Publications by authors named "Shantaram S Joshi"

51 Publications

Fusion genes as biomarkers in pediatric cancers: A review of the current state and applicability in diagnostics and personalized therapy.

Cancer Lett 2021 02 25;499:24-38. Epub 2020 Nov 25.

Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center, Omaha, NE, 68198, USA. Electronic address:

The incidence of pediatric cancers is rising steadily across the world, along with the challenges in understanding the molecular mechanisms and devising effective therapeutic strategies. Pediatric cancers are presented with diverse molecular characteristics and more distinct subtypes when compared to adult cancers. Recent studies on the genomic landscape of pediatric cancers using next-generation sequencing (NGS) approaches have redefined this field by providing better subtype characterization and novel actionable targets. Since early identification and personalized treatment strategies influence therapeutic outcomes, survival, and quality of life in pediatric cancer patients, the quest for actionable biomarkers is of great value in this field. Fusion genes that are prevalent and recurrent in several pediatric cancers are ideally suited in this context due to their disease-specific occurrence. In this review, we explore the current status of fusion genes in pediatric cancer subtypes and their use as biomarkers for diagnosis and personalized therapy. We discuss the technological advancements made in recent years in NGS sequencing and their impact on fusion detection algorithms that have revolutionized this field. Finally, we also discuss the advantages of pairing liquid biopsy protocols for fusion detection and their eventual use in diagnosis and treatment monitoring.
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http://dx.doi.org/10.1016/j.canlet.2020.11.015DOI Listing
February 2021

Exosomes secreted under hypoxia enhance stemness in Ewing's sarcoma through miR-210 delivery.

Oncotarget 2020 Oct 6;11(40):3633-3645. Epub 2020 Oct 6.

Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA.

Intercellular communication between tumor cells within the hypoxic microenvironment promote aggressiveness and poor patient prognoses for reasons that remain unclear. Here we show that hypoxic Ewing's sarcoma (EWS) cells release exosomes that promote sphere formation, a stem-like phenotype, in EWS cells by enhancing survival. Analysis of the hypoxic exosomal miRNA cargo identified a HIF-1α regulated miRNA, miR-210, as a potential mediator of sphere formation in cells exposed to hypoxic exosomes. Knockdown of HIF-1α in hypoxic EWS cells led to decreased exosomal miR-210 levels and reduced the capacity of hypoxic exosomes to form spheres. Inhibition of miR-210 in hypoxic spheres attenuated sphere formation and overexpression of miR-210 in normoxic spheres significantly enhanced the number of EWS spheres. Our results indicate that hypoxic exosomal miR-210 targets the proapoptotic protein CASP8AP2 in recipient cells. Moreover, the suppression of CASP8AP2 led to a reduction in apoptotic cells and increased sphere formation. Together, the findings in this study suggest that hypoxic exosomes promote stemness in EWS cells by delivering enriched miR-210 that is capable of down-regulating apoptotic pathways, resulting in the survival of cells with increased sphere formation. Future studies will further investigate the effects of EWS derived exosomal miRNAs on target genes and the role these interactions play in driving aggressiveness in hypoxic EWS tumors.
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http://dx.doi.org/10.18632/oncotarget.27702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546758PMC
October 2020

Long non-coding RNA profiling of pediatric Medulloblastoma.

BMC Med Genomics 2020 06 26;13(1):87. Epub 2020 Jun 26.

Department of Pediatrics, Hematology and Oncology Division, University of Nebraska Medical Center, Omaha, NE, 986395, USA.

Background: Medulloblastoma (MB) is one of the most common malignant cancers in children. MB is primarily classified into four subgroups based on molecular and clinical characteristics as (1) WNT (2) Sonic-hedgehog (SHH) (3) Group 3 (4) Group 4. Molecular characteristics used for MB classification are based on genomic and mRNAs profiles. MB subgroups share genomic and mRNA profiles and require multiple molecular markers for differentiation from each other. Long non-coding RNAs (lncRNAs) are more than 200 nucleotide long RNAs and primarily involve in gene regulation at epigenetic and post-transcriptional levels. LncRNAs have been recognized as diagnostic and prognostic markers in several cancers. However, the lncRNA expression profile of MB is unknown.

Methods: We used the publicly available gene expression datasets for the profiling of lncRNA expression across MB subgroups. Functional analysis of differentially expressed lncRNAs was accomplished by Ingenuity pathway analysis (IPA).

Results: In the current study, we have identified and validated the lncRNA expression profile across pediatric MB subgroups and associated molecular pathways. We have also identified the prognostic significance of lncRNAs and unique lncRNAs associated with each MB subgroup.

Conclusions: Identified lncRNAs can be used as single biomarkers for molecular identification of MB subgroups that warrant further investigation and functional validation.
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http://dx.doi.org/10.1186/s12920-020-00744-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318516PMC
June 2020

A Novel Combination Approach Targeting an Enhanced Protein Synthesis Pathway in MYC-driven (Group 3) Medulloblastoma.

Mol Cancer Ther 2020 06 5;19(6):1351-1362. Epub 2020 May 5.

Department of Pediatrics, University of Nebraska Medical Center, Omaha, Nebraska.

The MYC oncogene is frequently amplified in patients with medulloblastoma, particularly in group 3 patients, who have the worst prognosis. mTOR signaling-driven deregulated protein synthesis is very common in various cancers, including medulloblastoma, that can promote MYC stabilization. As a transcription factor, MYC itself is further known to regulate transcription of several components of protein synthesis machinery, leading to an enhanced protein synthesis rate and proliferation. Thus, inhibiting enhanced protein synthesis by targeting the MYC and mTOR pathways together may represent a highly relevant strategy for the treatment of MYC-driven medulloblastoma. Here, using siRNA and small-molecule inhibitor approaches, we evaluated the effects of combined inhibition of MYC transcription and mTOR signaling on medulloblastoma cell growth/survival and associated molecular mechanism(s) in MYC-amplified (group 3) medulloblastoma cell lines and xenografts. Combined inhibition of MYC and mTOR synergistically suppressed medulloblastoma cell growth and induced G cell-cycle arrest and apoptosis. Mechanistically, the combined inhibition significantly downregulated the expression levels of key target proteins of MYC and mTOR signaling. Our results with RNA-sequencing revealed that combined inhibition synergistically modulated global gene expression including MYC/mTOR components. In addition, the combination treatment significantly delayed tumor growth and prolonged survival of MYC-amplified medulloblastoma xenografted mice by downregulating expression of MYC and the key downstream components of mTOR signaling, compared with single-agent therapy. Together, our findings demonstrated that dual inhibition of MYC (transcription) and mTOR (translation) of the protein synthesis pathway can be a novel therapeutic approach against MYC-driven medulloblastoma.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0996DOI Listing
June 2020

Pan-Cancer Analysis Reveals the Diverse Landscape of Novel Sense and Antisense Fusion Transcripts.

Mol Ther Nucleic Acids 2020 Mar 29;19:1379-1398. Epub 2020 Jan 29.

Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198, USA; Bioinformatics and Systems Biology Core. University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address:

Gene fusions that contribute to oncogenicity can be explored for identifying cancer biomarkers and potential drug targets. To investigate the nature and distribution of fusion transcripts in cancer, we examined the transcriptome data of about 9,000 primary tumors from 33 different cancers in TCGA (The Cancer Genome Atlas) along with cell line data from CCLE (Cancer Cell Line Encyclopedia) using ChimeRScope, a novel fusion detection algorithm. We identified several fusions with sense (canonical, 39%) or antisense (non-canonical, 61%) transcripts recurrent across cancers. The majority of the recurrent non-canonical fusions found in our study are novel, unexplored, and exhibited highly variable profiles across cancers, with breast cancer and glioblastoma having the highest and lowest rates, respectively. Overall, 4,344 recurrent fusions were identified from TCGA in this study, of which 70% were novel. Additional analysis of 802 tumor-derived cell line transcriptome data across 20 cancers revealed significant variability in recurrent fusion profiles between primary tumors and corresponding cell lines. A subset of canonical and non-canonical fusions was validated by examining the structural variation evidence in whole-genome sequencing (WGS) data or by Sanger sequencing of fusion junctions. Several recurrent fusion genes identified in our study show promise for drug repurposing in basket trials and present opportunities for mechanistic studies.
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http://dx.doi.org/10.1016/j.omtn.2020.01.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044684PMC
March 2020

Role of protein arginine methyltransferase 5 in group 3 (MYC-driven) Medulloblastoma.

BMC Cancer 2019 Nov 6;19(1):1056. Epub 2019 Nov 6.

Department of Pediatrics, Division of Hematology and Oncology, University of Nebraska Medical Center, Omaha, NE, 68198, USA.

Background: MYC amplification or overexpression is common in Group 3 medulloblastoma and is associated with the worst prognosis. Recently, protein arginine methyl transferase (PRMT) 5 expression has been closely associated with aberrant MYC function in various cancers, including brain tumors such as glioblastoma. However, the role of PRMT5 and its association with MYC in medulloblastoma have not been explored. Here, we report the role of PRMT5 as a novel regulator of MYC and implicate PRMT5 as a potential therapeutic target in MYC-driven medulloblastoma.

Methods: Expression and association between PRMT5 and MYC in primary medulloblastoma tumors were investigated using publicly available databases. Expression levels of PRMT5 protein were also examined using medulloblastoma cell lines and primary tumors by western blotting and immunohistochemistry, respectively. Using MYC-driven medulloblastoma cells, we examined the physical interaction between PRMT5 and MYC by co-immunoprecipitation and co-localization experiments. To determine the functional role of PRMT5 in MYC-driven medulloblastoma, PRMT5 was knocked-down in MYC-amplified cells using siRNA and the consequences of knockdown on cell growth and MYC expression/stability were investigated. In vitro therapeutic potential of PRMT5 in medulloblastoma was also evaluated using a small molecule inhibitor, EPZ015666.

Results: We observed overexpression of PRMT5 in MYC-driven primary medulloblastoma tumors and cell lines compared to non-MYC medulloblastoma tumors and adjacent normal tissues. We also found that high expression of PRMT5 is inversely correlated with patient survival. Knockdown of PRMT5 using siRNA in MYC-driven medulloblastoma cells significantly decreased cell growth and MYC expression. Mechanistically, we found that PRMT5 physically associated with MYC by direct protein-protein interaction. In addition, a cycloheximide chase experiment showed that PRMT5 post-translationally regulated MYC stability. In the context of therapeutics, we observed dose-dependent efficacy of PRMT5 inhibitor EPZ015666 in suppressing cell growth and inducing apoptosis in MYC-driven medulloblastoma cells. Further, the expression levels of PRMT5 and MYC protein were downregulated upon EPZ015666 treatment. We also observed a superior efficacy of this inhibitor against MYC-amplified medulloblastoma cells compared to non-MYC-amplified medulloblastoma cells, indicating specificity.

Conclusion: Our results reveal the regulation of MYC oncoprotein by PRMT5 and suggest that targeting PRMT5 could be a potential therapeutic strategy for MYC-driven medulloblastoma.
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http://dx.doi.org/10.1186/s12885-019-6291-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836472PMC
November 2019

Targeting cyclin-dependent kinase 9 sensitizes medulloblastoma cells to chemotherapy.

Biochem Biophys Res Commun 2019 12 5;520(2):250-256. Epub 2019 Oct 5.

Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA.

Medulloblastoma (MB) is a highly aggressive, malignant brain tumor in children with poor prognosis. Cyclin-dependent kinase 9 (CDK9), a serine-threonine kinase, is widely implicated in the control of basal gene expression by phosphorylating Serine 2 (Ser2) of the heptad repeat in the RNA Polymerase II (RNA Pol II) C-terminal domain (CTD). Although CDK9 plays a pathogenic role in various cancers, its function in MB remains unknown. Here, we show that CDK9 is highly expressed in MB tumors and increased CDK9 expression is correlated with high risk MB patients. CDK9 expression along with phospho-Ser2 RNA Pol II (pRNA Pol II ser2) and bromodomain-binding protein 4 (BRD4), which recruits CDK9, were elevated in multiple MB cell lines and in MB tumors originated spontaneously from Ptch1p53 mice. Inhibition of CDK9 with LDC067 suppressed MB cell growth, reduced pRNA Pol II ser2 level and expression of oncogenic markers, including MYC. Moreover, LDC067 treatment synergistically sensitizes MB cells to chemotherapeutic agent cisplatin. Further, LDC067 in combination with BRD4 inhibitor decreased MB cells growth, delayed cell migration and attenuated pRNA Pol II ser2 occupancy to CCND1 and BCL2 gene promoters as revealed by chromatin immunoprecipitation assay (ChIP). Together, these findings highlight the importance of CDK9 in MB pathogenesis and suggest that it may serve as a promising therapeutic target for the treatment of MB.
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http://dx.doi.org/10.1016/j.bbrc.2019.09.118DOI Listing
December 2019

A novel approach to eliminate therapy-resistant mantle cell lymphoma: synergistic effects of Vorinostat with Palbociclib.

Leuk Lymphoma 2019 05 14;60(5):1214-1223. Epub 2018 Nov 14.

b Department Genetics, Cell Biology and Anatomy , University of Nebraska Medical Center , Omaha , NE , USA.

Mantle cell lymphoma (MCL) represents an aggressive B-cell lymphoma with frequent relapse and poor survival. Recently, dysregulated histone-deacetylases (HDACs) and cell cycle CDK-Rb pathway have been shown to be commonly associated with MCL pathogenesis, and are considered promising targets for relapsed-lymphoma therapy. Therefore, we investigated the single agents and combination efficacy of HDACs inhibitor Vorinostat, CDK4/6 dual-inhibitor Palbociclib on MCL cell growth/survival and underlying molecular mechanism(s) using MCL cell lines including therapy-resistant MCL cell lines. Our results showed that both inhibitors as single agents or combined, significantly suppressed the cell growth and induced apoptosis in therapy-resistant and parental MCL lines. In addition, the combination of Vorinostat and Palbociclib significantly inhibited the activation of the key molecules of the CDK4/6-Rb pathway and HDAC activity and subsequently decreased the expression of Cyclin-D1 and Bcl-2. These studies demonstrated the potential for combining these two inhibitors as a novel therapeutic approach in refractory MCL therapy.
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http://dx.doi.org/10.1080/10428194.2018.1520986DOI Listing
May 2019

Improved therapy for medulloblastoma: targeting hedgehog and PI3K-mTOR signaling pathways in combination with chemotherapy.

Oncotarget 2018 Mar 30;9(24):16619-16633. Epub 2018 Mar 30.

Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 69198, USA.

Aberrant activation and interactions of hedgehog (HH) and PI3K/AKT/mTOR signaling pathways are frequently associated with high-risk medulloblastoma (MB). Thus, combined targeting of the HH and PI3K/AKT/mTOR pathways could be a viable therapeutic strategy to treat high-risk patients. Therefore, we investigated the anti-MB efficacies of combined HH inhibitor Vismodegib and PI3K-mTOR dual-inhibitor BEZ235 together or combined individually with cisplatin against high-risk MB. Using non-MYC- and MYC-amplified cell lines, and a xenograft mouse model, the and efficacies of these therapies on cell growth/survival and associated molecular mechanism(s) were investigated. Results showed that combined treatment of Vismodegib and BEZ235 together, or with cisplatin, significantly decreased MB cell growth/survival in a dose-dependent-fashion. Corresponding changes in the expression of targeted molecules following therapy were observed. Results demonstrated that inhibitors not only suppressed MB cell growth/survival when combined, but also significantly enhanced cisplatin-mediated cytotoxicity. Of these combinations, BEZ235 exhibited a significantly greater efficacy in enhancing cisplatin-mediated MB cytotoxicity. Results also demonstrated that the MYC-amplified MB lines showed a higher sensitivity to combined therapies compared to non-MYC-amplified cell lines. Therefore, we tested the efficacy of combined approaches against MYC-amplified MB growing in NSG mice. results showed that combination of Vismodegib and BEZ235 or their combination with cisplatin, significantly delayed MB tumor growth and increased survival of xenografted mice by targeting HH and mTOR pathways. Thus, our studies lay a foundation for translating these combined therapeutic strategies to the clinical setting to determine their efficacies in high-risk MB patients.
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http://dx.doi.org/10.18632/oncotarget.24618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908274PMC
March 2018

Suppression of STAT3 NH -terminal domain chemosensitizes medulloblastoma cells by activation of protein inhibitor of activated STAT3 via de-repression by microRNA-21.

Mol Carcinog 2018 04 25;57(4):536-548. Epub 2018 Jan 25.

Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska.

Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/SHH tumors. Treatment of MB cells with S3-NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3-NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial-mesenchymal transition (EMT) marker expressions and reduced cancer stem-cell associated protein expressions in MB-spheres. To elucidate mechanisms, we showed that S3-NTDi induce expression of pro-apoptotic gene, C/EBP-homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3-NTDi downregulated microRNA-21, which in turn, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3-NTDi and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3-NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high-risk patients.
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http://dx.doi.org/10.1002/mc.22778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6132241PMC
April 2018

Regulation of MAPK signaling and implications in chronic lymphocytic leukemia.

Leuk Lymphoma 2018 07 7;59(7):1565-1573. Epub 2017 Sep 7.

c Department of Genetics Cell Biology and Anatomy , University of Nebraska Medical Centre , Omaha , NE , USA.

Chronic lymphocytic leukemia (CLL) is a heterogeneous B cell malignancy that still remains incurable. Recent studies have highlighted cellular and non-cellular components of the tissue microenvironment in CLL that help nurture the growth of leukemic cells by providing the necessary stimuli for their proliferation and survival. The diverse stimuli in the specialized tissue microenvironment of CLL lead to constitutive activation of several signaling pathways that includes B cell receptor signaling and the associated mitogen-activated protein kinase (MAPK) signaling. Recent findings have described aberrant activation of MAPK signaling and its interactions with other cellular signaling pathways in the pathogenesis of CLL. These studies have shed light on the deregulated molecular mechanisms contributing to hyperactivation of MAPK signaling and provided avenues for therapeutic options for aggressive CLL. In this review, we describe and discuss the current status of our understanding into the role of MAPK signaling in the pathogenesis of CLL.
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http://dx.doi.org/10.1080/10428194.2017.1370548DOI Listing
July 2018

Treatment of a chemoresistant neuroblastoma cell line with the antimalarial ozonide OZ513.

BMC Cancer 2016 11 8;16(1):867. Epub 2016 Nov 8.

Department of Pharmacy Practice and Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA.

Background: Evaluate the anti-tumor activity of ozonide antimalarials using a chemoresistant neuroblastoma cell line, BE (2)-c.

Methods: The activity of 12 ozonides, artemisinin, and two semisynthetic artemisinins were tested for activity against two neuroblastoma cell-lines (BE (2)-c and IMR-32) and the Ewing's Sarcoma cell line A673 in an MTT viability assay. Time course data indicated that peak effect was seen 18 h after the start of treatment thus 18 h pre-treatment was used for all subsequent experiments. The most active ozonide (OZ513) was assessed in a propidium iodide cell cycle flow cytometry analysis which measured cell cycle transit and apoptosis. Metabolic effects of OZ513 in BE (2)-c cells was evaluated. Western blots for the apoptotic proteins cleaved capase-3 and cleaved PARP, the highly amplified oncogene MYCN, and the cell cycle regulator CyclinD1, were performed. These in-vitro experiments were followed by an in-vivo experiment in which NOD-scid gamma immunodeficient mice were injected subcutaneously with 1 × 10 BE (2)-c cells followed by immediate treatment with 50-100 mg/kg/day doses of OZ513 administered IP three times per week out to 23 days after injection of tumor. Incidence of tumor development, time to tumor development, and rate of tumor growth were assessed in DMSO treated controls (N = 6), and OZ513 treated mice (N = 5).

Results: It was confirmed that five commonly used chemotherapy drugs had no cytotoxic activity in BE (2)-c cells. Six of 12 ozonides tested were active in-vitro at concentrations achievable in vivo with OZ513 being most active (IC50 = 0.5 mcg/ml). OZ513 activity was confirmed in IMR-32 and A673 cells. The Ao peak on cell-cycle analysis was increased after treatment with OZ513 in a concentration dependent fashion which when coupled with results from western blot analysis which showed an increase in cleaved capase-3 and cleaved PARP supported an increase in apoptosis. There was a concentration dependent decline in the MYCN and a cyclinD1 protein indicative of anti-proliferative activity and cell cycle disruption. OXPHOS metabolism was unaffected by OZ513 treatment while glycolysis was increased. There was a significant delay in time to tumor development in mice treated with OZ513 and a decline in the rate of tumor growth.

Conclusions: The antimalarial ozonide OZ513 has effective in-vitro and in-vivo activity against a pleiotropic drug resistant neuroblastoma cell-line. Treatment with OZ513 increased apoptotic markers and glycolysis with a decline in the MYCN oncogene and the cell cycle regulator cyclinD1. These effects suggest adaptation to cellular stress by mechanism which remain unclear.
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http://dx.doi.org/10.1186/s12885-016-2872-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100253PMC
November 2016

Interferon regulatory factor 4 attenuates Notch signaling to suppress the development of chronic lymphocytic leukemia.

Oncotarget 2016 Jul;7(27):41081-41094

Department of Genetics Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA.

Molecular pathogenesis of Chronic Lymphocytic Leukemia (CLL) is not fully elucidated. Genome wide association studies have linked Interferon Regulatory Factor 4 (IRF4) to the development of CLL. We recently established a causal relationship between low levels of IRF4 and development of CLL. However, the molecular mechanism through which IRF4 suppresses CLL development remains unclear. Deregulation of Notch signaling pathway has been identified as one of the most recurrent molecular anomalies in the pathogenesis of CLL. Yet, the role of Notch signaling as well as its regulation during CLL development remains poorly understood. Previously, we demonstrated that IRF4 deficient mice expressing immunoglobulin heavy chain Vh11 (IRF4-/-Vh11) developed spontaneous CLL with complete penetrance. In this study, we show that elevated Notch2 expression and the resulting hyperactivation of Notch signaling are common features of IRF4-/-Vh11 CLL cells. Our studies further reveal that Notch signaling is indispensable for CLL development in the IRF4-/-Vh11 mice. Moreover, we identify E3 ubiquitin ligase Nedd4, which targets Notch for degradation, as a direct target of IRF4 in CLL cells and their precursors. Collectively, our studies provide the first in vivo evidence for an essential role of Notch signaling in the development of CLL and establish IRF4 as a critical regulator of Notch signaling during CLL development.
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http://dx.doi.org/10.18632/oncotarget.9596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5173044PMC
July 2016

Modulation of p73 isoforms expression induces anti-proliferative and pro-apoptotic activity in mantle cell lymphoma independent of p53 status.

Leuk Lymphoma 2016 12 13;57(12):2874-2889. Epub 2016 Apr 13.

a Department of Pathology and Microbiology , University of Nebraska Medical Center , Omaha , NE , USA.

Mantle cell lymphoma (MCL) is characterized by a clinically aggressive course with frequent relapse and poor survival. The p53 pathway is frequently dysregulated and p53 status predicts clinical outcome. In this report, we investigated whether modulation of p73 isoforms by diclofenac inhibits cell growth, induces apoptosis and/or cell cycle arrest in MCL relative to p53 status. Wild-type p53 [Granta-519 and JVM-2], mutant p53 [Jeko-1 and Mino-1] expressing cells, therapy resistant cell lines, and primary human cells isolated from MCL patients were used. Overexpression of pro-apoptotic TAp73 enhanced MCL cell apoptosis. Diclofenac induced a concentration- and duration-dependent increase in TAp73, cell cycle arrest, cell death, and inhibited MCL cell growth independent of p53 status. Diclofenac treatment was associated with increased activity of caspases 3, 7, and 8 and induction of p53 transcriptional target genes. These studies demonstrate the potential for diclofenac as novel therapeutic agent in MCL independent of p53 status.
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http://dx.doi.org/10.3109/10428194.2016.1165814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967247PMC
December 2016

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

Oncotarget 2016 Mar;7(12):15215-29

Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA.

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.
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http://dx.doi.org/10.18632/oncotarget.7714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924781PMC
March 2016

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

Blood 2016 05 25;127(19):2310-21. Epub 2016 Jan 25.

Department of Genetics, Cell Biology and Anatomy.

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.
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http://dx.doi.org/10.1182/blood-2015-09-669317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4865588PMC
May 2016

Absence of caveolin-1 leads to delayed development of chronic lymphocytic leukemia in Eμ-TCL1 mouse model.

Exp Hematol 2016 Jan 3;44(1):30-7.e1. Epub 2015 Oct 3.

Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE. Electronic address:

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United States. The tissue microenvironment, specifically the lymph nodes, influences the biological and clinical behavior of CLL cells. Gene expression profiling of CLL cells from peripheral blood, bone marrow, and lymph nodes revealed Cav-1 as one of the genes that might be involved in the pathogenesis of CLL. We have previously reported that the knockdown of Cav-1 in primary CLL cells exhibits a significant decrease in cell migration and immune synapse formation. However, the precise role of Cav-1 in CLL initiation and progression in vivo is not known. Therefore, we decreased the expression of Cav-1 in vivo by breeding Eμ-TCL1 with cav-1 knockout mice. We observed a significant decrease in the number of CLL cells and rate of proliferation of CLL cells in spleen, liver, and bone marrow from Eμ-TCL1-Cav1(-/+) and Eμ-TCL1-Cav1(-/-) mice as compared with Eμ-TCL1 mice. In addition, there was a significant increase in survival of Eμ-TCL1-Cav1(-/+) and Eμ-TCL1-Cav1(-/-) compared with Eμ-TCL1 mice. Mechanistically, we observed a decrease in MAPK-Erk signaling measured by p-Erk levels in Eμ-TCL1-Cav1(-/+) mice when compared with Eμ-TCL1-Cav(wt/wt). Together these results indicate that decreased Cav-1 in Eμ-TCL1 mice significantly delays the onset of CLL and decreases leukemic progression by inhibiting MAPK-Erk signaling, suggesting a role for Cav-1 in the proliferation and progression of CLL.
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http://dx.doi.org/10.1016/j.exphem.2015.09.005DOI Listing
January 2016

Experimental validation of predicted subcellular localizations of human proteins.

BMC Res Notes 2014 Dec 15;7:912. Epub 2014 Dec 15.

Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198, USA.

Background: Computational methods have been widely used for the prediction of protein subcellular localization. However, these predictions are rarely validated experimentally and as a result remain questionable. Therefore, experimental validation of the predicted localizations is needed to assess the accuracy of predictions so that such methods can be confidently used to annotate the proteins of unknown localization. Previously, we published a method called ngLOC that predicts the localization of proteins targeted to ten different subcellular organelles. In this short report, we describe the accuracy of these predictions using experimental validations.

Findings: We have experimentally validated the predicted subcellular localizations of 114 human proteins corresponding to nine different organelles in normal breast and breast cancer cell lines using live cell imaging/confocal microscopy. Target genes were cloned into expression vectors as GFP fusions and cotransfected with RFP-tagged organelle-specific gene marker into normal breast epithelial and breast cancer cell lines. Subcellular localization of each target protein is confirmed by colocalization with a co-expressed organelle-specific protein marker. Our results showed that about 82.5% of the predicted subcellular localizations coincided with the experimentally validated localizations. The highest agreement was found in the endoplasmic reticulum proteins, while the cytoplasmic location showed the least concordance. With the exclusion of cytoplasmic location, the average prediction accuracy increased to 90.4%. In addition, there was no difference observed in the protein subcellular localization between normal and cancer breast cell lines.

Conclusions: The experimentally validated accuracy of ngLOC method with (82.5%) or without cytoplasmic location (90.4%) nears the prediction accuracy of 89%. These results demonstrate that the ngLOC method can be very useful for large-scale annotation of the unknown subcellular localization of proteins.
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http://dx.doi.org/10.1186/1756-0500-7-912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301851PMC
December 2014

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

Mol Med 2014 Jul 15;20:290-301. Epub 2014 Jul 15.

Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

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.
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http://dx.doi.org/10.2119/molmed.2012.00303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107103PMC
July 2014

Novel treatment for mantle cell lymphoma including therapy-resistant tumor by NF-κB and mTOR dual-targeting approach.

Mol Cancer Ther 2013 Oct 20;12(10):2006-17. Epub 2013 Aug 20.

Corresponding Author: Shantaram S. Joshi, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, 986395 Nebraska Medical Center, Omaha, NE 68198-6395.

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.
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http://dx.doi.org/10.1158/1535-7163.MCT-13-0239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4124828PMC
October 2013

Role of CTLA4 in the proliferation and survival of chronic lymphocytic leukemia.

PLoS One 2013 1;8(8):e70352. Epub 2013 Aug 1.

Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska, USA.

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, (3)H-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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070352PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731360PMC
March 2014

A role for IRF4 in the development of CLL.

Blood 2013 Oct 7;122(16):2848-55. Epub 2013 Aug 7.

Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center, Omaha, NE; and.

Interferon regulatory factor 4 (IRF4) is a critical transcriptional regulator of B-cell development and function. A recent genome-wide single-nucleotide polymorphism (SNP) association study identified IRF4 as a major susceptibility gene in chronic lymphocytic leukemia (CLL). Although the SNPs located in the IRF4 gene were linked to a downregulation of IRF4 in CLL patients, whether a low level of IRF4 is critical for CLL development remains unclear. In rodents, CLL cells are derived from B1 cells whose population is dramatically expanded in immunoglobulin heavy chain Vh11 knock-in mice. We bred a Vh11 knock-in allele into IRF4-deficient mice (IRF4(-/-)Vh11). Here, we report that IRF4(-/-)Vh11 mice develop spontaneous early-onset CLL with 100% penetrance. Further analysis shows that IRF4(-/-)Vh11 CLL cells proliferate predominantly in spleen and express high levels of Mcl-1. IRF4(-/-)Vh11 CLL cells are resistant to apoptosis but reconstitution of IRF4 expression in the IRF4(-/-)Vh11 CLL cells inhibits their survival. Thus, our study demonstrates for the first time a causal relationship between low levels of IRF4 and the development of CLL. Moreover, our findings establish IRF4(-/-)Vh11 mice as a novel mouse model of CLL that not only is valuable for dissecting molecular pathogenesis of CLL but could also be used for therapeutic purposes.
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http://dx.doi.org/10.1182/blood-2013-03-492769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798999PMC
October 2013

Accelerated development of chronic lymphocytic leukemia in New Zealand Black mice expressing a low level of interferon regulatory factor 4.

J Biol Chem 2013 Sep 29;288(37):26430-40. Epub 2013 Jul 29.

From the Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska 68198.

A recent genome-wide SNP association study identified IRF4 as a major susceptibility gene for chronic lymphocytic leukemia (CLL). Moreover, the SNPs located in the 3' UTR of the IRF4 gene have been linked to a down-regulation of IRF4. However, whether a low level of IRF4 is critical for CLL development remains unclear. New Zealand Black (NZB) mice are a naturally occurring, late-onset mouse model of CLL. To examine the role of a reduced level of IRF4 in CLL development, we generated, through breeding, IRF4 heterozygous mutant mice in the NZB background (NZB IRF4(+/-)). Our results show that CLL development is accelerated dramatically in the NZB IRF4(+/-) mice. The average onset of CLL in NZB mice is 12 months, but CLL cells can be detected in NZB IRF4(+/-) mice at 3 months of age. By 5 months of age, 80% of NZB IRF4(+/-) mice developed CLL. CLL cells are derived from B1 cells in mice. Interestingly, NZB IRF4(+/-) B1 cells exhibit prolonged survival, accelerated self-renewal, and defects in differentiation. Although NZB IRF4(+/-) CLL cells are resistant to apoptosis, high levels of IRF4 inhibit their survival. High levels of IRF4 also reduce the survival of MEC-1 human CLL cells. Our analysis further reveals that high levels of IRF4 suppress Akt activity and can do so without the IRF4 DNA binding domain. Thus, our findings reveal a causal relationship between a low level of IRF4 and the development of CLL and establish IRF4 as a novel regulator in the pathogenesis of CLL.
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http://dx.doi.org/10.1074/jbc.M113.475913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772189PMC
September 2013

Amyloid precursor-like protein 2 suppresses irradiation-induced apoptosis in Ewing sarcoma cells and is elevated in immune-evasive Ewing sarcoma cells.

Cancer Biol Ther 2013 Aug 26;14(8):752-60. Epub 2013 Jun 26.

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE USA.

Despite surgery, chemotherapy, and radiotherapy treatments, the children, adolescents, and young adults who are diagnosed with metastasized Ewing sarcoma face a dismal prognosis. Amyloid precursor-like protein 2 (APLP2) has recently been implicated in the survival of cancer cells and in our current study, APLP2's contribution to the survival of Ewing sarcoma cells was examined. APLP2 was readily detected in all Ewing sarcoma cell lines analyzed by western blotting, with the TC71 Ewing sarcoma cells expressing the lowest level of APLP2 among the lines. While irradiation induces apoptosis in TC71 Ewing sarcoma cells (as we determined by quantifying the proportion of cells in the sub-G 1 population), transfection of additional APLP2 into TC71 decreased irradiation-induced apoptosis. Consistent with these findings, in parallel studies, we noted that isolates of the TC71 cell line that survived co-culture with lymphokine-activated killer (LAK) cells (which kill by inducing apoptosis in target cells) displayed increased expression of APLP2, in addition to smaller sub-G 1 cell populations after irradiation. Together, these findings suggest that APLP2 lowers the sensitivity of Ewing sarcoma cells to radiotherapy-induced apoptosis and that APLP2 expression is increased in Ewing sarcoma cells able to survive exposure to cytotoxic immune cells.
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http://dx.doi.org/10.4161/cbt.25183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841215PMC
August 2013

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

Br J Haematol 2012 Jul 10;158(2):216-231. Epub 2012 May 10.

Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA.

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.
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http://dx.doi.org/10.1111/j.1365-2141.2012.09148.xDOI Listing
July 2012

Establishment and characterization of therapy-resistant mantle cell lymphoma cell lines derived from different tissue sites.

Leuk Lymphoma 2012 Nov 13;53(11):2269-78. Epub 2012 Jun 13.

Department of Genetics, University of Nebraska Medical Center, Omaha, NE, USA.

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.
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http://dx.doi.org/10.3109/10428194.2012.691481DOI Listing
November 2012

Novel therapy for therapy-resistant mantle cell lymphoma: multipronged approach with targeting of hedgehog signaling.

Int J Cancer 2012 Dec 21;131(12):2951-60. Epub 2012 May 21.

Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198-6395, USA.

Mantle cell lymphoma (MCL) is one of the most aggressive B-cell lymphomas with a median patient survival of only 5-7 years. The failure of existing therapies is mainly due to disease relapse when therapy-resistant tumor cells remain after chemotherapy. Therefore, development and testing of novel therapeutic strategies to target these therapy-resistant MCL are needed. Here, we developed an in vivo model of therapy-resistant MCL by transplanting a patient-derived MCL cell line (Granta 519) into NOD/SCID mice followed by treatment with combination chemotherapy. Cytomorphologic, immunophenotypic, in vitro and in vivo growth analyses of these therapy-resistant MCL cells confirm their MCL origin and resistance to chemotherapy. Moreover, quantitative real-time PCR revealed the upregulation of GLI transcription factors, which are mediators of the hedgehog signaling pathway, in these therapy-resistant MCL cells. Therefore, we developed an effective therapeutic strategy for resistant MCL by treating the NOD/SCID mice bearing Granta 519 MCL with CHOP chemotherapy to reduce tumor burden combined with GLI-antisense oligonucleotides or bortezomib, a proteosome inhibitor, to target therapy-resistant MCL cells that remained after chemotherapy. This regimen was followed by treatment with MCL-specific cytotoxic T lymphocytes to eliminate all detectable leftover minimal residual disease. Mice treated with this strategy showed a significantly increased survival and decreased tumor burden compared to the mice in all other groups. Such therapeutic strategies that combine chemotherapy with targeted therapy followed by tumor-specific immunotherapy are effective and have excellent potential for clinical application to provide long-term, disease-free survival in MCL patients.
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http://dx.doi.org/10.1002/ijc.27602DOI Listing
December 2012

Optimized adoptive T-cell therapy for the treatment of residual mantle cell lymphoma.

Cancer Immunol Immunother 2012 Oct 23;61(10):1819-32. Epub 2012 Mar 23.

Department of Genetics, Cell Biology and Anatomy, Center for Research in Leukemia and Lymphoma, University of Nebraska Medical Center, Omaha, NE 68198-6395, USA.

Mantle cell lymphoma (MCL) is an aggressive B-cell neoplasm with few patients achieving long-term survival with current treatment regimens. High-dose therapy is effective in reducing the tumor burden; however, patients eventually relapse due to minimal residual disease. Having demonstrated efficacy in other malignancies, the effectiveness of dendritic cell-based immunotherapy for minimal residual MCL was examined. We demonstrated that dendritic cells (DC) primed with MCL antigens stimulated the activation of MCL-specific T cells that recognized and destroyed both MCL cell lines and primary MCL in vitro. In addition, in vivo studies demonstrated that adoptively transferred MCL-specific T cells were able to significantly inhibit tumor growth in mice with minimal residual MCL. Subsequently, when combined with CHOP chemotherapy, adoptive T-cell therapy was able to significantly extend the survival of the mice by further reducing the tumor burden. These results clearly show that MCL-specific cellular immunotherapy is effective in treating minimal residual MCL, paving the way for future clinical studies.
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http://dx.doi.org/10.1007/s00262-012-1229-1DOI Listing
October 2012

IRF4 is a suppressor of c-Myc induced B cell leukemia.

PLoS One 2011 27;6(7):e22628. Epub 2011 Jul 27.

Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

Interferon regulatory factor 4 (IRF4) is a critical transcriptional regulator in B cell development and function. We have previously shown that IRF4, together with IRF8, orchestrates pre-B cell development by limiting pre-B cell expansion and by promoting pre-B cell differentiation. Here, we report that IRF4 suppresses c-Myc induced leukemia in EμMyc mice. Our results show that c-Myc induced leukemia was greatly accelerated in the IRF4 heterozygous mice (IRF4(+/-)Myc); the average age of mortality in the IRF4(+/-)Myc mice was only 7 to 8 weeks but was 20 weeks in the control mice. Our results show that IRF4(+/-)Myc leukemic cells were derived from large pre-B cells and were hyperproliferative and resistant to apoptosis. Further analysis revealed that the majority of IRF4(+/-)Myc leukemic cells inactivated the wild-type IRF4 allele and contained defects in Arf-p53 tumor suppressor pathway. p27(kip) is part of the molecular circuitry that controls pre-B cell expansion. Our results show that expression of p27(kip) was lost in the IRF4(+/-)Myc leukemic cells and reconstitution of IRF4 expression in those cells induced p27(kip) and inhibited their expansion. Thus, IRF4 functions as a classical tumor suppressor to inhibit c-Myc induced B cell leukemia in EμMyc mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022628PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144921PMC
December 2011

Tumor-specific peptide-based vaccines containing the conformationally biased, response-selective C5a agonists EP54 and EP67 protect against aggressive large B cell lymphoma in a syngeneic murine model.

Vaccine 2011 Aug 1;29(35):5904-10. Epub 2011 Jul 1.

Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center, 986395 Nebraska Medical Center, Omaha, NE 68198-6395, United States.

Vaccines to large B cell lymphoma were made by the covalent attachment of an epitope from the gp70 glycoprotein (SSWDFITV) to the N-termini of the conformationally biased, response-selective C5a agonists EP54 (YSFKPMPLaR) and EP67 (YSFKDMP(MeL)aR). Syngeneic Balb/c mice were immunized with these EP54/EP67-containing vaccines and challenged with a lethal dose of the highly liver metastatic and gp70-expressing lymphoma cell line RAW117-H10 to evaluate the ability of these vaccines to induce protective immune outcomes. All mice immunized with SSWDFITVRRYSFKPMPLaR (Vaccine 2) and SSWDFITVRRYSFKDMP(MeL)aR (Vaccine 3) were protected to a lethal challenge of RAW117-H10 lymphoma (>170 days survival) and exhibited no lymphoma infiltration or solid tumor nodules in the liver relative to unvaccinated controls (<18 days survival). Vaccines 2 and 3 contained the protease-sensitive double-Arg (RR) linker sequence between the epitope and the EP54/EP67 moieties in order to provide a site for intracellular proteases to separate the epitope from the EP54/EP67 moieties once internalized by the APC and, consequently, enhance epitope presentation in the context of MHC I/II. These protected mice exhibited an immune outcome consistent with increased involvement of CD8(+) and/or CD4(+) T lymphocytes relative to controls and mice that did not survive or showed low survival rates as with Vaccines 1 and 4, which lacked the RR linker sequence. CD8(+) T lymphocytes activated in response to Vaccines 2 and 3 express cytotoxic specificity for gp70-expressing RAW117-H10 lymphoma cells, but not antigen-irrelevant MDA-MB231A human breast cancer cells. Results are discussed against the backdrop of the ability of EP54/EP67 to selectively target antigens to and activate C5a receptor-bearing antigen presenting cells and the prospects of using such vaccines therapeutically against lymphoma and other cancers.
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http://dx.doi.org/10.1016/j.vaccine.2011.06.070DOI Listing
August 2011