Ramakrishnan Gopalakrishnan

Targeting Myc in KSHV-associated primary effusion lymphoma with BET bromodomain inhibitors.

Primary effusion lymphoma (PEL) is an aggressive form of non-Hodgkin’s B-cell lymphoma associated with infection by Kaposi’s sarcoma-associated herpes virus (KSHV). (+)-JQ1 and I-BET151 are two recently described novel small-molecule inhibitors of BET bromodomain chromatin-associated proteins that have shown impressive preclinical activity in cancers in which MYC is overexpressed at the transcriptional level due to chromosomal translocations that bring the MYC gene under the control of a super-enhancer. PEL cells, in contrast, lack structural alterations in the MYC gene, but have deregulated Myc protein due to the activity of KSHV-encoded latent proteins. We report that PEL cell lines are highly sensitive to bromodomain and extra-terminal (BET) bromodomain inhibitors-induced growth inhibition and undergo G0/G1 cell-cycle arrest, apoptosis and cellular senescence, but without the induction of lytic reactivation, upon treatment with these drugs. Treatment of PEL cell lines with BET inhibitors suppressed the expression of MYC and resulted in a genome-wide perturbation of MYC-dependent genes. Silencing of BRD4 and MYC expression blocked cell proliferation and cell-cycle progression, while ectopic expression of MYC from a retroviral promoter rescued cells from (+)-JQ1-induced growth arrest. In a xenograft model of PEL, (+)-JQ1 significantly reduced tumor growth and improved survival. Taken collectively, our results demonstrate that the utility of BET inhibitors may not be limited to cancers in which genomic alterations result in extremely high expression of MYC and they may have equal or perhaps greater activity against cancers in which the MYC genomic locus is structurally intact and c-Myc protein is deregulated at the post-translational level and is only modestly overexpressed.

Immunomodulatory drugs target IKZF1-IRF4-MYC axis in primary effusion lymphoma in a cereblon-dependent manner and display synergistic cytotoxicity with BRD4 inhibitors.

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma localized predominantly in body cavities. Kaposi’s sarcoma-associated herpes virus (KSHV) is the causative agent of PEL. PEL is an incurable malignancy and has extremely poor prognosis when treated with conventional chemotherapy. Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide are Food and Drug Administration-approved drugs for the treatment of various ailments. IMiDs display pronounced antiproliferative effect against majority of PEL cell lines within their clinically achievable concentrations, by arresting cells at G0/G1 phase of cell cycle and without any induction of KSHV lytic cycle reactivation. Although microarray examination of PEL cells treated with lenalidomide revealed activation of interferon (IFN) signaling, blocking the IFN pathway did not block the anti-PEL activity of IMiDs. The anti-PEL effects of IMiDs involved cereblon-dependent suppression of IRF4 and rapid degradation of IKZF1, but not IKZF3. Small hairpin RNA-mediated knockdown of MYC enhanced the cytotoxicity of IMiDs. Bromodomain (BRD) and extra-terminal domain (BET) proteins are epigenetic readers, which perform a vital role in chromatin remodeling and transcriptional regulation. BRD4, a widely expressed transcriptional coactivator, belongs to the BET family of proteins, which has been shown to co-occupy the super enhancers associated with MYC. Specific BRD4 inhibitors were developed, which suppress MYC transcriptionally. Lenalidomide displayed synergistic cytotoxicity with several structurally distinct BRD4 inhibitors (JQ-1, IBET151 and PFI-1). Furthermore, combined administration of lenalidomide and BRD4 inhibitor JQ-1 significantly increased the survival of PEL bearing NOD–SCID mice in an orthotopic xenograft model as compared with either agent alone. These results provide compelling evidence for clinical testing of IMiDs alone and in combination with BRD4 inhibitors for PEL.

Constitutive NF-κB Activation Confers Interleukin 6 (IL6) Independence and Resistance to Dexamethasone and Janus Kinase Inhibitor INCB018424 in Murine Plasmacytoma Cells

Myeloma cells are dependent on IL6 for their survival and proliferation during the early stages of disease, and independence from IL6 is associated with disease progression. The role of the NF-κB pathway in the IL6-independent growth of myeloma cells has not been studied. Because human herpesvirus 8-encoded K13 selectively activates the NF-κB pathway, we have used it as a molecular tool to examine the ability of the NF-κB pathway to confer IL6 independence on murine plasmacytomas. We demonstrated that ectopic expression of K13, but not its NF-κB-defective mutant or a structural homolog, protected plasmacytomas against IL6 withdrawal-induced apoptosis and resulted in emergence of IL6-independent clones that could proliferate long-term in vitro in the absence of IL6 and form abdominal plasmacytomas with visceral involvement when injected intraperitoneally into syngeneic mice. These IL6-independent clones were dependent on NF-κB activity for their survival and proliferation but were resistant to dexamethasone and INCB018424, a selective Janus kinase 1/2 inhibitor. Ectopic expression of human T cell leukemia virus 1-encoded Tax protein, which resembles K13 in inducing constitutive NF-κB activation, similarly protected plasmacytoma cells against IL6 withdrawal-induced apoptosis. Although K13 is known to up-regulate IL6 gene expression, its protective effect was not due to induction of endogenous IL6 production but instead was associated with sustained expression of several antiapoptotic members of the Bcl2 family upon IL6 withdrawal. Collectively, these results demonstrate that NF-κB activation cannot only promote the emergence of IL6 independence during myeloma progression but can also confer resistance to dexamethasone and INCB018424.

A Purine Scaffold HSP90 Inhibitor BIIB021 Has Selective Activity against KSHV-Associated Primary Effusion Lymphoma and Blocks vFLIP K13-Induced NF-κB

Purpose: Kaposi sarcoma–associated herpes virus (KSHV)–associated primary effusion lymphomas (PEL) have extremely poor prognosis when treated with conventional chemotherapy. KSHV-encoded viral FLICE-inhibitory protein (vFLIP) K13 binds to the IkappaB kinase (IKK) complex to constitutively activate the NF-κB pathway, which has been shown to be essential for the survival and proliferation of PEL cells. The molecular chaperone HSP90 is a component of the IKK complex and is required for its activity. Experimental Design: We have analyzed the effect of HSP90 inhibitors on the survival and proliferation of PEL cells and on the activity of the NF-κB pathway. Results: We show that BIIB021, a purine scaffold–based orally administrable HSP90 inhibitor, shows preferential cytotoxicity toward PEL cells as compared with non-PEL cells. The cytotoxic effect of BIIB021 against PEL was associated with induction of cell-cycle arrest and apoptosis. BIIB021 blocked the expression of a number of cellular proteins involved in the regulation of cell cycle and apoptosis. BIIB021 also blocked constitutive NF-κB activity present in PEL cells, in part, by blocking the interaction of vFLIP K13 with the IKK complex subunits. In a xenograft model of PEL, BIIB021 significantly reduced tumor growth. Conclusion: BIIB021 blocks constitutive NF-κB activity in PEL and shows preferential antitumor activity against PEL in vitro and in vivo. BIIB021 may be a promising agent for treatment of PEL. Clin Cancer Res; 19(18); 5016–26. ©2013 AACR.

Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein K13 activates NF-κB pathway independent of TRAF6, TAK1 and LUBAC.

Background Kaposi’s sarcoma associated herpesvirus encoded viral FLICE inhibitory protein (vFLIP) K13 activates the NF-κB pathway by binding to the NEMO/IKKγ subunit of the IκB kinase (IKK) complex. However, it has remained enigmatic how K13-NEMO interaction results in the activation of the IKK complex. Recent studies have implicated TRAF6, TAK1 and linear ubiquitin chains assembled by a linear ubiquitin chain assembly complex (LUBAC) consisting of HOIL-1, HOIP and SHARPIN in IKK activation by proinflammatory cytokines. Methodology/Principal Findings Here we demonstrate that K13-induced NF-κB DNA binding and transcriptional activities are not impaired in cells derived from mice with targeted disruption of TRAF6, TAK1 and HOIL-1 genes and in cells derived from mice with chronic proliferative dermatitis (cpdm), which have mutation in the Sharpin gene (Sharpincpdm/cpdm). Furthermore, reconstitution of NEMO-deficient murine embryonic fibroblast cells with NEMO mutants that are incapable of binding to linear ubiquitin chains supported K13-induced NF-κB activity. K13-induced NF-κB activity was not blocked by CYLD, a deubiquitylating enzyme that can cleave linear and Lys63-linked ubiquitin chains. On the other hand, NEMO was required for interaction of K13 with IKK1/IKKα and IKK2/IKKβ, which resulted in their activation by “T Loop” phosphorylation. Conclusions/Significance Our results demonstrate that K13 activates the NF-κB pathway by binding to NEMO which results in the recruitment of IKK1/IKKα and IKK2/IKKβ and their subsequent activation by phosphorylation. Thus, K13 activates NF-κB via a mechanism distinct from that utilized by inflammatory cytokines. These results have important implications for the development of therapeutic agents targeting K13-induced NF-κB for the treatment of KSHV-associated malignancies.

NEMO Is Essential for Kaposi's Sarcoma-Associated Herpesvirus-Encoded vFLIP K13-Induced Gene Expression and Protection against Death Receptor-Induced Cell Death, and Its N-Terminal 251 Residues Are Sufficient for This Process

ABSTRACT Kaposis sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 was originally believed to protect virally infected cells against death receptor-induced apoptosis by interfering with caspase 8/FLICE activation. Subsequent studies revealed that K13 also activates the NF-κB pathway by binding to the NEMO/inhibitor of NF-κB (IκB) kinase gamma (IKKγ) subunit of an IKK complex and uses this pathway to modulate the expression of genes involved in cellular survival, proliferation, and the inflammatory response. However, it is not clear if K13 can also induce gene expression independently of NEMO/IKKγ. The minimum region of NEMO that is sufficient for supporting K13-induced NF-κB has not been delineated. Furthermore, the contribution of NEMO and NF-κB to the protective effect of K13 against death receptor-induced apoptosis remains to be determined. In this study, we used microarray analysis on K13-expressing wild-type and NEMO-deficient cells to demonstrate that NEMO is required for modulation of K13-induced genes. Reconstitution of NEMO-null cells revealed that the N-terminal 251 amino acid residues of NEMO are sufficient for supporting K13-induced NF-κB but fail to support tumor necrosis factor alpha (TNF-α)-induced NF-κB. K13 failed to protect NEMO-null cells against TNF-α-induced cell death but protected those reconstituted with the NEMO mutant truncated to include only the N-terminal 251 amino acid residues [the NEMO(1-251) mutant]. Taken collectively, our results demonstrate that NEMO is required for modulation of K13-induced genes and the N-terminal 251 amino acids of NEMO are sufficient for supporting K13-induced NF-κB. Finally, the ability of K13 to protect against TNF-α-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-κB. IMPORTANCE Kaposis sarcoma-associated herpesvirus-encoded vFLIP K13 is believed to protect virally infected cells against death receptor-induced apoptosis and to activate the NF-κB pathway by binding to adaptor protein NEMO/IKKγ. However, whether K13 can also induce gene expression independently of NEMO and the minimum region of NEMO that is sufficient for supporting K13-induced NF-κB remain to be delineated. Furthermore, the contribution of NEMO and NF-κB to the protective effect of K13 against death receptor-induced apoptosis is not clear. We demonstrate that NEMO is required for modulation of K13-induced genes and its N-terminal 251 amino acids are sufficient for supporting K13-induced NF-κB. The ability of K13 to protect against TNF-α-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-κB. Our results suggest that K13-based gene therapy approaches may have utility for the treatment of patients with NEMO mutations and immunodeficiency.

A20 Is Induced by Kaposi Sarcoma-associated Herpesvirus-encoded Viral FLICE Inhibitory Protein (vFLIP) K13 and Blocks K13-induced Nuclear Factor-κB in a Negative Feedback Manner

Expression of A20, a negative regulator of the NF-κB pathway, is frequently lost in several subtypes of Hodgkin and non-Hodgkin lymphoma. We report that A20 is expressed in Kaposi sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma cell lines, and its expression correlates closely with the expression of KSHV-encoded viral FLICE inhibitory protein K13. Ectopic expression of K13 induced A20 expression through NF-κB-mediated activation of A20 promoter. In turn, A20 blocked K13-induced NF-κB activity and up-regulation of proinflammatory cytokines CCL20 and IL-8 in a negative feedback fashion. Both the N-terminal deubiquitinating domain and the C-terminal zinc finger domain of A20 were involved in the inhibition of K13-induced NF-κB activity. Overexpression of A20 blocked K13-induced IκBα phosphorylation, NF-κB nuclear translocation, and cellular transformation. Consistent with the above, K13-induced IκBα phosphorylation and NF-κB transcriptional activation were enhanced in A20-deficient cells. Finally, A20 was found to interact physically with K13. Taken collectively, these results demonstrate that K13 is a key determinant of A20 expression in KSHV-infected cells, and A20 is a key negative regulator of K13-induced NF-κB activity. A20 might serve to control the inflammatory response to KSHV infection and protect KSHV-infected cells from apoptosis.

Development and characterization of a novel luciferase based cytotoxicity assay

A simple, accurate, sensitive and robust assay that can rapidly and specifically measure the death of target cells would have applications in many areas of biomedicine and particularly for the development of novel cellular- and immune-therapeutics. In this study, we describe a novel cytotoxicity assay, termed the Matador assay, which takes advantage of the extreme brightness, stability and glow-like characteristics of recently discovered novel marine luciferases and their engineered derivatives. The assay involves expression of a luciferase of interest in target cells in a manner so that it is preferentially retained within the healthy cells but is either released from dead and dying cells or whose activity can be preferentially measured in dead and dying cells. We demonstrate that this assay is highly sensitive, specific, rapid, and can be performed in a single-step manner without the need for any expensive equipment. We further validate this assay by demonstrating its ability to detect cytotoxicity induced by several cellular and immune-therapeutic agents including antibodies, natural killer cells, chimeric antigen receptor expressing T cells and a bispecific T cell engager.