Shruti Bhatt

Efficacy of bortezomib in a direct xenograft model of primary effusion lymphoma

Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma most commonly diagnosed in HIV-positive patients and universally associated with Kaposis sarcoma-associated herpesvirus (KSHV). Chemotherapy treatment of PEL yields only short-term remissions in the vast majority of patients, but efforts to develop superior therapeutic approaches have been impeded by lack of animal models that accurately mimic human disease. To address this issue, we developed a direct xenograft model, UM-PEL-1, by transferring freshly isolated human PEL cells into the peritoneal cavities of NOD/SCID mice without in vitro cell growth to avoid the changes in KSHV gene expression evident in cultured cells. We used this model to show that bortezomib induces PEL remission and extends overall survival of mice bearing lymphomatous effusions. The proapoptotic effects of bortezomib are not mediated by inhibition of the prosurvival NF-κB pathway or by induction of a terminal unfolded protein response. Transcriptome analysis by genomic arrays revealed that bortezomib down-regulated cell-cycle progression, DNA replication, and Myc-target genes. Furthermore, we demonstrate that in vivo treatment with either bortezomib or doxorubicin induces KSHV lytic reactivation. These reactivations were temporally distinct, and this difference may help elucidate the therapeutic window for use of antivirals concurrently with chemotherapy. Our findings show that this direct xenograft model can be used for testing novel PEL therapeutic strategies and also can provide a rational basis for evaluation of bortezomib in clinical trials.

CD30 targeting with brentuximab vedotin: a novel therapeutic approach to primary effusion lymphoma

Primary effusion lymphoma (PEL) is an aggressive subtype of non-Hodgkin lymphoma characterized by short survival with current therapies, emphasizing the urgent need to develop new therapeutic approaches. Brentuximab vedotin (SGN-35) is an anti-CD30 monoclonal antibody (cAC10) conjugated by a protease-cleavable linker to a microtubule-disrupting agent, monomethyl auristatin E. Brentuximab vedotin is an effective treatment of relapsed CD30-expressing Classical Hodgkin and systemic anaplastic large cell lymphomas. Herein, we demonstrated that PEL cell lines and primary tumors express CD30 and thus may serve as potential targets for brentuximab vedotin therapy. In vitro treatment with brentuximab vedotin decreased cell proliferation, induced cell cycle arrest, and triggered apoptosis of PEL cell lines. Furthermore, in vivo brentuximab vedotin promoted tumor regression and prolonged survival of mice bearing previously reported UM-PEL-1 tumors as well as UM-PEL-3 tumors derived from a newly established and characterized Kaposis sarcoma-associated herpesvirus- and Epstein-Barr virus-positive PEL cell line. Overall, our results demonstrate for the first time that brentuximab vedotin may serve as an effective therapy for PEL and provide strong preclinical indications for evaluation of brentuximab vedotin in clinical studies of PEL patients.

Efficacious proteasome/HDAC inhibitor combination therapy for primary effusion lymphoma

Primary effusion lymphoma (PEL) is a rare form of aggressive B cell lymphoma caused by Kaposis sarcoma-associated herpesvirus (KSHV). Current chemotherapy approaches result in dismal outcomes, and there is an urgent need for new PEL therapies. Previously, we established, in a direct xenograft model of PEL-bearing immune-compromised mice, that treatment with the proteasome inhibitor, bortezomib (Btz), increased survival relative to that after treatment with doxorubicin. Herein, we demonstrate that the combination of Btz with the histone deacetylase (HDAC) inhibitor suberoylanilidehydroxamic acid (SAHA, also known as vorinostat) potently reactivates KSHV lytic replication and induces PEL cell death, resulting in significantly prolonged survival of PEL-bearing mice. Importantly, Btz blocked KSHV late lytic gene expression, terminally inhibiting the full lytic cascade and production of infectious virus in vivo. Btz treatment led to caspase activation and induced DNA damage, as evidenced by the accumulation of phosphorylated γH2AX and p53. The addition of SAHA to Btz treatment was synergistic, as SAHA induced early acetylation of p53 and reduced interaction with its negative regulator MDM2, augmenting the effects of Btz. The eradication of KSHV-infected PEL cells without increased viremia in mice provides a strong rationale for using the proteasome/HDAC inhibitor combination therapy in PEL.

Identification of LMO2 transcriptome and interactome in diffuse large B-cell lymphoma

LMO2 regulates gene expression by facilitating the formation of multipartite DNA-binding complexes. In B cells, LMO2 is specifically up-regulated in the germinal center (GC) and is expressed in GC-derived non-Hodgkin lymphomas. LMO2 is one of the most powerful prognostic indicators in diffuse large B-cell (DLBCL) patients. However, its function in GC B cells and DLBCL is currently unknown. In this study, we characterized the LMO2 transcriptome and transcriptional complex in DLBCL cells. LMO2 regulates genes implicated in kinetochore function, chromosome assembly, and mitosis. Overexpression of LMO2 in DLBCL cell lines results in centrosome amplification. In DLBCL, the LMO2 complex contains some of the traditional partners, such as LDB1, E2A, HEB, Lyl1, ETO2, and SP1, but not TAL1 or GATA proteins. Furthermore, we identified novel LMO2 interacting partners: ELK1, nuclear factor of activated T-cells (NFATc1), and lymphoid enhancer-binding factor1 (LEF1) proteins. Reporter assays revealed that LMO2 increases transcriptional activity of NFATc1 and decreases transcriptional activity of LEF1 proteins. Overall, our studies identified a novel LMO2 transcriptome and interactome in DLBCL and provides a platform for future elucidation of LMO2 function in GC B cells and DLBCL pathogenesis.

MiR-181a negatively regulates NF-κB signaling and affects activated B-cell-like diffuse large B-cell lymphoma pathogenesis

Distinct subgroups of diffuse large B-cell lymphoma (DLBCL) genetically resemble specific mature B-cell populations that are blocked at different stages of the immune response in germinal centers (GCs). The activated B-cell (ABC)-like subgroup resembles post-GC plasmablasts undergoing constitutive survival signaling, yet knowledge of the mechanisms that negatively regulate this oncogenic signaling remains incomplete. In this study, we report that microRNA (miR)-181a is a negative regulator of nuclear factor κ-light-chain enhancer of activated B-cells (NF-κB) signaling. miR-181a overexpression significantly decreases the expression and activity of key NF-κB signaling components. Moreover, miR-181a decreases DLBCL tumor cell proliferation and survival, and anti-miR-181a abrogates these effects. Remarkably, these effects are augmented in the NF-κB dependent ABC-like subgroup compared with the GC B-cell (GCB)-like DLBCL subgroup. Concordantly, in vivo analyses of miR-181a induction in xenografts results in slower tumor growth rate and prolonged survival in the ABC-like DLBCL xenografts compared with the GCB-like DLBCL. We link these outcomes to relatively lower endogenous miR-181a expression and to NF-κB signaling dependency in the ABC-like DLBCL subgroup. Our findings indicate that miR-181a inhibits NF-κB activity, and that manipulation of miR-181a expression in the ABC-like DLBCL genetic background may result in a significant change in the proliferation and survival phenotype of this malignancy.

Chlamydophila psittaci-negative ocular adnexal marginal zone lymphomas express self polyreactive B-cell receptors

The pathogenesis of Chlamydophila psittaci-negative ocular adnexal extranodal marginal zone lymphomas (OAEMZLs) is poorly understood. OAEMZLs are monoclonal tumors expressing a biased repertoire of mutated surface immunoglobulins. Antigenic activation of the B-cell receptor (BCR) may have a role in the pathogenesis of these lymphomas. We have analyzed the reactivity of recombinant OAEMZL immunoglobulins. OAEMZL antibodies reacted with self-human antigens, as demonstrated by enzyme-linked immunosorbent assays, HEp-2 immunofluorescence and human protein microarrays. All the analyzed recombinant antibodies (rAbs) exhibited polyreactivity by comprehensive protein array antibody reactivity and some rAbs also demonstrated rheumatoid factor activity. The identity of several reactive antigens was confirmed by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometry. The tested rAbs frequently reacted with shared intracellular and extracellular self-antigens (for example, galectin-3). Furthermore, these self-antigens induced BCR signaling in B cells expressing cognate surface immunoglobulins derived from OAEMZLs. These findings indicate that interactions between self-antigens and cognate OAEMZL tumor-derived BCRs are functional, inducing intracellular signaling. Overall, our findings suggest that self-antigen-induced BCR stimulation may be implicated in the pathogenesis of C. psittaci-negative OAEMZLs.

Inhibition of USP10 induces degradation of oncogenic FLT3

Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.

Direct and immune-mediated cytotoxicity of interleukin-21 contributes to antitumor effects in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a distinct subtype of non-Hodgkin lymphoma characterized by overexpression of cyclin D1 in 95% of patients. MCL patients experience frequent relapses resulting in median survival of 3 to 5 years, requiring more efficient therapeutic regimens. Interleukin (IL)-21, a member of the IL-2 cytokine family, possesses potent antitumor activity against a variety of cancers not expressing the IL-21 receptor (IL-21R) through immune activation. Previously, we established that IL-21 exerts direct cytotoxicity on IL-21R-expressing diffuse large B-cell lymphoma cells. Herein, we demonstrate that IL-21 possesses potent cytotoxicity against MCL cell lines and primary tumors. We identify that IL-21-induced direct cytotoxicity is mediated through signal transducer and activator of transcription 3-dependent cMyc upregulation, resulting in activation of Bax and inhibition of Bcl-2 and Bcl-XL. IL-21-mediated cMyc upregulation is only observed in IL-21-sensitive cells. Further, we demonstrate that IL-21 leads to natural killer (NK)-cell-dependent lysis of MCL cell lines that were resistant to direct cytotoxicity. In vivo treatment with IL-21 results in complete FC-muMCL1 tumor regression in syngeneic mice via NK- and T-cell-dependent mechanisms. Together, these data indicate that IL-21 has potent antitumor activity against MCL cells via direct cytotoxic and indirect, immune-mediated effects.

Interleukin 21 – its potential role in the therapy of B-cell lymphomas

Abstract Interleukin-21 (IL-21), a member of IL-2 cytokine family, has pleotropic biological effects on lymphoid and myeloid cells. During the past 15 years, since the discovery of IL-21, great advances have been made regarding its biological activity and the mechanisms controlling IL-21-mediated cellular responses, especially in hematological malignancies. Preclinical studies have shown that IL-21R is expressed on healthy and neoplastic B-cells and exogenous IL-21 can induce direct apoptosis of IL-21R expressing B-cell non-Hodgkin lymphomas (NHL), making it a potentially attractive anti-lymphoma therapy. However, in some hematological malignancies such as multiple myeloma, Hodgkin lymphoma and Burkitt lymphoma, IL-21 can induce proliferation of neoplastic B-cells. In NHL, the underlying mechanism of cell death was found to be different between the various subtypes, including activation of different JAK/STAT signal transduction pathways or other factors. Immunomodulatory effects of IL-21 have also been reported to contribute to its anti-tumor effects as described by earlier studies in solid tumors and B-cell associated malignancies. These effects are predominantly mediated by IL-21’s ability to activate cytolytic activities by NK-cells and CD4+/CD8+ T-cells. In this review, we provide an overview of IL-21’s effects in NHL, results from clinical trials utilizing IL-21, and propose how IL-21 can be therapeutically exploited for treating these lymphomas.

PPM1D truncating mutations confer resistance to chemotherapy and sensitivity to PPM1D inhibition in hematopoietic cells

Truncating mutations in the terminal exon of protein phosphatase Mg2+/Mn2+ 1D (PPM1D) have been identified in clonal hematopoiesis and myeloid neoplasms, with a striking enrichment in patients previously exposed to chemotherapy. In this study, we demonstrate that truncating PPM1D mutations confer a chemoresistance phenotype, resulting in the selective expansion of PPM1D-mutant hematopoietic cells in the presence of chemotherapy in vitro and in vivo. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease mutational profiling of PPM1D in the presence of chemotherapy selected for the same exon 6 mutations identified in patient samples. These exon 6 mutations encode for a truncated protein that displays elevated expression and activity due to loss of a C-terminal degradation domain. Global phosphoproteomic profiling revealed altered phosphorylation of target proteins in the presence of the mutation, highlighting multiple pathways including the DNA damage response (DDR). In the presence of chemotherapy, PPM1D-mutant cells have an abrogated DDR resulting in altered cell cycle progression, decreased apoptosis, and reduced mitochondrial priming. We demonstrate that treatment with an allosteric, small molecule inhibitor of PPM1D reverts the phosphoproteomic, DDR, apoptotic, and mitochondrial priming changes observed in PPM1D-mutant cells. Finally, we show that the inhibitor preferentially kills PPM1D-mutant cells, sensitizes the cells to chemotherapy, and reverses the chemoresistance phenotype. These results provide an explanation for the enrichment of truncating PPM1D mutations in the blood of patients exposed to chemotherapy and in therapy-related myeloid neoplasms, and demonstrate that PPM1D can be a targeted in the prevention of clonal expansion of PPM1D-mutant cells and the treatment of PPM1D-mutant disease.