Sujan Piya

The E1B19K oncoprotein complexes with Beclin 1 to regulate autophagy in adenovirus-infected cells.

The mechanisms underlying adenovirus-mediated autophagy are currently unknown. Recently, members of the Bcl-2 protein family have been associated with autophagy. It was also reported that the Bcl-2 homology-3 (BH3) domain encompassed by both Beclin 1 and Bcl-2-like proteins is essential for their pro-autophagy or anti-autophagy functions. Here, we report for the first time that E1B19K, the adenovirus BH3 domain protein, interacts with Beclin 1 to initiate autophagy. Using immunoprecipitation assays we showed that expression of E1B19K in the host cell disrupted the physical interactions between Beclin 1 and Bcl-2 proteins. The displacement of Bcl-2 was coincident with the recruitment of PI3KC3 to the Beclin 1/E1B19K complexes. As a result of the changes in the components of the Beclin 1 interactome, there was activation of PI3KC3, as showed by the identification of PI3K-mediated lipid phosphorylation, and subsequent formation of autophagosomes. Importantly, the BH3 functional domain of E1B19K protein was required for the heterodimerization with Beclin 1. We also showed that transfer of E1B19K was sufficient to trigger autophagy in cancer cells. Consistent with these data, mutant adenoviruses encompassing a deletion of the E1B19K gene produced a marked deficiency in the capability of the virus to induce autophagy as showed by examining the lipidation and cleavage of LC3-I as well as the subcellular localization of LC3-II, the decrease in the levels of p62, and the formation of autophagosomes. Our work offers new information on the mechanisms of action of the adenoviral E1B19K protein as partner of Beclin 1 and positive regulator of autophagy.

Targeting autophagy to overcome chemoresistance in acute myleogenous leukemia

ABSTRACT Therapeutic inhibition of macroautophagy/autophagy is expected to increase chemosensitivity of cancers and alter tumor-stroma interdependence. The hypoxic, metabolically challenged bone marrow microenvironment confers chemoresistance to leukemia cells. The impact of autophagy inhibition in the context of microenvironment-mediated resistance in leukemia is less explored. Our recent studies demonstrated that co-culture of acute myelogenous leukemia (AML) cells with marrow-derived mesenchymal stromal cells (MSC) induces autophagy in AML cells and increases resistance to genotoxic agents (cytarabine and idarubicin). Genetic silencing of ATG7 in AML enhances the sensitivity to these genotoxic agents, an effect that was more pronounced with concomitant silencing of ATG7 in AML and MSCs. Mechanistically, the increased sensitivity of AML cells to genotoxic agents is associated with alteration of BCL2 family proteins, particularly transcriptional upregulation of PMAIP1/NOXA. In a disseminated AML model in immunocompromised mice, ATG7 knockdown in AML cells results in better survival compared to control mice when treated with chemotherapy. Our studies support the therapeutic role of autophagy inhibition, specifically ATG7 inhibition, in AML.

Atg7 suppression enhances chemotherapeutic agent sensitivity and overcomes stroma-mediated chemoresistance in acute myeloid leukemia.

Autophagy is a cellular adaptive mechanism to stress, including that induced by chemotherapeutic agents. Reverse phase protein array suggested that high expression of the essential autophagy-related protein, Atg7, was associated with shorter remission in newly diagnosed acute myeloid leukemia (AML) patient samples, indicating a role in chemoresistance. Knockdown of Atg7 in AML cells using short hairpin RNA markedly increased apoptosis and DNA damage following treatment with cytarabine and idarubicin. Interestingly, coculture of AML cells with stromal cells increased autophagy and chemoresistance in the AML cells exposed to chemotherapeutic agents, and this was reversed following Atg7 knockdown. This effect was further enhanced by concomitant knockdown of Atg7 in both AML and stromal cells. These findings strongly suggest that Atg7, and likely microenvironment autophagy in general, plays an important role in AML chemoresistance. Mechanistic studies revealed that Atg7 knockdown induced a proapoptotic phenotype in AML cells, which was manifested by an increased NOXA expression at the transcriptional level. Finally, in a mouse model of human leukemia, Atg7 knockdown extended overall survival after chemotherapy. Thus, the inhibition of Atg7 appears to be a valid strategy to enhance chemosensitivity, and it could indeed improve outcomes in AML therapy.

MDM2 Inhibitor, Nutlin 3a, Induces p53 Dependent Autophagy in Acute Leukemia by AMP Kinase Activation

MDM2 (mouse double minute 2) inhibitors that activate p53 and induce apoptosis in a non-genotoxic manner are in clinical development for treatment of leukemias. P53 can modulate other programmed cell death pathways including autophagy both transcriptionally and non-transcriptionally. We investigated autophagy induction in acute leukemia by Nutlin 3a, a first-in-class MDM2 inhibitor. Nutlin 3a induced autophagy in a p53 dependent manner and transcriptional activation of AMP kinase (AMPK) is critical, as this effect is abrogated in AMPK -/- mouse embryonic fibroblasts. Nutlin 3a induced autophagy appears to be pro-apoptotic as pharmacological (bafilomycin) or genetic inhibition (BECLIN1 knockdown) of autophagy impairs apoptosis induced by Nutlin 3a.

Targeting Unc-51 Like Autophagy Activating Kinase 1 (ULK1) in Acute Myeloid Leukemia (AML)

AML-032 Targeting Unc-51 Like Autophagy Activating Kinase 1 (ULK1) in Acute Myeloid Leukemia (AML) Seemana Bhattacharya, Sujan Piya, Teresa McQueen, Marina Konopleva, Michael Andreeff, Gautam Borthakur Department of Leukemia and Section of Molecular Hematology and Therapy, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

Abstract 726: Oncolytic adenoviruses modulate autophagy in cancer cells via sumoylation of LC3

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Oncolytic adenoviruses are being examined as potential therapeutic agents for malignant gliomas and other solid tumors. We have reported that autophagy is a key component of the lysis process in adenovirus-infected cells, influencing the adenoviral spread. We hypothesize that understanding the regulation of autophagy should result in the generation of more potent oncolytic adenoviruses. Our group showed that the function of adenoviral early E1B-proteins include regulation of autophagy. Thus, E1B19k, a Bcl2-like protein, interacts with Beclin 1 and activates autophagy in adenovirus-infected cells. (Piya et al., PLOS One 2011). In this work, we sought to examine the capability of the E1B55K to modulate the autophagic process. Using mutant viruses deficient for the expression of E1B55k, E4orf6 or E1B55k/E4orf6, we observed that viruses lacking the expression of E1B55k were attenuated for the induction of autophagy. Upon examining the sequence of E1B55k, we discovered the presence of an LC3-interacting motif, termed LIR. Co-immunoprecipation assays revealed that E1B55k heterodimerized with LC3. As expected, mutations in the LIR sequence of E1B55k abrogated the physical interaction with LC3. Importantly, we showed that disruption of E1B55k/LC3 complexes had functional repercussions resulting in attenuation of the adenovirus-mediated autophagy. Because the complex of E1B55k and E4orf6 functions as a sumo ligase, we examined whether E1B55k/E4orf6 complexes were involved in LC3 sumoylation. Using co-immunoprecipitation studies in HeLa-6X-his-Sumo-1 infected with wild type and mutant adenoviruses, we showed that LC3 was sumoylated during adenoviral infection and that E1B55k/LC3 interactions were required for this posttranslational modification. Importantly, inhibition of the sumoylation process drastically reduced adenovirus replication. This is the first report showing that adenovirus induced sumoylation of cellular proteins to regulate autophagy. Preliminary studies of cytoplasmic and nuclear contents of LC3 and sumoylated LC3 suggest that adenovirus-mediated sumoylation of LC3 prolongs the presence of LC3 in the cytoplasm by modifying subcellular localization and decreasing protein degradation. We conclude that adenoviral E1B proteins cooperate in the induction and maintenance of autophagy in host cells. Our data provide new insights about the regulation of autophagy and the intracellular environment generated in cells infected with adenoviruses. It is hoped that these findings will propel the development of more potent and selective oncolytic adenoviruses. Citation Format: Sujan Piya, Hong Jiang, Sarah Klein, W.K.Alfred Yung, Raymond Sawaya, Candelaria Gomez-Manzano, Juan Fueyo. Oncolytic adenoviruses modulate autophagy in cancer cells via sumoylation of LC3. [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 726. doi:10.1158/1538-7445.AM2014-726

Advances in Oncolytic Virotherapy for Brain Tumors

Lessons from the history of cancer therapy inform that a multimodal approach offers the best potential to eradicate the tumor mass and prevent the recurrence after therapy. One of the most promising experimental therapies for cancer is the use of oncolytic adenoviruses as therapeutic agents. These biologic agents exert their function by directly infecting and killing tumor cells. The new progeny generated after the first infection round will spread, generating a therapeutic wave that will optimally eliminate every cancer cell. Recently, modifications in the virus genome have allowed for the designing of viruses that infect with more potency than wild-type adenoviruses, replicate exclusively in tumor cells, selectively target cellular receptors or molecular defects in the cancer cells, and deliver prodrug genes. In addition, these last-generation viruses can be combined with chemotherapy and other forms of cancer therapies to enhance the tumor killing capability. With the steady and fast pace of the progress in our knowledge of the genetics of tumors, we may soon fulfill our hope of reaching the final objective of an adenovirus-based personalized medicine for cancer.

Abstract B97: The role of JNK isoforms in adenovirus-induced autophagy: Implications for cancer immunotherapy using oncolytic adenoviruses.

Glioblastoma multiforme is the most common and lethal form of primary brain tumor in adults. Targeted immunotherapy using recombinant oncolytic viruses is a promising strategy for the treatment of brain tumors. Delta-24-RGD is a highly selective and potent oncolytic adenovirus that induces autophagy in malignant gliomas. Data from our ongoing clinical trial suggested Delta-24-RGD initiated an anti-tumor immune response in patients. Importantly, autophagy promotes the presentation of viral antigens for activation of immune cells; therefore, we sought to elucidate the molecular mechanisms by which adenoviruses induce autophagy. Adenoviral replication induced phosphorylation of JNK that was required for autophagy. Our results indicated that JNK regulates autophagy through Bcl-2 phosphorylation and consequent dissociation of Bcl-2 and beclin 1. Contrary to starvation-induced autophagy, adenovirus-mediated autophagy was facilitated by both JNK1 and JNK2 isoforms of JNK. Moreover, our data indicated that adenoviral antigens were presented at the cell surface, which was ablated upon down-regulation of JNK or autophagy. We propose a JNK/autophagy-dependent mechanism for the immune presentation of viral antigens that can be leveraged when developing recombinant adenoviruses for the advancement of anti-tumor immunotherapy. Thus, we developed a genetically-modified Delta-24 adenovirus expressing an EGFRvIII epitope that is aberrantly expressed in gliomas. The majority (>80%) of cells infected with Delta-24-FvIII presented the fiber-bound EGFRvIII epitope on their surface, an effect that was diminished upon inhibition of autophagy. Our data demonstrated that oncolytic adenoviruses can be modified to present cancer antigens for an anti-tumor immune response that may be enhanced by positively regulating autophagy. Citation Format: Sarah R. Klein, Hong Jiang, Sujan Piya, Zhimin Lu, Candelaria Gomez-Manzano, Juan Fueyo. The role of JNK isoforms in adenovirus-induced autophagy: Implications for cancer immunotherapy using oncolytic adenoviruses. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B97.

Abstract 1020: Recruitment of Tie2-expressing monocytes is associated with the heightened invasive phenotype of gliomas after antiangiogenic therapies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Gliomas are highly vascularized tumors, and preclinical data have suggested that glioma growth critically depends on the generation of tumor associated blood vessels. Among multiple factors controlling the complex process of angiogenesis, VEGF (Vascular Endothelial Growth Factor) and its associated signaling cascade are considered of central importance. Glioma cells are a major source of VEGF, and high levels of VEGF generation have been reported to correlate with high grade malignancy and poor prognosis. Antiangiogenic therapy has been recently added to the battery of treatments to newly diagnosed malignant gliomas. However, preclinical data from our laboratory and others, as some clinical results, shown concomitantly elicit tumor adaptation and progression to stages of greater malignancy, with heightened invasiveness. These results warrant preclinical investigation. Here, we present evidence of accumulation of Tie2-expressing monocytes (TEMs) in the brain tumor/normal interphase regions in tumor-bearing animals treated with anti-VEGF agents. Immunohistochemistry using F4/80 or Iba1 antibodies revealed an increase number of microglia/macrophages in central and peripheral tumoral areas after antiangiogenesis treatment. Moreover, the recruitment of these tumor-associated microglia/macrophages (TAMs) was associated with an overrepresentation of TEMs. Almost 50% of Tie2-positive cells co-localized with microglia/macrophages after bevacizumab or prolonged VEGF-Trap/Aflibercept therapies. To determine the role of TEMs in the invasive phenotype of gliomas upon antiangiogenic therapies, we use monocytes isolated from buffy coats and sorted positively for Tie2 expression, and monocytic cultures polarized to M2 phenotype and forced to express Tie2. We found that TEMs express high level of MMP2, MMP9, CD44, and CXCR4, molecules related with extracellular matrix remodeling, using q-PCR, ELISA, FACS analysis and double immunofluorescence. Gelatin zymography, as well as collagenase assay confirmed the upregulation of MMP-2 and MMP-9 activity. Of interest, knocking down Tie2 by siRNA suppresses MMP9 activity emphasizes a role of Tie2 signaling in the regulation of MMP9. Additionally, in vitro invasion assay showed a pivotal role of TEMs in development of an invasive phenotype in glioma cells. Together, our results suggest a novel mechanism of escape of GBMs to anti-VEGF therapies and the necessity of targeting the TEM population in combination with anti-VEGF therapies to improve clinical outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1020. doi:1538-7445.AM2012-1020

Abstract 2871: JNK1-phosphorylation of Bcl-2 is essential for adenoviral-induced autophagic cell death in glioma cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: Delta-24-RGD is a highly selective and potent oncolytic adenovirus that is currently being tested in a clinical trial for patients with malignant gliomas. The mechanism of oncolysis for Delta-24-RGD is defined as autophagic cell death, yet the signal transduction and molecular interactions responsible remain uncharacterized. Methods: Utilizing JNK1- and JNK2-null mouse embryonic fibroblasts, we analyzed the involvement of JNK in Bcl-2 phosphorylation and degradation, and adenoviral-induced autophagy. Mutations at the phosphorylation sites of Bcl-2 were employed to determine which are necessary for release of Beclin 1, degradation of Bcl-2, and induction of autophagy by adenovirus. Results: In this report, we reveal that activation of c-Jun N-terminal kinase 1 (JNK1) is required to initiate and maintain autophagy induced by wild-type and Delta-24-RGD adenoviruses in the host cell. In addition, JNK1-dependent phosphorylation of Bcl-2 at serine 87 (but not serine 70) and subsequent inhibition of Bcl-2:Beclin 1 heterodimerization is necessary for autophagy to occur in infected cells. Upon phosphorylation and release from Beclin 1, Bcl-2 is degraded by the proteosome. Finally, the activation of JNK leads to its translocation to the nucleus and results in the transcription of autophagy-related (ATG) genes for the maintenance of autophagy, as yet undocumented in mammalian cells. Conclusion: Adenoviruses have thus evolved a highly complex mechanism of regulating autophagy which differs from the typical cellular stress response that may be exploited in the treatment of gliomas. 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 2871. doi:10.1158/1538-7445.AM2011-2871