelium. Unexpectedly, nuclear p53 and MYC clustered cell expression was also identified in the uterine luminal epithelium, possibly from intraepithelial metastasis from the fallopian tube epithelium. Extracted tumor cells exhibited strong loss of heterozygosity at the p53 locus, leaving the mutant allele. Copy number alterations in these cancer cells were prevalent, disrupting a large fraction of genes. Transcriptome profiles most closely matched human HGSOC and serous endometrial cancer. Histological and transcriptomic findings are consistent with the hypothesis that serous uterine cancer may originate from the fallopian tube epithelium. Citation Format: Alexandra Blackman, Amy Rees, Rob Bowers, Christian Jones, Silvia Vaena, Shelby Carter, Evan Villamor, Della Evans, George Fullbright, David Long, Laura Spruill, Martin Romeo, Kristi Helke, Anthony Emanuel, Joe Delaney. MYC is sufficient to generate mid-life high-grade serous ovarian and serous endometrial carcinomas in a BRCA wild type p53-R270H mouse model [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B018.

Mutant p53 murine oviductal epithelial cells induce progression of high-grade serous carcinoma and are most sensitive to simvastatin therapy in vitro and in vivo

High-grade serous carcinoma (HGSC) is the most common and aggressive subtype of epithelial ovarian cancer, characterized by gain-of-function TP53 mutations originating in the fallopian tube epithelium. Therapeutic intervention occurs at advanced metastatic disease, due to challenges in early-stage diagnosis, with common disease recurrence and therapy resistance despite initial therapy success. The mevalonate pathway is exploited by many cancers and is potently inhibited by statin drugs. Statins have shown anti-cancer activity in many, but not all cancers. Here, we investigated the role of p53 status in relation to mevalonate pathway signaling in murine oviductal epithelial (OVE) cells and identified OVE cell sensitivity to statin inhibition. We found that p53R175H mutant and Trp53 knockout OVE cells have increased mevalonate pathway signaling compared to p53 wild-type OVE cells. Through orthotopic implantation to replicate the fallopian tube origin of HGSC, p53R175H mutant cells upregulated the mevalonate pathway to drive progression to advanced-stage ovarian cancer, and simvastatin treatment abrogated this effect. Additionally, simvastatin was more efficacious at inhibiting cell metabolic activity in OVE cells than atorvastatin, rosuvastatin and pravastatin. In vitro, simvastatin demonstrated potent effects on cell proliferation, apoptosis, invasion and migration in OVE cells regardless of p53 status. In vivo, simvastatin induced ovarian cancer disease regression through decreased primary ovarian tumor weight and increased apoptosis. Simvastatin also significantly increased cytoplasmic localization of HMG-CoA reductase in ovarian tumors. Downstream of the mevalonate pathway, simvastatin had no effect on YAP or small GTPase activity. This study suggests that simvastatin can induce anti-tumor effects and could be an important inhibitor of ovarian cancer progression.

DNAJA1 promotes proliferation and metastasis of breast cancer by activating mutant P53/NF-κB pathway

PurposeBreast cancer is one of the most common tumors with high malignancy and metastatic rate. DNAJA1 is closely related to tumor progress in several tumors. However, the role and mechanisms of DNAJA1 in the metastasis and proliferation of breast cancer are unknown. MethodsImmunohistochemistry and western blot were used to detect the protein expression genes. In vivo and vitro experiments were performed to evaluate the proliferation, invasive and metastatic abilities of breast cancer cells. ResultsDNAJA1 was high expressed in 234 cases of breast cancer tissues and associated with metastasis, p53 expression and poor survival for patients. Knock down of DNAJA1 decreased the number of plate clone formation and the OD value of CCK8 assays in breast cancer cells. Depletion of DNAJA1 also in decreased the invasive abilities of breast cancer cells. In vivo, knock down DNAJA1 decreased the growth of subcutaneous tumor and lung metastatic nodes. Mechanically, DNAJA1 could bind with P53-R175H and reduced its degradation. Up regulation of DNAJA1 in mutant P53-R175H breast cancer cell promoted the nuclear translocation of p65, activated NF-κB pathway and enhanced the transcription of its downstream genes such as MMP9, CXCL10 et al. Blockade of NF-κB pathway effectively rescued the effects of DNAJA1 on proliferation and metastasis in breast cancer. ConclusionOur study reveals that DNAJA1 is up regulated in breast cancer and promotes breast cancer cells proliferation and metastasis via P53-R175H/NF-κB pathway. It might be a potential prognosis marker for the breast cancer patients.

Biological Effects of HDAC Inhibitors Vary with Zinc Binding Group: Differential Effects on Zinc Bioavailability, ROS Production, and R175H p53 Mutant Protein Reactivation.

The coordination of zinc by histone deacetylase inhibitors (HDACi), altering the bioavailability of zinc to histone deacetylases (HDACs), is key to HDAC enzyme inhibition. However, the ability of zinc binding groups (ZBGs) to alter intracellular free Zn+2 levels, which may have far-reaching effects, has not been explored. Using two HDACis with different ZBGs, we documented shifts in intracellular free Zn+2 concentrations that correlate with subsequent ROS production. Next, we assayed refolding and reactivation of the R175H mutant p53 protein in vitro to provide greater biological context as the activity of this mutant depends on cellular zinc concentration. The data presented demonstrates the differential activity of HDACi in promoting R175H response element (RE) binding. After cells are treated with HDACi, there are differences in R175H mutant p53 refolding and reactivation, which may be related to treatments. Collectively, we show that HDACis with distinct ZBGs differentially impact the intracellular free Zn+2 concentration, ROS levels, and activity of R175H; therefore, HDACis may have significant activity independent of their ability to alter acetylation levels. Our results suggest a framework for reevaluating the role of zinc in the variable or off-target effects of HDACi, suggesting that the ZBGs of HDAC inhibitors may provide bioavailable zinc without the toxicity associated with zinc metallochaperones such as ZMC1.

Triple-negative breast tumors are dependent on mutant p53 for growth and survival

The TP53 tumor suppressor gene is mutated early in the majority of patients with triple-negative breast cancer (TNBC). The most frequent TP53 alterations are missense mutations that contribute to tumor aggressiveness. We developed an autochthonous somatic K14-Cre driven TNBC mouse model with p53R172H and p53R245W mutations in which mutant p53 can be toggled on and off genetically while leaving the tumor microenvironment intact and wild-type for p53. These mice develop TNBCs with a median latency of 1 y. Deletion of mutant p53R172H or p53R245W in vivo in these tumors blunts their tumor growth and significantly extends survival of mice. Downstream analyses revealed that deletion of mutant Trp53 activated the cyclic GMP-AMP Synthase-Stimulator of Interferon Genes pathway but did not cause apoptosis implicating other mechanisms of tumor regression. Furthermore, we determined that only tumors with stable mutant p53 are dependent on mutant p53 for growth.

TP53 gain-of-function mutation modulates the immunosuppressive microenvironment in non-HPV-associated oral squamous cell carcinoma

BackgroundTP53, the most mutated gene in solid cancers, has a profound impact on most hallmarks of cancer. Somatic TP53 mutations occur in high frequencies in head and neck cancers, including...

E2F7 drives autotaxin/Enpp2 transcription via chromosome looping: Repression by p53 in murine but not in human carcinomas.

Dysregulation of the autotaxin (ATX, Enpp2)-lysophosphatidic acid (LPA) signaling in cancerous cells contributes to tumorigenesis and therapy resistance. We previously found that ATX activity was elevated in p53-KO mice compared to wild-type (WT) mice. Here, we report that ATX expression was upregulated in mouse embryonic fibroblasts from p53-KO and p53R172H mutant mice. ATX promoter analysis combined with yeast one-hybrid testing revealed that WT p53 directly inhibits ATX expression via E2F7. Knockdown of E2F7 reduced ATX expression and chromosome immunoprecipitation showed that E2F7 promotes Enpp2 transcription through cooperative binding to two E2F7 sites (promoter region -1393 bp and second intron 996 bp). Using chromosome conformation capture, we found that chromosome looping brings together the two E2F7 binding sites. We discovered a p53 binding site in the first intron of murine Enpp2, but not in human ENPP2. Binding of p53 disrupted the E2F7-mediated chromosomal looping and repressed Enpp2 transcription in murine cells. In contrast, we found no disruption of E2F7-mediated ENPP2 transcription via direct p53 binding in human carcinoma cells. In summary, E2F7 is a common transcription factor that upregulates ATX in human and mouse cells but is subject to steric interference by direct intronic p53 binding only in mice.

DNA-delivered monoclonal antibodies targeting the p53 R175H mutant epitope inhibit tumor development in mice

The tumor suppressor p53 is the most common mutated gene in cancer, with the R175H as the most frequent p53 missense mutant. However, there are currently no approved targeted therapies or immunotherapies against mutant p53. Here, we characterized and investigated a monoclonal antibody (mAb) that recognizes the mutant p53-R175H for its affinity, specificity, and activity against tumor cells in vitro. We then delivered DNA plasmids expressing the anti-R175H mAb or a bispecific antibody (BsAb) into mice to evaluate their therapeutic effects. Our results showed that the anti-R175H mAb specifically bound to the p53-R175H antigen with a high affinity and recognized the human mutant p53-R175H antigen expressed on HEK293T or MC38 cells, with no cross-reactivity with wild-type p53. In cultured cells, the anti-R175H mAb showed higher cytotoxicity than the control but did not induce antibody-dependent cellular cytotoxicity. We made a recombinant MC38 mouse cell line (MC38-p53-R175H) that overexpressed the human p53-R175H after knocking out the endogenous mutant p53 alleles. In vivo, administration of the anti-R175H mAb plasmid elicited a robust anti-tumor effect against MC38-p53-R175H in mice. The administration of the anti-R175H BsAb plasmid showed no therapeutic effects, yet potent anti-tumor activity was observed in combination with the anti-PD-1 antibody. These results indicate that targeting specific mutant epitopes using DNA-delivered mAbs or BsAbs presents a form of improved natural immunity derived from tumor-infiltrating B cells and plasma cells against intracellular tumor antigens.

P53 inhibitor iASPP is an unexpected suppressor of KRAS and inflammation-driven pancreatic cancer

Oncogenic KRAS activation, inflammation and p53 mutation are key drivers of pancreatic cancer (PC) development. Here we report iASPP, an inhibitor of p53, as a paradoxical suppressor of inflammation and oncogenic KRASG12D-driven PC tumorigenesis. iASPP suppresses PC onset driven by KRASG12D alone or KRASG12D in combination with mutant p53R172H. iASPP deletion limits acinar-to-ductal metaplasia (ADM) in vitro but accelerates inflammation and KRASG12D-induced ADM, pancreatitis and PC tumorigenesis in vivo. KRASG12D/iASPPΔ8/Δ8 tumours are well-differentiated classical PCs and their derivative cell lines form subcutaneous tumours in syngeneic and nude mice. Transcriptomically, either iASPP deletion or p53 mutation in the KRASG12D background altered the expression of an extensively overlapping gene set, comprised primarily of NF-κB and AP1-regulated inflammatory genes. All these identify iASPP as a suppressor of inflammation and a p53-independent oncosuppressor of PC tumorigenesis.

Unique Transcriptional Profiles Underlie Osteosarcomagenesis Driven by Different p53 Mutants.

The p53 DNA binding domain contact mutant p53R245W, but not the structural mutant p53R172H, interacts with KLF15 to drive metastasis in somatic osteosarcoma, providing a potential vulnerability in tumors expressing p53R245W mutation.

Butein inhibits cancer cell growth by rescuing the wild-type thermal stability of mutant p53

p53 is a transcription factor that activates the expression of various genes involved in the maintenance of genomic stability, and more than 50% of cancers harbor inactivating p53 mutations, which are indicative of highly aggressive cancer and poor prognosis. Pharmacological targeting of mutant p53 to restore the wild-type p53 tumor-suppressing function is a promising strategy for cancer therapy. In this study, we identified a small molecule, Butein, that reactivates mutant p53 activity in tumor cells harboring the R175H or R273H mutation. Butein restored wild-type-like conformation and DNA-binding ability in HT29 and SK-BR-3 cells harboring mutant p53-R175H and mutant p53-R273H, respectively. Moreover, Butein enabled the transactivation of p53 target genes and decreased the interactions of Hsp90 with mutant p53-R175H and mutant p53-R273H proteins, while Hsp90 overexpression reversed targeted p53 gene activation. In addition, Butein induced thermal stabilization of wild-type p53, mutant p53-R273H and mutant p53-R175H, as determined via CETSA. From docking study, we further proved that Butein binding to p53 stabilized the DNA-binding loop-sheet-helix motif of mutant p53-R175H and regulated its DNA-binding activity via an allosteric mechanism, conferring wild-type-like the DNA-binding activity of mutant p53. Collectively, the data suggest that Butein is a potential antitumor agent that restores p53 function in cancers harboring mutant p53-R273H or mutant p53-R175H. SignificanceButein restores the ability of mutant p53 to bind DNA by reversing its transition to the Loop3 (L3) state, endows p53 mutants with thermal stability and re-establishes their transcriptional activity to induce cancer cell death.

Abstract 619: TP53 gain-of-function mutation modulates the immunosuppressive microenvironment in non-HPV associated oral squamous cell carcinoma

Abstract Background: TP53 is the most common mutated gene in head and neck cancer, including oral squamous cell carcinoma (OSCC), a lethal disease with limited therapeutic options. In OSCC, for example, TP53 R175H is a hot-spot, high-risk, functional mutation that inhibits the function of WT p53, however these correlations and functional roles of high-risk p53 mutations had not been experimentally addressed using syngeneic oral tumor models. To understand the functional role of TP53 mutations in modulating the tumor immune microenvironment (TIME) and response to immunotherapy, we used a syngeneic oral tumor model (ROC3 cell) derived from a genetically engineered mouse model expressing the p53 R172H (human, R175H). Methods: Orthotopic tongue injections were performed to (i) characterize tumor growth rate (ii) the TIME landscape by flow cytometry and opal multiplex immunofluorescence (mIF); and (iii) immunotherapy drug studies. We inactivated mutant p53 expression by shRNA and CRISPR Cas9, profiled gene expression changes by HTG EdgeSeq array, and validated these findings by qPCR, Western blot and immunochemistry. Tumor response was achieved by anti-PD1 and STING agonist therapies. Patients’ gene expression analyzed by CIBERSORT and TIME landscape performed by mIF. The spatial distribution of TIME was evaluated by Visiopharm software and UMAP. Results: We demonstrated that disruption of mutant p53R172H in ROC3 cells altered cytokine and chemokine expression levels and consequently affected the maturation and infiltration of different immune subsets, including cytotoxic CD8+ T cells, helper CD4+ T cells, Tregs, G-MDSCs, tumor-associated macrophages and neutrophils in ROC3 tumors. In addition, our spatial TIME analysis revealed that p53R172H mutation promoted the infiltration of Tregs, M2 macrophages, and exhausted CD8+ T cells, and excluded active CD8+ and CD11+ cells in both the tumor cores and on the tumor edges. Interestingly, it seems that p53R172H also regulates the spatial distribution of immunocyte populations, and their distribution between central and peripheral intratumoral locations. Moreover, we found that p53R172H mutation not only disrupted the phosphorylation of STING pathway which supported the recently reported finding, but also decreased the absolute amount of STING protein. Furthermore, we detected that p53-mutated tumor-infiltrating CD8+ and CD4+ T cells expressed programmed cell death protein 1 (PD-1), and that these tumors responded to anti-PD-1 and STING agonist therapy. Furthermore, Human OSCC samples also revealed associations between immune cell populations and the TP53R175H mutation, which paralleled the findings from our ROC3 tumor model. Conclusions: Our findings collectively demonstrate that the mutant TP53 GOF mutation modulates the tumor immune landscape and is associated with reduced overall survival in patients with OSCC. Citation Format: Yewen Shi, Xiaoyong Ren, Shaolong Cao, Tongxin Xie, Xi Chen, Chieko Michikawa, Abdullah A. Osman, Andrew G. Sikora, Jeffrey N. Myers, Roberto Rangel. TP53 gain-of-function mutation modulates the immunosuppressive microenvironment in non-HPV associated oral squamous cell carcinoma [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 619.

Specific regulation of BACH1 by the hotspot mutant p53R175H reveals a distinct gain-of-function mechanism.

Although the gain of function (GOF) of p53 mutants is well recognized, it remains unclear whether different p53 mutants share the same cofactors to induce GOFs. In a proteomic screen, we identified BACH1 as a cellular factor that recognizes the p53 DNA-binding domain depending on its mutation status. BACH1 strongly interacts with p53R175H but fails to effectively bind wild-type p53 or other hotspot mutants in vivo for functional regulation. Notably, p53R175H acts as a repressor for ferroptosis by abrogating BACH1-mediated downregulation of SLC7A11 to enhance tumor growth; conversely, p53R175H promotes BACH1-dependent tumor metastasis by upregulating expression of pro-metastatic targets. Mechanistically, p53R175H-mediated bidirectional regulation of BACH1 function is dependent on its ability to recruit the histone demethylase LSD2 to target promoters and differentially modulate transcription. These data demonstrate that BACH1 acts as a unique partner for p53R175H in executing its specific GOFs and suggest that different p53 mutants induce their GOFs through distinct mechanisms.

Proteasome inhibitor bortezomib stabilizes and activates p53 in hematopoietic stem/progenitors and double-negative T cells in vivo

We have previously shown that proteasome inhibitor bortezomib stabilizes p53 in stem and progenitor cells within gastrointestinal tissues. Here, we characterize the effect of bortezomib treatment on primary and secondary lymphoid tissues in mice. We find that bortezomib stabilizes p53 in significant fractions of hematopoietic stem and progenitor cells in the bone marrow, including common lymphoid and myeloid progenitors, granulocyte-monocyte progenitors, and dendritic cell progenitors. The stabilization of p53 is also observed in multipotent progenitors and hematopoietic stem cells, albeit at lower frequencies. In the thymus, bortezomib stabilizes p53 in CD4-CD8- T cells. Although there is less p53 stabilization in secondary lymphoid organs, cells in the germinal center of the spleen and Peyer's patch accumulate p53 in response to bortezomib. Bortezomib induces the upregulation of p53 target genes and p53 dependent/independent apoptosis in the bone marrow and thymus, suggesting that cells in these organs are robustly affected by proteasome inhibition. Comparative analysis of cell percentages in the bone marrow indicates expanded stem and multipotent progenitor pools in p53R172H mutant mice compared with p53 wild-type mice, suggesting a critical role for p53 in regulating the development and maturation of hematopoietic cells in the bone marrow. We propose that progenitors along the hematopoietic differentiation pathway express relatively high levels of p53 protein, which under steady-state conditions is constantly degraded by Mdm2 E3 ligase; however, these cells rapidly respond to stress to regulate stem cell renewal and consequently maintain the genomic integrity of hematopoietic stem/progenitor cell populations.

Glioblastoma extracellular vesicles influence glial cell hyaluronic acid deposition to promote invasiveness.

Infiltration of glioblastoma (GBM) throughout the brain leads to its inevitable recurrence following standard-of-care treatments, such as surgical resection, chemo-, and radiotherapy. A deeper understanding of the mechanisms invoked by GBM to infiltrate the brain is needed to develop approaches to contain the disease and reduce recurrence. The aim of this study was to discover mechanisms through which extracellular vesicles (EVs) released by GBM influence the brain microenvironment to facilitate infiltration, and to determine how altered extracellular matrix (ECM) deposition by glial cells might contribute to this. CRISPR was used to delete genes, previously established to drive carcinoma invasiveness and EV production, from patient-derived primary and GBM cell lines. We purified and characterized EVs released by these cells, assessed their capacity to foster pro-migratory microenvironments in mouse brain slices, and evaluated the contribution made by astrocyte-derived ECM to this. Finally, we determined how CRISPR-mediated deletion of genes, which we had found to control EV-mediated communication between GBM cells and astrocytes, influenced GBM infiltration when orthotopically injected into CD1-nude mice. GBM cells expressing a p53 mutant (p53R273H) with established pro-invasive gain-of-function release EVs containing a sialomucin, podocalyxin (PODXL), which encourages astrocytes to deposit ECM with increased levels of hyaluronic acid (HA). This HA-rich ECM, in turn, promotes migration of GBM cells. Consistently, CRISPR-mediated deletion of PODXL opposes infiltration of GBM in vivo. This work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue.

Abstract B055: Rac1 is essential for the maintenance of established KrasG12D-driven pancreatic ductal adenocarcinoma through senescence escape

Abstract The most prominent KRAS variants (G12D, G12V, G12R) that together represent around 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are so far undruggable. While the role of several Kras mediators have been characterized in the initiation of PDAC, very little is known about the hierarchy of Kras effectors in the maintenance of the tumor. In recent years, strategies targeting the effectors downstream of mutant KRAS have offered scope for combined inhibition of EGFR and CRAF or MEK/RAF. However, these studies, unfortunately, have been limited by either narrow interrogation of downstream effectors in mouse models, or by the use of two-dimensional cell culture systems that may not recapitulate dependencies of the tumor in situ, respectively. To systematically interrogate the potential molecular dependencies in pancreatic tumor maintenance across several combinations of driver mutations, we have deployed in vitro and in vivo approaches in which we have selectively targeted key mediators of known KRAS-dependent pathways. These include Craf, Braf, PI3K, RalA, RalB, and Rac1, for which we have used inducible GFP-coupled shRNAs in 3D mouse and human tumor organoids harboring KrasG12D and p53R172H mutation. Using competition, cell cycle, and volumetric assays, we have uncovered that Rac1, Kras, and Craf are essential to the growth of PDAC organoids, whereas Braf, PI3K, RalA and RalB are dispensable. Interestingly, Rac1 depletion led to the strongest phenotype among the Kras mediators with a reduction of macropinocytosis, cell migration and colony formation in vitro. In an orthotopic pancreatic injection model, we observed that Rac1 inhibition in vivo led to diminished primary tumor growth, improved survival, and a reduction of metastatic incidence and outgrowth. In parallel, we performed RNA-sequencing on Kras-, Craf-, Rac1-, RalA- and Renilla-depleted organoids and found that Rac1 depletion rewires tumor cells to acquire a more PanIN-like phenotype, highlighting the importance of these proteins for the maintenance of PDAC cells. We also identify in Rac1- depleted cells evidence of deregulation of reactive oxygen species (ROS) and induction of a senescence-associated secretory phenotype (SASP) compared to control organoids. With a cytokine and chemokine array, we confirmed the increase of SASP chemokines (Csf3, Cxcl1, Cxcl2 and Cxcl5) and also detected bona fide senescence via SA-βgal staining. Finally, using several Rac1 inhibitors, we recapitulate the importance of Rac1 in PDAC growth. These data suggest that among the pleiotropic signaling downstream of mutant Kras, Rac1 is a critical node in PDAC maintenance that promotes tumor cell proliferation and senescence escape. Our findings point towards future efforts to couple Rac1 inhibition to define therapeutic synergies with immunotherapy and/or radiation. All together, we anticipate these findings can inform the subsequent development of novel therapies to address these vulnerabilities. Citation Format: Adrien Grimont, David J. Falvo, Paul Zumbo, Grace Pan, John Nguyen, Rhonda K. Yantiss, Doron Betel, Laura Martin, Steven D. Leach, Rohit Chandwani. Rac1 is essential for the maintenance of established KrasG12D-driven pancreatic ductal adenocarcinoma through senescence escape [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B055.

LncRNA H19 Promotes Lung Adenocarcinoma Progression via Binding to Mutant p53 R175H.

Simple SummaryThis research explored the association and interaction between lncRNA H19 and mutant p53 (R175H) in lung adenocarcinoma development and progression. H19 over-expression may induce the elevated expression of mtp53 and interact with mtp53, which prolongs the p53 half-life and promotes transcriptional activity, leading to the progression of lung adenocarcinoma. The simultaneous inhibition of H19 and mtp53 may provide a novel strategy.Background: Accumulating data suggest that long non-coding RNA (lncRNA) H19 and p53are closely related to the prognosis of lung cancer. This study aims to analyze the association and interaction betweenH19 and mutant p53 R175H in lung adenocarcinoma (LAC). Methods: Mutant-type (Mt) p53 R175H was assessed by using RT-PCR in LAC cells and 100 cases of LAC tissue samples for association with H19 expression. Western blot, RNA-pull down, immunoprecipitation-Western blot and animal experiments were used to evaluate the interaction between H19 and mtp53. Results: Mtp53 R175H and H19 were over-expressed in LAC tissues and cells, while H19 over-expression extended the p53 half-life and enhanced transcriptional activity. Combined with anti-p53, ShH19 can significantly inhibit tumor growth in vivo. Conclusions: H19 over-expression may induce the elevated expression of mtp53 and interact with mtp53, leading to LAC progression. In addition, the high expression of mtp53 R175H is associated with poor overall survival inpatients. The simultaneous inhibition of H19 and mtp53 may provide a novel strategy for the effective control of LAC clinically.