T-cell Lymphoma Cell Research Articles

PIM1 overexpression in T-cell lymphomas protects tumor cells from apoptosis and confers doxorubicin resistance by upregulating c-myc expression.

T-cell lymphomas (TCLs) are a malignancy characterized by tumor aggression and resistance to traditional chemotherapy. Disruption of the extrinsic cell death pathway is essential for resistance to chemotherapy. PIM1 serves as a crucial modulator in cancers. However, the role of PIM1 in TCLs remains unclear. In this study, we studied the roles of PIM1 in established T-lymphoma cell lines Jurkat and HUT-78. CCK-8 assay was conducted to evaluate cell survival and flow cytometry was performed to evaluate cell death of TCL cells. siRNAs were used to knockdown the expression of PIM1 and c-myc. qRT-PCR was used to evaluate the mRNA expression levels of c-myc and PIM1. Western blot analysis was used to evaluate the protein expression levels of PIM1, c-myc, STAT3, and phospho-STAT3. Doxorubicin was used to determine the effect of PIM1 on apoptosis. Our results showed that PIM1 expression was markedly enhanced and induced c-myc expression in TCL cells. Doxorubicin inhibited the expressions of c-myc and PIM1, and triggered the extrinsic cell death of TCLs by suppressing the JAK-STAT3 signaling pathway. Moreover, PIM1 silencing via siRNA suppressed c-myc expression, promoted the cell death of TCLs, and increased doxorubicin sensitivity. Conversely, PIM1 overexpression in TCL cells induced c-myc expression, suppressed TCL cell death, and promoted doxorubicin resistance. Collectively, our results demonstrate that PIM1 overexpression in TCLs participates in cancer cell protection from apoptosis and leads to doxorubicin resistance by inducing c-myc expression, indicating that PIM1 may be a promising target in TCL treatment.

Abstract 2557: Assessing the therapeutic efficacy of disease-specific T-cell biofactories

Abstract Current protein-based cancer therapies have several disadvantages, which include general toxicity, lack of response at the administered dose and differing responses from patients to patient. To circumvent these issues, we propose a platform in which engineered T-cells specifically recognize tumors and secrete therapeutic peptides directly at the disease site leading to targeted cancer cell killing. This approach will increase the specificity of the therapy and decrease toxicity to healthy cells. We transformed acute T-cell lymphoma cells into biofactories for site-specific synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. The effector T-cell line was engineered by stably introducing a chimeric T-cell receptor to recognize Folate-Receptor alpha (FRα) or Mesothelin (MSLN) protein. Upon binding of the effector T-cells to the receptor, an intracellular cascade directed expression of non-human proteins is induced. Specific T-cell binding to human ovarian cancer cell lines and signaling was measured by in vitro co-culture using luciferase production as a surrogate for therapeutic peptide secretion. We demonstrated that T-cells can be genetically programed to synthesize and secrete proportionate amounts of engineered proteins upon engaging the tumor-associated antigens (FRα or MSLN) on a human ovarian cancer cell line, OVCAR3 (FRα+MSLN+). A FRα-MSLN- ovarian cancer cell line, A2780cis, was used as the non-targeted negative control. The difference in protein secretion following stimulation by the two cell lines, as measured by luciferase activity, was statistically significant within 1 hour. It reached ~35-fold within 1 to 3 days, and we observed stable expression for at least 10 days. The luminescent signal was proportionate to the number of OVCAR3 cells. To further validate the specificity of target engagement, we generated A2780cis-FRα positive and A2780cis-MSLN positive cell lines and demonstrated selective binding and activation of the corresponding effector cells in co-culture assays. No binding was detected to the A2780cis-vector control cells. In vivo results for T-cell biofactories targeting OVCAR3 tumors 24 hours post-stimulation validated the in vitro results. Currently, we are engineering T-cell biofactories to release cytotoxic peptides and are assessing their therapeutic efficacy against cancer cells in vitro using co-culture assays and supernatant transfer. Our results show that T-cells can be genetically reprogrammed to serve as biofactories for the synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. Importantly, these studies demonstrate the feasibility of developing the next generation of adoptively transferred T-cell therapies to target tumors that express FRα (e.g., ovarian, breast, lung) and/or MSLN (e.g. ovarian, lung, pancreatic) on their cell surfaces for cancer therapy. Citation Format: Claire E. Repellin, Puja Patel, Lucia Beviglia, Harold Javitz, Lidia C. Sambucetti, Parijat Bhatnagar. Assessing the therapeutic efficacy of disease-specific T-cell biofactories [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2557.

Abstract 798: Pathway-directed high-throughput drug screen identifies PI3K inhibitors that synergistically potentiate antitumor activity of HDAC inhibitors in cutaneous T-cell lymphoma

Abstract Background: Cutaneous T-cell lymphoma (CTCL) represents a type of malignancy in the skin that is incurable. Recent studies demonstrated dysregulation of several signaling pathways in CTCL, including PI3K/AKT, JAK/STAT, and NFκB pathways. We performed a high-throughput drug screen to determine the potential of novel agents targeting these pathways for the treatment of CTCL. Methods: We compiled a library of 94 compounds targeting pathways known to be relevant in cancer biology, such as kinases involved in growth factor receptor signaling, HDACs, proteasome, DNA repair and regulators of apoptosis. The compounds were screened for antiproliferative activity against four CTCL cell lines in high-throughput proliferation assays. PI3K inhibitors were selected from the “hits” of the screen and were further studied in xenograft models of CTCL and in primary T-cell lymphomas cells from patients. To examine which PI3K isoform mediates antitumor effect in CTCL, isoform-specific siRNAs were used to knock down each isoforms as well as the combination of doublet isoforms. Based on this experiment, we tested antitumor activity of isoform-specific inhibitors. Promising candidates were also tested in combination assays using a matrix block method designed to detect synergistic activities. Results: From the high-throughput screen, we identified 14 compounds with antiproliferative activity in CTCL, including multiple inhibitors of the PI3K pathway. Validation assays confirmed the class effect of PI3K inhibitors in growth inhibition. From this class, a pan-PI3K inhibitor BKM120 was selected for in vivo studies. In a xenograft model of CTCL, BKM120 exhibited striking antitumor activity measured by a marked suppression of tumor growth and prolonged survival of tumor-bearing mice compared with vehicle control. To investigate which isoform of the PI3K mediates the antitumor effect in CTCL, we performed isoform-specific siRNA assays and demonstrated that knocking down isoform ð exhibited the most potent growth inhibition. Copanlisib targets α and δ isoforms of PI3K and is newly approved by the FDA for low-grade B-cell lymphoma. We demonstrated that copanlisib exhibits potent antiproliferation activity in CTCL cell lines. Furthermore, combination of copanlisib and LBH, a HDAC inhibitor, display synergistic antiproliferative effects across a broad dose gradient for each agent. Conclusion: A pan-PI3k inhibitor, BKM120, is highly active in CTCL cell lines and exnograft models. Inhibition of Isoform δ exhibits the most potent growth inhibition. Furthermore, an isoform-specific inhibitor, copanlisib, synergistically potentiates the effect of HDAC inhibitors against CTCL. These are highly promising approaches for the treatment of CTCL and warrant clinical investigation. Citation Format: Chi-Heng Wu, Chen-Yen Yang, Taha Rakhshandhroo, Laura Pincus, Sourav Bandyopadhyay, Frank McCormick, Weiyun Ai. Pathway-directed high-throughput drug screen identifies PI3K inhibitors that synergistically potentiate antitumor activity of HDAC inhibitors in cutaneous T-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 798.

Genome-Wide Analysis of Interchromosomal Interaction Probabilities Reveals Chained Translocations and Overrepresentation of Translocation Breakpoints in Genes in a Cutaneous T-Cell Lymphoma Cell Line.

In classical models of tumorigenesis, the accumulation of tumor promoting chromosomal aberrations is described as a gradual process. Next-generation sequencing-based methods have recently revealed complex patterns of chromosomal aberrations, which are beyond explanation by these classical models of karyotypic evolution of tumor genomes. Thus, the term chromothripsis has been introduced to describe a phenomenon, where temporarily and spatially confined genomic instability results in dramatic chromosomal rearrangements limited to segments of one or a few chromosomes. Simultaneously arising and misrepaired DNA double-strand breaks are also the cause of another phenomenon called chromoplexy, which is characterized by the presence of chained translocations and interlinking deletion bridges involving several chromosomes. In this study, we demonstrate the genome-wide identification of chromosomal translocations based on the analysis of translocation-associated changes in spatial proximities of chromosome territories on the example of the cutaneous T-cell lymphoma cell line Se-Ax. We have used alterations of intra- and interchromosomal interaction probabilities as detected by genome-wide chromosome conformation capture (Hi-C) to infer the presence of translocations and to fine-map their breakpoints. The outcome of this analysis was subsequently compared to datasets on DNA copy number alterations and gene expression. The presence of chained translocations within the Se-Ax genome, partly connected by intervening deletion bridges, indicates a role of chromoplexy in the etiology of this cutaneous T-cell lymphoma. Notably, translocation breakpoints were significantly overrepresented in genes, which highlight gene-associated biological processes like transcription or other gene characteristics as a possible cause of the observed complex rearrangements. Given the relevance of chromosomal aberrations for basic and translational research, genome-wide high-resolution analysis of structural chromosomal aberrations will gain increasing importance.

T-cell lymphomas associated gene expression signature: Bioinformatics analysis based on gene expression Omnibus.

Due to the low incidence and the heterogeneity of subtypes, the biological process of T-cell lymphomas is largely unknown. Although many genes have been detected in T-cell lymphomas, the role of these genes in biological process of T-cell lymphomas was not further analyzed. Two qualified datasets were downloaded from Gene Expression Omnibus database. The biological functions of differentially expressed genes were evaluated by gene ontology enrichment and KEGG pathway analysis. The network for intersection genes was constructed by the cytoscape v3.0 software. Kaplan-Meier survival curves and log-rank test were employed to assess the association between differentially expressed genes and clinical characters. The intersection mRNAs were proved to be associated with fundamental processes of T-cell lymphoma cells. These intersection mRNAs were involved in the activation of some cancer-related pathways, including PI3K/AKT, Ras, JAK-STAT, and NF-kappa B signaling pathway. PDGFRA, CXCL12, and CCL19 were the most significant central genes in the signal-net analysis. The results of survival analysis are not entirely credible. Our findings uncovered aberrantly expressed genes and a complex RNA signal network in T-cell lymphomas and indicated cancer-related pathways involved in disease initiation and progression, providing a new insight for biotargeted therapy in T-cell lymphomas.

Characteristics and Diagnostic Values of Bone Marrow Cell Morphology and Immunophenotyping in Lymphoma Cell Leukemia

To explore the characteristics and diagnostic values of bone marrow cell morphology and immunophenotyping in lymphoma cell leukemia patients. Data of the bone marrow cell morphology and immunophenotyping of 35 patients with lymphoma cell leukemia admitted from January 2012 to January 2017 were analyzed retrospectively, and the value of bone marrow cell morphology and immunophenotype in the diagnosis of lymphoma cell leukemia was evaluated. Bone marrow cell morphological examination showed the typical lymphoma cells in all the patients. The expression of differentiation antigens in lymphoma cell leukemia was consistent with that of original pathological diagnosis. In T-cell lymphoma cell leukemia, the expression of CD7, CD3, CD2, CD5, CD11b, CD34, and HLA-DR were present predominantly, among them the CD7 was the most sensitive antigen and its positive expression rate was 69.2%. In B-cell lymphoma cell leukemia, the expression of CD19, CD20, CD22, CD79a, Skappa, and early antigen HLA-DR were observed predominantly, among them the positive expression rate of CD19 was the highest (89.5%). Out of 35 cases, 28 cases showed that the percentage of lymphoma cells on bone marrow smears was consistent with that of bone marrow immunophenotyping, and 7 cases showed that the percentage of lymphoma cells between bone marrow smears and immunophenotyping differed by more than 1.5-fold. Bone marrow slides combined with immunophenotyping may be helpful for judging lymphoma cell marrow invasion and making early diagnosis of lymphoma cell leukemia.

Clinical and misdiagnosed analysis of primary pulmonary lymphoma: a retrospective study

BackgroundThe primary pulmonary lymphoma (PPL), with a low incidence, was highly misdiagnosed in clinic. The present study analyzes the clinical features, laboratory and imaging data, pathologic characteristics, and summarizes misdiagnosis reasons of PPL cases, aims to provide a better understanding and increase the accuracy of early diagnosis and minimize the misdiagnosis of PPL.MethodsThe clinical data of 19 cases were collected from the first affiliated hospital of Wenzhou medical university (PRC) from April 2010 to May 2016. All cases were confirmed by pathology. The process of misdiagnosis was described. This study retrospectively analyzed the incidence, clinical presentation, laboratory examination, Chest CT scan and diagnosis of the cases.ResultsThe symptoms of the 19 cases were dyspnea, fever, hemoptysis, chest pain or physical findings without obvious symptoms. Five patients were pneumonia-like, nine patients had lung single nodule or mass and four patients got pleural effusion, which were reported by computed tomography (HRCT) scan. There were 2 cases of Hodgkin lymphoma (HL), and 17 cases of non-Hodgkin lymphoma (NHL). In NHL cases, 12 cases were confirmed mucosa associated lymphoid tissue B lymphoma type, 3 cases were confirmed diffuse large B-cell lymphoma, angioimmunoblastic T-cell lymphoma and ALK positive anaplastic large cell lymphoma were one case separately. Clinical and imaging manifestation of PPL is untypical, but there are still some hints: 1) Fuzzy shadow at the edge of lung mass with air bronchogram; 2) Lung mass shadow stable for a long time; 3) Pneumonia-like changing without infections clinical and lab manifestation. Thirteen patients (68.4%) were misdiagnosed as pneumonia, lung cancer and tuberculosis initially. The term between initial diagnosis and final diagnosis lasted for half a month up to 2 years, with median time of 6 months. Two cases were misdiagnosed as tuberculosis. One case was misdiagnosed as small cell lung cancer.ConclusionClinical and imaging manifestation of PPL is untypical. Biopsies should be taken actively if the imaging findings don’t match the symptoms or the anti-infection treatments to “lung infection” don’t work. Accurate diagnosis requires adequate tissue sampling with appropriate ancillary pathologic studies. If clinical manifestation and the diagnosis don’t match, repeated biopsy should be ordered.

Checkpoint molecule expression by B and T cell lymphomas in dogs.

Immunotherapies targeting checkpoint molecule programmed cell death 1 (PD-1) protein were shown to be effective for treatment of non-Hodgkin lymphoma in people, but little is known about the expression of PD-1 or its ligand PD-L1 by canine lymphoma. Therefore, flow cytometry was used to analyse expression of PD-1 and PD-L1 in canine lymphoma, using fine-needle aspirates of lymph nodes from 34 dogs with B cell lymphoma (BCL), 6 dogs with T cell lymphoma (TCL) and 11 dogs that had relapsed. Furthermore, fine-needle aspirates were obtained from 17 healthy dogs for comparison. Lastly, the impact of chemotherapy resistance on expression of PD-1 and PD-L1 was assessed in vitro. These studies revealed increased expression of PD-L1 by malignant B cells compared to normal B cells. In the case of TCL, tumour cells and normal T cells both showed low to negative expression of PD-1 and PD-L1. In addition, tumour infiltrating lymphocytes from both BCL and TCL had increased expression of both PD-1 and PD-L1 expression compared to B and T cells from lymph nodes of healthy animals. In vitro, chemotherapy-resistant BCL and TCL cell lines exhibited increases in both PD-1 and PD-L1 expression, compared to non-chemotherapy selected tumour cells. These findings indicate that canine lymphomas exhibit upregulated checkpoint molecule expression, though the impact of checkpoint molecule expression on tumour biological behaviour remains unclear.

Effect of dihydroarteminin combined with siRNA targeting Notch1 on Notch1/c-Myc signaling in T-cell lymphoma cells.

The effectiveness of therapy combining dihydroartemisinin (DHA) and small interfering RNA targeting Notch1 (siNotch1) in T-cell lymphoma remains unknown. The present study explored the potential and possible mechanisms of combined dihydroarteminin, and siNotch1 therapy for T-cell lymphoma. It was demonstrated that the viability rates of siRNA-DHA-treated cells was significantly suppressed in comparison with those in control cells, control siRNA cells, siRNA-treated cells and DHA-treated cells (P<0.01). Additionally, there was a significant increase in cell apoptosis of siRNA-DHA-treated cells in comparison with those of control cells, control siRNA cells, siRNA-treated cells, DHA-treated cells (P<0.05). Furthermore, Notch1 and c-Myc mRNA and protein expression were decreased in siRNA-DHA-treated cells (P<0.05). The present study demonstrated that DHA combined with siNotch1 is able to suppress proliferation and promote apoptosis, and downregulate the expression of Notch1 and c-Myc mRNA and protein in T-cell lymphoma cells. Targeting Notch1/c-Myc signaling with siRNA and DHA may represent a novel strategy for treating human T-cell lymphoma.

Expression and clinical significance of centrosomal protein 55 in T-cell lymphoma.

T-cell lymphomas (TCLs) have been heterogeneous lymphoid malignancies with aggressive clinical phenotype and poor prognosis. Centrosomal protein 55 (CEP55) played a critical role in cytokinesis and served as a centrosome- and midbody-associated protein. Previous studies have reported the overexpression and clinical significance of CEP55 in various human malignancies, but the exact biological roles of CEP55 in TCLs remained unclear. In this study, we aimed to evaluate the CEP55 expression in patients with TCL and reactive hyperplasia of lymph nodes. The correlation between CEP55 levels and clinical characteristics was also explored for TCL patients. For further investigation, the cell viability of TCL cell lines after CEP55 inhibition was also assessed. Immunohistochemistry was applied to assess the elevated level of CEP55 in TCLs. After siRNA treatment, cell viability and apoptotic rate of TCL cell lines were observed with CCK-8 assay and flow cytometry, respectively. The Pearson's Chi-square test or Fisher's exact test was applied to analyze the correlations between CEP55 overexpression and clinical characteristics. All statistical tests were two-sided, and P < 0.05 was considered to be statistically significant. CEP55 was upregulated in TCL patients and significantly correlated with Ki-67 label index. Consistently, cell viability was decreased, and apoptosis was increased after the suppression of CEP55 in TCL cell lines. These results suggested that target CEP55 would be a novel therapeutic strategy for the TCL.

Dual Role of EZH2 in Cutaneous Anaplastic Large Cell Lymphoma: Promoting Tumor Cell Survival and Regulating Tumor Microenvironment

Primary cutaneous anaplastic T-cell lymphoma, characterized by the CD30+ anaplastic large T cells, comprises the second most common group of cutaneous T-cell lymphoma. Little is known about the mechanisms of disease progression. Here we report that enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 that mediates histone H3 lysine 27 trimethylation, is overexpressed in CD30+ anaplastic cells in primary cutaneous anaplastic T-cell lymphoma and large-cell transformed cutaneous T-cell lymphoma. Silencing EZH2 or inhibiting its histone methyltransferase activity conferred increased apoptosis and G1 cell-cycle arrest in primary cutaneous anaplastic T-cell lymphoma cells invitro and a xenograft model invivo. This was mediated by the de-repression of thioredoxin-interacting protein, a major redox control molecule, and consequent formation of reactive oxygen species. Silencing thioredoxin-interacting protein abrogated reactive oxygen species accumulation in EZH2 suppressed cells and rescued cell growth disadvantage. Moreover, EZH2 suppression de-repressed C-X-C motif chemokine ligand 10 and facilitated the recruitment of effector CD4+ and CD8+ T cells into the tumor microenvironment via a C-X-C motif chemokine ligand 10/receptor 3 interaction. These results demonstrate a dual role for polycomb repressive complex 2-mediated epigenetic silencing in tumor progression and antitumor immunity in primary cutaneous anaplastic T-cell lymphoma, and provide a rationale for the pharmacologic inhibition of EZH2 activity in large-cell transformed cutaneous T-cell lymphoma.

Histone Deacetylase Inhibitors Downregulate CCR4 Expression and Decrease Mogamulizumab Efficacy in CCR4-Positive Mature T-Cell Lymphomas

Background: Histone deacetylase inhibitors (HDACis) are promising agents for various T-cell lymphomas, including cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and adult T-cell lymphoma/leukemia (ATLL). CCR4 is an important therapeutic target molecule because mogamulizumab, an anti-CCR4 antibody, has shown promising efficacy against CTCL, PTCL, and ATLL. However, their combined effects and interactions have not been examined thus far. We previously showed that CCR6, a chemokine receptor, is overexpressed in cutaneous T-cell lymphomas (Ito et al., 2014 Blood). Moreover, we recently demonstrated that HDACis downregulate CCR6 expression in advanced cutaneous T-cell lymphomas (Abe et al., 2017 Oncotarget). These reports lead us to hypothesize that HDACis might also downregulate CCR4 in various T-cell lymphomas. In this study, we clarify the effect of the combined use of mogamulizumab and HDACis on various T-cell and NK-cell lymphomas. Based on our findings, we discuss what benefits or adverse effects might be assumed for patients if these molecular targeting agents are used in clinical practice.Methods: We evaluated changes in CCR4 expression and antibody-dependent cell-mediated cytotoxicity (ADCC) activities against mogamulizumab- and HDACi-treated T-cell and NK-cell lymphoma lines and primary cases. To determine which HDAC mainly regulated CCR4 expression, we used isoform-specific HDACis and induced knockdown of respective HDACs for T-cell lymphoma cell lines. To examine the effect of CCR4 downregulation by HDACis in clinical cases, we examined the CCR4 expression of CTCL skin samples, which were obtained from the same patients before and after HDACi treatment (n = 6).Results: We first examined the expression of CCR4 for 15 T-cell and NK-cell lymphoma cell lines and a peripheral blood mononuclear cell (PBMC) sample derived from healthy donors to investigate the effect of vorinostat, a pan-HDACi, on CCR4 expression. The expression of CCR4 was mostly expressed in the (11 out of 15) cell lines: ATLL (MT-1, MT-2, MT-4, and TL-Su), CTCL (My-La, HH, and MJ), and NK/T-cell lymphoma cell lines (Kai3, SNK6, HANK1, and SNK10). We found that vorinostat decreases mRNA expression and surface expression of CCR4 except for the cell lines without CCR4 expression. Next, we used isoform-specific HDACis to examine which isoform of HDAC is involved in the regulation of CCR4. We used the following class-specific HDACis: romidepsin as a class I selective HDACi, CI-994 as an HDAC1/HDAC2-selective inhibitor, RGFP966 as an HDAC3-selective inhibitor, ricolinostat as an HDAC6-selective inhibitor, and PCI-34051 as an HDAC8-selective inhibitor. When these drugs were exposed to T-cell lymphoma cells, romidepsin and CI-994 strongly suppressed CCR4 expression. These results suggest that class I HDACs might controls CCR4 expression. We further performed knockdown experiments using siRNAs against HDAC1, HDAC2, and HDAC3. When we compared the expression change of CCR4 in HDAC-knockdown cells, HDAC2 knockdown cells showed the most significantly decreased expression of CCR4. These results suggest that class I HDACs, especially HDAC2, might be deeply involved in CCR4 expression regulation. When we examined the CCR4 expression in skin samples from primary CTCL, obtained from the same patients before and after vorinostat treatment, we found that CCR4 expression was greatly reduced after vorinostat treatment. Finally, when we conducted an ADCC assay with mogamulizumab by using various lymphoma cell lines and primary T-cell lymphoma samples, we found that the efficacy of mogamulizumab was significantly reduced by pre-treatment with vorinostat.Conclusion: Our results suggest that the primary use of HDACis before treatment of mogamulizumab might not be suitable to obtain synergistic effects. Moreover, these results provide potential implications for optimal therapeutic sequences in various CCR4 positive T-cell and NK-cell lymphomas. DisclosuresKitadate:Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Eisai: Research Funding; Otsuka: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Asahi Kasei: Research Funding; Chugai: Research Funding; Toyama kagaku: Research Funding. Abe:Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Toyama Kagaku: Research Funding; Chugai: Research Funding; Asahi Kasei: Research Funding; Eisai: Research Funding. Tagawa:TaNeDS (Daiichi Sankyo): Research Funding.

In Vitro, In Vivo, and Parallel Phase I Evidence Support the Safety and Activity of Duvelisib, a PI3K-δ,γ Inhibitor, in Combination with Romidepsin or Bortezomib in Relapsed/Refractory T-Cell Lymphoma

In Vitro, In Vivo, and Parallel Phase I Evidence Support the Safety and Activity of Duvelisib, a PI3K-δ,γ Inhibitor, in Combination with Romidepsin or Bortezomib in Relapsed/Refractory T-Cell Lymphoma

Identification of autofluorescent cells in human angioimmunoblastic T-cell lymphoma.

Endogenous cell autofluorescence is a common nuisance that complicates the use of fluorescence microscopy. When using fluorescence-labeled antibodies for specific cell labeling in tissue sections of human angioimmunoblastic T-cell lymphoma (AITL), we encountered with a problematic autofluorescence of multiple cells. These cells emitted fluorescence signals in the green, red and deep-red spectral range. Characterization of these autofluorescent cells solely on the basis of their autofluorescence failed. To identify these enigmatic cells residing the lymphoma tissue, we combined two imaging techniques-fluorescence and brightfield microscopy. Combined fluorescence/brightfield imaging of cells immunolabeled with a panel of CD antibodies raised against diverse cellular components allowed us to identify the autofluorescent cells in the AITL as eosinophils. These cells tended to accumulate in the vicinity of capillaries and arterioles apparently mediating the process of angiogenesis resembling other angiogenesis-associated diseases.

Targeting T-cell malignancies using anti-CD4 CAR NK-92 cells.

Peripheral T-cell lymphomas (PTCLs) are a group of very aggressive non-Hodgkin's lymphomas (NHLs) with poor prognoses and account for a majority of T-cell malignancies. Overall, the standard of care for patients with T-cell malignancies is poorly established, and there is an urgent clinical need for a new approach. As demonstrated in B-cell malignancies, chimeric antigen receptor (CAR) immunotherapy provides great hope as a curative treatment regimen. Because PTCLs develop from mature T-cells, these NHLs are commonly CD4+, and CD4 is highly and uniformly expressed. Therefore, CD4 is an ideal target for PTCL CAR immunotherapy. To that effect, we created a robust third-generation anti-CD4 CAR construct (CD4CAR) and introduced it into clonal NK cells (NK-92). CD4CAR NK-92 cells specifically and robustly eliminated diverse CD4+ human T-cell leukemia and lymphoma cell lines (KARPAS-299, CCRF-CEM, and HL60) and patient samples ex vivo. Furthermore, CD4CAR NK-92 cells effectively targeted KARPAS-299 cells in vivo that modeled difficult-to-access lymphoma nodules, significantly prolonging survival. In our study, we present novel targeting of CD4 using CAR-modified NK cells, and demonstrate efficacy. Combined, our data support CD4CAR NK cell immunotherapy as a potential new avenue for the treatment of PTCLs and CD4+ T-cell malignancies.

Isolation of Chlorogenic Acid from Soil Borne Fungi Screlotium rolfsii, their Reversal of Multidrug Resistance and Anti-proliferative in Mouse Lymphoma Cells.

Fungi performing a wide range of function in soil by secreting low molecular weight compound known as secondary metabolites. S. rolfsii is a soil borne phytopathogenic fungi was used for the production of bioactive compounds. The present study belongs to evaluate the anticancer potentials of a secondary metabolites isolated from S. rolfsii, their multidrug resistance (MDR), and molecular docking study. (1S,3R,4R,5R,E)-3-(3-(3,4-Dihydroxyphenyl)acryloyloxy)-1,4,5 trihydroxycyclohexanecarboxylic acid (1), or best known as chlorogenic acid, was isolated from the ethyl acetate fraction of crude secondary metabolites produced by the soil borne Fungus Screlotium rolfsii. Structure of chlorogenic acid (1) was confirmed by means of FT-IR, 1H NMR, 13C NMR, and mass spectrometry as well as by melting point. Effect of compound 1 on the reversion of multidrug resistant (MDR) mediated by Pglycoprotein (P-gp) against cancer cells was evaluated with a rhodamine-123 exclusion screening test on human mdr1 gene transfected mouse gene transfected L5178 and L5178Y mouse T-cell lymphoma. Compound 1 was also evaluated for Anti-proliferative effect on the L5178 mouse Tcell lymphoma cell line. Results from the present investigation revealed that compound 1 exhibits excellent MDR reversing effect in a dose-dependent manner against mouse T-lymphoma cell line. Compound 1 also showed anti-proliferative effect on L5178Y mouse T-lymphoma cell line.

Involvement of oncogenic tyrosine kinase NPM-ALK in trifluoperazine-induced cell cycle arrest and apoptosis in ALK+ anaplastic large cell lymphoma

ABSTRACTObjectives: Trifluoperazine (TFP) has potential anticancer activity that was demonstrated in different types of cancer cells. However, little is known about its effects in the T-cell ALK+ anaplastic large cell lymphoma (ALCL). In this study, we investigated the effects of TFP in this aggressive type of cancer.Methods: The cytotoxicities of TFP on DEL and SUP-M2 cells were measured by trypan blue staining. The effects of TFP on cell cycle and apoptosis in DEL and SUP-M2 cells were determined by flow cytometry. The underlying anticancer mechanism of TFP was investigated by real-time PCR, Western Blotting, and nucleophosmin-ALK (NPM-ALK) tyrosine kinase assay in DEL cells.Results: Our results show that TFP did not significantly affect cell viability of human normal lymphocytes, whereas it reduced the cell viability of the ALK+ T-cell lymphoma cells, DEL and SUP-M2, in concentration- and time-dependent manners. TFP also induced cell cycle arrest at G0/G1 phase, and caused cell apoptosis in these cells. These effects could be explained by the involvement of NPM-ALK and its downstream cell survival regulatory proteins.Conclusions: Our results demonstrate for the first time that TFP is capable of inducing degradation and inhibition of kinase activity of NPM-ALK, as well as the increased level of phospho-NPM-ALK, leading to the inhibition of cell growth in ALK+ ALCL cells. TFP may have a potential to be utilized to treat this aggressive lymphoma.

Histone deacetylase inhibitors downregulate CCR4 expression and decrease mogamulizumab efficacy in CCR4-positive mature T-cell lymphomas.

Histone deacetylase inhibitors are promising agents for various T-cell lymphomas, including cutaneous T-cell lymphoma, peripheral T-cell lymphoma, and adult T-cell lymphoma/leukemia. CCR4 is an important therapeutic target molecule because mogamulizumab, an anti-CCR4 antibody, has shown promising efficacy against various T-cell lymphomas. In this study, we examined the in vitro synergistic effects of mogamulizumab and histone deacetylase inhibitors against various T-cell lymphomas. First, we examined the expression of CCR4 mRNA and surface CCR4 in various T-cell lymphoma cell lines and found that it was downregulated upon treatment with vorinostat, a pan-histone deacetylase inhibitor. Next, we used isoform-specific histone deacetylase inhibitors and short-interfering RNA to determine the histone deacetylase isoform involved in the regulation of CCR4, and demonstrated that romidepsin, a class I selective histone deacetylase inhibitor, reduced CCR4 most efficiently. Moreover, among class I histone deacetylases, histone deacetylase 2 knockdown led to a reduction of CCR4 in lymphoma cells, suggesting that CCR4 expression is mainly regulated by histone deacetylase 2. When we examined the CCR4 expression in skin samples from primary cutaneous T-cell lymphoma, obtained from the same patients before and after vorinostat treatment, we found that CCR4 expression was greatly reduced after treatment. Finally, when we conducted an antibody-dependent cell-mediated cytotoxicity assay with mogamulizumab by using various lymphoma cells, we found that the efficacy of mogamulizumab was significantly reduced by pretreatment with vorinostat. Altogether, our results suggest that the primary use of histone deacetylase inhibitors before treatment with mogamulizumab might not be suitable to obtain synergistic effects. Moreover, these results have potential implications for optimal therapeutic sequences in various CCR4-positive T-cell lymphomas.

522 Blocking CD137-CD137L interactions inhibits proliferation and survival of cutaneous T-cell lymphoma cells via hampering several signaling pathways

522 Blocking CD137-CD137L interactions inhibits proliferation and survival of cutaneous T-cell lymphoma cells via hampering several signaling pathways

AN-7, a butyric acid prodrug, sensitizes cutaneous T-cell lymphoma cell lines to doxorubicin via inhibition of DNA double strand breaks repair.

We previously found that the novel histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) had greater selectivity against cutaneous T-cell lymphoma (CTCL) than SAHA. AN-7 synergizes with doxorubicin (Dox), an anthracycline antibiotic that induces DNA breaks. This study aimed to elucidate the mechanism underlying the effect of AN-7 on Dox-induced double-strand DNA breaks (DSBs) in CTCL, MyLa and Hut78 cell lines. The following markers/assays were employed: comet assay; western blot of γH2AX and p-KAP1; immunofluorescence of γH2AX nuclear foci; Western blot of repair protein; quantification of DSBs-repair through homologous recombination. DSB induction by Dox was evidenced by an increase in DSB markers, and DSBs-repair, by their subsequent decrease. The addition of AN-7 slightly increased Dox induction of DSBs in MyLa cells with no effect in Hut78 cells. AN-7 inhibited the repair of Dox-induced DSBs, with a more robust effect in Hut78. Treatment with AN-7 followed by Dox reduced the expression of DSB-repair proteins, with direct interference of AN-7 with the homologous recombination repair. AN-7 sensitizes CTCL cell lines to Dox, and when combined with Dox, sustains unrepaired DSBs by suppressing repair protein expression. Our data provide a mechanistic rationale for combining AN-7 with Dox or other DSB inducers as a therapeutic modality in CTCL.