Nagendra K. Chaturvedi

Chronic lymphocytic leukemia cells in a lymph node microenvironment depict molecular signature associated with an aggressive disease.

Chronic lymphocytic leukemia (CLL) cells survive longer in vivo than in vitro, suggesting that the tissue microenvironment provides prosurvival signals to tumor cells. Primary and secondary lymphoid tissues are involved in the pathogenesis of CLL, and the role of these tissue microenvironments has not been explored completely. To elucidate host-tumor interactions, we performed gene expression profiling (GEP) of purified CLL cells from peripheral blood (PB; n = 20), bone marrow (BM; n = 18), and lymph node (LN; n = 15) and validated key pathway genes by real-time polymerase chain reaction, immunohistochemistry and/or TCL1 trans-genic mice. Gene signatures representing several pathways critical for survival and activation of B cells were altered in CLL cells from different tissue compartments. Molecules associated with the B-cell receptor (BCR), B cell-activating factor/a proliferation-inducing ligand (BAFF/APRIL), nuclear factor (NF)-κB pathway and immune suppression signature were enriched in LN-CLL, suggesting LNs as the primary site for tumor growth. Immune suppression genes may help LN-CLL cells to modulate antigen-presenting and T-cell behavior to suppress antitumor activity. PB CLL cells overexpressed chemokine receptors, and their cognate ligands were enriched in LN and BM, suggesting that a chemokine gradient instructs B cells to migrate toward LN or BM. Of several chemokine ligands, the expression of CCL3 was associated with poor prognostic factors. The BM gene signature was enriched with antiapoptotic, cytoskeleton and adhesion molecules. Interestingly, PB cells from lymphadenopathy patients shared GEP with LN cells. In Eμ-TCL1 transgenic mice (the mouse model of the disease), a high percentage of leukemic cells from the lymphoid compartment express key BCR and NF-κB molecules. Together, our findings demonstrate that the lymphoid microenvironment promotes survival, proliferation and progression of CLL cells via chronic activation of BCR, BAFF/APRIL and NF-κB activation while suppressing the immune response.

Human Prostatic Acid Phosphatase: Structure, Function and Regulation

Human prostatic acid phosphatase (PAcP) is a 100 kDa glycoprotein composed of two subunits. Recent advances demonstrate that cellular PAcP (cPAcP) functions as a protein tyrosine phosphatase by dephosphorylating ErbB-2/Neu/HER-2 at the phosphotyrosine residues in prostate cancer (PCa) cells, which results in reduced tumorigenicity. Further, the interaction of cPAcP and ErbB-2 regulates androgen sensitivity of PCa cells. Knockdown of cPAcP expression allows androgen-sensitive PCa cells to develop the castration-resistant phenotype, where cells proliferate under an androgen-reduced condition. Thus, cPAcP has a significant influence on PCa cell growth. Interestingly, promoter analysis suggests that PAcP expression can be regulated by NF-κB, via a novel binding sequence in an androgen-independent manner. Further understanding of PAcP function and regulation of expression will have a significant impact on understanding PCa progression and therapy.

Lymph node‐induced immune tolerance in chronic lymphocytic leukaemia: a role for caveolin‐1

Emerging evidence indicates that the tumour microenvironment (TME) regulates the behaviour of chronic lymphocytic leukaemia (CLL). However, the precise mechanism and molecules involved in this process remain unknown. Gene expression profiles of CLL cells from lymph node (LN), bone marrow (BM) and peripheral blood (PB) indicate overexpression of a tolerogenic signature in CLL cells in lymph nodes (LN‐CLL). Based on their role in B cell biology, the progression of CLL, or immune regulation, a few genes of this 83‐gene signature were selected for further analyses. We observed a significant correlation between the clinical outcomes and the expression of CAV1 (P = 0·041), FGFR1 isoform 8 (P = 0·032), PTPN6 (P = 0·031) and ZWINT (P < 0·001). CAV1, a molecule involved in the regulation of tumour progression in other cancers, was seven‐fold higher in LN‐CLL cells compared to BM‐ and PB‐CLL cells. Knockdown of CAV1 expression in CLL cells resulted in significantly decreased migration (P = 0·016) and proliferation (P = 0·04). When CAV1 was knocked down in B and T cell lines, we observed an inability to form immune synapses. Furthermore, CAV1 knockdown in CLL cells impaired their ability to form immune synapses with autologous T lymphocytes and allogeneic, healthy T cells. Subsequent analyses of microarray data showed differential expression of cytoskeletal genes, specifically those involved in actin polymerization. Therefore, we report a novel role for CAV1 in tumour‐induced immunosuppression during the progression of CLL.

Histone deacetylase inhibitor valproic acid suppresses the growth and increases the androgen responsiveness of prostate cancer cells.

We identified the molecular target by histone deacetylase (HDAC) inhibitors for exploring their potential prostate cancer (PCa) therapy. Upon HDAC inhibitors-treatment, LNCaP cell growth was suppressed, correlating with increased cellular prostatic acid phosphatase (cPAcP) expression, an authentic protein tyrosine phosphatase. In those cells, ErbB-2 was dephosphorylated, histone H3/H4 acetylation and methylation increased and cyclin proteins decreased. In PAcP shRNA-transfected C-81 cells, valproic acid (VPA) efficacy of growth suppression was diminished. Further, VPA pre-treatment enhanced androgen responsiveness of C-81, C4-2 and MDA PCa2b-AI cells. Thus, cPAcP expression is involved in growth suppression by HDAC inhibitors in PCa cells, and VPA pre-treatments increase androgen responsiveness.

Role of CTLA4 in the Proliferation and Survival of Chronic Lymphocytic Leukemia

Earlier, we reported that CTLA4 expression is inversely correlated with CD38 expression in chronic lymphocytic leukemia (CLL) cells. However, the specific role of CTLA4 in CLL pathogenesis remains unknown. Therefore, to elucidate the possible role of CTLA4 in CLL pathogenesis, CTLA4 was down-regulated in primary CLL cells. We then evaluated proliferation/survival in these cells using MTT, 3H-thymidine uptake and Annexin-V apoptosis assays. We also measured expression levels of downstream molecules involved in B-cell proliferation/survival signaling including STAT1, NFATC2, c-Fos, c-Myc, and Bcl-2 using microarray, PCR, western blotting analyses, and a stromal cell culture system. CLL cells with CTLA4 down-regulation demonstrated a significant increase in proliferation and survival along with an increased expression of STAT1, STAT1 phosphorylation, NFATC2, c-Fos phosphorylation, c-Myc, Ki-67 and Bcl-2 molecules. In addition, compared to controls, the CTLA4-downregulated CLL cells showed a decreased frequency of apoptosis, which also correlated with increased expression of Bcl-2. Interestingly, CLL cells from lymph node and CLL cells co-cultured on stroma expressed lower levels of CTLA4 and higher levels of c-Fos, c-Myc, and Bcl-2 compared to CLL control cells. These results indicate that microenvironment-controlled-CTLA4 expression mediates proliferation/survival of CLL cells by regulating the expression/activation of STAT1, NFATC2, c-Fos, c-Myc, and/or Bcl-2.

Novel Treatment for Mantle Cell Lymphoma Including Therapy-Resistant Tumor by NF-κB and mTOR Dual-Targeting Approach

Mantle cell lymphoma (MCL) is one of the most aggressive B-cell non-Hodgkin lymphomas with a median survival of approximately five years. Currently, there is no curative therapy available for refractory MCL because of relapse from therapy-resistant tumor cells. The NF-κB and mTOR pathways are constitutively active in refractory MCL leading to increased proliferation and survival. Targeting these pathways is an ideal strategy to improve therapy for refractory MCL. Therefore, we investigated the in vitro and in vivo antilymphoma activity and associated molecular mechanism of action of a novel compound, 13-197, a quinoxaline analog that specifically perturbs IκB kinase (IKK) β, a key regulator of the NF-κB pathway. 13-197 decreased the proliferation and induced apoptosis in MCL cells including therapy-resistant cells compared with control cells. Furthermore, we observed downregulation of IκBα phosphorylation and inhibition of NF-κB nuclear translocation by 13-197 in MCL cells. In addition, NF-κB–regulated genes such as cyclin D1, Bcl-XL, and Mcl-1 were downregulated in 13-197–treated cells. In addition, 13-197 inhibited the phosphorylation of S6K and 4E-BP1, the downstream molecules of mTOR pathway that are also activated in refractory MCL. Further, 13-197 reduced the tumor burden in vivo in the kidney, liver, and lungs of therapy-resistant MCL-bearing nonobese diabetic severe-combined immunodeficient (NOD/SCID) mice compared with vehicle-treated mice; indeed, 13-197 significantly increased the survival of MCL-transplanted mice. Together, results suggest that 13-197 as a single agent disrupts the NF-κB and mTOR pathways leading to suppression of proliferation and increased apoptosis in malignant MCL cells including reduction in tumor burden in mice. Mol Cancer Ther; 12(10); 2006–17. ©2013 AACR.

Stromal Tumor Microenvironment in Chronic Lymphocytic Leukemia: Regulation of Leukemic Progression

Chronic Lymphocytic Leukemia (CLL), the most prevalent adult leukemia in western countries, which is highly heterogeneous with a very variable clinical outcome. Emerging evidence indicates that the stromal tumor microenvironment (STME) and stromal associated genes (SAG) play important roles in the pathogenesis and progression of CLL. However, the precise mechanisms by which STME and SAG are involved in this process remain unknown. In an attempt to explore the role of STME in this process, we examined the expression levels of stromal associated genes using gene expression profiling (GEP) of CLL cells from lymph nodes (LN) (n=15), bone marrow (BM) (n=18), and peripheral blood (PB) (n=20). Interestingly, LUM, MMP9, MYLK, ITGA9, CAV1, CAV2, FBN1, PARVA, CALD1, ITGB5 and EHD2 were found to be overexpressed while ITGB2, DLC1 and ITGA6 were under expressed in LN-CLL compared to BM-CLL and PB-CLL. This is suggestive of a role for LN-mediated TME in CLL cell survival/progression. Among these genes, expression of MYLK, CAV1 and CAV2 correlated with clinical outcome as determined by time to first treatment. Together, our studies show that members of the stromal signature, particularly in the CLL cells from lymph nodes, regulate CLL cell survival and proliferation and thus leukemic progression.

Establishment and characterization of therapy-resistant mantle cell lymphoma cell lines derived from diff erent tissue sites

Abstract Mantle cell lymphoma (MCL) is a rare but aggressive form of B cell non-Hodgkin lymphoma in which therapy resistance is common. New therapeutic options have extended survival in refractory MCL but have not provided durable remission. Tools are needed to assess the molecular and genetic changes associated with therapy resistance. Therefore, therapy-resistant MCL cell lines were established from the liver, kidney and lungs of human Granta 519-bearing NOD-SCID (non-obese diabetic-severe combined immunodeficiency) mice following treatment with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy in combination with bortezomib. The cytomorphologies, immunophenotypes, growth patterns in semi-solid agar, cytogenetic profiles and gene expression differences between these cell lines were characterized to identify major changes associated with therapy resistance. Therapy-resistant cell lines exhibit more aggressive growth patterns and markedly different gene expression profiles compared to parental Granta 519 cells. Thus, these stable therapy-resistant cell lines are useful models to further study the molecular basis of drug resistance and to identify clinically relevant molecular targets in MCL.

Improved therapy for neuroblastoma using a combination approach: superior efficacy with vismodegib and topotecan.

Aberrant activation/expression of pathways/molecules including NF-kB, mTOR, hedgehog and polo-like-kinase-1 (PLK1) are correlated with poor-prognosis neuroblastoma. Therefore, to identify a most efficacious treatment for neuroblastoma, we investigated the efficacy of NF-kB/mTOR dual-inhibitor 13-197, hedgehog inhibitor vismodegib and PLK1 inhibitor BI2536 alone or combined with topotecan against high-risk neuroblastoma. The in vitro efficacy of the inhibitors alone or combined with topotecan on cell growth/apoptosis and molecular mechanism(s) were investigated. Results showed that as single agents 13-197, BI2536 and vismodegib significantly decreased neuroblastoma cell growth and induced apoptosis by targeting associated pathways/molecules. In combination with topotecan, 13-197 did not show significant additive/synergistic effects against neuroblastoma. However, BI2536 or vismodegib further significantly decreased neuroblastoma cell growth/survival. These results clearly showed that vismodegib combination with topotecan was synergistic and more efficacious compared with BI2536 in combination. Together, in vitro data demonstrated that vismodegib was most efficacious in potentiating topotecan-induced antineuroblastoma effects. Therefore, we tested the combined efficacy of vismodegib and topotecan against neuroblastoma in vivo using NSG mice. This resulted in significantly (p<0.001) reduced tumor growth and increased survival of mice. Together, the combination of vismodegib and topotecan showed a significant enhanced antineuroblastoma efficacy by targeting associated pathways/molecules which warrants further preclinical evaluation for translation to the clinic.

Sprouty 2: a novel attenuator of B-cell receptor and MAPK-Erk signaling in CLL

Clinical heterogeneity is a major barrier to effective treatment of chronic lymphocytic leukemia (CLL). Emerging evidence suggests that constitutive activation of various signaling pathways like mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-Erk) signaling plays a role in the heterogeneous clinical outcome of CLL patients. In this study, we have investigated the role of Sprouty (SPRY)2 as a negative regulator of receptor and nonreceptor tyrosine kinase signaling in the pathogenesis of CLL. We show that SPRY2 expression is significantly decreased in CLL cells, particularly from poor-prognosis patients compared with those from good-prognosis patients. Overexpression of SPRY2 in CLL cells from poor-prognosis patients increased their apoptosis. Conversely, downregulation of SPRY2 in CLL cells from good-prognosis patients resulted in increased proliferation. Furthermore, CLL cells with low SPRY2 expression grew more rapidly in a xenograft model of CLL. Strikingly, B-cell-specific transgenic overexpression of spry2 in mice led to a decrease in the frequency of B1 cells, the precursors of CLL cells in rodents. Mechanistically, we show that SPRY2 attenuates the B-cell receptor (BCR) and MAPK-Erk signaling by binding to and antagonizing the activities of RAF1, BRAF, and spleen tyrosine kinase (SYK) in normal B cells and CLL cells. We also show that SPRY2 is targeted by microRNA-21, which in turn leads to increased activity of Syk and Erk in CLL cells. Taken together, these results establish SPRY2 as a critical negative regulator of BCR-mediated MAPK-Erk signaling in CLL, thereby providing one of the molecular mechanisms to explain the clinical heterogeneity of CLL.