Date of Graduation

12-2013

Document Type

Dissertation (PhD)

Program Affiliation

Cancer Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Juan Fueyo, M.D.

Committee Member

David Johnson, Ph.D.

Committee Member

Zhimin Lu, M.D., Ph.D.

Committee Member

Luiz Vence, Ph.D.

Committee Member

Joya Chandra, Ph.D.

Abstract

Targeted immunotherapy with recombinant, oncolytic adenoviruses is under investigation for the treatment of cancer. Evidence indicates adenoviruses induce autophagy that is required for oncolysis, but the molecular regulation of autophagy in infected cells remains under investigation. Our data suggested the canonical pathway regulating starvation-induced autophagy was not implemented in adenovirus-induced autophagy; however, adenovirus infection triggered phosphorylation of c-Jun N-terminal kinases (JNK) that was essential for autophagy. Adenoviral replication within the host cell elicited JNK pathway activation leading to B cell lymphoma-2 (Bcl-2) phosphorylation. JNK-dependent Bcl-2 phosphorylation stimulated the dissociation of Bcl-2/beclin 1 heterodimers, enabling beclin 1 to initiate autophagy. Moreover, phosphorylation of both JNK and Bcl-2 was observed in human glioma xenografts after infection with the oncolytic, tropism-enhanced Delta-24-RGD adenovirus, which is currently under investigation in a clinical trial for the treatment of recurring malignant gliomas. Contrary to starvation-induced autophagy, inhibiting adenovirus-mediated autophagy requires genetic knock-out of both JNK1 and JNK2 isoforms, suggesting these proteins have overlapping roles for regulating autophagy in infected cells. Therefore, we report a unique mechanism for the regulation of adenovirus-mediated autophagy that differs from the conventional paradigm.

Adenoviruses are highly immunogenic; and thus, potentially useful vectors for immunotherapeutic strategies. Importantly, autophagy promotes the presentation of self and foreign antigens for activation of immune cells. Our data demonstrated for the first time that adenoviral epitopes were presented at the surface of cells through JNK- and autophagy-dependent mechanisms. Rapamycin, a chemical activator of autophagy, was able to enhance adenoviral antigen presentation. This investigation proposes JNK signaling and autophagy promote adenoviral antigen presentation to the immune system which can be leveraged in developing recombinant adenoviruses for the advancement of anti-tumor immunotherapy. With this knowledge, we developed a novel adenovirus expressing the immunogenic, tumor-specific epidermal growth factor receptor variant III (EGFRvIII) peptide inserted in the adenoviral fiber protein. We confirmed that the EGFRvIII epitope was displayed at the surface of cells in an autophagy-dependent manner that could be enhanced by the pro-inflammatory cytokine, interferon gamma. Thus, we established a new mechanism for the regulation of adenovirus-mediated autophagy and antigen presentation that we employed for the advancement of adenovirus-based vaccines.

Keywords

Adenovirus, JNK, autophagy, antigen presentation

Download

Share

COinS