Loss Of P53 Function Research Articles

Effects of YM155 on survivin levels and viability in neuroblastoma cells with acquired drug resistance.

Resistance formation after initial therapy response (acquired resistance) is common in high-risk neuroblastoma patients. YM155 is a drug candidate that was introduced as a survivin suppressant. This mechanism was later challenged, and DNA damage induction and Mcl-1 depletion were suggested instead. Here we investigated the efficacy and mechanism of action of YM155 in neuroblastoma cells with acquired drug resistance. The efficacy of YM155 was determined in neuroblastoma cell lines and their sublines with acquired resistance to clinically relevant drugs. Survivin levels, Mcl-1 levels, and DNA damage formation were determined in response to YM155. RNAi-mediated depletion of survivin, Mcl-1, and p53 was performed to investigate their roles during YM155 treatment. Clinical YM155 concentrations affected the viability of drug-resistant neuroblastoma cells through survivin depletion and p53 activation. MDM2 inhibitor-induced p53 activation further enhanced YM155 activity. Loss of p53 function generally affected anti-neuroblastoma approaches targeting survivin. Upregulation of ABCB1 (causes YM155 efflux) and downregulation of SLC35F2 (causes YM155 uptake) mediated YM155-specific resistance. YM155-adapted cells displayed increased ABCB1 levels, decreased SLC35F2 levels, and a p53 mutation. YM155-adapted neuroblastoma cells were also characterized by decreased sensitivity to RNAi-mediated survivin depletion, further confirming survivin as a critical YM155 target in neuroblastoma. In conclusion, YM155 targets survivin in neuroblastoma. Furthermore, survivin is a promising therapeutic target for p53 wild-type neuroblastomas after resistance acquisition (neuroblastomas are rarely p53-mutated), potentially in combination with p53 activators. In addition, we show that the adaptation of cancer cells to molecular-targeted anticancer drugs is an effective strategy to elucidate a drug's mechanism of action.

Targeting p53-deficient chronic lymphocytic leukemia cells in vitro and in vivo by ROS-mediated mechanism.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. Loss of p53 function in CLL cells due to chromosome 17p deletion or p53 mutations often leads to a more malignant disease phenotype and is associated with drug resistance and poor clinical outcome. Thus, development of novel therapeutic strategies to effectively target CLL cells with p53 deficiency is clinically important. Here we showed that p53-null CLL cells were highly sensitive to ROS-mediated cell killing due to their intrinsic ROS stress. We further demonstrated that a natural compound phenethyl isothiocyanate (PEITC) was able to effectively kill CLL cells with loss of p53, even under the protection of stromal cells. In p53-defficient CLL cells, PEITC induced a rapid depletion of glutathione and a severe accumulation of ROS, leading to massive leukemia cell death in the stromal microenvironment. The drug-induced cell death was associated with a significant decrease of in MCL-1 survival molecule. We further showed that ROS-mediated cell death was the key mechanism by which PEITC induced cytotoxicity, since such cell death could be prevented by addition of antioxidant NAC. Importantly, in vivo study showed that PEITC was able to induce substantial leukemia cell death in mice. Treatment of CLL mice harboring TCL1-Tg:p53−/− genotype with PEITC significantly prolonged the median survival time of the animals. Our study identifies a vulnerability of p53-null CLL cells with high sensitivity to ROS-generating agents, and suggests that PEITC may potentially be useful for clinical treatment of CLL with 17p deletion and p53 mutations.

Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells

O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked β-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma.

P53 functional deficiency in human colon cancer cells promotes fibroblast-mediated angiogenesis and tumor growth.

Cancer-associated fibroblasts (CAFs) create a microenvironment that contributes to tumor growth; however, the mechanism by which fibroblasts are phenotypically altered to CAFs remains unclear. Loss or mutation of the tumor suppressor p53 plays a crucial role in cancer progression. Herein, we analyzed how the p53 status of cancer cells affects fibroblasts by investigating the in vivo and in vitro effects of loss of p53 function in cancer cells on phenotypic changes in fibroblasts and subsequent tumor progression in human colon cancer cell lines containing wild-type p53 and in cells with a p53 functional deficiency. The growth of p53-deficient tumors was significantly enhanced in the presence of fibroblasts compared with that of p53-wild-type tumors or p53-deficient tumors without fibroblasts. p53-deficient cancer cells produced reactive oxygen species, which activated fibroblasts to mediate angiogenesis by secreting vascular endothelial growth factor (VEGF) both in vivo and in vitro Activated fibroblasts significantly contributed to tumor progression. Deletion of fibroblast-derived VEGF or treatment with N-acetylcysteine suppressed the growth of p53-deficient xenograft tumors. The growth effect of blocking VEGF secreted from cancer cells was equivalent regardless of p53 functional status. Human colon cancer tissues also showed a significant positive correlation between p53 cancer cell staining activated fibroblasts and microvessel density. These results reveal that fibroblasts were altered by exposure to p53-deficient epithelial cancer cells and contributed to tumor progression by promoting neovascularization. Thus, p53 acts as a modulator of the tumor microenvironment.

Abstract 2157: Enhanced in vitro activity of dianhydrogalactitol (VAL-083) in combination with platinum drugs: Impact of p53 and platinum-resistance

Abstract Cisplatin is an important frontline drug for ovarian carcinoma and non-small cell lung cancer (NSCLC). However, the initial response rates of up to 70% are usually followed by relapse due to the onset of drug resistance. Mechanistically, platinum resistance is multifactorial, with loss of p53 function (by mutation or inhibitory protein binding to wild-type (wt) p53) playing a central role. The appearance of drug resistance is a major clinical barrier and, therefore, new agents are needed to overcome this limitation. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent that induces DNA interstrand cross-links at N7-guanine, a mechanism that is distinct from the intrastrand cross-links by platinum-based drugs. VAL-083 is clinically approved in China for lung cancer, and is undergoing clinical trial in the US for glioma. In the present study, we have examined the in vitro cytotoxicity of VAL-083 as a single agent and in combination with cisplatin or oxaliplatin using the 5-day MTT assay. In the isogenic HCT-116p53+/+ and HCT-116p53-/- colorectal models, loss of p53 increased IC50 of (or resistance to) cisplatin and oxaliplatin by 3- to 6-fold, whereas resistance to VAL-083 was increased only 1.7-fold by loss of p53. These results indicate that the cytotoxicity of VAL-083 is less impacted than the platinum drugs by loss of p53. When tested in cisplatin-sensitive (A2780) vs. cisplatin-resistant wt p53 ovarian tumor models (2780CP-16, OVCAR-10, Hey and OVCA-433) there was a 10- to 40-fold increase in IC50 for cisplatin, while the corresponding increase in IC50 for VAL-083 was only 4- to 7-fold. This indicates only partially cross-resistance between VAL-083 and cisplatin and thus suggests a distinct mode of action for VAL-083 as compared to cisplatin. To further investigate, immunoblots were developed after a 24-h exposure of isogenic A2780 or 2780CP-16 models to cisplatin or VAL-083. The two drugs were equally effective at stabilizing and activating p53 in A2780 cells. However, in cisplatin-resistant 2780CP-16 cells, VAL-083 was more effective than cisplatin at increasing p53 and p21 levels, and at inducing Ser-15 and Ser-20 phosphorylation of p53. This is consistent with the ability of VAL-083 to circumvent cisplatin resistance and demonstrated that this alkylating agent also has the capacity to partially restore wt p53 function in ovarian tumor cells. The independent mode of actions of these drugs suggested the potential for combining VAL-083 with cisplatin or oxaliplatin. These combinations in wt (H460 and A549) and mutant (H1975 and H157) p53 NSCLC models demonstrated significant super-additivity (p<0.05) and/or synergy (CI < 1). Taken together, these results demonstrate the antitumor activity of VAL-083 against both wt and mutant p53 cancers and raise the clinical potential for treatment in a combination setting with platinum drugs. Citation Format: Anne Steino, Guanghan He, Michelle Martinez-Rivera, Jeffrey A. Bacha, Dennis M. Brown, Zahid H. Siddik. Enhanced in vitro activity of dianhydrogalactitol (VAL-083) in combination with platinum drugs: Impact of p53 and platinum-resistance. [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 2157.

Pharmacological activation of wild-type p53 in the therapy of leukemia.

The tumor suppressor p53 is inactivated by mutations in the majority of human solid tumors. Conversely, p53 mutations are rare in leukemias and are only observed in a small fraction of the patient population, predominately in patients with complex karyotype acute myeloid leukemia or hypodiploid acute lymphoblastic leukemia. However, the loss of p53 function in leukemic cells is often caused by abnormalities in p53-regulatory proteins, including overexpression of MDM2/MDMX, deletion of CDKN2A/ARF, and alterations in ATM. For example, MDM2 inhibits p53-mediated transcription, promotes its nuclear export, and induces proteasome-dependent degradation. The MDM2 homolog MDMX is another direct regulator of p53 that inhibits p53-mediated transcription. Several small-molecule inhibitors and stapled peptides targeting MDM2 and MDMX have been developed and have recently entered clinical trials. The clinical trial results of the first clinically used MDM2 inhibitor, RG7112, illustrated promising p53 activation and apoptosis induction in leukemia cells as proof of concept. Side effects of RG7112 were most prominent in suppression of thrombopoiesis and gastrointestinal symptoms in leukemia patients. Predictive biomarkers for response to MDM2 inhibitors have been proposed, but they require further validation both invitro and invivo so that the accumulated knowledge concerning pathological p53 dysregulation in leukemia and novel molecular-targeted strategies to overcome this dysregulation can be translated safely and efficiently into novel clinical therapeutics.

Targeting cancer stem cells with p53 modulators.

Cancer stem cells (CSC) typically over-express aldehyde dehydrogenase (ALDH). Thus, ALDHbright tumor cells represent targets for developing novel cancer prevention/treatment interventions. Loss of p53 function is a common genetic event during cancer development wherein small molecular weight compounds (SMWC) that restore p53 function and reverse tumor growth have been identified. Here, we focused on two widely studied p53 SMWC, CP-31398 and PRIMA-1, to target ALDHbright CSC in human breast, endometrial and pancreas carcinoma cell lines expressing mutant or wild type (WT) p53. CP-31398 and PRIMA-1 significantly reduced CSC content and sphere formation by these cell lines in vitro. In addition, these agents were more effective in vitro against CSC compared to cisplatin and gemcitabine, two often-used chemotherapeutic agents. We also tested a combinatorial treatment in methylcholantrene (MCA)-treated mice consisting of p53 SMWC and p53-based vaccines. Yet using survival end-point analysis, no increased efficacy in the presence of either p53 SMWC alone or with vaccine compared to vaccine alone was observed. These results may be due, in part, to the presence of immune cells, such as activated lymphocytes expressing WT p53 at levels comparable to some tumor cells, wherein further increase of p53 expression by p53 SMWC may alter survival of these immune cells and negatively impact an effective immune response. Continuous exposure of mice to MCA may have also interfered with the action of these p53 SMWC, including potential direct interaction with MCA. Nonetheless, the effect of p53 SMWC on CSC and cancer treatment remains of great interest.

O‐GlcNAc Regulates p53 in Ovarian Cancer

O‐GlcNAcylation is a dynamic post‐translational modification (PTM) that consists of an addition of a single N‐acetylglucosamine sugar to serine and threonine residues in nuclear, cytoplasmic and mitochondrial proteins. The enzyme O‐linked β‐N‐acetylglucosamine transferase (OGT) adds the modification, while enzyme O‐GlcNAcase (OGA) removes the modification. O‐GlcNAc regulates enzymatic activity, localization, stability and protein‐protein interactions on a wide range of proteins such as transcription factors, structural proteins and kinases. In cancer, when comparing O‐GlcNAc levels in normal and tumor cells, tumor samples present higher O‐GlcNAcylation in vitro and in vivo. This aberrant O‐GlcNAc pattern contributes to changes in important signaling pathways and could contribute to the progression of the disease. Interestingly, the tumor suppressor p53 is modified by O‐GlcNAc. The transcription factor p53 regulates diverse cellular processes like cell survival and the cell cycle. Most solid tumors contain mutations in p53 leading to increased degradation or loss of p53 function, which promotes malignant tumor growth. Ovarian cancer has one of the highest frequencies of p53 mutation rates. The goal of this work is to understand the relationship between O‐GlcNAcylation and p53 function in ovarian cancer. First, O‐GlcNAcylation, OGT and OGA protein and mRNA levels were measured in a series of human ovarian cancer cell lines and no correlation was found between p53 genetic status and O‐GlcNAcylation. Overexpression or silencing of p53 in a p53 wild‐type cell line (A2780) had little effect on O‐GlcNAcylation. On the other hand, pharmacological inhibition of OGA with the inhibitor Thiamet‐G (TMG) leads to an increase in nuclear localization of p53 and increased mRNA levels of p53‐target genes (p21 and Bax). Overexpression of OGA or OGT leads to increased p53 protein but not mRNA levels and a concurrent increase in p53‐targets (Mdm‐2, p21 and Bax) expression. Also, p53 acetylation on lysine 382, a transcriptional activating modification, is higher in OGA/OGT overexpressing cells. In conclusion, O‐GlcNAcylation regulates p53 function through stabilization and activation.Support or Funding InformationSupport and funding: Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Science Without Borders Program, NIH NIDDK R01 DK 100595.

Inactivation of p53 in pterygium influence miR-200a expression resulting in ZEB1/ZEB2 up-regulation and EMT processing

Loss of p53 function has been linked to progression of pterygium. MiR-200a is known to be controlled by p53. Here, we hypothesize that expression of miR-200a and downstream ZEB1/ZEB2 genes are regulated epithelial–mesenchymal transition (EMT) involved in the pathogenesis and recurrence of pterygium. For this study, 120 primary pterygial samples were collected. Immunohistochemistry and real-time RT-PCR were performed to determine the expression of p53, p53 down-stream EMT associated protein and miR-200a. The molecular correlation of p53, miR-200a and downstream genes were confirmed using primary pterygium cells (PECs). Expression of miR-200a in pterygium tissues was significantly lower than in conjunctiva controls (p = 0.015). Up-regulated miR-200a levels were positively correlated with and p53 protein expression (p < 0.001). The miR-200a downstream ZEB1/ZEB1 protein expression were negative correlated with miR-200a expression. Cell model studies demonstrated that miR-200a controlled the EMT of PECs through up-regulated ZEB1, ZEB2 and Snail gene expression. Our study demonstrated that inactivation of p53 in pterygium may influence miR-200a, resulting in ZEB1/ZEB2 up-regulation and EMT processing of pterygium. Therefore, we suggest that expression of miR-200a play an important role in EMT processing and recurrence of pterygium.

A porcine model of osteosarcoma.

We previously produced pigs with a latent oncogenic TP53 mutation. Humans with TP53 germline mutations are predisposed to a wide spectrum of early-onset cancers, predominantly breast, brain, adrenal gland cancer, soft tissue sarcomas and osteosarcomas. Loss of p53 function has been observed in >50% of human cancers. Here we demonstrate that porcine mesenchymal stem cells (MSCs) convert to a transformed phenotype after activation of latent oncogenic TP53R167H and KRASG12D, and overexpression of MYC promotes tumorigenesis. The process mimics key molecular aspects of human sarcomagenesis. Transformed porcine MSCs exhibit genomic instability, with complex karyotypes, and develop into sarcomas on transplantation into immune-deficient mice. In pigs, heterozygous knockout of TP53 was sufficient for spontaneous osteosarcoma development in older animals, whereas homozygous TP53 knockout resulted in multiple large osteosarcomas in 7–8-month-old animals. This is the first report that engineered mutation of an endogenous tumour-suppressor gene leads to invasive cancer in pigs. Unlike in Trp53 mutant mice, osteosarcoma developed in the long bones and skull, closely recapitulating the human disease. These animals thus promise a model for juvenile osteosarcoma, a relatively uncommon but devastating disease.

Inactivation of the p53-KLF4-CEBPA Axis in Acute Myeloid Leukemia.

In acute myeloid leukemia (AML), the transcription factors CEBPA and KLF4 as well as the universal tumor suppressor p53 are frequently deregulated. Here, we investigated the extent of dysregulation, the molecular interactions, and the mechanisms involved. One hundred ten AML patient samples were analyzed for protein levels of CEBPA, KLF4, p53, and p53 modulators. Regulation of CEBPA gene expression by KLF4 and p53 or by chemical p53 activators was characterized in AML cell lines. We found that CEBPA gene transcription can be directly activated by p53 and KLF4, suggesting a p53-KLF4-CEBPA axis. In AML patient cells, we observed a prominent loss of p53 function and concomitant reduction of KLF4 and CEBPA protein levels. Assessment of cellular p53 modulator proteins indicated that p53 inactivation in leukemic cells correlated with elevated levels of the nuclear export protein XPO1/CRM1 and increase of the p53 inhibitors MDM2 and CUL9/PARC in the cytoplasm. Finally, restoring p53 function following treatment with cytotoxic chemotherapy compounds and p53 restoring non-genotoxic agents induced CEBPA gene expression, myeloid differentiation, and cell-cycle arrest in AML cells. The p53-KLF4-CEBPA axis is deregulated in AML but can be functionally restored by conventional chemotherapy and novel p53 activating treatments.

P53 Deletion or Hotspot Mutations Enhance mTORC1 Activity by Altering Lysosomal Dynamics of TSC2 and Rheb.

The activity of mammalian target of rapamycin complex 1 (mTORC1) is frequently enhanced in carcinomas, an effect thought to contribute to the malignant phenotype. Here, it is demonstrated that either deletion or mutation of TP53 in colon or lung carcinoma cells substantially enhances mTORC1 kinase activity by an effect downstream of and independent of AMPK. Mechanistically, it was determined that loss or mutation of p53 decreased expression of TSC2 and Sestrin2 (SESN2). Complementation of p53 null cells with TSC2 or Sestrin2 reduced mTORC1 activity to levels found in p53 wild-type (wt) cells, whereas their genetic depletion enhanced mTORC1 activity in p53 wt cells. However, the primary causal event in enhanced mTORC1 activity upon loss of p53 appeared to be a diminished distribution of TSC2 to lysosomal membranes containing mTOR. Subsequently, there was increased Rheb in the lysosomal compartment, and a higher mTOR association with Raptor. Transfection of TSC2 into p53 null cells replaced TSC2 and diminished Rheb at the lysosome, recapitulating cells with wt p53. In contrast, transfection of Sestrin2 decreased mTOR in lysosomes, but the lower levels of Sestrin2 in p53 null cells did not change lysosomal mTOR. In summary, loss of the transcriptional activity of p53, either by deletion or by key mutations in the DNA-binding domain, diminishes expression of TSC2 and Sestrin2, thus, shifting membrane-bound TSC2 out of lysosomal membranes, increasing lysosomal Rheb and increasing the kinase activity of mTORC1. This study establishes that loss of p53 function decreases lysosomal TSC2 and increases lysosomal Rheb resulting in hyperactive mTORC1, findings that are consistent with a more malignant phenotype.

P53 functional abnormality in mesenchymal stem cells promotes osteosarcoma development.

It has been shown that p53 has a critical role in the differentiation and functionality of various multipotent progenitor cells. P53 mutations can lead to genome instability and subsequent functional alterations and aberrant transformation of mesenchymal stem cells (MSCs). The significance of p53 in safeguarding our body from developing osteosarcoma (OS) is well recognized. During bone remodeling, p53 has a key role in negatively regulating key factors orchestrating the early stages of osteogenic differentiation of MSCs. Interestingly, changes in the p53 status can compromise bone homeostasis and affect the tumor microenvironment. This review aims to provide a unique opportunity to study the p53 function in MSCs and OS. In the context of loss of function of p53, we provide a model for two sources of OS: MSCs as progenitor cells of osteoblasts and bone tumor microenvironment components. Standing at the bone remodeling point of view, in this review we will first explain the determinant function of p53 in OS development. We will then summarize the role of p53 in monitoring MSC fidelity and in regulating MSC differentiation programs during osteogenesis. Finally, we will discuss the importance of loss of p53 function in tissue microenvironment. We expect that the information provided herein could lead to better understanding and treatment of OS.

Targeted Deletion of p53 in Lgr5-Expressing Intestinal Stem Cells Promotes Colon Tumorigenesis in a Preclinical Model of Colitis-Associated Cancer.

p53 has been shown to mediate cancer stem-like cell function by suppressing pluripotency and cellular dedifferentiation. However, there have been no studies to date that have addressed the specific effects of p53 loss in colonic adult stem cells. In this study, we investigated the consequences of conditionally ablating p53 in the highly relevant Lgr5(+) stem cell population on tumor initiation and progression in the colon. In a mouse model of carcinogen (AOM)-induced colon cancer, tamoxifen-inducible Lgr5-driven deletion of p53 reduced apoptosis and increased proliferation of crypt stem cells, but had no effect on tumor incidence or size. Conversely, in a mouse model of colitis-associated cancer, in which mice are exposed to AOM and the potent inflammation inducer DSS, stem cell-specific p53 deletion greatly enhanced tumor size and incidence in the colon. These novel findings suggest that the loss of p53 function in stem cells enables colonic tumor formation only when combined with DNA damage and chronic inflammation. Furthermore, we propose that stem cell targeting approaches are valuable for interrogating prevention and therapeutic strategies that aim to specifically eradicate genetically compromised stem cells.

Cytotoxic Exposure to Both Hematopoietic Stem and Progenitor Cells and the Bone Marrow Niche Cooperate to Promote Leukemogenesis in a Mouse Model of Therapy-Related Myeloid Neoplasms with a Del(5q)

A del(5q) is frequently noted in MDS, AML, and therapy-related myeloid neoplasms (t-MN) following alkylating agent therapy. Mutation/loss of the TP53 is gene found in 80% of t-MNs with a del(5q). Recent studies suggest that TP53 mutations are found at a low frequency in hematopoietic stem/progenitor cells (HSPCs) in adults (Xie et al., Nat Med 20:1472, 2014; Genovese et al., NEJM 371:2477, 2014; Jaiswal et al., NEJM 371:2488, 2014), and chemotherapy confers a selective growth advantage to these rare clones (Wong et al., Nature 518:552, 2015). We previously established a mouse model for t-MN with a del(5q) and showed that haploinsufficiency of two del(5q) genes, Egr1 and Apc, cooperate with loss of function of the Trp53 (p53) gene to induce myeloid neoplasms in mice. Specifically, transplantation of Egr1+/-, Apcdel/+ bone marrow (BM) cells transduced with p53 shRNAs into wild type (WT) recipients resulted in the development of a transplantable AML, characterized by a complex karyotype and genetic instability, in 17% of mice.There is growing evidence that microenvironment perturbations play a major role in the malignant process; however, the effect of cytotoxic therapy on HSPCs as well as the BM niche is not well understood. Using our mouse model of t-MN with a del(5q), we explored the effects of ENU, an alkylating agent, on both HSPCs and the BM microenvironment by exposing both donor and recipient mice to ENU (Panel 1 in the figure). In mice transplanted with Egr1+/-, Apcdel/+, p53 shRNA HSPCs, exposure to ENU strikingly decreased survival (median survival: 200d vs. not reached) and increased the incidence of AML or MDS with multilineage dysplasia (73% vs. 17%). In the absence of p53 knockdown (i.e., control shRNA), mice survived longer (370d vs. 200d, P = 0.0014); however, 100% of mice developed MDS with dyserythropoiesis. None developed AML, suggesting that loss of p53 function is critical for leukemic transformation (Panel 2). Loss of both del(5q) genes, EGR1 and APC, was necessary to develop AML. Compared to mice transplanted with Egr1+/-, Apcdel/+, p53 shRNA HSPCs, mice transplanted with Egr1+/-, p53 shRNA HSPCs survived longer (369 d vs. 200 d, P = 0.0117) and only 40% of mice developed MDS with dysgranulopoiesis and/or dyserythropoiesis (Panel 3). None developed AML. Thus, severity of disease increases with loss of more than one del(5q) gene. Finally, to determine the separate effects of alklating agent therapy on HSPCs vs. the niche, we treated either the recipient or donor mice with ENU. Whereas ENU exposure to both donor and recipient resulted in a profound expansion of p53 shRNA+ cells and the development of MDS/AML in 73% of mice, ENU exposure of either donor or recipient led to only modest expansion of p53 shRNA+ cells and none of the mice developed MDS or AML. This suggests that the clonal expansion of cells with loss of multiple 5q genes and p53 is likely promoted by cytotoxic exposure to the cells themselves, as well as exposure to the surrounding niche cells.t-MN patients with a del(5q) typically present with trilineage dysplasia implicating all three hematopoietic cell lineages (erythroid, myeloid, and megakaryocytic) in the dysplastic process. Our mouse models shed light on some of the key genes on 5q, as well as the environmental exposure, that contributes to trilineage dysplasia in patients. Finally, our data suggests that t-MN is a "disease of the tissue", and the expansion of mutant HSPCs, e.g., with TP53 mutations, likely results from the combined effects of cytotoxic therapy on the hematopoietic cells themselves, as well as the BM microenvironment that supports hematopoiesis [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Mitochondrial Metabolism Inhibitor Cpi-613 in Combination with High Dose Ara-C (HDAC) and Mitoxantrone Is Highly Active in Poor Risk Relapsed or Refractory Acute Myeloid Leukemia (AML)

Background: CPI-613 is a first in class agent that inhibits pyruvate dehydrogenase (PDH) and α-ketogluterate dehydrogenase. As a single agent CPI-613 was found to be well tolerated with possible activity in several patients with myeloid malignancies.This trial determined the maximum tolerated dose (MTD), safety, and efficacy of CPI-613 in combination with HDAC and mitoxantrone in patients with relapsed or refractory AML.Methods: CPI-613 was given daily on days 1 through 5 starting at a dose of 500 mg/m2. Beginning on day 3, HDAC at 3,000 mg/m2 (or 1,500 mg/m2 for age ≥60) is administered every 12 hours for 5 total doses and mitoxantrone at 6 mg/m2 is given daily for 3 doses. If residual disease is present on day 14 re-induction with the same or a three day abbreviated course could be given. Patients who achieved a complete remission with or without complete count recovery (CR or CRi) could receive up to a total of two additional consolidation cycles with the goal to get responders to stem cell transplant whenever possible.Results: A total of 67 patients have been enrolled and 65 are evaluable. The median age is 60 (range 21-79). Nineteen patients had refractory disease and 14 received at least 1 previous line of salvage therapy. In patients with relapsed disease the median duration of CR1 was 5 months. Cytogenetics were poor risk in 30 patients, intermediate in 30 and good in 6. One patient had CML in myeloid blast crisis. The overall intention to treat response rate was 48% (26CR+5CRi) with a median survival of 6.4 months. In patients ≥60 years old the CR/CRi rate was 42% (15/36). Surprisingly, the response rate for patients with poor risk cytogenetics was 47% (11CR+3CRi) with a median survival of 5.2 months. In a historical cohort treated with HDAC, mitoxantrone and asparaginase at our institution, only 19% (3/16) of patients with poor risk cytogenetics responded with a median survival of 2.8 months. The MTD of CPI-613 in combination with HDAC and mitoxantrone is 2,500 mg/m2. The dose limiting toxicities were diarrhea and nausea. Nine patients (13%) died on or before day 30. The most common toxicities attributed to CPI-613 were diarrhea and nausea, mainly grade 1 or 2. At the time of this submission thirteen patients have gone on to allogeneic stem cell transplantation. Several patients with circulating blasts had blood samples taken before and after CPI-613 infusion. Three of these patients had increased phosphorylation of PDH consistent with its inhibition. Additionally, two patients demonstrated a robust phosphorylation of adenosine monophosphate-activated protein kinase consistent with depletion of ATP. To explore if loss of p53 function (common in poor risk cytogenetics) affected response we tested CPI-613 alone or with an anthracycline against an AML cell line with and without p53 knockdown. Suppression of p53 function resulted in significant resistance to the anthracycline but no change in response to CPI-613; the combination enhanced cell kill over either agent alone.Conclusions: CPI-613 in combination with HDAC and mitoxantrone is a promising salvage regimen, especially in patients with poor risk cytogenetics. DisclosuresPardee:Novartis: Speakers Bureau; Celgene: Speakers Bureau. Off Label Use: CPI-613 is a novel anticancer agent not currently approved by the FDA. Ellis:Alexion: Speakers Bureau. Manuel:Novartis: Speakers Bureau. Hurd:Merck: Equity Ownership; Pfizer: Equity Ownership; Medtronic: Equity Ownership; Procter and Gamble: Equity Ownership; Bristol Myers Squib: Equity Ownership. Powell:Celgene: Speakers Bureau; Cornerstone Pharmaceuticals: Consultancy.

Dysregulated Nucleotide Excision Repair (NER) Is a New Target in Multiple Myeloma

Abstract Nucleotide excision repair (NER) is involved in the removal of bulky adducts and DNA crosslinks induced by various genotoxins including alkylating agents. In cancer, somatic mutations, genes' up-regulation, and epigenetic silencing of NER may lead to abnormal DNA damage responses. Little is known about the role of NER in biology of multiple myeloma (MM), a heterogeneous disease characterized by genomic instability which is often treated by alkylating agents. In our genome sequencing data we did not observe recurrent mutation in NER although non silent mutations affected 13 genes involved in NER in 20out of 272 distinct patients. Using a functional assay based on the purified DNA-Damage Binding protein 2 (DDB2) proteo-probe complex that allows monitoring of photoproducts removal after UV irradiation, we observed NER variability in MM cell lines (MMCL) allowing to separate the cell lines in 2 groups according to their ability to repair: namely rapid (repair&gt;90% after 2 hours) versus slow (repair &lt; 85% after 2 hours). The separation in two NER phenotypes did not correlate with P53 loss of function or any cytogenetic event with the exception of t (4;14) translocation which was associated with the group showing a repair rate &gt; 90%. Interestingly, the LR5 melphalan resistant cell line harbored a rapid repair phenotype as compared to the parental RPMI-8226 cell line which exhibited a slow repair. We evaluated melphalan sensitivity in relationship to NER activity in rapid and slow MMCL and observed that MMCL with slow NER were more sensitive to melphalan. We next evaluated whether inhibiting NER modified drug sensitivity. We utilized spironolactone which is known to inhibit NER. With the DDB2 proteo-probe assay, we confirmed the ability of spironolactone to inhibit NER in MMCL by degrading the Xeroderma Pigmentosum group B (XPB) protein. Combination of melphalan with NER inhibition in the 20 MMCL induced a significant increase in melphalan sensitivity in MMCL (increase from 15 to 79%). We have also confirmed similar NER activity in CD138 positive patient plasma cells. We then developed a gene expression signature using rapid and slow NER cell lines. In a multivariate analysis, the rapid NER signature was significantly associated with low survival in the IFM 2005-01 trial dataset that included 170 patients treated with high-dose melphalan. In conclusion, we have observed that NER affects MM cells and plays a significant role in drug sensitivity; we have also shown that NER can be targeted to increase melphalan sensitivity and potentially improve patient outcome. Disclosures Avet-Loiseau: onyx: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; Oncopep: Equity Ownership; Acetylon: Equity Ownership.

Abstract IA21: Pediatric medulloblastoma: Drug screens and preclinical studies

Abstract Medulloblastoma (MB) is the most common malignant brain tumor that develops in the cerebellum. MB occurs mostly in children between the ages of 3-7 years. Human MBs are classified into four subgroups: Wingless (WNT), Sonic Hedgehog (SHH), Group 3 (G3) and G4, each of which with distinct molecular signatures. SHH MBs with MYCN amplification and TP53 mutations and G3 MBs represent the most aggressive subgroups and the least curable with current therapeutic regimen. Whole genome sequencing and gene expression analysis of human G3 MBs demonstrated C-MYC (MYC) overexpression, from gene amplification in ~17% of cases, stem like properties and high levels of EZH2 and histone 3 lysine 27 trimethylation (H3K27me3) marks. G3 MBs have few, recurrent oncogenic point mutations that might be targeted therapeutically but contain large chromosomal copy number changes and an aberrant epigenome. We developed a mouse model of G3 MB using orthotopic transplantation as well as in utero electroporation approaches by enforcing the expression of Myc with loss of p53 function that shares many characteristics of its human counterpart including high levels of Ezh2 and H3K27me3 marks. A high throughput screen of FDA-approved drugs on mouse G3 MB neuropsheres identified Pemetrexed and Gemcitabine that, in combination with standard of care chemotherapy, greatly increased the survival of mice bearing mouse or human G3 MBs. Because genetic alterations found in human G3 MBs occur in epigenetic regulators, we tested several drugs that target the epigenome to re-establish normal gene expression profiles, including those of tumor suppressor genes. Using a preclinical drug development pipeline, we evaluated nine compounds that are believed to modulate the epigenome. Of the nine compounds tested we found that the nucleoside analog 5-fluoro-2′-deoxycytidine (FdCyd) markedly reduced the proliferation of G3 MBs in vitro at nanomolar concentrations. Detailed intracranial PK studies confirmed that systemically administered FdCyd exceeded concentrations in the tECF (tumor extra cellular fluid) necessary to inhibit tumor cell proliferation in vivo. However, despite promising in vitro activity and in vivo PK properties, FdCyd was completely ineffective in treating mouse and human G3 MBs in vivo. Our studies highlight the requirement for a comprehensive and integrated preclinical drug development pipeline with mouse and human medulloblastoma to identify the best candidate drug to be translated into the clinic and to avoid taking a drug forward that looks feasible in in vitro screens but which efficacy does not translate into an in vivo setting. Citation Format: Marie Morfouace, Yogesh Patel, Anang Shelat, Daisuke Kawauchi, Giles W. Robinson, Kip Guy, Richard J. Gilbertson, Clinton Stewart, Amar Gajjar, Martine F. Roussel. Pediatric medulloblastoma: Drug screens and preclinical studies. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA21.

Regulation of endoplasmic reticulum functions.

The endoplasmic reticulum (ER) is a multifunctional organelle that is specialized for lipid synthesis, Ca2+ homeostasis, and protein synthesis. Protein synthesis is a multistep process involving folding and manipulation of proteins as well as the exportation of proteins to an appropriate location. Several stress responses, such as metabolic and anaerobic stress, induce ER dysfunction and protein unfolding, which accelerates the disruption of ER homeostasis. To prevent the production of unfolded proteins, cells induce the unfolded protein response (UPR), which is a highly regulated protein quality surveillance system. UPR maintains and restores ER homeostasis to increase the secretory function of the ER via the induction of the ER chaperone protein. This stimulates re-folding or degradation of unfolded proteins. However, irreparable ER stress induces a cell death response to eliminate these damaged cells [1]. Three types of ER transmembrane proteins, a protein-kinase and site-specific endoribonuclease, inositol-requiring enzyme 1 α (IRE1α), protein kinase R-like ER kinase/pancreatic eukaryotic initiation factor 2 kinase, and activating transcription factor 6 are essential for the mammalian UPR system. In particular, IRE1α is an essential signal transducer involved in the maintenance of ER function. The IRE1α pathway induces X-box binding protein 1(S) [XBP1(S)], which is the active form of XBP1 generated by IRE1α-dependent splicing of the XBP1 mRNA. XBP1(S) is a transcription factor that increases the expression of the ER chaperone, thus enhancing the secretory function of the ER and suppressing ER stress-mediated cell death [1]. Cancer cells alternate between glycolysis and protein synthesis pathways to survive and proliferate during stress associated with tumor growth and prolonged metabolic and anaerobic stress [2]. Cancer cells produce a lot of new proteins to satisfy their rapid growth using an energy source, such as the cancer-specific aerobic glycolysis pathway. Thus, the augmented function of the ER and resistance of ER stress-mediated cell death is essential for tumor proliferation and survival. Many reports have shown the enhancement of ER function in a variety of human cancers, but the mechanism for the cancer cell-specific regulation of ER function remains unresolved. Our recent study reveals that the tumor suppression gene, p53, is a key regulator of ER function [3]. To the best of our knowledge, this is the first report to connect the cancer-related gene to ER function. p53 is mutated in at least half of human cancers, and defects in the p53 response lead to tumor development. The functions of p53 influence many types of cell signaling transduction pathways, but the role of p53 in the ER is largely unknown [4]. We found that the loss of p53 function increased IRE1α, XBP1(S), and ER chaperone expression in non-stressed and ER-stressed conditions and also increased ER function. Interestingly, IRE1α was abundantly expressed in mutant p53 cell lines compared with wild-type p53 cell lines. The upregulation of IRE1α expression triggered the activation of the IRE1α/XBP1 pathway. Further, our experiments identified the mechanism for increased IRE1 expression with p53 deficiency, and our results revealed that suppressed p53-dependent IRE1α-synoviolin-1 (SYVN1) association induced IRE1α proteasomal degradation. Therefore, wild-type p53 suppresses ER function and adapts to ER stress by keeping IRE1α expression levels low, which highlights the anti-cancer effects of p53. However, loss of p53 function activates the IRE1α/XBP1 pathway and stimulates resistance to ER stress-mediated cell death. Thus, up-regulation of IRE1α expression via the loss of p53 function is one of the mechanisms by which a cancer-specific increase in ER function and activation of the IRE1α/XBP1 pathway is related to some parts of the aggressive cancer phenotype found in p53-mutant cancers. Another important finding of our current study is that an inhibitor of IRE1 selectively prevents the growth of p53-null tumors in vivo compared with that of wild type p53 tumors [3]. Several reports suggested that the inhibition of the IRE1α/XBP1 pathway is a new anti-cancer target. Cubillos-Ruiz et al. reported that the inhibition of activated XBP1 stimulates tumor-infiltrating dendritic cell-induced anti-tumor immunity [5]. The IRE1α inhibitor has an anti-cancer effect on human multiple myeloma and pancreatic cancers [6, 7]. Furthermore, DNA-damaging agents are widely used as treatments for various forms of cancer in the clinic, and its anti-cancer effects depend on the appropriate function of wild-type p53. Thus, mutant p53 cancer cells have been reported to show chemoresistance to many anti-cancer agents, and there is a need for novel therapies against mutant p53 cancers. Therefore, preventing the activation of the IRE1α/XBP1 pathway is a promising target for anti-tumor agents of mutant p53 cancers. In summary, cancer cells need to increase ER function to survive stressful environments. Loss of p53 function augments ER function via the activation of the IRE1α/XBP1 pathway by the disruption of the p53-SYVN1-IRE1α complex. Thus, targeting the IRE1α/XBP1 pathway has potential as a novel therapeutic strategy for malignancy and chemo-resistance of mutant p53 cancers.

Losing p53 loosens up ER-stress.

The endoplasmic reticulum (ER) is the central intracellular organelle responsible for proper folding, localization, and post-translational modifications of proteins in eukaryotic cells. During conditions of cellular stress, incorrectly folded proteins can accumulate in the ER and trigger the unfolded protein response (UPR), which involves the activation of signal transduction pathways designed to restore correct protein folding and recovery from ER stress [1]. In rapidly dividing tumor cells, UPR can become constitutively activated, presumably because higher levels of proliferation require increased protein synthesis [1, 2]. In addition, the tumor micro-environment is often host to additional adverse conditions including nutrient and oxygen deprivation, leading to further perturbations of ER function. Therefore, it may come as no surprise that UPR has been shown to play a crucial cytoprotective role in tumor cells [2]. We have recently unveiled an unexpected role for the tumor suppressor p53 in regulating ER function via the IRE1α/XBP1 pathway [3]. IRE1α is an ER stress sensor that activates the UPR to restore ER function and maintain cellular homeostasis. It was observed that wild-type p53 suppresses IRE1α expression and activation, while the loss of active p53 potentiates the IRE1α/XBP1 pathway, enhancing ER function [3]. This finding suggests a survival benefit for cancer cells that can inactivate p53. Because cancer cells with high protein load on the ER would need enhanced ER function to cope with constant ER stress, losing p53 function would assist tumors to grow in adverse conditions by increasing IRE1α/XBP1 pathway activation. In addition, we found that a p53 target, CDIP1, acts as a proapototic signal mediator in conditions of ER stress [4]. The CDIP1/BAP31 complex was shown to transduce ER stress-mediated apoptotic signaling to the mitochondria and activate the intrinsic apoptosis pathway, suggesting that loss of p53 function may contribute to the survival of cancer cells by enhancing ER function as well as hindering ER stress-mediated apoptosis signaling. The loss of p53 function in relation to ER stress is likely to have important consequences for the sustenance of cancer cell proliferation. Previous studies have demonstrated that the activation of p53 suppresses mTOR activity and consequently inhibits protein translation [5, 6]. The elimination of wild-type p53 function may therefore provide an opportunity for cancer cells to further promote unregulated proliferation, via elevated protein synthesis and a subsequent increase in ER function. We have revealed that wild-type p53 directly interacts with synoviolin (SYVN1) and can thereby stimulate SYVN1-dependent degradation of IRE1α. The loss of p53 disrupts SYVN1-mediated IRE1α downregulation, producing an ER stress-resistant phenotype by increased IRE1α/XBP1 pathway activity [3]. However, although the loss of p53 function facilitates enhanced ER function to manage high ER stress, this also generates an overreliance on IRE1α in cancer cells with p53 mutations. Indeed, we have found that pharmacological inhibition of IRE1α strongly suppresses protein secretion and potently induces cell death in such p53-deficient cancer cells [3]. Importantly, the IRE1α inhibitor exhibited significant inhibitory effects against p53-deficient human tumors in vivo compared to those with wild-type p53. These results illuminate an important proof-of-concept for targeting tumors with mutant p53 via inhibition of the IRE1α/XBP1 pathway. Malignant transformation occurs through a combination of oncogenic pathway activation and the loss of tumor suppression. Understanding the consequences and identifying vulnerabilities caused by such changes can provide promising opportunities to selectively target cancer cells. Mutation in the p53 gene is the most frequent genetic alteration found in human cancer and inactivation of p53 is known as one of the strongest driving factors in cancer development and chemoresistance [7]. The new finding that loss of p53 activates the IRE1α/XBP1 pathway leading to enhanced ER function broadens our understanding of how cancer cells with mutant p53 survive, and also highlights a possibility for the development of novel therapeutic strategies.