Wt P53 Research Articles

Cancer-derived C-terminus-extended p53 mutation confers dominant-negative effect on its wild-type counterpart.

ABSTRACTThe vast majority of p53 missense mutants lose the wild-type (wt) function and/or exert ‘dominant-negative’ effects on their wt counterpart. Here, we identify a novel form of p53 mutation with an extended C-terminus (p53 long C-terminus, p53LC) in a variety of human cancers. Interestingly, the two representative mutants (named ‘p53-374*48’ and ‘p53-393*78’) as tested in this study show both loss-of-function and dominant-negative phenotypes in cell proliferation and colony formation assays. Mechanistically, p53LCs interact with and retain wt p53 in the cytoplasm and prevent it from binding to the promoters of target genes, consequently inhibiting its transcriptional activity. Also, p53LCs are very stable, though not acetylated in cells. Remarkably, the p53LCs can desensitize wt p53-containing cancer cells to p53-activating agents. Together, our results unveil a longer form of p53 mutant that possesses a dominant-negative effect on its wt counterpart, besides losing its wt activity.

CBIO-11. HISTONE H3.3 G34R/V MUTATIONS STIMULATE PEDIATRIC HIGH-GRADE GLIOMA FORMATION THROUGH THE INDUCTION OF CHROMOSOMAL INSTABILITY

Abstract H3.3 G34R/V mutations are drivers of high-grade pediatric glioma (pHGG). H3.3 G34R/V mutations are linked to altered H3.3 K36 trimethylation (H3K36me3); implicating epigenetic gene regulation as a possible contributor to pHGG formation. Here we show that H3.3 G34R/V also induces chromosomal instability (CIN); a hallmark of pHGG. If CIN promotes pHGG formation is unknown. We observed that H3.3 G34 mutant pHGG cells have reduced mitotic H3.3 S31 phosphorylation compare to WT H3.3 cell lines. And, H3.3 G34R reduced Chk1 phosphorylation at S31 by >90% in an in vitro kinase assay. Chk1 regulates chromosome segregation through phosphorylation of pericentromeric H3.3 S31 during early mitosis. Overexpression of H3.3 G34R or non-phosphorylatable S31A in H3.3 WT, diploid cells caused a significant increase in CIN. Likewise, H3.3 G34 mutant pHGG cells have significantly elevated rates of CIN as compare to H3.3 WT pHGG cells. During normal cell division, phospho-S31 is lost in anaphase. However, following chromosome missegregation, phospho-S31 spreads and stimulates p53-induced cell cycle arrest. Here we show that WT p53 cells expressing mutant G34 fail to arrest following chromosome mis-segregation. These studies demonstrate that H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferating CIN cells. To determine if this process contributes to tumorigenesis, we used RCAS Nestin-TVA mice to overexpress H3.3 WT, G34R, or S31A – P2A-linked to PDGFB expression in glial precursor cells of newborn mice. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free. Our work implicates CIN as a driver of H3.3 G34 mutant pHGG formation. Our ongoing studies utilize K36M and double mutants to further define the contributions of S31 phosphorylation (CIN) and H3K36me3 (epigenetic gene regulation) to tumorigenesis.

CBIO-27. INTEGRATED FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF THE INTRINSIC APOPTOTIC MACHINERY IDENTIFIES THERAPEUTIC VULNERABILITIES IN GLIOBLASTOMA

Abstract Molecular profiling frequently fails to predict therapeutic outcomes in cancer. This is in part due to the myriad of genetic alterations comprising a tumor which enable rapid rewiring of cancer signaling pathways. Functional profiling ascertains signaling dynamics and has emerged as an alternative method to predict drug responses. It is unknown whether incorporating molecular with functional profiling offers superior insight into specific tumor dependencies and consequently therapeutic susceptibilities. Here we performed integrated molecular and functional characterization (via BH3 profiling) of the intrinsic apoptotic machinery in 50 GBM patient specimens. We found that treatment with standard of care (e.g., temozolomide or radiation) rewired the apoptotic machinery in a p53 dependent manner, eliciting an exclusive survival dependency on BCL-xL in a genetically defined subset of GBM tumors. However, functional BH3 profiling of ‘apoptotic priming’ was required to predict response to combined IR/TMZ and BCL-xL inhibition in p53 WT GBM tumors. Accordingly, a composite of genetic and functional profiling predicted the response of patient derived GBM models to IR/TMZ with a novel GBM specific BCLxL antagonist. Collectively, these studies identify the mechanisms of intrinsic apoptosis resistance in GBM and demonstrate how functional and molecular data can be complementary to robustly predict therapy-induced cell death.

Focal Adhesion Kinase Drives Resistance to Therapy in HPV-Negative Head and Neck Squamous Cell Carcinoma in a p53-Dependent Manner

Our group has previously demonstrated focal adhesion kinase (FAK) expression is associated with radioresistance and worse outcome in human papillomavirus negative (HPV-) head and neck squamous cell carcinoma (HNC). However, the relative importance of FAK in driving therapeutic resistance is unclear. FAK function can be inhibited by wild type (wt) p53, but p53 is mutated in over 80% of HPV(-) HNC. Based on these observations, we tested the hypothesis that mutant (mut) p53 permits or even drives FAK-mediated radioresistance.Two cohorts of HPV(-) HNC tumors treated with radiation (RT), single institution (n = 81) and TCGA (n = 324), were stratified by TP53 mutational status (wt versus mut) and FAK copy number/mRNA expression. In vivo shRNA screening in 5 human HNSCC xenograft flank models was performed using libraries focused on known "druggable" proteins and DNA damage repair combined with RT or carboplatin. Correlation between FAK protein expression and sensitivity to cisplatin was evaluated in 20 TP53 mut HNC cell lines. The relationship between p53, FAK and radioresponse was examined in vitro using UMSCC (p53 null) HNC cells forced to express wt or mut TP53. FAK function was modulated in these cells using shRNA or chemical inhibition (defactinib) and response to radiation or carboplatin was evaluated via immunoblot for DNA damage repair proteins and clonogenic assay. FAK and p53 interactions were evaluated via immunoprecipitation. An in vivo immunodeficient flank model using HN31 cells expressing FAK shRNA was used to assess in vivo radioresponse.High levels of FAK copy number and gene expression were associated with worse disease-free survival in TP53 mut HPV(-) HNC, but not TP53 wt tumors. In vivo shRNA screening identified FAK as an excellent target for either radio- or chemosensitization. FAK protein expression was correlated with cisplatin IC50 (P = 0.0083, r = 0.572) in our HNC cell line panel. Inhibition of FAK via shRNA in the HN31(TP53 mut) cell line led to radiosensitization both in vitro and in a xenograft flank model. Defactinib treatment led to increased sensitivity to radiation or carboplatin in TP53 mut (HN31) but not TP53 wt (HN30) tumor cell lines. In UMSCC1 cells pretreated with defactinib, TP53 mut expression resulted in significant radiosensitization, but TP53 wt expression did not. To examine the physical interaction between p53 and FAK, different TP53 constructs were expressed in UMSCC1 cells. Interestingly, missense mutation does not impair the ability of p53 to bind to FAK. However, FAK phosphorylation was higher in cells expressing mut p53 compared to wt. Moreover, FAK inhibition combined with radiation led to higher levels of ɣ-H2AX in mut p53 expressing cells.FAK is a potentially important target for therapeutic sensitization, primarily in TP53 mutated HNSCC.

Anticancer effect of AZD2461 PARP inhibitor against colon cancer cells carrying wt or dysfunctional p53

Colon cancer is one of the most common cancers, currently treated with traditional chemotherapies or alternative therapies. However, these treatments are still not enough effective and induce several side effects, so that the search of new therapeutic strategies is needed. The use of Poly-(ADP-ribose)-polymerase (PARP) inhibitors, although originally approved against BRCA-1 or BRCA-2 mutated cancers, has been extended, particularly in combination with other treatments, to cure cancers that do not display defects in DNA repair signaling pathways. The role of p53 oncosuppressor in the regulating the outcome of PARP inhibitor treatment remains an open issue. In this study, we addressed this topic by using a well-tolerated PARP 1/2/3 inhibitor, namely AZD2461, against colon cancer cell lines with different p53 status. We found that AZD2461 reduced cell proliferation in wtp53 and p53−/− cancer cells by increasing ROS and DNA damage, while R273H mutant (mut) p53 counteracted these effects. Moreover, AZD2461 improved the reduction of cell proliferation by low dose radiation (IR) in wtp53 cancer cells, in which a down-regulation of BRCA-1 occurred. AZD2461 did not affect cell proliferation of mutp53 colon cancer cells also in combination with low dose radiation, suggesting that only wt p53 or p53 null colon cancer cells could benefit AZD2461 treatment.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Inhibitors Targeting CDK9 Show High Efficacy against Osimertinib and AMG510 Resistant Lung Adenocarcinoma Cells.

Simple SummaryNon-small cell lung cancer accounts for 80% of all lung cancer cases. While a subset of non-small cell lung cancer patients respond to immunotherapy, those who are treated with chemotherapy or targeted therapy develop resistance to the drugs. Thus, novel therapeutic strategies are needed to combat this disease. Here we show that inhibitors of the cyclin-dependent kinase 9 are highly effective in preventing the growth of a variety of lung cancer cell lines and lung cancer organoids with high potency. These inhibitors suppressed the expression of several genes like Sox2, Sox9, and Mcl1 that promote tumor growth, facilitating growth arrest. Since inhibitors of cyclin-dependent kinase 9 are undergoing clinical trials for hematological malignancies, our studies suggest that these inhibitors would be attractive candidates to combat non-small cell lung cancer.Non-small cell lung cancer has a 5-year survival rate of less than 12–15%, calling for the development of additional therapeutic strategies to combat this disease. Here we tested the efficacy of inhibiting cyclin-dependent kinase 9 (CDK9) on lung cancer cell lines with K-Ras and EGFR mutations and on lung cancer organoids. Three different CDK9 inhibitors reduced the viability and anchorage-independent growth of lung cancer cell lines at very low nanomolar to micromolar concentrations. CDK9 inhibition suppressed the expression of the anti-apoptotic protein, Mcl1, as well as the embryonic stem cell transcription factors, Sox2 and Sox9, which are pro-tumorigenic. In contrast, treatment with CDK9 inhibitors increased the levels of WT p53 and its downstream target p21 in K-Ras mutant cell lines. Furthermore, the CDK9 inhibitors could markedly reduce the viability of Osimertinib-resistant PC9 and AMG510-resistant H23 and H358 cells with comparable efficacy as the parental cells. CDK9 inhibitors could also significantly reduce the growth and viability of lung cancer organoids with high potency. Taken together, the data presented here strongly suggest that CDK9 inhibitors would be efficacious against K-Ras mutant and EGFR mutant NSCLCs, including those that develop resistance to targeted therapies.

Abstract 2160: Tumor suppressor p53 down-regulates programmed cell death protein 4 (PDCD4) expression

Abstract Programmed Cell Death Protein 4 (PDCD4), a well-known tumor suppressor, acts to inhibit translation initiation and cap-dependent translation by inhibiting the helicase activity of EIF4A. EIF4A tends to target mRNAs with structured 5'-UTR. PDCD4 can also prevent tumorigenesis by inhibiting tumor promoter-induced neoplastic transformation, and studies indicate that PDCD4 may inhibit translation of certain mRNAs via directly binding. A previous study has demonstrated that PDCD4 inhibits translation of p53 mRNA and treatment with DNA-damaging agents down-regulates PDCD4 expression but activates p53 expression. The study further demonstrated that treatment with DNA-damaging agents resulted in down-regulation of PDCD4 expression and an increase in p53 expression, suggesting a potential mechanism in which p53 regulates expression of Pdcd4. However, whether p53 directly regulates PDCD4 remains unknown. Herein, we demonstrate for the first time that p53 regulates PDCD4 expression. First, we found that overexpression of p53 in p53-null cells (H1299 and Saos2 cells) decreased PDCD4 protein level. Secondly, p53 dose-dependently decreased PDCD4 promoter activity in gene reporter assays. Moreover, we demonstrated that mutations on p53 (R273H: contact mutant, hotspot and R175H: conformational mutant, hotspot) abolished p53-mediated PDCD4 repression. Furthermore, using Tet-on expression system, PDCD4 protein level was decreased when WT p53, but not hotspot mutants of p53, was expressed in H1299 cells. Finally, mutations on DNA-binding domain, but not C-terminal regulatory domain, of p53 disrupted p53-mediated PDCD4 repression. Taken together, our results suggest that p53 functions as a novel regulator of PDCD4 and the relationship between of p53 and PDCD4 may be involved in tumor development and progression. Citation Format: Wei-Hsiung Yang, Andrew P. George, William H. Yang, Chiung-Min Wang, Richard H. Yang. Tumor suppressor p53 down-regulates programmed cell death protein 4 (PDCD4) expression [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 2160.

MAGE-a Inhibits Apoptosis in Multiple Myeloma through Regulation of Bim and PUMA

MAGE-a Inhibits Apoptosis in Multiple Myeloma through Regulation of Bim and PUMA

Ywhae Located on Chromosome 17p13 Promotes Myeloma Cells Proliferation Via Induction of Anti-Apoptosis and Survival Signaling Pathways

Patients with multiple myeloma (MM) carrying 17p deletion (del17p) have poor outcome. Around a third of MM patients with del(17p) are also reported to have a TP53 mutation. The acquisition of TP53 mutations seems to occur after allelic loss of 17p, and confers worse clinical outcomes. However, a recent publication has raised possibility that other additional genes resident at 17p13 may play a role in the clinical outcome of this subgroup of MM patients.The YWHAE gene is located at 17p13.3 and encodes the 14-3-3ε protein, one of the mammalian 14-3-3 protein family members that are highly conserved in eukaryotes. Altered 14-3-3ε expression is associated with development and progression of cancer. It has a context dependent pleotropic effect both as oncogene or tumor suppressor gene. However, its precise role in tumorigenesis has yet to be fully discovered.Using a large cohort of MM patients (n=369) RNA sequencing and SNP array data with complete clinical annotation, we observed a significant positive correlation (r=0.7) between YWHAE and TP53 at gene and copy number levels. In MM patients with del17p, YWHAE expression was significantly lower compared to normal plasma cells. Importantly, low expression of YWHAE correlated with poor clinical outcome (both EFS and OS), suggesting that YWHAE may be important in this specific myeloma clinical setting. However, in 2 independent MM datasets, we observed that overall YWHAE expression is higher in CD138+ MM cells compared to normal plasma cells. Moreover, we confirmed high expression of 14-3-3ε at the protein level in a large panel of MM cell lines and primary MM cells.To clarify this apparent paradox and shed light on the role of 14-3-3ε in MM, we used gene-specific CRISPR screen as well as shRNA knock down (KD) to study the functional impact of perturbation of YWHAE in MM. For the loss-of-function studies, we selected a large panel of MM cell lines based on their p53 status (WT, HD, Mu). We found a significant inhibitory effect of YWHAE KD on MM cell growth and survival over time, and induction of apoptosis via PARP cleavage and BAX increase. The antiproliferative effects were even more pronounced in p53 WT myeloma cell lines, such as MM1S and H929.Gene expression profiling (GEP) analysis showed a significant number of differentially expressed genes in YWHAE KD cells compared to control cells. Gene enrichment analysis revealed a major impact of YWHAE KD on a large set of genes involved in the mTORC1 signaling pathway. WB analysis confirmed a reduction of p-mTOR along with a decreased of p-p70 S6 kinase and p-4E BP1 after YWHAE KD. Considering the crucial role of mTOR pathway in the regulation of autophagy, we are now in the process of analyzing the effect of YWHAE on the autophagic response. Moreover, we found that YWHAE KD exerted a significant suppression of known E2F1 gene targets. Notably, a significant decrease in the expression of genes involved in the unfolded protein response was observed upon silencing/knock-out of YWHAE. As a consequence, myeloma cell sensitivity to bortezomib was increased after 14-3-3ε KD.In conclusion, YWHAE and its coding protein 14-3-3ε are constitutively expressed in MM cells. Perturbation of 14-3-3ε expression impacts MM cell growth via induction of apoptosis and inhibition of mTOR pathway. 14-3-3ε KD increase cell sensitivity to bortezomib. Ongoing efforts will further elucidate the potential molecular mechanism of YWHAE/14-3-3ε action in MM. DisclosuresAnderson:Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

HGG-31. HISTONE H3.3 G34R/V MUTATIONS STIMULATE PEDIATRIC HIGH-GRADE GLIOMA FORMATION THROUGH THE INDUCTION OF CHROMOSOMAL INSTABILITY

The histone H3.3 G34R/V mutations are known drivers of high-grade pediatric glioma (pHGG). However, the mechanism(s) for H3.3 G34R/V induced tumor formation are unclear. Chk1 phosphorylates H3.3 S31 at the pericentromere during early mitosis, suggesting a novel mitotic function. We observed that H3.3 G34 mutant pHGG cells have reduced mitotic H3.3 S31 phosphorylation compare to WT H3.3 cell lines. The H3.3 G34R mutation reduced Chk1 phosphorylation at S31 by >90% in an in vitro kinase assay. Overexpression of either H3.3 G34R or non-phosphorylatable S31A in H3.3 WT, diploid cells led to a significant increase in chromosome mis-segregations. Likewise, H3.3 G34 mutant pHGG cells have significantly elevated rates of mis-segregation as compare to H3.3 WT pHGG cells. During normal cell division, phospho-S31 is lost in late anaphase. However, when chromosome missegregation occurs, phospho-S31 spreads and stimulates p53 accumulation in G1 – thus suppressing aneuploid cell proliferation. Here we show that cells expressing mutant G34 fail to arrest following mis-segregation, despite having WT p53. These studies demonstrate that the H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferative, chromosomally instable cells. To determine if this process contributes to tumorigenesis, we used the RCAS/TVA mouse model to overexpress H3.3 WT, G34R, or S31A in the glial precursor cells of mice pups. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT mice). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free and did not develop high-grade tumors. Our work strongly indicates that a major factor in H3.3 G34R pHGG formation is the induction of chromosomal instability – which occurs directly through the suppression of H3.3 S31 phosphorylation.

PSMD9 ribosomal protein network maintains nucleolar architecture and WT p53 levels

Capitalizing on an unexpected observation that multiple free ribosomal proteins co-purify/pull-down with PSMD9, we report here for the first time that PSMD9 is necessary to maintain the morphology and integrity of the nucleolus. As seen by NPM1 immunofluorescence and electron microscopy, the nucleolar structure is clearly disrupted in PSMD9 null MCF7 breast cancer cells. The resultant stress is pronounced leading to the accumulation of WT p53 and slow growth. A dual insult with Actinomycin D exasperates the nucleolar stress in these cells which fail to recover in stipulated time. This double insult in the WT cells enhances the interaction of PSMD9 with ribosomal subunits. Our data also reveals that in PSMD9 null cells, ribosomal proteins RPS25 and RPL15 fail to localise in the nucleolus. We speculate that the interaction of PSMD9 with multiple free ribosome subunits has at least two important implications: a) PSMD9 plays a role in trafficking of ribosomal proteins into the nucleolus, therefore contributing to the maintenance of structural and morphological organization of the membrane-less nucleolar compartment; b) under conditions that induce nucleolar stress, PSMD9-Ribosomal Protein interaction protects WT MCF7 breast cancer cells from slow growth and eventual death. This possibility renders the domains of PSMD9 to be attractive drug targets in the context of cancer and other multiple ribosome-associated disorders.

Upregulation of wild-type p53 by small molecule-induced elevation of NQO1 in non-small cell lung cancer cells.

The tumor suppressor p53 is usually inactivated by somatic mutations in malignant neoplasms, and its reactivation represents an attractive therapeutic strategy for cancers. Here, we reported that a new quinolone compound RYL-687 significantly inhibited non-small cell lung cancer (NSCLC) cells which express wild type (wt) p53, in contract to its much weaker cytotoxicity on cells with mutant p53. RYL-687 upregulated p53 in cells with wt but not mutant p53, and ectopic expression of wt p53 significantly enhanced the anti-NSCLC activity of this compound. RYL-687 induced production of reactive oxygen species (ROS) and upregulation of Nrf2, leading to an elevation of the NAD(P)H:quinoneoxidoreductase-1 (NQO1) that can protect p53 by inhibiting its degradation by 20S proteasome. RYL-687 bound NQO1, facilitating the physical interaction between NQO1 and p53. NQO1 was required for RYL-687-induced p53 accumulation, because silencing of NQO1 by specific siRNA or an NQO1 inhibitor uridine, drastically suppressed RYL-687-induced p53 upregulation. Moreover, a RYL-687-related prodrug significantly inhibited tumor growth in NOD-SCID mice inoculated with NSCLC cells and in a wt p53-NSCLC patient-derived xenograft mouse model. These data indicate that targeting NQO1 is a rational strategy to reactivate p53, and RYL-687 as a p53 stabilizer bears therapeutic potentials in NSCLCs with wt p53.

P53 Gain-of-Function Mutants and Steroids in Ovarian Cancer Cell Metastasis

High-grade serous ovarian cancer (HGSOC) is a heterogeneous disease for which there currentlyis no cure. Because p53 is mutated in >90% of all ovarian cancer, we studied specific gain-of-function (GOF) p53 mutants and steroid hormones for tumor morphology and metastasis in vivo. For this, we analyzed ALST (WT p53), SKOV3 (p53 null), TYK-NU (p53-R175H), OVCAR3 (p53-R248Q) and OVCA420 (p53-R273H) cell line xenografts in Foxn1-/- mice. ALST cells failed tometastasize, likely due to the known apoptotic effects of WT p53. SKOV3 and the p53-GOF celllines metastasized to the omentum and exhibited distinct morphologies: SKOV3, epithelial-like;TYK-Nu, vascular-like; OVCAR3, epithelial/mesenchymal; and OVCA420 epithelial exclusive. Despite different morphologies and p53 status, each tumor type contained large, Polyploid GiantCancer Cells (PGCCs) that are stem-like cells undergoing endoreplication. A specificphosphorylated, active form of β−catenin (pCTNNB1-S31/S37/T41; pCTNNB1) co-localizedselectively with GOF p53 and the mitotic stress regulatory kinase pMSK1-T581 in mitotic cells andPGCCs, indicating that in addition to GOF p53 mutants, pCTNNB1 and pMSK1 play a role intumor progression. To determine if ALST cells could be rendered metastatic, the p53 GOFmutants R175H and R273H were stably expressed in these cells. Remarkably, the ALST(WT/R273H) cells, but not the ALST (WT/R175H) cells, formed solid tumors on the ovary, visceralfat and uterus; but not on the omentum where OVCA420 (p53-R273H) cells formed tumors. TheALST (WT/R273H) tumors harbored discreet populations of pCTNNB1+ mitotic cells and PGCCsand exhibited distinct growth-promoting responses to estradiol while showing growth-inhibitoryeffects of DHT and nuclear AR in the tumor-associated stromal cells. Stably expressing p53-R175H or p53-R273H GOF mutants in SKOV3 (p53 null) cells enhanced tumor progression withthe R273H mutant being most aggressive. The parental SKOV3 tumors also contained pCTNNB1that was associated with mitotic cells and PGCCs. In the SKOV3 p53-R175H cells - and moreimpressively in the p53-R273H cells - pCTNNB1 co-localized with p53 in PGCCs. Whereas ESR1staining was diffuse in the ALST-R273H cells, it was nuclear in the SKOV3 cell lines. However,tumor promotion by estrogen and inhibition by DHT were observed in each SKOV3 cell line,suggesting that stromal cells may also contribute to the steroid dependent effects. Collectively, 1)elevated levels of specific phosphorylated forms of CTNNB1 and MSK1 identify mitotic cells andPGCCs within the tumors and new targets for therapeutic approaches; 2) knowing the status ofp53, and the presence and localization of pCTNNB1 and pMSK1 and steroid hormone receptorsin ovarian tumors suggest combinatorial approaches should be considered to combat the mostlethal gynecological cancer. NIH-CA181808; NIH-HD097321 (JSR).

Oncogenic WIP1 phosphatase attenuates the DNA damage response and sensitizes p53 mutant Jurkat cells to apoptosis

Wild-type (wt) p53-induced phosphatase 1 (Wip1), encoded by the protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D) gene, is a serine/threonine phosphatase induced upon genotoxic stress in a p53-dependent manner. Wip1/PPM1D is frequently overexpressed, amplified and mutated in human solid tumors harboring wt p53 and is thus currently recognized as an oncogene. Oncogenic Wip1 dampens cellular stress responses, such as cell cycle checkpoints, apoptosis and senescence, and consequently increases resistance to anticancer therapeutics. Targeting Wip1 has emerged as a therapeutic strategy for tumors harboring wt p53. However, little is known about the efficacy of Wip1-targeted therapies in tumors lacking p53. The present study aimed to investigate the potential role of oncogenic Wip1 in p53 mutant (mt) Jurkat cells. In the present study, it was demonstrated that p53 mt Jurkat cells exhibited PPM1D/Wip1 gene amplification and expressed relatively high levels of Wip1, as confirmed by gene copy number and RNA expression analysis. In addition, Jurkat cells underwent G2 cell cycle arrest, apoptotic cell death and senescence in response to etoposide and doxorubicin, although the phosphorylation levels of DNA damage response (DDR) elements, including ataxia-telangiectasia mutated, ataxia-telangiestasia and Rad3-related, checkpoint kinase (Chk)1 and Chk2 were significantly low. Accordingly, the targeting of Wip1 phosphatase by RNA interference increased the phosphorylation of DDR elements, but decreased the rate of apoptosis in response to etoposide or doxorubicin in Jurkat cells. The induction of senescence or cell cycle arrest was not affected by the knockdown of Wip1. The results suggest that increased Wip1 expression enhances the apoptotic sensitivity of Jurkat cells in response to chemotherapeutic agents by attenuating DDR signaling. The present study highlights the possible pro-apoptotic role of Wip1 in a p53 mt T-cell acute lymphoblastic leukemia cell line. The data suggest the careful consideration of future treatment strategies aiming to manipulate or target Wip1 in human cancers lacking p53.

The TCGA Molecular Classification of Endometrial Cancer and Its Possible Impact on Adjuvant Treatment Decisions.

Simple SummaryThe Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) is a recently developed tool to identify four distinct molecular subgroups of endometrial cancer. Patients identified as Polymerase Epsilon exonuclease domain mutated (POLE EDM) or p53-mutated have significantly altered prognosis compared to patients allocated to the mismatch repair deficient (MMRd) or p53 wt groups. The aim of this review is to give a broad overview over the initial development and refinement of the classifier as well as possible effects on the recommended adjuvant treatment. We have summarized the clinical data of 8 studies including 3650 endometrial cancer patients and analyzed the distribution of tumor stage and adjuvant treatment received in respect to the molecular subgroups. Based on the findings of the summarized studies treatment de-escalation might be feasible for POLE EDM patients while p53 abn patients should receive adjuvant (chemo-)radiotherapy.Adjuvant treatment decisions for endometrial cancer (EC) are based on stage, the histological grade of differentiation, histological subtype, and few histopathological markers. The Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) identified four risk groups of EC patients using a combination of immunohistochemistry and mutation analysis: Polymerase Epsilon exonuclease domain mutated (POLE EDM), mismatch repair deficient (MMRd), p53 wild-type/copy-number-low (p53 wt), and p53-mutated/copy-number-high (p53 abn). Patients allocated to the POLE or abnormal p53 expression subtype are faced with a significantly altered outcome possibly requiring a modified adjuvant treatment decision. Within this review, we summarize the development of ProMisE, characterize the four molecular subtypes, and finally discuss its value in terms of a patient-tailored therapy in order to prevent significant under or overtreatment.

TP53 drives abscopal effect by secretion of senescence-associated molecular signals in non-small cell lung cancer

BackgroundRecent developments in abscopal effect strongly support the use of radiotherapy for the treatment of metastatic disease. However, deeper understanding of the molecular mechanisms underlying the abscopal effect are required to best benefit a larger proportion of patients with metastasis. Several groups including ours, reported the involvement of wild-type (wt) p53 in radiation-induced abscopal effects, however very little is known on the role of wtp53 dependent molecular mechanisms.MethodsWe investigated through in vivo and in vitro approaches how wtp53 orchestrates radiation-induced abscopal effects. Wtp53 bearing (A549) and p53-null (H1299) NSCLC lines were xenotransplanted in nude mice, and cultured in 2D monolayers and 3D tumor spheroids. Extracellular vesicles (EVs) were isolated from medium cell culture by ultracentrifugation protocol followed by Nanoparticle Tracking Analysis. Gene expression was evaluated by RT-Real Time, digital qRT-PCR, and dot blot technique. Protein levels were determined by immunohistochemistry, confocal anlysis, western blot techniques, and immunoassay.ResultsWe demonstrated that single high-dose irradiation (20 Gy) induces significant tumor growth inhibition in contralateral non-irradiated (NIR) A549 xenograft tumors but not in NIR p53-null H1299 or p53-silenced A549 (A549sh/p53) xenografts. We further demonstrates that irradiation of A549 cells in vitro induces a senescence-associated secretory phenotype (SASP) producing extracellular vesicles (EVs) expressing CD63 and carrying DNA:RNA hybrids and LINE-1 retrotransposon. IR-A549 EVs also hamper the colony-forming capability of recipient NIR A549 cells, induce senescent phenotype, nuclear expression of DNA:RNA hybrids, and M1 macrophage polarization.ConclusionsIn our models, we demonstrate that high radiation dose in wtp53 tumors induce the onset of SASP and secretion of CD63+ EVs loaded with DNA:RNA hybrids and LINE-1 retrotransposons that convey senescence messages out of the irradiation field triggering abscopal effect in NIR tumors.

DIPG-05. HISTONE H3.3 K27M IMPAIRS SER31 PHOSPHORYLATION, RESULTING IN CHROMOSOMAL INSTABILITY, LOSS OF CELL CYCLE CHECKPOINT CONTROL, AND TUMOR FORMATION

Diffuse midline gliomas with the H3.3 K27M mutation are lethal brain tumors in children. H3 K27M causes global loss of Lys27 triple methylation (Lys27me3), inducing epigenetic reprograming. Here we show that H3.3 K27M also causes decreased H3.3 Ser31 phosphorylation on mitotic chromosomes. We show that H3.3 K27M DIPG cells have reduced pericentromeric phospho-Ser31 and increased rates of chromosome missegregation compared to normal, diploid human cells. CRISPR-editing K27M to M27K restored phospho-Ser31 to WT levels and dramatically decreased the rate of chromosome missegregation. We confirm that Chk1 is the H3.3 Ser31 kinase: K27M mutant H3.3 protein exhibits ~60% reduced Chk1 phosphorylation of Ser31 in vitro. Chk1 knockdown completely abolishes phospho-Ser31 in cells and these have increased rates of chromosome missegregation. In normal, diploid cells, expression of K27M or an S31A non-phosphorylatable mutant increased chromosome missegregation; this is suppressed by expressing a phosphomimetic double mutant (K27M/S31E) that restores phospho-Ser31. WT cells arrest following chromosome missegregation. However, cells expressing H3.3 K27M or S31A fail to arrest - despite having WT p53. Finally, we expressed H3F3AS31A and PDGFb in an RCAS/TVA mouse model of DIPG and ~80% developed diffuse high-grade brain tumors and show significantly decreased survival. Our results suggest that loss of phospho-Ser31 alone is oncogenic because H3.3 S31A-expressing cells are WT for K27me3. Our results demonstrate that H3.3 K27M inhibits Ser31 phosphorylation both in vitro and in vivo, leading to both chromosome missegregation and loss of subsequent G1 arrest – thus creating diffuse midline gliomas with both dynamic, complex karyotypes and epigenetic reprogramming.

Dual Modulators of p53 and Cyclin D in ER Alpha Signaling by Albumin Nanovectors Bearing Zinc Chaperones for ER-positive Breast Cancer Therapy.

CDATA[The inherited mutations and underexpression of BRCA1 in sporadic breast cancers resulting in the loss or functional inactivation of BRCA1 may contribute to a high risk of breast cancer. Recent researchers have identified small molecules (BRCA1 mimetics) that fit into a BRCA1 binding pocket within Estrogen Receptor alpha (ERα), mimic the ability of BRCA1 to inhibit ERα activity, and overcome antiestrogen resistance. Studies indicate that most of the BRCA1 breast cancer cases are associated with p53 mutations. It indicates that there is a potential connection between BRCA1 and p53. Most p53 mutations are missense point mutations that occur in the DNA-binding domain. Structural studies have demonstrated that mutant p53 core domain misfolding, especially p53-R175H, is reversible. Mutant p53 reactivation with a new class of zinc metallochaperones (ZMC) restores WT p53 structure and functions by restoring Zn2+ to Zn2+ deficient mutant p53. Considering the role of WT BRCA1 and reactivation of p53 in tumor cells, our hypothesis is to target both tumor suppressor proteins by a novel biomolecule (ZMC). Since both proteins are present in the same cell and are functionally inactive, this state may be a novel efficacious therapeutic regime for breast cancer therapy. In addition, we propose to use Albumin Nanovector (ANV) formulation for target drug release.

CBIO-16. SUPPRESSION OF HISTONE H3.3 MITOTIC PHOSPHORYLATION DRIVES DEVELOPMENT OF H3K27M-MUTANT DIFFUSE MIDLINE GLIOMAS

Abstract Pediatric midline gliomas – including DIPG – are lethal brain tumors in children, with poor prognosis and limited treatment options that provide only short-term benefits. The majority have a lysine-to-methionine substitution at residue 27 (H3K27M) in genes expressing histone H3 – predominantly in the H3.3 variant. This causes a global reduction in H3 Lys27 tri-methylation (H3K27Me3), comprehensive epigenetic reprogramming, and is a key driver in gliomagenesis. We show that the H3.3K27M mutation also induces chromosome segregation defects, which in high-grade tumors, results in extensive copy number alterations (CNAs). Ser31 is one of five amino acid substitutions differentiating H3.3 from canonical H3.1. Mitotic phosphorylation of H3.3 Ser31 by Chk1 kinase is restricted to pericentromeric heterochromatin, where it plays a role in chromosome segregation. We show that the K27M mutation affects neighboring Ser31 phosphorylation and pericentromeric heterochromatin organization. We demonstrate that (i) H3.3 K27M protein is defective for Ser31 phosphorylation by Chk1 kinase in vitro; (ii) DIPG cell lines have significantly decreased mitotic Ser31 phosphorylation, and are chromosomally unstable; and (iii) CRISPR-reversion of H3.3K27M to Lys27 restores phospho-Ser31 (and Lys27 tri-methylation) and significantly decreases chromosome instability. Expression of H3.3K27M or non-phosphorylatable H3.3S31A mutants in WT cells results in chromosome missegregation; this is suppressed by co-expression of phospho-mimetic H3.3K27M/S31E. In normal cells, chromosome missegregation stimulates p53-dependent cell cycle arrest in G1 to prevent the proliferation of aneuploid daughters. However, cells expressing H3.3 K27M or S31A failed to arrest following missegregation - despite having WT p53. Finally, in a novel mouse model of glioma, mean survival of mice with tumors induced with H3.3K27M and H3.3S31A was 81 and 68 days: 100% of H3.3S31A mice developed high-grade tumors. H3.3 WT controls developed only low-grade tumors and all survived 100 days. H3.3S31A is WT for Lys27 tri-methylation and thus, loss of Ser31 phosphorylation alone is oncogenic.