Gina Ney

The Immunomodulatory Benzodiazepine Bz-423 Inhibits B-Cell Proliferation by Targeting c-Myc Protein for Rapid and Specific Degradation

Myc proteins regulate cell growth and are oncogenic in many cancers. Although these proteins are validated molecular anticancer targets, new therapies aimed at modulating myc have yet to emerge. A benzodiazepine (Bz-423) that was discovered in efforts to find new drugs for lupus was found recently to have antiproliferative effects on Burkitts lymphoma cells. We now show that the basis for the antiproliferative effects of Bz-423 is the rapid and specific depletion of c-myc protein, which is coupled to growth-suppressing effects on key regulators of proliferation and cell cycle progression. c-Myc is depleted as a result of signals coupled to Bz-423 binding its molecular target, the oligomycin sensitivity-conferring protein subunit of the mitochondrial F(1)F(o)-ATPase. Bz-423 inhibits F(1)F(o)-ATPase activity, blocking respiratory chain function and generating superoxide, which at growth-inhibiting concentrations triggers proteasomal degradation of c-myc. Bz-423-induced c-myc degradation is independent of glycogen synthase kinase but is substantially blocked by mutation of the phosphosensitive residue threonine 58, which when phosphorylated targets c-myc for ubiquitination and subsequent proteasomal degradation. Collectively, this work describes a new lead compound, with drug-like properties, which regulates c-myc by a novel molecular mechanism that may be therapeutically useful.

ATDC/TRIM29 phosphorylation by ATM/ MAPKAP kinase 2 mediates radioresistance in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is characterized by therapeutic resistance for which the basis is poorly understood. Here, we report that the DNA and p53-binding protein ATDC/TRIM29, which is highly expressed in PDAC, plays a critical role in DNA damage signaling and radioresistance in pancreatic cancer cells. Ataxia-telangiectasia group D-associated gene (ATDC) mediated resistance to ionizing radiation in vitro and in vivo in mouse xenograft assays. ATDC was phosphorylated directly by MAPKAP kinase 2 (MK2) at Ser550 in an ATM-dependent manner. Phosphorylation at Ser-550 by MK2 was required for the radioprotective function of ATDC. Our results identify a DNA repair pathway leading from MK2 and ATM to ATDC, suggesting its candidacy as a therapeutic target to radiosensitize PDAC and improve the efficacy of DNA-damaging treatment.

ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis.

The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of β-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT.

ATDC/TRIM29 Drives Invasive Bladder Cancer Formation through miRNA-Mediated and Epigenetic Mechanisms

Bladder cancer is a common and deadly malignancy but its treatment has advanced little due to poor understanding of the factors and pathways that promote disease. ATDC/TRIM29 is a highly expressed gene in several lethal tumor types, including bladder tumors, but its role as a pathogenic driver has not been established. Here we show that overexpression of ATDC in vivo is sufficient to drive both noninvasive and invasive bladder carcinoma development in transgenic mice. ATDC-driven bladder tumors were indistinguishable from human bladder cancers, which displayed similar gene expression signatures. Clinically, ATDC was highly expressed in bladder tumors in a manner associated with invasive growth behaviors. Mechanistically, ATDC exerted its oncogenic effects by suppressing miR-29 and subsequent upregulation of DNMT3A, leading to DNA methylation and silencing of the tumor suppressor PTEN. Taken together, our findings established a role for ATDC as a robust pathogenic driver of bladder cancer development, identified downstream effector pathways, and implicated ATDC as a candidate biomarker and therapeutic target.

ATDC (Ataxia Telangiectasia Group D Complementing) Promotes Radioresistance through an Interaction with the RNF8 Ubiquitin Ligase.

Background: ATDC/TRIM29 promotes resistance to ionizing radiation, but the factor(s) that mediate this effect are incompletely understood. Results: ATDC/TRIM29 binds to RNF8, promoting DNA repair and resistance to IR. Conclusion: Following DNA damage, ATDC/TRIM29 is phosphorylated and interacts with RNF8, promoting DNA repair and cell survival. Significance: The interaction between ATDC/TRIM29 and RNF8 is novel and is important for the DNA damage response. Induction of DNA damage by ionizing radiation (IR) and/or cytotoxic chemotherapy is an essential component of cancer therapy. The ataxia telangiectasia group D complementing gene (ATDC, also called TRIM29) is highly expressed in many malignancies. It participates in the DNA damage response downstream of ataxia telangiectasia-mutated (ATM) and p38/MK2 and promotes cell survival after IR. To elucidate the downstream mechanisms of ATDC-induced IR protection, we performed a mass spectrometry screen to identify ATDC binding partners. We identified a direct physical interaction between ATDC and the E3 ubiquitin ligase and DNA damage response protein, RNF8, which is required for ATDC-induced radioresistance. This interaction was refined to the C-terminal portion (amino acids 348–588) of ATDC and the RING domain of RNF8 and was disrupted by mutation of ATDC Ser-550 to alanine. Mutations disrupting this interaction abrogated ATDC-induced radioresistance. The interaction between RNF8 and ATDC, which was increased by IR, also promoted downstream DNA damage responses such as IR-induced γ-H2AX ubiquitination, 53BP1 phosphorylation, and subsequent resolution of the DNA damage foci. These studies define a novel function for ATDC in the RNF8-mediated DNA damage response and implicate RNF8 binding as a key determinant of the radioprotective function of ATDC.

Oncogenic N-Ras and Tet2 haploinsufficiency collaborate to dysregulate hematopoietic stem and progenitor cells

Concurrent genetic lesions exist in a majority of patients with hematologic malignancies. Among these, somatic mutations that activate RAS oncogenes and inactivate the epigenetic modifier ten-eleven translocation 2 (TET2) frequently co-occur in human chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias, suggesting a cooperativity in malignant transformation. To test this, we applied a conditional murine model that endogenously expressed oncogenic NrasG12D and monoallelic loss of Tet2 and explored the collaborative role specifically within hematopoietic stem and progenitor cells (HSPCs) at disease initiation. We demonstrate that the 2 mutations collaborated to accelerate a transplantable CMML-like disease in vivo, with an overall shortened survival and increased disease penetrance compared with single mutants. At preleukemic stage, N-RasG12D and Tet2 haploinsufficiency together induced balanced hematopoietic stem cell (HSC) proliferation and enhanced competitiveness. NrasG12D/+/Tet2+/- HSCs displayed increased self-renewal in primary and secondary transplantations, with significantly higher reconstitution than single mutants. Strikingly, the 2 mutations together conferred long-term reconstitution and self-renewal potential to multipotent progenitors, a pool of cells that usually have limited self-renewal compared with HSCs. Moreover, HSPCs from NrasG12D/+/Tet2+/- mice displayed increased cytokine sensitivity in response to thrombopoietin. Therefore, our studies establish a novel tractable CMML model and provide insights into how dysregulated signaling pathways and epigenetic modifiers collaborate to modulate HSPC function and promote leukemogenesis.

Case 2: Respiratory distress and abdominal tenderness in 21-month-old girl.

1. Heather Lesage-Horton, MD* 2. Gina Ney, MD, PhD* 3. Jennifer Stojan, MD* 1. *Department of Pediatrics, University of Michigan, Ann Arbor, MI. A 21-month-old girl with asthma and eczema (treated by albuterol and topical hydrocortisone as needed) presents in January with 5 days of cough, rhinorrhea, and fever. One day before presentation, she developed emesis and diarrhea with a decrease in oral intake and urine output. On presentation, her temperature is 101.7oF (38.7oC), with a respiratory rate of 60 breaths per minute, a pulse of 160 beats per minute, blood pressure of 110/68 mm Hg, and an oxygen saturation of 91% on room air. Mucous membranes are dry, and capillary refill is delayed. She is responsive but in moderate respiratory distress with intercostal and subcostal retractions. Crackles and coarse breath sounds are auscultated bilaterally. She has abdominal distension with significant right upper quadrant tenderness and guarding. Laboratory results are remarkable for a blood glucose level of 40 mg/dL (2.2 mmol/L), venous pH 7.15, lactate level of 73.9 mg/dL (8.2 mmol/L), bicarbonate level of 16 mEq/L (16 mmol/L) with an anion gap of 17 mEq/L (17 mmol/L), aspartate aminotransaminase (AST) level of 789 IU/L, and alanine aminotransaminase (ALT) of 301 IU/L. Resuscitation with 25% dextrose achieved normal blood glucose levels, and further fluid resuscitation with 2 normal saline boluses followed by 5% dextrose and 0.45% normal saline was administered. Her total bilirubin level is 1.0 mg/dL (17.1 μmol/L) with an alkaline phosphatase level of 271 IU/L. Serum albumin, protein, …

Abstract 313: ATDC drives bladder cancer formation by promoting methylation of the PTEN promoter and inhibition of p53 function.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Bladder cancer is the 5th most common malignancy and a significant cause of morbidity and mortality, but the molecular events leading to its development are incompletely understood. We have identified an oncogene, Ataxia-Telangiectasia Group D Complementing (ATDC) gene, which drives formation and progression of pancreatic and bladder tumors. To better characterize the oncogenic function of ATDC, we generated transgenic (tg) mice which overexpress ATDC and the predominant phenotype of these mice was the development of non-invasive and muscle-invasive urothelial carcinomas. Tg bladder tumors were histologically identical to human tumors and displayed a similar gene expression signature to human invasive bladder tumors. ATDC expression was assessed in a tissue microarray and was highly up-regulated in 70% of human invasive bladder tumors (173/252). High expression of ATDC correlated with more advanced stage disease and worse survival after chemotherapy (p = 0.002). ATDC knockdown in human bladder cancer cell lines correlated with increased sensitivity to chemotherapy in vitro and decreased proliferation and invasion both in vitro and in vivo in a human bladder cancer xenograft model. To understand the mechanism of ATDC-mediated tumor formation, we modulated ATDC expression in normal and bladder cancer cells lines. ATDC overexpression down-regulated PTEN levels via DNA promoter methylation by DNMT3A in both tg mouse tumors and human cell lines. IHC analysis of ATDC, PTEN and DNMT3A expression in human bladder tumors specimens demonstrated similar findings. ATDC was also found to bind to p53 and inhibit p53 mediated functions. These findings establish ATDC as a novel determinant of bladder cancer initiation, progression and resistance to treatment which mediates tumorigenesis by down-regulation of PTEN expression and inhibition of p53. Citation Format: Phillip Palmbos, Lidong Wang, Huibin Yang, Taylor Detzler, Gina Ney, Justin Hart, Stephanie Daignault-Newton, L. Priya Kunju, Chandan Kumar-Sinha, Monica Liebert, Mats Ljungman, Diane Simeone. ATDC drives bladder cancer formation by promoting methylation of the PTEN promoter and inhibition of p53 function. [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 313. doi:10.1158/1538-7445.AM2013-313

ATDC as a novel oncogene in bladder cancer.

269 Background: Bladder cancer is a common and deadly disease, but the molecular events leading to its initiation and progression are incompletely understood. We recently identified Ataxia-Telangiectasia Group D Associated (ATDC) as a novel oncogene which drives tumor proliferation and invasion in pancreatic carcinoma (Cancer Cell, 2009). In this study, we describe the role of ATDC as an oncogene in bladder cancer. METHODS To further determine the oncogenic role of ATDC, we generated ATDC transgenic (tg) mice in which ATDC expression was driven by a CMV promoter and characterized the resulting tumors. RESULTS The dominant phenotype in these mice was the development of both papillary and invasive urothelial carcinomas (9% and 20% respectively, average age of onset 10-12 months of age). Histologically, these tumors were indistinguishable from human urothelial carcinomas. Gene expression profiling of invasive tumors derived from ATDC tg mice demonstrated a marked overlap with gene signatures of human invasive bladder cancers. Analysis of a human bladder cancer tissue microarray (311 samples) showed elevated expression in 70% (173/252) of muscle-invasive carcinomas, whereas normal bladder had no expression. 22% (5/23) of papillary tumors also expressed elevated levels of ATDC. ATDC was the 11th most highly up-regulated gene in bladder cancers represented in the Oncomine gene expression database. ATDC tg mouse bladder tumors demonstrated loss of p53 signaling and down-regulation of PTEN expression, which was determined to be due to ATDC abrogation of p53 function by cytoplasmic sequestration and ATDC-mediated methylation of the PTEN promoter. Furthermore, ATDC knock-down in invasive cancer cell lines resulted in decreased proliferation, invasion and reactivation of p53-mediated signaling and PTEN expression. CONCLUSIONS ATDC is a novel oncogene that is highly expressed in human bladder cancers and is sufficient to drive the development of invasive bladder tumors in tg mice. The mechanism by which ATDC drives bladder cancer formation involves alterations in p53 and PTEN pathways known to be important in bladder tumorigenesis.

Abstract A93: Radiation-induced phosphorylation of ATDC via ATM/MAPKAP kinase 2 signaling mediates radioresistance of pancreatic cancer cells.

Pancreatic cancer is a highly lethal disease characterized by chemotherapy and radiation resistance. We have previously shown that Ataxia-Telangiectasia Group D-Associated gene (ATDC) is highly expressed in pancreatic cancer and stimulates cell proliferation via activation of the β-catenin pathway (Cancer Cell, 2009). In addition, ATDC expression induces cellular resistance to ionizing radiation and ATDC harbors a putative SQ motif (unpublished data). The ATM signaling pathway is induced in response to ionizing radiation (IR) and activates the DNA damage response (DDR) by phosphorylation of downstream targets including H2AX, p53 and other cell cycle checkpoint and DNA repair proteins having SQ motifs. We postulated that ATDC induces cellular resistance to ionizing radiation via participation in the ATM-induced DDR and sought to define the mechanism(s) by which this occurs. To explore the role of ATDC in radio- resistance, ATDC was either overexpressed in HEK293 cells or knocked down in several pancreatic cancer cell lines and sensitivity to IR was measured by clonogenic and apoptosis assays. ATDC overexpression in HEK293 cells, that lack endogenous expression of ATDC, promoted resistance to IR and ATDC knockdown sensitized pancreatic cell lines with high endogenous levels of ATDC to IR. IR induces DNA double strand breaks (DSBs) which are marked by γ-H2AX foci in cellular nuclei. ATDC overexpression correlated with more rapid resolution of γ-H2AX foci consistent with faster repair of DSBs. To test whether ATDC induces radioresistance in vivo, we treated mice with established orthotopic BxPC-3 xenograft tumors with ATDC shRNA-targeting nanovectors which reduced ATDC levels in the tumors. Treatment of orthotopic pancreatic tumors with ATDC shRNA-targeting nanovectors decreased tumor growth, and consistent with in vitro data, also sensitized tumors to fractionated IR, confirming the importance of ATDC in pancreatic tumor radioresistance. To explore the mechanism by which ATDC induces resistance to IR, we next interrogated the ATM signaling pathway. ATDC harbors serine residues at 550 and 552 and S550 is a putative SQ motif that may be phosphorylated by ATM. Consistent with a role in the ATM-induced DDR signaling pathway, ATDC was phosphorylated on S550 following exposure to IR in an ATM-dependent manner. To determine if phosphorylation of S550 was responsible for the radioresistant phenotype, S550 and S552 were replaced with alanines. Overexpression of ATDC S550A, but not S552A, blocked ATDC- associated radioresistance, and left β-catenin-stimulated cell proliferation by ATDC intact, suggesting a separation of function between ATDC-dependent induction of the β-catenin pathway and radioresistance. Although ATDC S550 phosphorylation was dependent upon ATM activation, ATM was unable to directly phosphorylate ATDC on S550 as determined in an in vitro kinase assay. MK2 also plays an important role in ATM-induced DDR in p53 mutant cells and its kinase activity is induced by ATM after IR. MK2 was able to phosphorylate ATDC on S550 in vitro and siRNA-mediated knockdown of MK2 blocked ATDC’s radioprotective effect in cell lines. Moreover, MK2 colocalized with ATDC in cells following IR. In summary, these results suggest that ATDC is a member of the DDR and is phosphorylated directly at its S550 site by MK2 kinase in an ATM-dependent manner following exposure to ionizing radiation. Phosphorylation at S550 is specifically required for the radio-protective function of ATDC. Our data suggest that ATDC, and potentially MK2, represent promising new therapeutic targets for the sensitization of pancreatic adenocarcinoma to radiation therapy. Citation Format: Lidong Wang, Theodore Lawrence, Liang Xu, Chris Canman, Mats Ljungman, Diane Simeone, Huibin Yang, Phillip Palmbos, Gina Ney, Taylor Ann Detzler, Dawn Coleman, Mary Davis, Kevin Hicks, Corey M. Helchowski. Radiation-induced phosphorylation of ATDC via ATM/MAPKAP kinase 2 signaling mediates radioresistance of pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A93.