Publication Date

2014-05-08

Availability

Embargoed

Embargo Period

2016-05-07

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2014-04-02

First Committee Member

Izidore S. Lossos

Second Committee Member

Kerry Burnstein

Third Committee Member

Vladlen Z. Slepak

Fourth Committee Member

Thomas Malek

Fifth Committee Member

Robert B. Levy

Abstract

B-cell non-Hodgkin lymphoma (NHL) is the fifth most common cancer comprised of indolent and aggressive subtypes. Current therapeutic approaches result in short term survival, urging the development of better therapeutics. This dissertation involves design and evaluation of novel therapeutic approaches for the treatment of B-cell lymphomas. Primary effusion lymphoma (PEL), a rare form of aggressive NHL is caused by Kaposi’s sarcoma-associated herpesvirus (KSHV) and/or Epstein-barr virus (EBV). An attractive option for treating PEL and other γ-herpesvirus induced cancers is targeting endogenous latent viruses with drugs that reactivate their lytic replication, thereby eradicating virally-infected reservoirs. Here we demonstrate that the combination of proteasome inhibitor bortezomib (Btz) with the HDAC inhibitor, vorinostat (SAHA), potently reactivates KSHV lytic replication and induces PEL cell death resulting in significantly prolonged survival of PEL-bearing mice (p<0.001, compared to single agent). Importantly, Btz blocks KSHV late lytic gene expression thus terminally inhibiting the full lytic cascade and production of infectious virus in vivo. Btz also led to caspase activation, and induced DNA damage and in synergy with SAHA induces early acetylation of p53 resulting in reduced interaction with its negative regulator MDM2 to facilitate p53 stabilization. Consequently we propose the antineoplastic and lytic-inductive paradigm combining proteasome and HDAC inhibitors to eradicate KSHV-infected PEL without increased viremia. In a second targeting strategy, we first demonstrate that majority of PEL tumors express CD30 antigen, an attractive target for immunotherapy. Brentuximab vedotin, anti-CD30 monoclonal antibody conjugated to anti-mitotic agent is clinically approved for against CD30-expressing Hodgkin’s and systemic anaplastic large cell lymphomas. In our study we showed for the first time that brentuximab vedotin has anti-lymphoma activity against PEL. In vitro treatment with brentuximab vedotin decreases cell proliferation, induces cell cycle arrest and triggers apoptosis of PEL cell lines. Furthermore, in vivo brentuximab vedotin promotes tumor regression and prolongs survival of UM-PEL-1 as well as UM-PEL-3 xenograft mice. In third approach, we evaluated the anti-tumor potential of interleukin 21 (IL-21), in mantle cell lymphoma (MCL), a distinct subtype of NHL which is highly chemoresistant. IL-21 is a member of the IL-2 cytokine family, possesses potent anti-tumor activity against a variety of cancers not expressing IL-21 receptor (IL-21R) through activation of the immune system. Previously study from the lab has established that apart from immuno-stimulatory effects, IL-21 exerts direct cytotoxicity on IL-21R-expressing diffuse large B cell lymphoma cells (DLBCL). Here we found that IL-21 also possess potent cytotoxicity against MCL tumors. IL-21-induced direct cytotoxicity is mediated through STAT3-dependent cMyc upregulation resulting in activation of Bax and inhibition of Bcl-2 and Bcl-XL. Importantly, IL-21-mediated cMyc upregulation is only observed in IL-21-sensitive MCL cell lines and primary tumors. We also discover that IL-21 leads to NK-cell dependent lysis of the MCL cell lines that showed resistance to direct cytotoxicity and also demonstrate potent in vivo activity of IL-21 in MCL mouse model (FcmuMCL1). To further improve anti-tumor potential of IL-21, we designed a fusion protein comprising IL-21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL-21 fusokine). We demonstrate that cytotoxic effects induced by αCD20-IL-21 fusokine in DLBCL and MCL cell lines and primary tumors markedly increase compared to its individual components (IL-21 and parent αCD20-IgG1 antibody). Importantly, fusokine treatment results into cell death of MCL cell lines that are found to be resistant to IL-21 alone treatment. Notably, αCD20-IL-21 fusokine has shown increased ADCC activity in comparison to parent antibody plus IL-21 in NHL cell lines and primary tumors. These data strongly suggest that together with direct apoptotic potential, αCD20- IL-21 fusokine retains the ability to trigger indirect cell killing via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, this dissertation demonstrates pre-clinical efficacy of novel therapeutic approaches for the treatment of B-cell lymphomas which is crucial for clinical intervention.

Keywords

Lymphoma, PEL, Interleukin-21, MCL, DLBCL, Antibody

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