Mutant P53 Tumor Cells Research Articles

Elevated type I interferon signaling defines the proliferative advantage of ARF and p53 mutant tumor cells.

The tumor suppressors p53 and ARF collaborate to prevent unwarranted cell proliferation and as such are two of the most frequently mutated genes in human cancer. Concomitant loss of functional p53 and ARF leads to massive gains in cell proliferation and transformation and is often observed in some of the most aggressive human cancer subtypes. These phenotypic gains are preceded by increased type I interferon (IFN) signaling that involves canonical STAT1 activation and a subsequent IFN-stimulated gene (ISG) signature. Here, we show that cells lacking p53 and ARF require active JAK1 to phosphorylate STAT1 on Y701 to maintain their high rate of proliferation. In fact, the use of selective JAK1 inhibitors ruxolitinib or baricitinib inhibited the induction of ISG's and the proliferation of p53 and ARF deleted cells. We identify a group of solid human tumors that lack functional p53 and ARF, show an expression signature of the upregulated type I IFN response genes, and are sensitive to selective JAK1 inhibitors. These data suggest that the type I IFN response acts as a positive driver of proliferation in the absence of p53 and ARF and, as such, presents itself as a potential therapeutic target in aggressive solid tumors.

CCRG-06. PP2A DEFICIENCY INDUCES CELLULAR SENESCENCE AND SENSITIZES P53 MUTANT TUMORS TO SENOLYTIC THERAPY

Abstract Loss of p53 can promote tumor progression by preventing induction of senescence, a critical tumor-suppressive mechanism that limits tumor proliferation. Promotion of senescence in a p53-independent manner can be a novel strategy to overcome treatment resistance in p53-mutant brain tumors. Protein-Phosphatase-2A (PP2A), a ubiquitous serine/threonine phosphatase, regulates various cellular processes including cell-cycle progression. We hypothesize that PP2A-deficiency can induce tumor senescence independent of p53-mutational-status and sensitize tumors to senolytic therapy. CRISPR-KO of PP2A was generated in multiple p53-mutant tumor cell lines: D425 (human medulloblastoma), SB28 and GL261 (murine glioma). Hallmarks of senescence such as b-galactosidase, p21, and p16 were assessed via flow cytometry, RT-PCR and Western-Blot. Secretion of senescence-associated secretory phenotype (SASP), such as IL8, was assessed through ELISA. b-galactosidase, p21, p16 and IL8 were dramatically increased in PP2A-KO compared to WT at baseline. The effect was further enhanced when combined with radiation therapy (RT), a known inducer of senescence, demonstrating PP2A-deficiency synergizes with RT and significantly increases cellular senescence. Expression of CD1d, a MHC1-like molecule associated with senescence and required for invariant NKT cell-activation (iNKT), was dramatically increased in PP2A-KO in comparison to WT via flow-cytometry. In-vitro, sensitivity to WT and PP2A-KO cells to senolytic treatments, such as ABT-737 and iNKT-cell therapy, was tested using an XCelligence-assay system and flow-cytometry-based cytotoxicity assay. PP2A-deficient cells were sensitized to ABT-737, a BCL-xL inhibitor that preferentially targets and kills senescent cells. Co-culture revealed increased cytotoxicity of iNKT-cells against PP2A-deficient tumors compared to WT. In vivo, D425 WT or PP2A-KO cells were injected intracranially into nude NU/J mice, radiated 2 days post-injection, and monitored till endpoint. At baseline, PP2A-KO mice survived longer than WT, and those with PP2A-KO and RT survived the longest. In conclusion, we report that PP2A-deficiency in tumor cells can induce p53-independent senescence and sensitize brain tumors to senolytic therapy.

Rab11-FIP1 mediates epithelial-mesenchymal transition and invasion in esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) is the most common subtype of esophageal cancer worldwide. The most commonly mutated gene in ESCC is TP53. Using a combinatorial genetic and carcinogenic approach, we generate a novel mouse model of ESCC expressing either mutant or null p53 and show that mutant p53 exhibits enhanced tumorigenic properties and displays a distinct genomic profile. Through RNA-seq analysis, we identify several endocytic recycling genes, including Rab Coupling Protein (Rab11-FIP1), which are significantly downregulated in mutant p53 tumor cells. In 3-dimensional (3D) organoid models, genetic knockdown of Rab11-FIP1 results in increased organoid size. Loss of Rab11-FIP1 increases tumor cell invasion in part through mutant p53 but also in an independent manner. Furthermore, loss of Rab11-FIP1 in human ESCC cell lines decreases E-cadherin expression and increases mesenchymal lineage-specific markers, suggesting induction of epithelial-mesenchymal transition (EMT). Rab11-FIP1 regulates EMT through direct inhibition of Zeb1, a key EMT transcriptional factor. Our novel findings reveal that Rab11-FIP1 regulates organoid formation, tumor cell invasion, and EMT.

A thiol‐bound drug reservoir enhances APR‐246‐induced mutant p53 tumor cell death

The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. The compound APR‐246 (PRIMA‐1Met/Eprenetapopt) is converted to methylene quinuclidinone (MQ) that targets mutant p53 protein and perturbs cellular antioxidant balance. APR‐246 is currently tested in a phase III clinical trial in myelodysplastic syndrome (MDS). By in vitro, ex vivo, and in vivo models, we show that combined treatment with APR‐246 and inhibitors of efflux pump MRP1/ABCC1 results in synergistic tumor cell death, which is more pronounced in TP53 mutant cells. This is associated with altered cellular thiol status and increased intracellular glutathione‐conjugated MQ (GS‐MQ). Due to the reversibility of MQ conjugation, GS‐MQ forms an intracellular drug reservoir that increases availability of MQ for targeting mutant p53. Our study shows that redox homeostasis is a critical determinant of the response to mutant p53‐targeted cancer therapy.

Abstract IA11: Overcoming immune evasion in pediatric brain tumors

Abstract Many immunotherapies act by enhancing T-cell killing of tumor cells. Cytotoxic T cells recognize antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. Our studies suggest that medulloblastomas and high-grade gliomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. Treatment with tumor necrosis factor or lymphotoxin beta receptor agonist rescues expression of Erap1, Tap1, and MHC-I on p53 mutant tumor cells. In vivo, TNF treatment prolongs survival and markedly augments the efficacy of the immune checkpoint inhibitor anti-PD-1. These studies identify p53 as a key regulator of immune evasion in vivo and suggest that TNF could be used to enhance sensitivity of p53-mutant tumors to immunotherapy. Citation Format: Alexandra Garancher, Hiromichi Suzuki, Svasti Haricharan, Meher B. Masihi, Jessica M. Rusert, Paula S. Norris, Florent Carrette, Megan M. Romero, Sorana A. Morrissy, Patryk Skowron, Florence M.G. Cavalli, Hamza Farooq, Vijay Ramaswamy, Alaide Morcavallo, Jacob J. Henderson, James M. Olson, Yoon-Jae Cho, Xiao-Nan Li, Louis Chesler, Marco A. Marra, Oren J. Becher, Linda M. Bradley, Carl F. Ware, Michael D. Taylor, Robert J. Wechsler-Reya. Overcoming immune evasion in pediatric brain tumors [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA11.

Tumor necrosis factor overcomes immune evasion in p53-mutant medulloblastoma.

Many immunotherapies act by enhancing the ability of cytotoxic T cells to kill tumor cells. Killing depends on T cell recognition of antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. In this study, we showed that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. In vitro, tumor necrosis factor (TNF) or lymphotoxin-β receptor agonist can rescue expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, low doses of TNF prolong survival and synergize with immune checkpoint inhibitors to promote tumor rejection. These studies identified p53 as a key regulator of immune evasion and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.

Abstract 4441: Discovery of potent and selective inhibitors of USP7 with anti-tumor activity in vitro and in vivo

Abstract USP7 is a deubiquitinase that regulates the levels of multiple downstream targets with roles in cancer progression and immune response. Inhibitors of USP7 may thus decrease oncogene function, increase tumor suppressor function, enhance immune function and sensitize tumor cells to DNA damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple p53-wild type cell lines, including several blood cancer and MYCN-amplified neuroblastoma cell lines, as well as a subset of p53-mutant tumor cell lines in vitro. Further, oral administration of our USP7 inhibitors inhibits MM.1S (multiple myeloma; p53-wild type) and H526 (small cell lung cancer; p53-mutant) tumor growth in vivo. Our work confirms that USP7 is a pharmacologically tractable target and future studies will aim to further understand the mechanism of action of USP7 inhibitors in p53-mutant cancers. Citation Format: Yamini M. Ohol, Michael Sun, Paul Leger, Dennis Hu, Berenger Biannic, Payal Rana, Cynthia Cho, Scott Jacobson, Steve Wong, Jerick Sanchez, Xinping Han, Kyle Young, Akinori Okano, Jack Maung, Gene Cutler, Nick Shah, Lavanya Adusumilli, Deepika Kaveri, Oezcan Talay, Deepa Pookot, Betty Abraham, Delia Bradford, Nathan Kozon, Christophe Colas, Andrea Kim, Jacob Schwarz, David Wustrow, Dirk Brockstedt, Paul Kassner. Discovery of potent and selective inhibitors of USP7 with anti-tumor activity in vitro and in vivo [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 4441.

TNF superfamily cytokines overcome immune evasion in medulloblastoma

Abstract Many immunotherapies act by enhancing T cell killing of tumor cells. CD8+ cytotoxic T cells recognize antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. Here we show that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. Treatment with tumor necrosis factor (TNF) or lymphotoxin beta receptor agonist (LTβRag) rescues expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, TNF treatment prolongs survival and markedly augments the efficacy of the immune checkpoint inhibitor anti-PD-1. These studies identify p53 as a key regulator of immune evasion in vivo, and suggest that TNF could be used to enhance sensitivity of p53-mutant tumors to immunotherapy.

Abstract 3043: Survivin-mediated radio-sensitization response in p53 mutant tumor cells

Abstract Doses of ionizing radiation ≤100 mGy induce changes in radiation sensitivity expressed by cells exposed to subsequent higher doses. This is referred to as an adaptive effect. We describe a unique survivin-associated adaptive response in which increased radiation resistance or sensitization of cells can be induced by exposure to doses as low as 5 mGy. Experiments were performed using two murine sarcomas, SA-NH and FSa,grown either in culture or as tumors, and two human cancer cell lines, Panc-1 (pancreatic epithelioid carcinoma) and MDA-MB-231 (breast adenocarcinoma). Doses of 5 mGy were used to induce changes in the response of these tumor cells to higher therapy doses using a multi-dosing paradigm. Effects on radiation sensitivity were associated with changes in both survivin concentration and its translocation to the cytoplasm (SA-NH, p53 wild type) or nucleus (FSa, Panc-1 and MDA-MB-231, p53 mutant). In vitro survival assays (2 Gy per fraction, two once daily fractions) and tumor growth delay (TGD) (5 Gy per fraction, five once daily fractions) studies were performed on SA-NH and FSa cells. Intracellular localization of survivin was determined by ELISA and correlated to survival response. 2 Gy alone had no effect on intracellular translocation of survivin. When preceded 15 min earlier by a 5 mGy exposure, survivin increased in the cytoplasm of SA-NH as compared to the nuclei of FSa, Panc-1 and MDA-MB-231 cells. SA-NH tumors were protected by 5 mGy exposures (1.9 day decrease in TGD, P = 0.032) while FSa tumors were sensitized (4.5 day increase in TGD, P < 0.001). Panc-1 and MDA-MB-231 tumor cells were also sensitized to ionizing radiation following 5 mGy exposures. Exposure of SA-NH to 4 mM of the free radical scavenger N-acetylcysteine inhibited the effect of 5 mGy on inducing this adaptive response. We have identified a very low radiation dose-induced survivin-mediated radio-sensitization response that could enhance therapeutic outcomes associated with the increased usage of imaging procedures in radiation therapy. This work was supported by NIH/NCI grant R01 CA132998 and DOE Low Dose Program/Project Grant DE-SC0001271 (D.J.G.) Citation Format: David J. Grdina, Jeffrey S. Murley, Richard C. Miller, Ralph R. Weichselbaum, Alfred W. Rademaker, Gayle E. Woloschak, Jian Jian Li. Survivin-mediated radio-sensitization response in p53 mutant tumor cells. [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 3043.

Abstract LB-C11: HTLV-1 Tax-induced apoptosis to counteract tumor drug resistance

Abstract p53 mutations have been identified in over 50% of human cancers. As p53-mutant tumor cells have an inherent resistance to therapy-induced apoptosis, such a mechanism of resistance is an important problem in cancer therapy. Interestingly, Tax, a protein expressed by the human T-cell lymphotropic virus type 1 (HTLV-1), increases the sensitivity of p53-mutant and p53-null cells to DNA-damaging drugs. Tax-induced chemosensitivity presents therefore a novel strategy that can be exploited to counteract p53-mutant tumor resistance. The objective of this work is to harness the chemosensitizing property of Tax and use it to develop a chemotherapeutic approach that can overcome the drug resistance observed in p53-mutant/p53-null cancers. We hypothesize that Tax initiates a proapoptotic response in cancer cells by modulating cyclin-dependent kinase 4 (CDK4) activity. We use a combination of in vitro and in silico analyses to determine the mechanism of Tax-induced chemosensitivity and to identify small molecules that can phenocopy the CDK4-activating and the proapoptotic effects of Tax. Immunoblot analyses and in vitro kinase assays reveal that Tax alters CDK4 expression and activity. In addition, flow cytometry and gene expression studies respectively indicate that Tax expression promotes G1-to-S phase transition and leads to increased expression of the TP73 gene, a known homolog of TP53. Moreover, our in silico analyses, using an N-terminal construct of Tax and a published structure of CDK4 (PDB 3G33), reveal that CDK4 interacts with the Tax construct via a C-terminal domain that is distinct from its nucleotide-binding and D-type cyclin-binding sites. Further analyses identify ten small molecule mimics of Tax and putative CDK4 activators, which we show have a proliferative effect on treated cells. Although the focus of ongoing studies in the field has been the development of kinase inhibitors, several tumors develop strategies allowing them to override the effect of those inhibitors. Our work adopts a different strategy that will maximize the cytotoxic effect of anticancer drugs to overcome acquired drug resistance. Citation Format: Vanessa C. Ducas, Gary M. Kupfer. HTLV-1 Tax-induced apoptosis to counteract tumor drug resistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C11.

Abstract 847: Suppression of p53R2 but not R2 radiosensitizes mutant p53 tumor cells

Abstract Gemcitabine (2’,2’-difluroro-2’-deoxycytidine;dFdCyd) is a potent radiosensitizer in tumor cells in vitro and in vivo. dFdCyd elicits cytotoxicity primarily via incorporation of its triphosphate, dFdCTP, into DNA, whereas inhibition of ribonucleotide reductase (RR) by dFdCDP produces a profound depletion of dATP which correlates to radiosensitization. We have demonstrated that dNTP imbalances generated by dFdCyd produce mismatches in DNA, which augment sensitivity to subsequent ionizing radiation (IR) but are not required to elicit cytotoxicity. We have proposed that RR suppression would be as effective as dFdCyd for radiosensitization. RR is a heterodimeric tetramer composed of the regulatory and active site subunit R1 paired with either R2 or its p53-inducible homolog, p53R2, as the catalytic and rate-limiting subunit. We used a RNAi approach to suppress either R2 or p53R2 (≥ 90% suppression of either R2 or p53R2, with little effect (≤20%) on expression of its homolog) in two p53 wild type cell lines, MCF7 breast carcinoma and A549 lung carcinoma. This approach produced equivalent radiosensitization, dATP depletion, cytotoxicity and increase in DNA mismatches compared to inactivation of RR by dFdCyd (IC50). These results reinforce our prior finding that radiosensitization with dFdCyd is the result of inhibition of RR and not incorporation into DNA or cytotoxicity. We then proposed that R2 but not p53R2 suppression would radiosensitize mutant p53 tumor cells. Interestingly, R2 shRNA suppression did not radiosensitize mutant p53 MCF7/ADR cells (radiation enhancement ratio (RER) = 1.07 ± 0.06). Despite the mutant p53 status of MCF7/ADR cells, p53R2 was elevated (≥ 50%) following suppression of R2 compared to untreated cells (no shRNA). We hypothesized that this increase in p53R2 permits RR to continue producing dNTPs for DNA replication and repair in the absence of R2, thus preventing radiosensitization. Indeed, simultaneous suppression of R2 and p53R2 produced excellent radiosensitization (RER = 1.68 ± 0.04 (shRNAs) vs. 1.6 ± 0.01 (dFdCyd (IC50)), and similar cytotoxicity (surviving fraction (SF) = 76.5 ± 8.3% (shRNA) vs. 71.3 ± 6.7% (dFdCyd)). Impressively, suppression of p53R2 alone produced excellent radiosensitization (RER= 1.76 ± 0.30) with similar cytotoxicity (SF = 84 ± 20%) compared to the double knockdown or dFdCyd. Considering that most solid tumors express mutant p53, suppression of p53R2 instead of R2 may be a more effective method for radiosensitization. These studies suggest that deoxynucleotide biosynthesis is regulated differently in p53 wild type compared to mutant p53 tumor cells, and may be used to maintain DNA replication and repair and prevent anticancer efficacy after damage induced by IR or other DNA damaging agents. The mechanism by which mutant p53 tumor cells upregulate p53R2 warrants further investigation. Note: This abstract was not presented at the meeting. Citation Format: Sheryl A. Flanagan, Jeffrey J. Ackroyd, Sudha Mannava, Mikhail A. Nikiforov, Donna S. Shewach. Suppression of p53R2 but not R2 radiosensitizes mutant p53 tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 847. doi:10.1158/1538-7445.AM2014-847

Abstract 4334: Greater resistance of mutant p53 tumor cells to hypoxia or chloroquine with glucose or pyruvate supplementation is diminished under glucose starvation

Abstract BACKGROUND. Poorly vascularized solid tumors are exposed to regions of low oxygen and glucose deprivation. These restrictions may promote either defensive autophagy or cell death through excessive reactive oxygen species (ROS), mediated by activated NADPH oxidases (NOX) which transport electrons from NADPH on the cytoplasmic side of plasma membranes to reduce molecular oxygen at the extracellular space, increasing superoxide anion radical (O2•-). .OBJECTIVES. To investigate in genetically-matched human melanoma cells with wt or mutant p53, responses to: a) autophagy inhibitor chloroquine and to glucose-lowering metformin or to hypoxia in glucose-supplemented medium; b) pyruvate in glucose-free medium under normoxia or hypoxia; c) sub-physiological glucose. EXPERIMENTAL. Responses to normoxia or hypoxia were assesed by live-dead fluorometric assays, or by crystal violet staining of surviving cells. Mechanisms influencing response to glucose limitation were studied by specific immune blotting. RESULTS. Lower susceptibility to chloroquine ± metformin involving greater basal AMPK activation was seen in physiological 5 mM glucose in mutant p53 cells. These cells were also preferentially protected from prolonged hypoxia by glucose supplementation or by pyruvate supplementation of glucose-free medium under normoxia, but not under hypoxia. Sub-physiological glucose without pyruvate induced apoptosis-associated PARP cleavage irrespective of p53 status. However, treatment with impermeable exogenous superoxide dismutase (SOD) to remove extracellular superoxide signalling across cell membranes, preferentially counteracted PARP cleavage only in wt p53 cells with lower LDHA expression after 48 hr in 2.5 mM glucose. CONCLUSIONS. Glucose availability increases resistance to hypoxia or to chloroquine ± metformin in mutant p53 cells. However, glucose starvation promotes toxicity linked to extracellular (O2•-), preferentially in mutant p53 cells. Citation Format: Manuel Rieber. Greater resistance of mutant p53 tumor cells to hypoxia or chloroquine with glucose or pyruvate supplementation is diminished under glucose starvation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4334. doi:10.1158/1538-7445.AM2014-4334

Hypoxia and hypoxia mimetic cooperate to counteract tumor cell resistance to glucose starvation preferentially in tumor cells with mutant p53

We demonstrated that exogenous pyruvate promotes survival under glucose depletion in aerobic mutant p53 (R175H) human melanoma cells. Others subsequently indicated that mutant p53 tumor cells undergo p53 degradation and cell death under aerobic glucose-free conditions. Since glucose starvation occurs in hypoxic gradients of poorly vascularized tumors, we investigated the role of p53 siRNA under hypoxia in wt p53 C8161 melanoma using glucose starvation or 5mM physiological glucose. p53 Silencing decreased survival of glucose-starved C8161 melanoma with pyruvate supplementation under hypoxia (⩽1% oxygen), but increased resistance to glycolytic inhibitors oxamate and 2-deoxyglucose in 5mM glucose, preferentially under normoxia. Aiming to counteract hypoxic tumor cell survival irrespective of p53 status, genetically-matched human C8161 melanoma harboring wt p53 or mutant p53 (R175H) were used combining true hypoxia (⩽1% oxygen) and hypoxia mimetic CoCl2. No significant decrease in metabolic activity was evidenced in C8161 melanoma irrespective of p53 status in 2.5mM glucose after 48h of physical hypoxia. However, combining the latter with 100μM CoCl2 was preferentially toxic for mutant p53 C8161 melanoma, and was enhanced by catalase in wt p53 C8161 cells. Downregulation of MnSOD and LDHA accompanied the toxicity induced by hypoxia and CoCl2 in 5mM glucose, and these changes were enhanced by oxamate or 2-deoxyglucose. Our results show for the first time that survival of malignant cells in a hypoxic microenvironment can be counteracted by hypoxia mimetic co-treatment in a p53 dependent manner.

Selective radiosensitization of p53 mutant pancreatic cancer cells by combined inhibition of Chk1 and PARP1

We have recently shown that inhibition of HRR (homologous recombination repair) by Chk1 (checkpoint kinase 1) inhibition radiosensitizes pancreatic cancer cells and others have demonstrated that Chk1 inhibition selectively sensitizes p53 mutant tumor cells. Furthermore, PARP1 [poly (ADP-ribose) polymerase-1] inhibitors dramatically radiosensitize cells with DNA double strand break repair defects. Thus, we hypothesized that inhibition of HRR (mediated by Chk1 via AZD7762) and PARP1 [via olaparib (AZD2281)] would selectively sensitize p53 mutant pancreatic cancer cells to radiation. We also used 2 isogenic p53 cell models to assess the role of p53 status in cancer cells and intestinal epithelial cells to assess overall cancer specificity. DNA damage response and repair were assessed by flow cytometry, γH2AX, and an HRR reporter assay. We found that the combination of AZD7762 and olaparib produced significant radiosensitization in p53 mutant pancreatic cancer cells and in all of the isogenic cancer cell lines. The magnitude of radiosensitization by AZD7762 and olaparib was greater in p53 mutant cells compared with p53 wild type cells. Importantly, normal intestinal epithelial cells were not radiosensitized. The combination of AZD7762 and olaparib caused G2 checkpoint abrogation, inhibition of HRR, and persistent DNA damage responses. These findings demonstrate that the combination of Chk1 and PARP1 inhibition selectively radiosensitizes p53 mutant pancreatic cancer cells. Furthermore, these studies suggest that inhibition of HRR by Chk1 inhibitors may be a useful strategy for selectively inducing a BRCA1/2 ‘deficient-like’ phenotype in p53 mutant tumor cells, while sparing normal tissue.

Restoring p53 active conformation by zinc increases the response of mutant p53 tumor cells to anticancer drugs

Absence of p53 expression or expression of mutant p53 (mtp53) are common in human cancers and are associated with increased cancer resistance to chemo- and radiotherapy. Therefore, significant efforts towards pharmaceutical reactivation of defective p53 pathways are underway. We previously reported that, in HIPK2 knockdown background, p53 undergoes misfolding with inhibition of DNA binding and transcriptional activities that correlate with increased chemoresistance, and that zinc rescues wild-type p53 activity. Zinc has a crucial role in the biology of p53, in that p53 binds to DNA through a structurally complex domain stabilized by zinc atom. In this study, we explored the role of zinc in p53 reactivation in mutant p53-expressing cancer cells. We found that zinc re-established chemosensitivity in breast cancer SKBR3 (expressing R175H mutation) and glioblastoma U373MG (expressing R273H mutation) cell lines. Biochemical studies showed that zinc partly induced the transition of mutant p53 protein (reactive to conformation-sensitive PAb240 antibody for mutant conformation) into a functional conformation (reactive to conformation-sensitive PAb1620 antibody for wild-type conformation). Zinc-mediated p53 reactivation also reduced the mtp53/p73 interaction restoring both wtp53 and p73 binding to target gene promoters by ChIP assay with in vivo induction of wtp53 target gene expression, which rendered mutant p53 cells more prone to drug killing in vitro. Finally, zinc administration in U373MG tumor xenografts increased drug-induced tumor regression in vivo, which correlated with increased wild-type p53 protein conformation. These results show that the use of zinc might restore drug sensitivity and inhibit tumor growth by reactivating mutant p53.

Abstract 2657: Radiosensitization of pancreatic cancer cells by combined inhibition of Chk1 and PARP1 using AZD7762 and olaparib

Abstract Small molecule inhibitors targeted to key DNA damage response/repair proteins, such as Chk1 (checkpoint kinase 1) and PARP1 (poly (ADP-ribose) polymerase-1) are being developed to sensitize tumor cells to chemo- and radio- therapy. We have recently shown that inhibition of HRR (homologous recombination repair) by the Chk1/2 inhibitor, AZD7762 (AstraZeneca) is a mechanism of radiosensitization in pancreatic cancer cells. Others have demonstrated that AZD7762 selectively sensitizes p53 mutant tumor cells. PARP inhibitors have demonstrated preferential efficacy as radiation sensitizers in cells with other DNA damage repair defects, including HRR. Thus, we hypothesized that inhibition of HRR (mediated by Chk1 via AZD7762) and PARP1 (via olaparib, AZD2281) would selectively sensitize p53 mutant pancreatic cancer cells to radiation. To test this hypothesis we assessed radiosensitization by clonogenic survival in two p53 mutant pancreatic cancer cell lines, MiaPaCa-2 and MPanc96. Cells were treated with AZD7762 (100nM) and/or olaparib (1uM) for 1 hour before radiation (0-8Gy), and for 23 hours after radiation, followed by processing for clonogenic survival. We verified that treatment with olaparib inhibited poly (ADP-ribose). We found that olaparib or AZD7762 alone produced similar magnitudes of radiosensitization in MiaPaCa-2 cells (1.5 ± 0.1 and 1.5 ± 0.01, respectively). However, the combination of olaparib and AZD7762 produced significantly greater radiosensitization than either agent alone (2.4 ± 0.1, P<0.05), which appeared to be greater than additive. Similarly, in a second cell line, MPanc96, the combination of olaparib and AZD7762 produced significantly greater radiosensitization (3.0 ± 0.4, P<0.05) than either olaparib (1.5 ± 0.1) or AZD7782 alone (2.0 ± 0.1). The combination of olaparib with AZD7762 produced minimal toxicity with clonogenic surviving fractions in MiaPaCa-2 cells of 1.1 ± 0.1 (olaparib), 0.8 ± 0.1 (AZD7762), and 0.7 ± 0.1 (olaparib + AZD7762) and in MPanc96 cells of >1.0 ± 0.2 (olaparib), 0.5 ± 0.1 (AZD7762), and 0.6 ± 0.2 (olaparib + AZD7762). Radiosensitization by olaparib and AZD7762 was accompanied by an increase in γH2AX and S345 Chk1 suggesting that DNA damage was increased under these conditions. Taken together, these findings demonstrate that the combination of olaparib and AZD7762 induces profound radiosensitization of p53 mutant pancreatic cancer cell lines. Furthermore, these studies suggest that inhibition of HRR by Chk1 inhibitors may be a useful strategy for selectively inducing a BRAC1/2 deficient-like phenotype in p53 mutant tumor cells, thus resulting in synergistic radiosensitization when used in combination with PARP1 inhibition. Supported by R01CA78554. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2657. doi:10.1158/1538-7445.AM2011-2657

Targeting wild-type and mutant p53 with small molecule CP-31398 blocks the growth of rhabdomyosarcoma by inducing reactive oxygen species-dependent apoptosis.

Rhabdomyosarcoma (RMS) is a common soft-tissue sarcoma of childhood in need of more effective therapeutic options. The expression of p53 in RMS is heterogeneous such that some tumors are wild-type whereas others are p53 mutant. The small molecule CP-31398 modulates both the wild-type and the mutant p53 proteins. Here, we show that CP-31398 blocks the growth of RMS cells that have either wild-type or mutant p53 status. In wild-type A204 cells, CP-31398 increased the expression of p53 and its downstream transcriptional targets, p21 and mdm2; enhanced the expression of apoptosis-related proteins; and reduced proliferation biomarkers. Flow profiling of CP-31398-treated cells indicated an enhancement in sub-G(0) and G(1) populations. CP-31398 inhibited proliferation in a manner associated with co-induction of SOX9 and p21. Apoptosis induced by CP-31398 occurred with translocation of p53 to mitochondria, leading to altered mitochondrial membrane potential, cytochrome c release, and reactive oxygen species release. In vivo, CP-31398 decreased the growth of tumor xenografts composed of wild-type or mutant p53 tumor cells, increasing tumor-free host survival. Our findings indicate that the ability of CP-31398 to modulate wild-type and mutant p53 results in the inhibition of RMS growth and invasiveness.

Abstract C94: Deubiquitylases as novel anticancer targets: Discovery and development of novel USP7 inhibitors

Abstract The ubiquitin-proteasome pathway is a complex system consisting of enzymes that conjugate or deconjugate ubiquitin to/from target proteins upstream of the 26S proteasome. In particular, ubiquitin deconjugation is performed by families of proteases known as deubiquitylating enzymes (DUBs). The therapeutic validation of the ubiquitin-proteasome system was provided by the marketing approval of bortezomib (PS-341) for the treatment of multiple myeloma (MM). In our search for alternative approaches for the treatment of MM, we investigated the therapeutic potential of inhibiting another component of the ubiquitin-proteasome pathway, the DUB USP7. Our rationale for targeting USP7 is as follows: 1) Blockade of USP7 using siRNA or homologous recombination results in elevated levels of the tumor suppressor p53 and cell cycle arrest in cancer cells. In contrast to the situation in the majority of human tumors, mutations or deletions of p53 in MM are relatively rare and thus the activation of p53 is likely to offer a therapeutic benefit in MM. 2) Knockdown of USP7 destabilizes the adaptor protein Claspin, inhibiting activation of Chk1, an effector kinase in the DNA damage response, abrogating the G2M cell cycle checkpoint and providing a mechanism for inducing tumor cell death in a p53 independent manner. Thus inhibition of USP7 would be predicted to activate pro-apoptotic and inactivate pro-survival pathways in multiple myeloma, resulting in tumor suppression. Recently, using its proprietary screening technology, Progenra discovered and characterized a small molecule inhibitor of USP7, P5091. Specifically, P5091 selectively inhibits USP7 relative to other proteases and exhibits cytotoxic activity against both p53 wild type and p53 mutant tumor cell lines. Furthermore, P5091 destabilizes the pharmacodynamic markers HDM2 and Claspin in tumor cells and induces cell death synergistically in combination with genotoxic agents. Medicinal chemistry has identified a series of potent and selective novel chemical entities. Initial preclinical studies at the Dana-Farber Cancer Institute demonstrate that P5091 induces apoptosis and is cytotoxic against human MM cells but not normal plasma cells. In conclusion, these data demonstrate that 1) USP7 is a viable target for the treatment of MM and other cancers; and 2) P5091 is an efficacious inhibitor of USP7. Preclinical development of the P5091 series continues, augmented by a comprehensive medicinal chemistry program designed to identify a clinical candidate from this series in 2010. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C94.

Specific activation of the p53 pathway by low dose actinomycin D: A new route to p53 based cyclotherapy

The activation of p53 has been proposed as a novel anti-cancer treatment in two distinct contexts. In the first activation of p53 in tumor cells can promote apoptosis and senescence and enhance the anti-tumor activity of cytotoxic chemotherapeutic drugs. In the second application activation of p53 in normal tissues can cause a reversible cell cycle arrest that can be used to protect normal cells from the action of anti-mitotics. In this cyclotherapy role p53 mutant tumor cells are not arrested and remain sensitive to anti-mitotics. The advent of specific p53 activating molecules such as nutlin-3 has encouraged both approaches. We have sought for a clinically approved drug that can mimic nutlin-3. We show here that low doses of actinomycin D mimic nutlin-3 in the highly specific activation of p53 dependant transcription, in the induction of a reversible protective growth arrest in normal cells and in the enhancement of the activity of chemotherapeutic drug induced killing of p53 positive human tumor cells. While high doses of actinomycin D reveal its more non-specific activities, low doses of the drug will allow exploration of the value of p53 activation in preclinical and clinical models before nutlin-3 like drugs are approved.

Ignition of p53 Bomb Sensitizes Tumor Cells to Granzyme K-Mediated Cytolysis

Inactivation of tumor suppressor p53 results in loss of the apoptosis-regulating function of the p53 protein in tumor cells. Restoration of wild-type p53 expression in p53 mutant tumor cells increases tumor susceptibility to CTL-mediated cytolysis. However, the direct role of p53 in regulating tumor sensitivity to NK cell-mediated lysis and the functional relationship between p53 and granzymes in the control of tumor killing are still poorly documented. In this study, we found that p53 can sensitize tumor-killing susceptibility to NK and granzyme K-mediated cytolysis. Granzyme K is constitutively expressed in high levels in NK cells and induces rapid caspase-independent cell death. Granzyme K may exert a critical role in NK cell-mediated tumor clearance. p53 associates with granzyme K and is a physiological substrate of granzyme K. p53 was processed to three cleavage products of p40, p35, and p13 fragments at Lys(24) and Lys(305). These three cleavage products harbor strong proapoptotic activities that amplify the proapoptotic action of p53 to potentiate tumor-killing sensitivity. Therefore, p53 is as a cytotoxic bomb that can be triggered by granzyme K, leading to potentiating killing efficacy.