Role Of Mutant P53 Research Articles

Abstract 136: Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer which lacks HER2 overexpression, as well estrogen and progesterone receptor expression. TNBC constitutes 10-15% of all breast cancers but has a worse prognosis due to limited treatment options and high rates of metastatic recurrence. Doxorubicin (dox) remains one of the most active chemotherapy agents for the treatment of TNBC, although de novo and acquired resistance to chemotherapy remains a major challenge. The purpose of this work was to characterize dox resistant cell phenotypes and investigate treatment-induced senescence in p53-mutated (Mut) and wildtype (WT) TNBC cell lines. Methods: A panel of 12 TNBC cell lines (p53 WT and p53 Mut) were exposed to dox (0-5 uM) for 72 hours and cellular proliferation was determined using the Cell Titer Glo assay. A subset of sensitive and resistant cell lines were treated with dox (0.1, 0.25 uM) or vehicle control and apoptosis was determined by flow cytometry (Annexin-V) at 24 and 48 hrs. Cells were treated with dox or control for 24 hrs and western blotting was performed for mediators of apoptosis. Senescence was analyzed by ß-galactosidase staining following treatment with dox for 3-14 days. shRNA knockdown (KD) of p53 was performed in the CAL-51 (p53 WT cell line) and cells were subject to investigations above. Results: Doxorubicin treatment resulted in decreased cellular proliferation and increased apoptosis as assessed by Annexin-V expression in a subset of p53 WT and p53 Mut cell lines. Treatment with dox resulted in an increase in the pro-apoptotic proteins BID and p21, as well as a decrease in VAMP in sensitive cell lines. In a subset of cell lines resistant to dox treatment, we observed an increase in cells demonstrating phenotypic features of senescence and ß -galactosidase staining. We observed a decrease in p16 with dox treatment in the CAL-51 (p53 WT cell line) compared to an increase following treatment in p53 Mut cell lines. KD of p53 resulted in an increase in senescent cells following treatment with low dose dox. Conclusions: Treatment with doxorubicin resulted in different terminal cell phenotypes in TNBC cell lines with apoptosis observed in p53 WT and Mut cell lines. Senescence was observed in resistant cells and KD of p53 increased dox-induced senescence, confirming a role for p53 in mediating terminal cell fate. Efforts are ongoing to understand the role of mutant p53 in mediating terminal cell fate in response to dox and rational combinations to overcome dox-induced senescence may be clinically active in metastatic TNBC. Citation Format: Stephen G. Smoots, Anna R. Schreiber, Betelehem Yacob, Adrian Dominguez, Cecilia Levandowski, Todd M. Pitts, Jennifer R. Diamond. Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 136.

Abstract P5-10-03: Mutant p53 and ERK1/2 MAPK cooperate with the production of TNBC inflammatory secretome

Abstract TP53 is the most frequently mutated gene in most types of human cancer, including breast cancer. The triple-negative breast cancer (TNBC) subtype in particular displays TP53 mutation in approximately 80% of patients. Unlike other breast cancer subtypes (e.g., ER/PR-positive or HER2-positive), TNBC patients currently lack an approved highly effective targeted therapy. Notably, most TNBC acquires TP53 mutations, which, in addition to the loss of canonical p53 functions, can result in gain-of-function, activating different cellular mechanisms involved in tumor phenotypes such as proliferation, metastasis, invasiveness, and angiogenesis. Here we evaluate the role of mutant p53 in cancer phenotypes of TNBC cell lines, in particular their inflammatory profile. As expected, loss of p53 protein by small interfering RNA depletion did not show a major impact on the cell viability of MDA-MB231 and Hs578t cells in MTT assay. However, depleting mutant p53 knockdown made MDA-MB231 cells more susceptible to treatment with methyl-methane sulfonate, a genotoxic alkylating agent. Interestingly, cell invasion as measured by the transwell assay demonstrated that depletion of mutant p53 depletion decreased the invasiveness potential of MDA-MB231 and Hs578t cells which was substantiated with decreased migration of MDA-MB231 cells in a scratch assay over 24 h. RNA sequencing of MDA-MB231 and Hs578t cells revealed that mutant p53 knockdown decreased the expression of several constitutively expressed pro-inflammatory cytokines such as IL8, IL6, CXCL2, and CXCL3, but not genes associated with survival to alkylating agents (NRF2 and Endoplasmic reticulum stress markers) or genes typically regulated by wild-type p53 when compared to control-silenced and MMS-treated cells as indicated by Pathway Enrichment Analysis using the Enrich R and DAVID tools. These results were confirmed by ELISA quantification of IL8, IL6, and CXCL2 in MDA-MB231 and Hs578t transfected with two sequences of siRNA targeting mutant p53. On the other hand, RNA sequencing revealed some constitutively expressed genes known to be involved in breast cancer cells malignancy, such as PTGS2 (COX-2 enzyme gene) and MMP1, which were not affected by p53 knockdown. We found that MMP1, and PGE2 (the product of PTGS2/COX-2 enzyme), are upregulated by the ERK1/2 MAPK signaling pathway as determined in MDA-MB231 cells treated with the MEK1/2 inhibitor UO126 and sorafenib. UO126 and sorafenib also decreased IL8 and IL6 production. Furthermore, combined mutant p53 knockdown with MEK/ERK1/2 pathway inhibition caused a more pronounced IL8 and IL6 inhibition when compared to either p53 knockdown or UO126/sorafenib alone, whereas MMP1 and PGE2 levels were only reduced by MEK/ERK1/2 inhibitor treatments. Interestingly, neither mutant p53 knockdown impacted ERK1/2 phosphorylation status nor did UO126/sorafenib alter mutant p53 immunocontent. Reporter assays showed that mutant p53 promotes NFkappaB reporter activation, and MEK/ERK1/2 controls both NFkappaB and AP-1 transcription factors, both associated with the expression of the secretome components evaluated herein. Functional cell assays showed that concomitant inhibition of mutant p53 and MEK/ERK1/2 pathways reduce cell proliferation, invasion, and migration, indicating that mutant p53 protein gain of function cooperates with ERK1/2 MAPK signaling pathway to promote secretome production and malignant phenotypes in TNBC cell models. Citation Format: Raquel Nascimento das Neves, Aparna Gorthi, Alexander James Roy Bishop, Alfeu Zanotto Filho. Mutant p53 and ERK1/2 MAPK cooperate with the production of TNBC inflammatory secretome [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-03.

Abstract 2489: A functional genomics approach to determine mutant p53 gain-of-function mechanisms and phenotypes in tumorigenesis

Abstract Two of the most common events in human tumors are mutation of the tumor suppressor gene TP53 and development of aneuploidy. In addition to losing their wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. Mechanistic understanding of mutant p53 GOF activities is complicated by the diversity and context-specific nature of reported GOF phenotypes. The study of mutant p53 GOF activities is especially challenging because mutations in p53 are positively correlated with the development of aneuploidy, which can increase heterogeneity through diverse chromosomal alterations and itself contributes to tumorigenesis. To study mutant p53 GOF mechanisms and phenotypes, we used CRISPR/Cas9-mediated genome editing and developed two isogenic epithelial cell line models (one non-transformed and one tumor-derived) that express the most frequently occurring p53 missense mutations (R175H and R273H), are deficient for functional p53 protein (null), or retain the wild-type (WT) protein. In these engineered models, endogenous p53 expression is regulated by the native p53 promoter, thus providing a controlled system for rigorous functional experimentation across different p53 states. Additionally, the use of clonally-derived cell lines originating from the same near diploid parental genetic background allows for assessment of the genomic alterations and resulting molecular heterogeneity following mutation of TP53. Through genomic, transcriptomic, and cellular based assays we have validated our cell line models and found that missense mutant and p53 null cells display loss of p53 function. Through functional genomics analyses comparing isogenic epithelial cells, which initially differed only by the TP53 genotype, we have evaluated the relationship between mutant p53 and aneuploidy and assessed whether our clonal cell lines display several previously reported mutant p53 GOF phenotypes such as altered gene expression, proliferation, metabolism, drug sensitivity, and migration. Further, using lentiviral mediated knockdown of p53 protein we evaluated the dependency of these phenotypes on expression of mutant p53 protein. Finally, data from The Cancer Genome Atlas (TCGA) was used for the analysis of manifestations of clinical mutant p53 GOF phenotypes. The dissection of mutant p53 GOF phenotypes will improve the current understanding of the role of mutant p53 in tumorigenesis. The results generated from these studies have the potential for clinical translation in major types of human cancer that have high-frequency p53 mutation. Citation Format: Lindsay N. Redman-Rivera, Timothy M. Shaver, Hailing Jin, Johanna M. Schafer, Quanhu Sheng, Rachel A. Hongo, Kathryn E. Beckermann, Brian D. Lehmann, Ferrin C. Wheeler, Jennifer A. Pietenpol. A functional genomics approach to determine mutant p53 gain-of-function mechanisms and phenotypes in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2489.

Abstract LT005: Tumor-intrinsic gain of function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment that promotes resistance to immunotherapy

Abstract KRASG12D mutations in tumor cells can modify the immune microenvironment of pancreatic ductal adenocarcinoma (PDAC), though the effects of secondary genetic alterations have been unexplored. After oncogenic KRAS, TP53 is the most frequently mutated gene in PDAC, however the immune effects of neomorphic TP53 mutations have not been defined. We sought to evaluate the non-cell-autonomous role of mutant p53 in modulating the immune microenvironment of pancreatic cancer by comparing the immune profiles generated by KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDEC) genetically engineered to express the Trp53R172H gain-of-function mutation to their p53 wildtype control. We found orthotopically implanted tumors derived from KrasG12D;Trp53R172H PDEC had a distinct immune profile in comparison to KrasG12D/+;Trp53+/+ tumors, characterized by an influx of intratumoral CD11b+Ly6G+ neutrophils and concomitant decreases in CD3+ T cells, CD8+ T cells, and CD4+ T helper 1 (Th1) cells. Analysis of publicly available human PDAC cohorts revealed enrichment of genes in neutrophil-related pathways in TP53-mutated tumors. Knockdown of CXCL2, a neutrophil chemoattractant, in the tumor epithelial compartment of KrasG12D;Trp53R172H PDEC tumors reversed the neutrophil phenotype. Depleting neutrophils in mice bearing KrasG12D;Trp53R172H PDEC tumors augmented sensitivity to combined CD40 immunotherapy and chemotherapy. Collectively, these data link mutant p53 to the presence of intratumoral neutrophils in pancreatic cancer and suggests that tumor genotypes could inform patient selection for immunotherapy. Citation Format: Despina Siolas, Emily Vucic, Emma Kurz, Cristina Hajdu, Dafna Bar-Sagi. Tumor-intrinsic gain of function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment that promotes resistance to immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT005.

Abstract PS16-19: Hotspot p53 mutations correlate with increased expression of stem cell markers in triple-negative breast cancer

Abstract p53 mutations occur in 80% of triple-negative breast cancer (TNBC) cases. Several types of p53 mutations have been reported, among which gain of function mutations have been associated with increased cancer stem cells, chemotherapy resistance, and disease relapse in several malignancies. Particularly, hotspot p53 mutations (mutations at amino acid location 157, 175, 248, 249, 273 predict worse overall survival in some cancer types. We have previously reported that Ganglioside GD2, a cell surface glycosphingolipid, identifies breast cancer stem cells and promotes tumorigenesis. We have also reported that GD3 synthase, a key enzyme which catalyzes the rate-limiting step of the GD2 biosynthesis pathway, is significantly upregulated in breast tumors with p53 mutations. Here, we hypothesize that mutant p53 promotes stemness in TNBC cells by regulating GD3 synthase and GD2 expression. To identify specific p53 mutations that contribute to TNBC stemness, we stratified TNBC cell lines (N=18) based on p53 mutation status including mutation type, location, and domain affected. We measured GD2 and GD3 synthase expression in the available TNBC cell lines by flow cytometry and RT-PCR, respectively. We found that GD3 synthase expression is significantly upregulated in TNBC cell lines with p53 hotspot mutations compared to cell lines with other p53 mutations (Median relative expression 0.0114 compared to 0.0003, p=0.005). Similarly, we found that GD2 expression is significantly higher in TNBC cell lines with hotspot mutations compared to non-hotspot counterparts (Median GD2+ cells 15.2% compared to 0.97%, p= 0.013) Additionally, GD2 and GD3 synthase are upregulated in basal-type TNBC cell lines and TCGA patient samples compared to other TNBC molecular subtypes. Interestingly, we also found that GD2 expression is not always directly correlated with GD3S expression, suggesting that GD2 synthase expression is also a key factor in the regulation of GD2 expression. To examine the effect of mutant p53 protein levels on TNBC stemness, we measured p53 expression in all the available TNBC cell lines by western blot, and found that p53 protein levels do not correlate with GD2 or GD3S expression. To validate these findings, we analyzed RPPA data from the MD Anderson Cancer Cell Lines Project and found similar results. These data suggest that it is the type of p53 mutation, but not the amount of p53 protein in the cells that determines GD2 and GD3 synthase expression in TNBC cells. For instance, p53 hotspot mutations involving amino acid position 248 have been shown to be strongly associated with increased cancer stemness. We found that cell lines with p53 mutations at this specific location have significantly higher GD3 synthase expression compared to other mutant p53 forms. We are currently examining the direct role of mutant p53 in the regulation of GD3S expression by stabilizing mutant p53 using MDM2 inhibitors as well as stable knockdown of p53 gene in multiple TNBC cell lines to establish a clear link between mutant p53 and stem cell marker expression in TNBC cell lines. In conclusion, stem cell markers are highly expressed in p53 mutant TNBC cell lines compared to wild type p53 counterparts. Specifically, cell lines with hotspot p53 mutations significantly correlate with increased TNBC stemness. The type of p53 mutation, rather than level of its expression correlates with stem cell marker expression in TNBC cells. Citation Format: Fouad El-Dana, Bin Yuan, Stanley Ly, Vivek Anand, Venkata Lokesh Battula. Hotspot p53 mutations correlate with increased expression of stem cell markers in triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-19.

Gain-of-"endocytic' function in mutant p53 cancer cells.

Beyond its well-known canonical function as a tumor suppressor, p53 is also involved in numerous cellular processes through altered transcription under both normal and pathological conditions. The functional diversity of p53 outputs is complex and dependent on cell context. However, the underlying mechanisms responsible for this diversity remain largely unclear. The emerging evidence of p53 mutations involved in regulating endocytic trafficking and signaling, in tandem to promote malignancy (invasion, exosome biogenesis and immune evasion), sheds light on possible mechanisms behind the p53-driven complexity. The interrelated nature of endocytic trafficking and receptor signaling that form dynamic and adaptable feedback loops - either positive or negative - functions to modulate multiple cellular outputs. Biasing the tunable endocytic trafficking and receptor signaling network by mutant p53 expands the purview of p53, allowing its contribution to diverse and aggressive phenotypes. In this review, we explore recent studies in which the novel role of mutant p53 in altering endocytic trafficking to bias receptor signaling and drive transforming phenotypes is revealed. Understanding the complex crosstalk of mutant p53, endocytic trafficking and receptor signaling will allow the development of therapies to selectively target p53-altered endocytic processes.

MBRS-18. TUMOR SUPPRESSOR p53 DEFINES THE THERAPEUTIC RESPONSES IN TREATMENT OF MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common primary pediatric malignant brain tumor. Current molecular analysis classifies MB into 4 groups, classic (WNT), sonic hedgehog (Shh), group 3, and group 4. Furthermore, atypical p53 signaling is associated with disease progression and confers poor prognosis. This study investigated the correlation of mutational status of p53 and iSO17q with disease progression and metastatic potential. In addition, we used small molecule inhibitors of PI3K (Buparlisib; BKM120) and HDAC (LBH-589) on a p53-mutant MB cell line to find novel therapeutic targets. Efficacy of these drugs were assessed using functional assays (cell proliferation, migration, cell cycle and drug resistance). MB tumors (n=53) were evaluated for GLI-1, GAB-1, NPR, KV1, YAP expression and mutant p53 via immunohistochemistry and correlated to patient outcomes. Results demonstrated that: 1) high expression of GAB-1 and YAP were found in the Shh group, while KV1 expression was present in all subtypes; 2) mutant p53 expression was present in various subsets of MB with no apparent correlation with metastasis or disease progression; 3) patients displaying iSO17q (determined by fluorescence in situ hybridization (FISH) technique) exhibited metastatic disease; 4) LBH-589 and BKM120 caused both time and dose-dependent inhibition of MB cell proliferation and migration; 5) combined treatment of BKM120 and LBH-589 had a synergistic effect; 6) MB cells demonstrated drug-resistance to BKM120. In conclusion, these findings underscore use of Buparlisib and LBH-589 in treatment of MB. Further, the role of mutant p53 in disease progression remains elusive, whereas presence of iSO17q defines metastatic potential.

Abstract B58: The role of mutant P53 in repetitive element regulation and the immune response in ovarian cancer

Abstract While therapies that activate the host immune system have shown tremendous promise for a wide variety of solid tumors, less than 10% of ovarian cancer (OC) patients have responded. Previous work from our group showed that DNA methyltransferase inhibitors (DNMTis) activate the interferon response in tumors through upregulation of repetitive elements (REs) including endogenous retroviruses (ERVs), remnants of exogenous retroviruses that integrated into the germline several million years ago. DNMTis increase ERVs in a mouse model of ovarian cancer, activating interferon signaling and recruiting CD8+ T cells to kill the tumors. Importantly, nearly all high-grade serous OCs harbor a mutation in the TP53 gene, encoding the tumor suppressor protein P53. p53 may transcriptionally repress ERVs to ensure genome stability; 30% of all p53 binding sites are in ERV-derived sequences. We treated seven human cancer cell lines, including four OC lines (3 TP53 wild-type, 4 TP53 mutant) with DNMTi and analyzed repetitive element expression (RNA-Seq), open chromatin (ATAC-Seq), and DNA methylation (MeDIP + MRE). Interestingly, RE upregulation was significantly higher in the P53 mutant cell lines. This was true for the SINE, LINE, and ERV groups of repetitive elements. The DNA methylation of these elements was not significantly different between TP53 wild-type and mutant cell lines, but the open chromatin peaks (ATAC-Seq) were significantly increased in the TP53 mutant cell lines. We thus hypothesize that wild-type TP53 may partner with DNA methylation to transcriptionally repress RE transcription. Treating TP53 wild-type cells with Nutlin-3A, which activates P53, represses transcription of some ERVs. When we overexpressed R175H or R273H mutant P53 constructs in the SKOV3 P53-null cell line and treated with DNMTi, we observed much higher upregulation of ERVs after DNMTi treatment than when we overexpressed the control wild type P53 vector. We predicted that higher ERVs would induce a higher interferon response and perhaps change the microenvironment in P53 mutant tumors. We used the ID8 MOSE mouse ovarian cancer model modified by the MacNeish laboratory to compare p53+/+ and p53-/- tumor response to DNMTi treatment. Characterization of the tumor microenvironment indicated a significantly higher frequency of lymphocytes, Treg cells, MDSCs, and myeloid cells in the p53-/- model in comparison to the p53 wild-type model. In current work, we are creating isogenic cell lines of human and mouse wild-type, mutant, and null p53 and comparing the response to DNMTi in vitro and in vivo. We will then determine whether P53 binding directly regulates repetitive elements transcriptionally, and more broadly, whether mutant P53 cells are more sensitive to DNMTi upregulation of repetitive elements and induction of an immune response. Results of this work will be particularly relevant for high-grade serous OC, which is driven by mutant P53 and is generally unresponsive to immunotherapies. Citation Format: Stephanie Gomez, James I. McDonald, Elisa Arthofer, Noor Diab, Aneil Srivastava, Paul Austin, Katherine B. Chiappinelli. The role of mutant P53 in repetitive element regulation and the immune response in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B58.

Tumor-Intrinsic Gain-of-Function p53 <sup>R172H</sup> Mutation Drives Accumulation of Neutrophils in the Pancreatic Tumor Microenvironment that Promotes Resistance to Immunotherapy

The immune microenvironment plays a critical role in cancer development, progression and resistance to therapy. Recent reports indicate different tumor genotypes can profoundly influence the composition of the immune microenvironment. After oncogenic KRAS, TP53 is the most frequently mutated gene in pancreatic ductal adenocarcinoma (PDAC). KRASG12D mutations in tumor cells can modify the immune landscape of PDAC, however, the immune effects of neomorphic TP53 mutations have not been defined. Focusing on the most common TP53 mutation in PDAC, we sought to evaluate the non-cell-autonomous role of mutant p53 in modulating the immune microenvironment of pancreatic cancer by comparing the immune profiles generated by KRASG12D-mutated mouse pancreatic ductal epithelial cells (PDEC) genetically engineered to express the Trp53 R172H gain-of-function mutation to their p53 wildtype control. We found orthotopically implanted tumors derived from KrasG12D/+;Trp53R172H/+ PDEC had a distinct immune profile in comparison to KrasG12D/+; Trp53 +/+ tumors, characterized by an influx of intratumoral CD11b+ Ly6G+ neutrophils and concomitant decreases in CD3+ T cells, CD8+ T cells, and CD4+ T helper 1 (Th1) cells. Analysis of publicly available human PDAC cohorts revealed enrichment of genes in neutrophil-related pathways in TP53 -mutated tumors. Knockdown of CXCL2, a neutrophil chemoattractant, in the tumor epithelial compartment of KrasG12D/+; Trp53R172H/+ PDEC tumors reversed the neutrophil phenotype. Depleting neutrophils in mice bearing G12D/+; Trp53R172H/+ PDEC tumors augmented sensitivity to combined CD40 immunotherapy and chemotherapy. Collectively, these data link mutant p53 to the presence of intratumoral neutrophils in pancreatic cancer and suggests that tumor genotypes could inform patient selection for immunotherapy.

Abstract TMIM-075: MUTANT P53 PROMOTES THE INITIATION OF HIGH-GRADE SEROUS OVARIAN CARCINOMA FROM FALLOPIAN TUBE TUMOR PRECURSORS THROUGH ENHANCING TYROSINE RECEPTOR KINASE B SIGNALING

Abstract Lesions in the fallopian tube (FT) fimbriae are hypothesized to be the precursors of high-grade serous ovarian carcinomas (HGSOC). Immunohistalchemical and sequencing analyses demonstrated that FT lesions shared the same TP53 mutation as the surrounding carcinomas. Analyses of mutations and chromosomal alterations in FT lesions, ovarian cancers, and metastases also revealed that the mutation of TP53 is an early event that might drive tumor initiation. Our study focuses on understanding the molecular mechanisms of mutant p53 promoting tumor initiation from the FT. Our previous data showed that the FT epithelial cells expressed TrkB. The ligand of TrkB, brain-derived neurotrophic factor (BDNF), is secreted by the ovary, omental adipocytes, peritoneal visceral epithelial cells, and immune cells in the omentum and peritoneum. BDNF suppresses anoikis, the apoptosis induced by lack of proper cell to extra-cellular matrix (ECM) attachment. BDNF/TrkB signaling has been associated to tumor progression. We hypothesize that mutant p53 enhances tyrosine receptor kinase B (TrkB) signaling in FT tumor precursors, which promotes tumor precursors to spread towards the ovary and peritoneal sites. We used two immortalized human FT cell lines, in which p53 was knocked down by shRNA. Mutant p53 or a control vector were introduced to these cell lines via a lentiviral expression system. The expression level of TrkB protein in FT cells expressing mutant p53 or a control vector was evaluated using Western blot and flow cytometry. CellTiter luminescent cell viability assays were performed to assess the effects of BDNF on the survival of FT cells with or without p53 mutation in 3D culture. Cellmate hydrogel was used as scaffold to support FT cells. And the cells migrated outside the hydrogel were quantified using CellTiter assay. Collagen I-coated beads were used in a 3D cell adhesion model to quantify the ability of FT cells to adhere to ECM and evaluate the effects of BDNF on the cell adhesion. The activation of TrkB downstream proteins was assessed using western blot. Our data demonstrated that the protein level of TrkB was higher in FT cells expressing mutant p53 (R175H, R248W, and R273H) compared to the control FT cells. Mutant p53 significantly enhanced the survival of FT cells in the serum-free 3D culture condition in the presence of BDNF. BDNF increased the ability of FT cells to migrate outside the hydrogel, and mutant p53 further enhance their migration. Mutant p53 also accelerated the BDNF-mediated attachment of FT cells to collagen I-coated beads. Western blot results showed that the BDNF-activated phosphorylations of TrkB, AKT, ERK, PLC-gamma1 CREB were all increased in the FT cells expressing mutant p53 comparing to the control cells without mutant p53. These results suggest the role of mutant p53 in promoting the initiation of HGSOC from FT tumor precursors. Through enhancing BDNF/TrkB signaling, mutant p53 can enhance the ability of FT tumor precursors to overcome anoikis and spread towards the ovary, peritoneum, and omentum. A better understanding of the underlying mechanisms will contribute to the development of new treatment and detection markers for HGSOC initiated from the FT. Citation Format: Min Kang, Kay Y. Chong, Tobias MP. Hartwich, Sarah Cady, Jonah Ncci, Yang Yang-Hartwich. MUTANT P53 PROMOTES THE INITIATION OF HIGH-GRADE SEROUS OVARIAN CARCINOMA FROM FALLOPIAN TUBE TUMOR PRECURSORS THROUGH ENHANCING TYROSINE RECEPTOR KINASE B SIGNALING [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-075.

Role of Mutant p53 in the Progression of Chronic Lymphocytic Leukemia

Patients with Chronic Lymphocytic Leukemia (CLL) have a variety of chromosomal abnormalities and mutations. At diagnosis, about 10% of CLL patients have deletions of chromosome 17 (Del17p) leading to the loss of one allele of tumor suppressor protein TP53, which increases to over 30% in relapsed/refractory disease. Additionally, 83% of patients with a Del17p acquire a mutation on their second TP53 allele at one of several sites within the DNA binding domain. While the consequence of some of these "hotspot" mutations (R175H, R179H, G245D, G248Q/W, Y220, R213X, R273H and R282H) has been described in solid tumors and AML, very little is known of their role in CLL. Clinically, patients with Del17p/Mutp53 have worse overall survival, increased disease progression and are more likely to relapse on the current targeted therapies such as ibrutinib. Although relapse to these treatments is largely due to acquired mutations in Bruton's Tyrosine Kinase (BTK) or its downstream target PLCg2, we hypothesize that the biology of mutant 53 bearing CLL is a key driver of resistance and progression. Specifically, we aim to determine the molecular signature and downstream effectors that allow mutant p53 to drive the adverse biology associated with this subtype. Conversely, we hypothesize that targeting the mutant p53 pathway will lead to better outcomes and overall survival for patients bearing this adverse prognosis marker. We performed high-throughput Sequencing of DNA from 270 CLL patients with high coverage in the exonic regions of TP53 prior to ibrutinib therapy as well as during progression. At baseline, 40% of patients had mutations found in the DNA binding region with the most frequent occurring in R248Q, R175H and R273H. We then characterized each p53 mutant (n=106) functionally in terms of their ability to ability to activate p21, PUMA, and Bax which serve as cell cycle checkpoint and apoptotic effectors of wild type p53 in response to DNA damage. Most mutants were incompetent in upregulating p21, PUMA or Bax at the transcript level. A few mutants upregulated p21 protein in a p53 independent fashion. We then evaluated the consequence of mutant p53 in CLL. We performed chromatin immunoprecipitation (ChIP-Seq), open chromatin signatures (ATAC-Seq) and expression analysis (RNA-Seq) on CLL samples with R248Q or R175H as well as in wild-type (WT) p53 samples. Integration of ChIP, ATAC and RNA Seq profiles indicated that mutant p53 activated a unique transcriptomic profile not shared by wt p53 bearing CLL. Several genes that facilitated survival or progression were downstream targets of mutant p53. Of these, we identified PRKCB (PKC-beta), BCL2L1 (Bcl-xL), EZH2, MLL and MALAT as a potential key downstream effectors of mutant p53. To determine whether mutations at R248Q and R175H in p53 were causal in the observed increases in PKC-beta, Bcl-xL, EZH2, MLL, and MALAT we used CRISPR/Cas9 editing to introduce mutations at R175H and R248Q in the p53 wildtype CLL cell lines HG-3 and PGA-1. These were accomplished by electroporating sgRNA-Cas9 ribonucleoprotein complexes (RNPs). Western blotting of mutants revealed an increase in the mRNA and protein expression of PKC-beta, and BCL-xL in mutant p53 compared to WT. Levels of EZH2 and MLL were not increased in these cells indicating that PKC-beta and Bcl-xL may be direct transcriptional targets upregulated by mutations at R248Q and R175H in p53. Ongoing efforts will characterize the transcriptional profile of all p53 mutants in our cohort to determine whether they all have a unifying transcriptomic profile that confers a gain of function phenotype to this subtype of CLL. Disclosures Byrd: Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Acerta: Research Funding; BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau. Woyach:Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding.

Abstract 5289: Gain-of-function mutant p53 predisposes head and neck keratinocytes and squamous cell carcinoma cells to replicative stress and genomic instability through minichromosome maintenance complex component 5

Abstract As the guardian of genome, p53 plays important roles in maintaining the genome integrity, and TP53 mutations thereby often lead to genomic instability in cancers. In addition to the loss-of-function, many TP53 mutations can lead to the gain-of-functions (GOF) that often promote tumor development and tumor progression. As an effort to identify the mechanisms involved in mutant p53 GOF activities in head and neck squamous cell carcinoma (HNSCC), we performed an unbiased proteomic screen to identify the mutant p53 G245D interactome in HNSCC cells by immunoprecipitation, and quantitative proteomics using stable isotope labeling with amino acids in cell culture and liquid chromatography coupled with tandem mass spectrometry. Our results identified that MCM5, a member of replication licensing heterohexameric origin recognition complex minichromosome maintenance 2-7 (MCM2-7), interacts with mutant p53s, which suggests a possible role of mutant p53 in regulation of DNA replication. Consistent with this, we show that overexpression of mutant p53 in p53-depelted oral keratinocytes and HNSCC cells predisposes cells susceptibility to replication stress by inhibiting the dormant origin firing, leading to increased chromosomal instability (CIN) under replication stress. Moreover, by overexpressing and downregulating MCM5, we demonstrate that MCM5 modulates GOF mutant p53-mediated susceptibility to replication stress and CIN. Given the importance of MCM2-7 in replication licensing, initiation, and elongation, our discovery of the relationship between mutant p53 and MCM5 suggests that the MCM2-7 complex is one of the key downstream effectors, through which GOF mutant p53s regulate DNA replication and genomic instability in HNSCC. Citation Format: Mei Zhao, Tianxiao Wang, Chen Zhen, Jing Wang, Curtis R. Pickering, Junjie Chen, Jeffrey N. Myers, Ge Zhou. Gain-of-function mutant p53 predisposes head and neck keratinocytes and squamous cell carcinoma cells to replicative stress and genomic instability through minichromosome maintenance complex component 5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5289.

Abstract 3548: Differential miRNA expression levels and risk of lung adenocarcinoma in transgenic mice

Abstract In 2018, 154,100 lung cancer deaths are projected to occur in the United States. Currently, there are very few predictors to identify lung cancer in its early stages. Recent studies have demonstrated a link between microRNAs (miRNAs) and specific cancers. Dysregulation of miRNAs in cancer can lead to upregulate oncogenes and downregulate tumor suppressors. Previously, we developed a line of transgenic mice expressing the human mutant p53 (273H) gene. The transgenic mice were observed to have accelerated risk of spontaneous adenocarcinomas at the age of 13-14 months. However the mechanisms involved in lung tumorigenesis at this age cohort is still unclear. Herein, we report our analysis on miRNA expression in lung tissue between a low risk cohort and high risk cohort. To investigate the role of mutant p53 in lung tumorigenesis, a p53(273H) transgenic mouse model was developed. Murine lung tissue were harvested from age groups of 7 months (low risk group with risk of 5% of lung cancer), and the 13.5 months (high risk group with risk of 27% of lung cancer). RNA was then extracted with trizol, and miRNAs were quantified using a Nanostring counter miRNA array. The miRNA expressions were normalized and averaged. The normalized miRNAs from high risk group (n=3) were compared with the miRNAs obtained from the low risk group (n=3). We screened 600 different miRNA expressions from these age cohorts. Total of 19 miRNAs that had an up-regulation of at least 5-fold in the high risk cohort as compared with the low risk cohort including some important miRNAs in human lung cancer (e.g mmu-miR-2133, mmu-miR-2140, mmu-miR-142-5p, mmu-miR-2138, mmu-miR-2134, mmu-miR-2135, mmu-miR-452, mmu-miR-703, mmu-miR-690, mmu-miR-206, mmu-miR-706, mmu-miR-691, mmu-miR-208a, mmu-miR-574-5p, mmu-miR-1). Four miRNAs had a down regulation at least 5-fold in the high risk cohort including mmu-miR-720, mmu-miR-1937c, and mmu-miR-1937a+mmu-miR-1937b.We found a group of microRNAs to be expressed abnormally high and another group microRNA to be expressed abnormally low in the high risk group as comparing to the low risk group. We compared the data to our murine lung cancer microRNA profile and found a group of miRNA expression potentially correlated to risk of lung cancer development in mice. Most human lung cancers are at an advanced stage when they are first found. These cancers are very hard to cure. However, if a lung cancer is found at an earlier stage when it is small and before it has spread, the early stage lung cancer can be successfully treated. Further studies are necessary to employ these miRNAs as early diagnostic biomarkers in prediction of human lung cancers. Citation Format: Myia Aiges, Abeer Almiman, Li Gao, Kathleen Dotts, Miguel A. Villalona-Calero, Wenrui Duan. Differential miRNA expression levels and risk of lung adenocarcinoma in transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3548.

MiR-135 suppresses glycolysis and promotes pancreatic cancer cell adaptation to metabolic stress by targeting phosphofructokinase-1

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. It thrives in a nutrient-poor environment; however, the mechanisms by which PDAC cells undergo metabolic reprogramming to adapt to metabolic stress are still poorly understood. Here, we show that microRNA-135 is significantly increased in PDAC patient samples compared to adjacent normal tissue. Mechanistically, miR-135 accumulates specifically in response to glutamine deprivation and requires ROS-dependent activation of mutant p53, which directly promotes miR-135 expression. Functionally, we found miR-135 targets phosphofructokinase-1 (PFK1) and inhibits aerobic glycolysis, thereby promoting the utilization of glucose to support the tricarboxylic acid (TCA) cycle. Consistently, miR-135 silencing sensitizes PDAC cells to glutamine deprivation and represses tumor growth in vivo. Together, these results identify a mechanism used by PDAC cells to survive the nutrient-poor tumor microenvironment, and also provide insight regarding the role of mutant p53 and miRNA in pancreatic cancer cell adaptation to metabolic stresses.

Abstract A31: Mutant p53 increases integrin-ECM interactions in early HGSOC

Abstract Until recently, it was thought that high-grade serous ovarian cancer (HGSOC) arises from the ovarian surface epithelium (OSE). Now, it has become clear that the epithelium of the fallopian tube fimbriae is the starting point of many HGSOCs. Serous tubal intraepithelial carcinomas (STICs) are suggested to be the earliest form of HGSOC and show mutations in the p53 gene. Part of the neoplastic transformation into invasive HGSOC may be the initial adhesion of STIC cells to the surface of the ovary as a first site of metastasis. To examine the role of mutant p53 immortalized fallopian tube secretory epithelial (FTSE) cells in the early onset of HGSOC, we used mechanically tunable hydrogels and developed a protocol to obtain decellularized human ovary scaffolds for 3D cell cultures. Stable expression of mutant p53 in FTSE cells promoted adhesion to hydrogel matrices and invasion into decellularized ovary scaffolds. Next, we sought to analyze the extracellular matrix (ECM) landscape of human fallopian tube and ovary tissues and identified proteins with a high affinity to integrin αvβ3. Mutant p53 FTSE cells had increased levels of integrin αvβ3 and decreased adhesive potential upon integrin αvβ3 blockage compared to wild-type FTSE cells. The results indicate that FTSE cells expressing mutant p53 upregulate integrin expression, which in turn alters their tissue-specific ECM interactions. Our findings give new insights in the early onset of HGSOC and may help to understand the role of p53 in the cells of origin of HGSOC. Citation Format: Laura SM Lecker, Caterina Trevisan, Robin Delaine-Smith, Eleni Maniati, Oliver Pearce, Christoph Meinert, Marcin Iwanicki, Ronny Drapkin, Daniela Loessner, Frances Balkwill. Mutant p53 increases integrin-ECM interactions in early HGSOC. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A31.

Abstract 269: Reactivation of mutant p53 in human prostate cancer cells as a critical mechanism for inhibition of tumor growth by phenethyl isothiocyanate

Abstract Mutations in the p53 gene occur in a wide variety of human cancers at remarkably high frequencies. Prostate cancer (PCa), the most commonly diagnosed cancer and the second leading cause of cancer-related death in men in the United States, show a significantly high frequency of p53 mutations (~30%-70%). However, the role of mutant p53 as a target for the treatment or prevention of PCa remained to be investigated. In this study, we showed that cruciferous vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutants, irrespective of its site of mutation, in PCa cells in vitro. Importantly, this study showed that dietary supplementation of PEITC can significantly inhibit DU145 p53 mutant xenograft tumor in a nu/nu Balb/c mice. Interestingly, the animals in the PEITC-treated group showed a significant decrease in the mutant p53 expression levels as compared to the animals in the control group. DU145 cells harboring p53V274F/P223L mutant display maximum sensitivity to PEITC-induced growth inhibition as compared to the wild-type p53 LNCaP and p53 null PC-3 cells. PEITC treatment also caused a significant decrease in the p53 expression level in DU145 cells. No significant inhibition of cell proliferation was detected for the normal prostate epithelial RWPE1 cells treated with PEITC. Mechanistic studies revealed that PEITC induces a conformation change and restores the transactivation functions to the p53 mutant. Accordingly, in PEITC-treated cells the reactivated p53 mutant(s) induces apoptosis by activating canonical wild-type p53 targets, inducing a G1 and S phase delay and by phosphorylating ATM. PEITC treatment of isogenic PC-3 p53 null cells revealed that PC-3 cells expressing p53P223L mutant exhibit maximum inhibition of cell proliferation, suggesting that PEITC acts in a p53P223L mutant-dependent manner. Furthermore, PEITC treatment restores the transactivation functions to p53P223L mutant as shown by the activation of p53 regulated downstream target genes. Interestingly p73, a member of the p53 gene family that shares high functional homology and regulate cell-growth/ cell-death pathways in a manner similar to p53, does not play a role in p53-mediated pathway activation in PEITC treated cells. We further showed that PEITC inhibits cell proliferation and induces apoptosis in prostate cancer cells with hotspot mutants R248W (VCaP) and R175H (LAPC-4). Importantly, PEITC can induce a conformation change, reactivates p53 mutants and induces G1 and S-phase delays in these cells. These results demonstrate that PEITC can reactivate mutant p53, irrespective of the site of p53 mutation, in human prostate cancer cells. These studies provide the first example of targeting mutant p53 in prostate cancer cells by a dietary-related compound that may have impact on the prevention and treatment strategies of this disease. Citation Format: Monika Aggarwal, Rahul Saxena, Nasir Asif, Elizabeth Sinclair, Judy Tan, Idalia Cruz, Deborah Berry, Bhaskar Kallakury, Quynhchi Pham, Thomas Ty Wang, Fung-Lung Chung. Reactivation of mutant p53 in human prostate cancer cells as a critical mechanism for inhibition of tumor growth by phenethyl isothiocyanate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 269.

Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction.

Cancer cells depend on glutamine to sustain their increased proliferation and manage oxidative stress, yet glutamine is often depleted at tumor sites due to excessive cellular consumption and poor vascularization. We have previously reported that p53 protein, while a well-known tumor suppressor, can contribute to cancer cell survival and adaptation to low glutamine conditions. However, the TP53 gene is frequently mutated in tumors, and the role of mutant p53 (mutp53) in response to metabolic stress remains unclear. Here, we demonstrate that tumor-associated mutp53 promotes cancer cell survival upon glutamine deprivation both in vitro and in vivo. Interestingly, cancer cells expressing mutp53 proteins are more resistant to glutamine deprivation than cells with wild type p53 (wtp53). Depletion of endogenous mutp53 protein in human lymphoma cells leads to cell sensitivity to glutamine withdrawal, while expression of mutp53 in p53 null cells results in resistance to glutamine deprivation. Furthermore, we found that mutp53 proteins hyper-transactivate p53 target gene CDKN1A upon glutamine deprivation, thus triggering cell cycle arrest and promoting cell survival. Together, our results reveal an unidentified mechanism by which mutp53 confers oncogenic functions by promoting cancer cell adaptation to metabolic stresses.

Abstract 3692: Transcriptome analysis of oral tongue cancer reveals novel insights into wild type and mutant TP53 transcription program

Abstract The p53 oncoprotein is a tumor suppressor that is stabilized upon various forms of cellular stresses to induce transcription of genes regulating cell cycle arrest or apoptosis. TP53 is the most frequently mutated gene in human cancers and majority of mutations are located in the region encoding the DNA binding domain compromising thereby its transcription activation ability and resulting in loss of function. Recent studies however suggest mutant p53 proteins to exhibit a gain of function property. Specific p53 missense mutations can result in an altered transcription program causing positive regulation of cell proliferation, metastasis and chemoresistance. In our previous studies, we performed comprehensive characterization of squamous cell carcinoma of the oral tongue (SCCOT) with respect to p53, EGFR, Wnt, MSI, LoH of several tumor suppressor loci and HPV status. Mutant p53 was a significant predictor of overall survival and the TP53 codon 72 Proline allele was significantly associated with SCCOT. In order to dissect the role of mutant p53 in tongue cancer, we performed genome wide DNA and RNA profiling of 26 and 40 SCCOT samples, respectively. Both mutant and wild type tumor samples appeared to exhibit comparable levels of DNA copy number alterations. Transcriptome data analyses using a combination of single sample gene set enrichment analysis and comparative marker selection revealed gene sets that could significantly distinguish p53 mutant and wild type tumor samples. Significance analysis of microarrays performed on all genes constituting the differentially enriched gene sets surprisingly identified only two genes to be upregulated in p53 mutant samples at a false discovery rate significantly lower than 10% namely TP53 itself and SMARCD1; the latter a member of the SWI/SNF chromatin remodelling complex. Elevated levels of TP53 transcript in tumors harbouring mutant p53 significantly correlated with levels of ZMAT3, itself induced by p53 and known to stabilize the TP53 transcript. In addition, the analysis revealed several known (ATF3 and others) and novel (GCHFR and others) targets of wild type p53. Differential expression of all targets was validated in additional tongue cancer samples. Ectopic expression of certain (but not all) p53 mutant proteins in p53 null cells induced SMARCD1 (but not canonical wild type p53 targets) while expression of wild type p53 induced GCHFR, ATF3, CDKN1A, etc. (but not SMARCD1). In contrast, p53 stabilization in cells harboring wild type p53 caused elevation of GCHFR, ATF3, CDKN1A, etc., but not of SMARCD1. Validation of novel targets using promoter-luciferase constructs, chromatin immunoprecipitation PCR and a tongue cancer tissue microarray is underway. This is perhaps the first evidence from Head and Neck tumor samples for a gain of function activity of mutant p53. Thus wild type and mutant p53 may support distinct transcription programs in tongue cancer. Citation Format: Raju SR Adduri, Padmavathi Kavadipula, Viswakalyan Kotapalli, Leena Bashyam, Anupama Shirke, Arun kumar Paripati, Swarnalata Gowrishankar, Mukta Srinivasulu, Mohammed Mujtaba Ali, Subramanyeshwar Rao, Snehalatha Dhagam, Mohana Vamsy Chigurupati, Shantveer G. Uppin, Vijaya Tourani, Murali D. Bashyam. Transcriptome analysis of oral tongue cancer reveals novel insights into wild type and mutant TP53 transcription program. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3692.

Genomic Instability: The Pivotal Role of Mutant P53 in Human Cancers

The tumor suppressor p53 plays a critical role to preserve DNA fidelity from diverse insults through the regulation of cell-cycle checkpoints, DNA repair, senescence and apoptosis. The TP53 is altered in more than half of human cancers. This leads to the production of mutant p53 proteins that loose wild type p53 tumor suppression functions and concomitantly acquire new oncogenic deleterious features implicated in: increased cell proliferation, increased chemoresistance, disruption of tissue architecture, promotion of migration, invasion and metastasis and several other pro-oncogenic properties. Accumulating evidences suggest that mutant p53 proteins drastically perturb the residual genome-stabilizing mechanisms during cancer progression, thereby increasing genomic instability of mutant p53 carrying human cancers. In this commentary we briefly summarize the most important evidences suggesting that mutant p53 plays a relevant role in promoting genomic instability in human cancers.

Abstract 3507: APR-246 reactivates mutant p53 in non-small cell lung cancer cell lines and sensitizes cells for CDDP treatment under normoxic and hypoxic conditions

Abstract Introduction: Tumors are often characterized by inactivating mutations in the TP53 gene. Mutations in this gene occur in about 50% of non-small cell lung cancers (NSCLC), and influence the response to common chemotherapeutics like CDDP (cisplatin). In addition, as hypoxic stress, which is often found in tumors, is able to induce p53 accumulation and p53 dependent apoptosis, the reactivation of mutant p53 might induce a strong cytotoxic effect in hypoxic tumor regions. Therefore, we treated NSCLC tumor cells with APR-246, a known activator of mutant p53, under normoxic and hypoxic conditions. In addition, we combined APR-246 with CDDP, to determine whether reactivation of mutant p53 could enhance the cytotoxic response of CDDP. Materials & Methods: The NSCLC cell line CRL-5908, harboring a R273H p53 mutation, was used. To determine the role of mutant p53 in response to APR-246, CRL-5908 was transduced with a p53 shRNA lentiviral vector to obtain a p53 deficient sub cell line. The SRB-assay was used to determine the cytotoxic effect of both mono-and combination therapies after which possible synergism was calculated by the Chou-Talalay method, both under normoxic and hypoxic conditions (0% O2, 5% CO2, 95% N2). In addition, we determined the p53 protein level as well as the mRNA and protein levels of its main transcription targets MDM2, p21, PUMA and BAX. The induction of apoptosis and cell cycle arrest were determined by flowcytometric analysis. Results: Our results show a clear response of CRL-5908 to APR-246, which was stronger under hypoxic conditions compared to normoxic conditions (IC50: 9.5 ± 2.5 vs. 16.3 ± 3.4). We observed that CRL-5908 was less sensitive to CDDP monotherapy than the p53 wild type cell line A549 (IC50: 15.2 ± 3.3 vs. 6.2 ± 1.6). We were able to overcome this resistance to CDDP by combining the drug with APR-246 (IC20, IC40, IC60), which resulted in CDDP IC50 values of respectively 11.5 ± 4.0; 5.0 ± 2.2 and 6.5 ± 2.0 under normoxic conditions. Discussion: Our initial results show that APR-246 monotherapy is promising for the treatment of p53 mutant NSCLC, particularly under hypoxic conditions. In addition, APR-246 seems to sensitize cells for CDDP treatment when combined simultaneously. We will further study the underlying mechanisms, like the activation of p53 and the transcription of its main transcription target MDM2, p21, PUMA and BAX as well as apoptosis and cell cycle arrest. Moreover, we will compare simultaneous treatment of CDDP and APR-246 with sequential treatment in which CDDP is followed by APR-246 treatment, and further sort out the response of this combination therapy under hypoxic conditions, as well as the role of mutant p53. Citation Format: Christophe Deben, Vanessa Deschoolmeester, An Wouters, Jolien Van den Bossche, Julie Jacobs, Filip Lardon, Christian Rolfo, Patrick Pauwels. APR-246 reactivates mutant p53 in non-small cell lung cancer cell lines and sensitizes cells for CDDP treatment under normoxic and hypoxic conditions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3507. doi:10.1158/1538-7445.AM2015-3507