Intact P53 Signaling Research Articles

Fasting improves therapeutic response in hepatocellular carcinoma through p53-dependent metabolic synergism.

Cancer cells voraciously consume nutrients to support their growth, exposing metabolic vulnerabilities that can be therapeutically exploited. Here, we show in hepatocellular carcinoma (HCC) cells, xenografts, and patient-derived organoids that fasting improves sorafenib efficacy and acts synergistically to sensitize sorafenib-resistant HCC. Mechanistically, sorafenib acts noncanonically as an inhibitor of mitochondrial respiration, causing resistant cells to depend on glycolysis for survival. Fasting, through reduction in glucose and impeded AKT/mTOR signaling, prevents this Warburg shift. Regulating glucose transporter and proapoptotic protein expression, p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting. p53 is also crucial for fasting-mediated improvement of sorafenib efficacy in an orthotopic HCC mouse model. Together, our data suggest fasting and sorafenib as rational combination therapy for HCC with intact p53 signaling. As HCC therapy is currently severely limited by resistance, these results should instigate clinical studies aimed at improving therapy response in advanced-stage HCC.

CBIO-25. INTEGRATED MOLECULAR AND BH3 PROFILING OF THE INTRINSIC APOPTOTIC MACHINERY IDENTIFIES THERAPEUTIC VULNERABILITIES IN GLIOBLASTOMA

Abstract Resistance to apoptosis is a hallmark of cancer. However, the underlying molecular mechanisms of intrinsic-apoptotic resistance in glioblastoma (GBM) are largely unknown. Here we performed integrated molecular and functional characterization (via BH3 profiling) of the intrinsic apoptotic machinery in 50 GBM patient specimens. We found that, despite significant genetic heterogeneity of our GBM samples, all GBMs have a cross compensatory reliance on BCLXL and MCL1 for basal survival. Treatment with standard of care (e.g., temozolomide or radiation) caused minimal apoptosis, yet ablated the MCL-1 block in a p53-dependent manner, thus creating an exclusive dependence on BCLXL for survival in p53 wild-type GBM tumors (65% of GBM tumors). Consequently, BCLXL inhibition caused synergistic cell death with IR/TMZ in GBM tumors with intact p53 signaling. Importantly, the degree of synergistic cell kill was best predicted by combining molecular features with BH3 profiling, providing an integrated predictive signature of response to this novel therapeutic approach. Collectively, these studies identify mechanisms of intrinsic apoptosis resistance in both basal and treatment states of GBM and demonstrate how functional and molecular data can be complementary to robustly predict therapy-induced cell death.

STEM-15. RADIATION-INDUCED MODULATION OF NEURODEVELOPMENTAL PATHWAYS IN CLASSICAL GLIOMA STEM CELLS

Abstract The current standard-of-care treatment for GBM is ineffective and fails to significantly prolong survival. Following exposure to aggressive multimodal treatment, GBMs have been shown to frequently shift their biological features upon recurrence, acquiring a more resistant phenotype. However, the temporal dynamics and molecular mechanisms that facilitate GBM recurrence are poorly understood. The objective of our study was to assess the acute response to ionizing radiation in glioma stem cells (GSCs) from the Classical subtype of GBM in vitro and in vivo. We find that Classical GSCs rapidly undergo dramatic molecular and cellular changes in response to single and fractionated doses of ionizing radiation, resulting in a heterogeneous cell population. Ionizing radiation causes a transient decrease in the expression of key stemness genes (e.g., SOX2) followed by drastic morphological changes and a concomitant significant increase in the levels of key cell fate markers expressed in adult quiescent neural stem cells. Radiation-induced alteration of SOX2 levels in Classical GSCs is dependent on intact p53 signaling. GSCs previously exposed to radiation are more radio-resistant upon re-treatment compared to their naïve, untreated counterparts – suggesting that the aforementioned phenotypic shift to a quiescent neural stem cell phenotype promotes treatment resistance. Our results suggest that cell-intrinsic factors dictate how GSCs respond to radiation, and that Classical GSCs are neurodevelopmentally predisposed to shift towards an astrocytic/neural stem cell identity in response to cellular stress.

Dino Is a DNA Damage-Induced Tumor Suppressing Long Noncoding RNA

While the CKDN1A locus is frequently suppressed in human cancers, the mechanism by which this finding contributes to tumor suppression has not been fully elucidated. We hypothesized that DINO, a novel DNA damage-induced long noncoding RNA encoded in the CDKN1A promoter, is essential to p53-dependent tumor suppression. We used a combination of in vivo mouse models, cell culture experiments and human tumor profiling to examine the contribution of DINO to p53-dependent tumor suppression. We crossed a Dino loss of function mouse to the E-mu-Myc model of B-cell lymphoma to examine p53-dependent, p21-independent tumor suppression. Rescue experiments to restore Dino function in Dino-/- lymphoma cells were performed to confirm its contribution to growth suppression. Finally, we analyzed methylation patterns of the DINO/CDKN1A locus in TCGA datasets to investigate the relationship of DINO inactivation and TP53 mutation in human cancers. We find that loss of Dino significantly accelerates tumorigenesis in the E-mu-Myc mouse model of B-cell lymphoma in a haplo-insufficient manner, while previous studies have shown that loss of Cdkn1a does not impact tumor development. Complementation with Dino suppresses growth of isolated Dino-/-, E-mu-Myc lymphoma cells and delays tumor development when re-engrafted into mice. We show that each of these effects requires an intact p53 signaling pathway. Human DINO and mouse Dino can each functionally restore growth suppression in cell culture, suggesting functional conservation through mammalian evolution. Finally, we find that DINO is hypermethylated in multiple types of human cancer and that DINO hypermethylation is correlated to DINO expression, but not CDNK1A expression. Hypermethylation of DINO is mutually exclusive with TP53 alteration in several specific human cancers. We provide direct evidence that the long noncoding RNA DINO is a tumor suppressor transcribed from the CDKN1A promoter. These findings provide insight into a mechanism by which human tumors inactivate p53-dependent functions and describe a novel mechanism of tumor suppression by a long noncoding RNA. We suggest that inactivation of DINO presents a potential biomarker of tumors with altered p53 function, and provides a future basis for targeted therapy and personalized treatment.

Abstract 5420: Ribosomal protein-p53-MDM2 signaling by nucleolar stress response and drug discovery

Abstract Ribosomal biogenesis is a major energy consuming cellular step, and tightly regulated by the demand of protein synthesis associated with cell growth and proliferation. Thus, defects that impair ribosomal biogenesis cause nucleolar stress response. This response initiates a cascade mediated by ribosomal proteins (RPs), particularly RPL5, RPL11, RPL23, and RPS7. These RPs are usually located in the nucleolus but are rapidly released into the nucleoplasm upon nucleolar stress, activating the tumor suppressor MDM2-p53 pathway. Thus, nucleolar stress response is considered as a quality control surveillance mechanism that regulates balance between proper protein synthesis and cell cycle progression for tumor suppression. However, it is unknown whether there are direct connections between p53-activating RP genes and cancer development in vivo, and whether this stress response might be target for cancer therapy. We identified nucleolar protein PICT1 as a novel key regulator of nucleolar stress response. To clarify PICT1 function, we generated PICT1-deficient mice and ES cells and carried out extensive analyses. Our results show that even without DNA damage, PICT1 loss results in p53-dependent growth arrest in vitro and in vivo. We further show that PICT1 binds to RPL11, and that RPL11 is released from nucleoli in the absence of PICT1. In PICT1-deficient cells, increased binding of RPL11 to MDM2 blocks MDM2-mediated ubiquitination of p53, leading to p53 accumulation and repression of cell proliferation. Human colon and esophageal cancer patients with low PICT1 expression have better prognoses. Similarly, when shRNA is used to deplete PICT1 in various tumor cell lines with intact p53 signaling, the cells slow in growth and accumulate p53. These data suggest that PICT1 is a novel key regulator of the MDM2-p53 pathway on nucleolar stress response and promotes tumor progression by retaining RPL11 in the nucleolus. Thus, the nucleolar stress response PICT1 regulates could play a definitive role in tumor suppression and, chemicals which induce nucleolar stress response might be lead to new cancer therapeutics. Therefore, we next constructed a reporter system to visualize and quantitate nucleolar stress response. We found that this reporter system allowed quantitative measurement depend on strength and duration of this stress. As this reporter system showed excellent accuracy, we next screened 400 inhibitors, 1,300 approved drug, and hundreds of natural products from marine species. We found hit compounds including RNA synthesis inhibitor Doxorubicin and Aclarubicin, and also positive control compound, ActinomycinD. In addition, inhibitors of CDK and HDAC were included here. Thus, the nucleolar stress reporter system may reveal unexpected physiological functions of the nucleolar stress response, such as mitotic progression and epigenetics regulation, and identify novel cancer therapeutics, especially for individuals with intact-p53 signaling. Citation Format: Kohichi Kawahara, Takuto Kawahata, Fumito Horikuchi, Yohei Kamijo, Masatatsu Yamamoto, Yoshinari Shinsato, Kentaro Minami, Kazunari Arima, Toshiyuki Hamada, Tatsuhiko Furukawa. Ribosomal protein-p53-MDM2 signaling by nucleolar stress response and drug discovery. [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 5420. doi:10.1158/1538-7445.AM2015-5420

The Role of Ribosomal Protein Deficiency in T-MDS Pathogenesis

Therapy-related myelodysplastic syndrome (t-MDS) is a lethal complication of cytotoxic cancer treatment and a leading cause of non-relapse mortality among patients undergoing autologous hematopoietic cell transplantation (aHCT) for lymphoma. The molecular abnormalities leading to t-MDS, however, are poorly understood. We are studying a prospective, longitudinal cohort of patients undergoing aHCT for lymphoma at our center with serial sample collection and banking. Using this approach we have identified impaired hematopoietic function and altered gene expression in CD34+ progenitor cells from pre-aHCT samples from patients who later developed t-MDS (cases) as compared to patients who did not develop t-MDS (controls) (Cancer Cell 2011, 20:591). These results indicate that genetic programs associated with t-MDS are perturbed long before disease onset. The alterations seen include down-regulation of genes related to ribosome biogenesis and translation, highlighting the potential contribution of altered ribosome function to disease development. Interestingly, a group of inherited ribosomal disorders including Diamond-Blackfan Anemia (DBA), called ribosomopathies, are characterized by cytopenias and a propensity for MDS development, features that closely resemble those of t-MDS patients. We evaluated the expression level of 79 ribosomal genes and a selected group of ribosome associated genes in cases and controls by multiplex Q-RT-PCR. Expression of DBA associated genes RPL10, RPL11, and RPL27 was significantly reduced in cases compared to controls. Each of these genes is reported to be associated with DBA. In addition, expression of p53, p21, c-Myc, and hdm2 were significantly upregulated in t-MDS cases. Altered expression of RPL11 is hypothesized to contribute to hematopoietic dysfunction by modulating the p53 ubiquitin ligase, HDM2, and activating p53-dependent apoptosis. We therefore characterized the effect of reduced RPL11 expression on hematopoietic function and determined the role of p53 activation in mediating the effects of RPL11 knockdown. We generated lentiviral vectors expressing shRNAs against RPL11 to reduce its expression in hematopoietic cell lines and CD34+ stem cell populations. Cells were cultured for 48 to 96 hours post-transduction followed by apoptosis analysis using Annexin-V staining, and proliferation by dye dilution assays. We confirmed >90% knockdown of RPL11 in shRNA expressing cells (MOLM13) by Q-RT-PCR and Western blot analysis. RPL11 knockdown resulted in 5-fold increase in apoptosis (Figure 1a) and 2-fold inhibition of proliferation of MOLM13 hematopoietic cells that express wild type p53, but did not affect survival and proliferation of p53 mutated HL60 cells. This suggests that cellular responses to RPL11 deficiency may depend at least in part on intact p53 signaling. This was confirmed in experiments showing that concomitant shRNA mediated knockdown of p53 and RPL11 (Figure 1a) resulted in a 2-fold reduction of apoptosis compared to RPL11 knockdown alone. Furthermore, RPL11 knockdown in CD34+ stem/progenitor cells resulted in 40% increase in apoptosis, 30% inhibition of proliferation and 90% decrease in colony forming cell (CFC) frequency in methylcellulose progenitor assays. We measured protein translation by measuring the intensity of an alkyne analog of puromycin (OPP) integrated into newly translated proteins, ligation to an azide dye, and flow cytometry analysis. We observed a 5-fold drop in global protein translation with RPL11 knockdown using the Click-iT Plus Alexa Fluor Protein Synthesis Assay (Figure 1b). Our results suggest that ribosomal abnormalities occur early in the course of t-MDS development and may lead to enhanced cell death and impaired proliferation. These results support a potential role of aberrant RPL11 expression in altered cell survival, proliferation, and differentiation, which may contribute to hematopoietic dysfunction seen early in the course of t-MDS development. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 2936: JNJ-47117096, a selective small molecule inhibitor of the MELK oncogene decreases DNA damage tolerance in highly proliferating cancer cells

Abstract Maternal embryonic leucine zipper kinase (MELK, hMP38, pEG3), a Ser/Thr protein kinase, is highly overexpressed in stem cells and cancer cells. The oncogenic role of MELK is attributed to disabling critical cell cycle check points as well as enhancing replication. Most functional studies have relied on the use of siRNA/shRNA mediated gene silencing, but this often can be associated with off-target effects. Here we want to present a novel, potent and selective small molecule inhibitor JNJ-47117096 that has enabled us to validate the biological function of MELK kinase. Outcome: MELK inhibition in cancer cells with an intact p53 signaling pathway is linked to a replicative senescence phenotype. This phenotype correlates with a rapid ATM activation and phosphorylation of CHK2 without any effects on the alternative ATR/ CHK1 DNA damage pathways. Furthermore, JNJ-47117096 induces strong phosphorylation of p53 and prolonged up-regulation of p21 without the induction of apoptosis. Strikingly, MELK inhibition in several p53 disabled cancer cells showed induction of a mitotic catastrophe phenotype followed by prominent cell killing. Finally, JNJ-47117096 triggers a rapid degradation of cellular MELK protein, independent of the cell cycle phase and its regulation by the E2F pathway. This observation can clearly be linked to a direct binding effect of JNJ-47117096 to cellular MELK protein confirmed by chemical proteomics. Conclusion: Our data generated with JNJ-47117096, confirmed by selective siRNAs, indicate MELK as a key stimulator of proliferation and replication by its ability to increase the threshold for DNA damage tolerance. Targeting MELK function by selective small molecule inhibitors might sensitise tumors to DNA-damaging agents or radiation therapy. Citation Format: Lijs Beke, Joannes T.m. Linders, Cenk Kig, An Boeckx, Erika van Heerde, Dirk Wuyts, Marc Parade, Lieven Meerpoel, Chris Johnson, Monique Beullens, Mathieu Bollen, Dirk Brehmer. JNJ-47117096, a selective small molecule inhibitor of the MELK oncogene decreases DNA damage tolerance in highly proliferating cancer 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 2936. doi:10.1158/1538-7445.AM2014-2936

Abstract 191: Methylseleninic acid super-activates p53 senescence axis as a preventive barrier to prostate tumorigenesis driven by Pten deficiency in vivo.

Abstract Prostate cancer (PCa) arises from progression of pre-invasive lesions such as prostatic intraepithelial neoplasia (PIN) to adenocarcinoma and metastatic disease. Due to the very long latency of lesion progression, chemoprevention of prostate adenocarcinogenesis is recognized as a plausible and essential approach to win the war against PCa. Whereas the results from recent clinical trials (SWOG 9917 and SELECT) did not support the protective function for seleno-methionine (SeMet) supplement in healthy men with or without high grade PIN, we and others have shown that supra-nutritional supplement of methylseleninic acid (MSeA), which differs, both chemically and metabolically, from SeMet, suppressed tumor growth in human prostate cancer xenografts, and in the transgenic adenocarcinoma mouse prostate (TRAMP) model. However, the former models did not mimic primary prevention, and the TRAMP mice vastly differ from the etiology and adenocarcinogenesis nature of the human prostate cancers. Therefore, a mouse model that closely recapitulates the clinic pathological features of human prostate cancer will be essential and necessary to predict the preventive efficacy and mechanisms of MSeA in men. As the loss of tumor suppressor gene Pten (phosphatase and tensin homolog deleted on chromosome 10) plays a causal role in human prostate cancers, the prostate epithelial-specific Pten-knockout mouse is such a preclinical model of choice. Here we utilized Probasin-Cre (Pb-Cre) transgenic mice expressing Cre to delete Pten in prostate epithelium to investigate whether administration of MSeA inhibited prostate tumorigenesis. We treated Pten-knockout and age-matched wild type male mice from 8 weeks of age with water or MSeA at a dose of 3 mg/kg body weight five times a week (Monday through Friday) for 27 weeks and sacrificed them for analyses. Our results show that MSeA significantly suppressed prostate tumorigenesis as evidenced by (1) suppression of tumor progression to high grade PIN or/and adenocarcinoma; (2) a 50% reduction in prostate tumor weight (P<0.01); (3) a significant reduction in cellular proliferation determined by ki67 immunostaining (P<0.01); and (4) attenuation of androgen receptor (AR) signaling. Mechanistically, we found that MSeA induced a super-activation of p53, p21 and cellular senescence. Because intact p53 signaling is preserved in most early prostate lesions, our findings provide compelling rationale to consider MSeA as a bioactive dietary supplement to activate p53-p21 mediated senescence as a barrier to prevent PIN from progressing to advanced prostate cancer. Citation Format: Lei Wang, Hua Xiong, Fengxia Wu, Tao Lin, Junxuan Lu, Yibin Deng. Methylseleninic acid super-activates p53 senescence axis as a preventive barrier to prostate tumorigenesis driven by Pten deficiency in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 191. doi:10.1158/1538-7445.AM2013-191

Abstract PR9: PICT1/GLTSCR2 is a critical nucleolar binding partner of RPL11 that regulates the MDM2-p53 pathway and tumor growth

Abstract The tumor-suppressive MDM2-p53 intracellular signaling pathway is activated by cellular stress. Such stress includes DNA damage, which triggers the ATM-Chk2 and ATR-Chk1 cascades; infection or oncogene activation, which induce the p19ARF pathway; and nucleolar stress, which initiates a cascade mediated by ribosomal proteins (RPs), particularly RPL5, RPL11, RPL23, and RPS7. These RPs are usually located in the nucleolus but are rapidly released into the nucleoplasm upon nucleolar stress, activating the MDM2-p53 pathway. However, it is unknown whether there are direct connections between p53-activating RP genes and cancer development in vivo, and which mechanism these RPs employ to translocate from the nucleolus to the nucleoplasm in response to stress. PICT1/GLTSCR2 was originally identified as a candidate tumor suppressor gene located at human chromosome 19q13.32, the site of deletions in human gliomas. Subsequent in vitro studies have demonstrated that PICT1 regulates the stability of PTEN, and that low PICT1 expression caused growth advantage. Based on these findings, PICT1 has been deemed a tumor suppressor. Other evidence, however, suggests that PICT1 may not always dampen cancer progression. In oligodendroglial tumors, loss of heterozygosity (LOH) at chromosome 19q13 is associated with longer disease-free survival after chemotherapy. These contradictory observations indicate that one or more genes mapped to chromosome 19q may contribute to brain cancer development. We speculated that PICT1 might be an important chromosome 19q–mapped gene that regulates tumor progression. To clarify PICT1 function, we generated PICT1-deficient mice and ES cells and carried out extensive biochemical and molecular analyses. Our results show that PICT1 is a nucleolar protein essential for murine embryogenesis and ES cell survival. Even without DNA damage, PICT1 loss results in p53-dependent G1 arrest and apoptosis in vitro and in vivo. Strikingly, PICT1-deficient cells accumulate p53 due to impaired MDM2 function. We further show that PICT1 binds to RPL11, and that RPL11 is released from nucleoli in the absence of PICT1. In PICT1-deficient cells, increased binding of RPL11 to MDM2 blocks MDM2-mediated ubiquitination of p53, leading to p53 accumulation and repression of cell proliferation. In cases of human colon and esophageal cancer, patients with low PICT1 expression have better prognoses. Similarly, when shRNA is used to deplete PICT1 in various tumor cell lines with intact p53 signaling, the cells grow more slowly and accumulate p53. These data suggest that PICT1 is a novel key regulator of the MDM2-p53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus. Thus, PICT1 is an important cancer-related gene that, at the very least, does not behave like a typical tumor suppressor and may instead have oncogenic effects. Studies on identification of factors which influence PICT1 expression or stability, or interfering with PICT1-RPL11 binding may lead to new cancer therapeutics, especially for individuals with intact-p53 signaling. This abstract is also presented as Poster B30. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR9.

Regulation of the MDM2-P53 pathway and tumor growth by PICT1 via nucleolar RPL11

PICT1 (also known as GLTSCR2) is considered a tumor suppressor because it stabilizes phosphatase and tensin homolog (PTEN), but individuals with oligodendrogliomas lacking chromosome 19q13, where PICT1 is located, have better prognoses than other oligodendroglioma patients. To clarify the function of PICT1, we generated Pict1-deficient mice and embryonic stem (ES) cells. Pict1 is a nucleolar protein essential for embryogenesis and ES cell survival. Even without DNA damage, Pict1 loss led to p53-dependent arrest of cell cycle phase G(1) and apoptosis. Pict1-deficient cells accumulated p53, owing to impaired Mdm2 function. Pict1 binds Rpl11, and Rpl11 is released from nucleoli in the absence of Pict1. In Pict1-deficient cells, increased binding of Rpl11 to Mdm2 blocks Mdm2-mediated ubiquitination of p53. In human cancer, individuals whose tumors express less PICT1 have better prognoses. When PICT1 is depleted in tumor cells with intact P53 signaling, the cells grow more slowly and accumulate P53. Thus, PICT1 is a potent regulator of the MDM2-P53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus.

The Hsp90 Inhibitor SNX7081 Restores the Fludarabine Sensitivity of Chronic Lymphocytic Leukemia (CLL) Cells Harbouring Mutations of ATM or TP53

AbstractAbstract 2473 Introduction:Resistance to fludarabine-based treatment represents a challenge in the clinical management of Chronic Lymphocytic Leukemia (CLL). Despite the unprecedented response rates seen with the fludarabine (F), cyclophosphamide (C), Rituximab (R) regimen novel treatment strategies are required that do not rely on an intact p53 signaling pathway. We recently described the activity of a novel, synthetic inhibitor of the molecular chaperone, heat-shock protein 90 (Hsp90) named SNX7081 (Serenex, now Pfizer) against CLL cells in vitro (Best et al., 2010 BJH). Here we explored the effect of this inhibitor on the fludarabine sensitivity of 3 haematological cell lines and 12 patient samples with mutations of ATM or TP53. Methods:Mononuclear cells were isolated by density centrifugation from CLL patients following informed consent. The 13 patient samples selected for study were determined to have mutations of either ATM or TP53 using a functional assay described in detail elsewhere (Best et al., 2008). The Mec1 (CLL), Mec2 (CLL) and U266 (B-ALL) cell lines were maintained under standard conditions in RPMI-1640 with 2mM L-glut and 1% pen/strep. Sensitivity to fludarabine, with and without SNX7081, was assessed using the MTT (3–4, 5-dimethylthiazol-2,5-diphenyl tetrazolium bromide) assay. Synergy between the agents, activation of caspase-3 and the induction of double stranded DNA (dsDNA) breaks following treatment were all assessed by flow cytometry using the mitochondrial membrane potential dye DilC1 (5) and propidium iodide (PI) or appropriate antibodies. Results:The IC50 for fludarabine was significantly higher in the 3 cell lines and 13 patient samples with ATM/TP53 lesions than in 4 cell lines or 10 patient samples defined as ATM/TP53 wild-type. Simultaneous exposure to a combination of fludarabine and SNX7081 at a ratio based on the IC50 of the compounds as single agent significantly reduced the IC50 for fludarabine (P<0.01); in 11 patient samples the IC50 for fludarabine was reduced to within a clinically achievable range (<5μM). Synergy between fludarabine and SNX7081 was evident as an effect on the distribution of the cell lines in the cell cycle and as a marked effect on the proportion of apoptotic cells (DilC (1)5 negative/PI negative) in cultures of both the cell lines and patient samples. Furthermore, we show that the combination of the compounds has a greater than additive effect on the activation of caspase-3 and on the formation of dsDNA breaks, as evidenced by the phosphorylation of g-H2Ax. Conclusions:Our studies suggest that inhibition of Hsp90 may overcome fludarabine resistance conferred by mutations of ATM or TP53. The mechanism of the synergy between these compounds appears to be via augmentation of fludarabine-induced dsDNA breaks and is concomitant with an increase in caspase-3 signaling. The data suggest that this combination may represent a promising regimen in the treatment of fludarabine-refractory CLL. Disclosures:Mulligan: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer Schering, now Genzyme: Honoraria.

P53 and aging: A fresh look at an old paradigm

Apoptosis and cellular senescence, two key tumor suppression mechanisms, are thought to be antagonistically pleiotropic. Antagonistic pleiotropy holds that functions that are advantageous for a young and reproductively fit organism (eg. cancer protection and proper development) can be deleterious when that same organism becomes old (eg. loss of stem cell proliferation and tissue degeneration leading to diseases associated with age) [1]. This theory predicts that in an older animal (or human), the activity of tumor suppressors would be associated with enhanced aging phenotypes. However, confirmation of a direct connection between apoptosis, senescence and aging remains elusive [2]. In fact, at least in the case of p53 there is mounting data challenging the antagonistic pleiotropy model. Multiple lines of evidence from animal models suggest that a functional p53 pathway favors prolonged survival. Aging mice show a decrease in p53 activity correlated with increased tumor incidence as well as an overall reduction in longevity [3]. On the other hand, mice with an extra gene dosage of Arf and p53 show significant tumor protection, decreased oxidative damage and delayed aging [4]. Animals expressing the p53S18A mutation present with accelerated aging, and cells from these mice undergo early senescence [5]. As phosphorylation of p53 at Ser18 (Ser15 in humans) is associated with activation, these results highlight a requirement for intact p53 signaling in longevity [6]. In the nematodeCaenorhabditis elegans, mutations that lead to longevity preferentially antagonize tumor growth, likely due to an increase in DAF-16/p53-dependentapoptosis [7]. The mTOR pathway is intimately connected with organismal aging. In fact inhibition of mTOR either by treatment with rapamycin or by the inhibition of upstream signaling molecules, extends lifespan in yeast, worms and flies suggesting that this pathway may be one of the main mechanisms that decrease lifespan [8]. p53 is able to regulate activity of mTOR following DNA damage or oncogenic stress by activation of PTEN, AMP kinase and TSC-2, each of which signals to diminish the activity of mTOR (Figure ​(Figure1)1) [9,10]. p53 may also function downstream of mTOR by activating antioxidant genes and thus protecting cells against increased ROS levels in cells, one of the consequences of heightened mTOR activity [11-13]. Figure 1. Signaling circuitry connecting Mdm2 with the regulation of longevity and metabolism. As the ability of p53 to increase longevity becomes more evident, we should consider the role of its negative regulator, Mdm2, if not in the process of aging directly, at least in its effects on the activity of mTOR. Growth factor and oncogene signaling activate PI-3 kinase and its downstream effector AKT, a protein kinase that activates mTOR via inhibition of TSC-1 protein. AKT also phosphorylates Mdm2 leading to enhanced Mdm2 ubiquitin ligase activity and more rapid degradation of p53 (Figure ​(Figure1)1) [8,14]. Additionally, AKT stimulates FOXO phosphorylation, which results in FOXO nuclear exportation and ubiquitin dependent proteasomal degradation [15]. FOXO proteins have conserved abilities to increase longevity in worms and flies [16]. Indeed, Mdm2 was reported to function as an E3 ubiquitin ligase to promote FOXO degradation, following activation of AKT, thus forging an additional link between Mdm2 and changes in longevity [17]. Additionally, mTOR is able to positively regulate Mdm2 through an increase in translation of Mdm2 mRNA. Consistently, an increase in p53 dependent apoptosis in the liver of mouse embryos treated with Rapamycin in utero is attributed mTOR's ability to control translation of Mdm2 [18]. However, because the interplay between apoptosis and aging is likely to be highly context specific, it is important to note that mice expressing only ~30% of the wild type Mdm2 levels do not have an aging phenotype, while exhibiting clear enhancement in tumor protection [19]. It would be of interest to look at the effects of nutrient deprivation on the mice carrying an Mdm2 hypomorphic allele. In conclusion, as our notion of p53 function in aging and senescence changes, it is very tempting to imagine that just a slight inhibition of Mdm2 function in cells could both prolong full tumor surveillance mechanisms of p53, and in some circumstances increase longevity. Numerous molecular inhibitors of Mdm2 are in various stages of development with the goal of reactivating p53 activity in cancer [20]. The idea that controlled pharmacological modulation of Mdm2 function might also have positive consequences in extension of human lifespan could be an unexpected benefit and an additional incentive for design of new compounds targeting Mdm2.

Caveolin-1 up-regulates IGF-I receptor gene transcription in breast cancer cells via Sp1- and p53-dependent pathways

The insulin-like growth factor (IGF) system plays an important role in the biology of breast cancer. Most of the biological actions of IGF-I and IGF-II are mediated by the IGF-I receptor (IGF-IR), a membrane-bound heterotetramer with potent antiapoptotic and cell survival activities. Caveolin-1 (Cav-1) is one of the main components of caveolae, and it has been shown to interact with multiple signaling molecules. In view of the important roles of IGF-IR and Cav-1 in oncogenically transformed mammary gland cells, in the present study we addressed the potential regulation of IGF-IR gene expression by Cav-1. The results obtained showed that MCF7/Cav-1 cells, expressing the Cav-1 gene in a stable manner, contain significantly higher levels of IGF-IR protein and mRNA than native MCF7 cells. These elevated levels of expression are mediated at the level of transcription, as shown by the results of experiments showing that the activity of the proximal IGF-IR promoter was higher in Cav-1-expressing MCF7 cells than in untransfected MCF7 cells. Furthermore, in subcellular localization studies, intensive IGF-IR staining in membrane ruffles and projections in MCF7/Cav-1 cells were noted, in contrast to typical membrane staining in MCF7 cells. In addition, we demonstrated that transcriptional activation of the IGF-IR gene by Cav-1 requires an intact p53 signaling pathway, since Cav-1 was unable to elevate IGF-IR levels in p53-null cells. Finally, the effect of Cav-1 was associated with an elevation in the levels of Sp1, a zinc-finger protein with important roles in IGF-IR gene transactivation. In summary, we identified the IGF-IR gene as a downstream target for Cav-1 action in breast cancer cells.

P53's believe it or not: lessons on transcription-independent death.

The requirement for an intact p53 signaling pathway to sense tumor-promoting DNA damage is evident from over 20 years of molecular, cellular, and whole animal studies. Without a doubt, p53's major contribution in maintaining the genomic integrity of multicellular organisms is through transcriptional regulation of genes required for cell cycle arrest, DNA repair, and apoptosis. Nonetheless, evidence is mounting that p53 has an extranuclear role in the cytoplasm to induce apoptosis, perhaps coupled to its transcriptional effects, or conceivably at instances when transcription is not optimal or possible. This phenomenon, transcription-independent p53-induced apoptosis (TIPA), has been described for almost 10 years, yet little is known about mechanisms by which p53 can directly engage the apoptotic cascade in the absence of transcription. Here we will explore what is currently known about TIPA learned from various p53 mutants and truncations, along with discussing several proposed mechanisms.

Human p14ARF-mediated cell cycle arrest strictly depends on intact p53 signaling pathways

The tumor suppressor ARF is transcribed from the INK4a/ARF locus in partly overlapping reading frames with the CDK inhibitor p16(Ink4a). ARF is able to antagonize the MDM2-mediated ubiquitination and degradation of p53, leading to either cell cycle arrest or apoptosis, depending on the cellular context. However, recent data point to additional p53-independent functions of mouse p19(ARF). Little is known about the dependency of human p14(ARF) function on p53 and its downstream genes. Therefore, we analysed the mechanism of p14(ARF)-induced cell cycle arrest in several human cell types. Wild-type HCT116 colon carcinoma cells (p53(+/+)p21(CIP1+/+) 14-3-3sigma(+/+)), but not p53(-/-) counterparts, underwent G(1) and G(2) cell cycle arrest following infection with a p14(ARF)-adenovirus. In p21(CIP1-/-) cells, p14(ARF) did not induce G(1) or G(2) arrest, while 14-3-3sigma(-/-) counterparts were mainly arrested in G(1), pointing to essential roles of p21(CIP1) in G(1) and G(2) arrest and cooperative roles of p21 and 14-3-3sigma in ARF-mediated G(2) arrest. Our data demonstrate a strict p53 and p21(CIP1) dependency of p14(ARF)-induced cell cycle arrest in human cells.