Meredith A. Steeves

Mitochondrial dysfunction in ataxia telangiectasia

Ataxia-telangiectasia mutated (ATM) plays a central role in DNA damage responses, and its loss leads to development of T-cell malignancies. Here, we show that ATM loss also leads to intrinsic mitochondrial abnormalities in thymocytes, including elevated reactive oxygen species, increased aberrant mitochondria, high cellular respiratory capacity, and decreased mitophagy. A fraction of ATM protein is localized in mitochondria, and it is rapidly activated by mitochondrial dysfunction. Unexpectedly, allelic loss of the autophagy regulator Beclin-1 significantly delayed tumor development in ATM-null mice. This effect was not associated with rescue of DNA damage signaling but rather with a significant reversal of the mitochondrial abnormalities. These data support a model in which ATM plays direct roles in modulating mitochondrial homeostasis and suggest that mitochondrial dysfunction and associated increases in mitochondrial reactive oxygen species contribute to the cancer-prone phenotype observed in organisms lacking ATM. Thus, ataxia-telangiectasia should be considered, at least in part, as a mitochondrial disease.

Hsp90-Cdc37 Chaperone Complex Regulates Ulk1- and Atg13-Mediated Mitophagy

Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologs of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1, and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13-directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.

Tristetraprolin Impairs Myc-Induced Lymphoma and Abolishes the Malignant State

Myc oncoproteins directly regulate transcription by binding to target genes, yet this only explains a fraction of the genes affected by Myc. mRNA turnover is controlled via AU-binding proteins (AUBPs) that recognize AU-rich elements (AREs) found within many transcripts. Analyses of precancerous and malignant Myc-expressing B cells revealed that Myc regulates hundreds of ARE-containing (ARED) genes and select AUBPs. Notably, Myc directly suppresses transcription of Tristetraprolin (TTP/ZFP36), an mRNA-destabilizing AUBP, and this circuit is also operational during B lymphopoiesis and IL7 signaling. Importantly, TTP suppression is a hallmark of cancers with MYC involvement, and restoring TTP impairs Myc-induced lymphomagenesis and abolishes maintenance of the malignant state. Further, there is a selection for TTP loss in malignancy; thus, TTP functions as a tumor suppressor. Finally, Myc/TTP-directed control of select cancer-associated ARED genes is disabled during lymphomagenesis. Thus, Myc targets AUBPs to regulate ARED genes that control tumorigenesis.

Targeting the autophagy pathway for cancer chemoprevention.

Autophagy is crucial for maintaining cellular homeostasis, coping with metabolic stress, and limiting oxidative damage. Several autophagy-deficient or knockout models show increased tumor incidence, implicating autophagy as a tumor suppressor. Autophagy is involved in multiple processes that may curb transformation, including the control of oncogene-induced senescence (OIS), which can limit progression to full malignancy, and efficient antigen presentation, which is crucial for immune cell recognition and elimination of nascent cancer cells. Activation of the autophagy pathway may therefore hold promise as a chemoprevention strategy. Caloric restriction, bioactive dietary compounds, or specific pharmacological activators of the autophagy pathway are all possible avenues to explore in harnessing the autophagy pathway in cancer prevention.

Ornithine decarboxylase regulates M1 macrophage activation and mucosal inflammation via histone modifications

Significance The pathogenesis of many bacteria is enhanced by the ability to establish persistent infection. Macrophages, particularly classically activated M1 macrophages, provide essential functions in the initiation of antibacterial immune responses. The regulation of macrophage activation is still poorly understood. Here, we demonstrate that ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, regulates M1 activation during Helicobacter pylori and Citrobacter rodentium infection. Deletion of Odc in macrophages resulted in increased inflammation and decreased bacterial persistence in mouse models. The enhanced M1 response was due to alterations in histone modifications, resulting in changes in chromatin structure and up-regulated transcription. These findings represent a novel mechanism by which ODC directly regulates macrophage activation and provides new insights into understanding bacterial persistence. Macrophage activation is a critical step in host responses during bacterial infections. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, has been well studied in epithelial cells and is known to have essential roles in many different cellular functions. However, its role in regulating macrophage function during bacterial infections is not well characterized. We demonstrate that macrophage-derived ODC is a critical regulator of M1 macrophage activation during both Helicobacter pylori and Citrobacter rodentium infection. Myeloid-specific Odc deletion significantly increased gastric and colonic inflammation, respectively, and enhanced M1 activation. Add-back of putrescine, the product of ODC, reversed the increased macrophage activation, indicating that ODC and putrescine are regulators of macrophage function. Odc-deficient macrophages had increased histone 3, lysine 4 (H3K4) monomethylation, and H3K9 acetylation, accompanied by decreased H3K9 di/trimethylation both in vivo and ex vivo in primary macrophages. These alterations in chromatin structure directly resulted in up-regulated gene transcription, especially M1 gene expression. Thus, ODC in macrophages tempers antimicrobial, M1 macrophage responses during bacterial infections through histone modifications and altered euchromatin formation, leading to the persistence and pathogenesis of these organisms.

Monitoring the autophagy pathway in cancer.

Autophagy is an ancient cell survival pathway that is induced by metabolic stress and that helps prevent bioenergetic failure. This pathway has emerged as a promising new target in cancer treatment, where agents that inhibit autophagic degradation have efficacy in preventing cancer and in treating resistant disease when combined with conventional chemotherapeutics, which generally activate the pathway. However, agents that specifically target the autophagy pathway are currently lacking, and monitoring the effects of therapeutics on the autophagy pathway raises several challenges. Here we review the potential roles of the autophagy pathway in tumor progression and in maintenance of the malignant state, and introduce novel methods that we have developed that allow one to monitor autophagic activity ex vivo and in vivo.

Abstract 261: Emerging roles for the CREB regulated transcription coactivators (CRTCs) in oncogenesis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Oncogenesis is a complex, multi-factorial process of cellular transformation that leads to the development of many types of cancers. The factors that contribute to this process reprogram normal cellular functions, including metabolic pathways, and allow uncontrolled cell growth. The CRTC family of CREB coactivators, in conjunction with CBP/p300, cooperate in the regulation of cAMP-inducible genes involved in cell survival, proliferation, glucose metabolism, and adaptive mitochondrial biogenesis. A subset of tumors share a common t(11;19)(q21;p13.1) translocation that forms a chimeric oncogene by fusing CRTC1 to the NOTCH coactivator MAML2. Consequently, the CRTC1/MAML2 translocation induces the aberrant expression of genes regulated by CREB and NOTCH and the deregulation of these target genes is believed to cause tumorigenesis. We demonstrate that a gain-of-function activity by the CRTC1/MAML2 oncoprotein promotes interactions with the MYC:MAX network. This interaction is specific as neither CRTC1 or MAML2 parental proteins activate MYC:MAX complexes. Specifically, RNA-seq analysis revealed that a significant proporation of genes involved in key aspects of cell growth, survival, and metabolism within the MYC:MAX and CREB transcription networks are induced by CRTC1/MAML2. Analysis of cellular transformation by RK3E foci formation assays identified a synergistic effect of MYC expression on CRTC1/MAML2-induced transformation and this can be blocked by a dominant negative MYC molecule. Furthermore, in-frame deletions of CRTC1/MAML2 that lack transforming activity are unable to induce the expression of MYC-responsive reporters revealing a critical role for MYC target genes in CRTC1/MAML2-induced cell growth and transformation. Collectively, these studies indicate that CRTC1/MAML2 promotes cellular transformation through cooperative activation of MYC and CREB pathways thereby challenging current paradigms which suggest that translocations function through aberrant activation of parental pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 261. doi:10.1158/1538-7445.AM2011-261

Abstract 4027: Myc controls mRNA stability through the agency of AU-binding proteins to promote lymphomagenesis

Myc oncoproteins regulate transcription by binding to target genes harboring E-boxes or Initiator elements, yet these responses explain only a fraction of the genes Myc affects. mRNA turnover is controlled via binding of AU-binding proteins (AUBPs) to AU-rich elements (AREs) found within many transcripts. Expression analyses of precancerous and malignant Myc-expressing B cells revealed Myc regulates hundreds of ARE-containing genes and several AUBPs. Notably, Myc directly suppresses transcription of Tristetraprolin (TTP), an mRNA-destabilizing AUBP, and TTP suppression is a hallmark of Myc-driven lymphoma in mice and man. Furthermore, TTP has profound effects on tumorigenesis, where restoring TTP impairs Myc-induced lymphoma development and abolishes maintenance of the malignant state; thus, TTP functions as a tumor suppressor. Finally, the Myc-to-TTP pathway regulates a very select cast of ARE-containing genes that control cell proliferation and transformation. Thus, Myc utilizes AUBPs to target ARE-containing genes in its efforts to control cell fate and transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4027. doi:10.1158/1538-7445.AM2011-4027