Cell Lymphoma Progression Research Articles

TET2 regulates extranodal NK/T cell lymphoma progression through regulation of DNA methylation.

The biological role of Ten-11 translocation 2 (TET2) and the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in the development of extra-nodal natural killer/T-cell lymphoma (ENKTL) remains unclear. The level of 5mC and 5hmC was detected in 112 cases of ENKTL tissue specimens by immunohistochemical (IHC) staining. Subsequently, TET2 knockdown and the overexpression cell models were constructed in ENKTL cell lines. Biochemical analyses were used to assess proliferation, apoptosis, cell cycle and monoclonal formation in cells treated or untreated with L-Ascorbic acid sodium salt (LAASS). Dot-Blots were used to detect levels of genome 5mC and 5hmC. Additionally, the ILLUMINA 850k methylation chip was used to analyze the changes of TET2 regulatory genes. RNA-Seq was used to profile differentially expressed genes regulated by TET2. The global level of 5hmC was significantly decreased, while 5mC was highly expressed in ENKTL tissue. TET2 protein expression was negatively correlated with the ratio of 5mC/5hmC (p<0.0001). The 5mC/5hmC status were related to the site of disease, clinical stage, PINK score and Ki-67 index, as well as the 5-year OS. TET2 knockdown prolonged the DNA synthesis period, increased the cloning ability of tumor cells, increased the level of 5mC and decreased the level of 5hmC in ENKTL cells. While overexpression of TET2 presented the opposite effect. Furthermore, treatment of ENKTL cells with LAASS significantly induced ENKTL cell apoptosis. These results suggest that TET2 plays an important role in ENKTL development via regulation of 5mC and 5hmC and may serve as a novel therapeutic target for ENKTL.

Retraction: MiR-525-5p inhibits diffuse large B cell lymphoma progression via the Myd88 /NF-κB signaling pathway

Retraction: MiR-525-5p inhibits diffuse large B cell lymphoma progression via the Myd88 /NF-κB signaling pathway

STAT5B Suppresses Ferroptosis by Promoting DCAF13 Transcription to Regulate p53/xCT Pathway to Promote Mantle Cell Lymphoma Progression.

The purpose of this study was to analyze the mechanism by which STAT5B inhibits ferroptosis in mantle cell lymphoma (MCL) by promoting DCAF13 transcriptional regulation of p53/xCT pathway. The correlations between STAT5B, DCAF13 and ferroptosis in MCL were analyzed using Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/index.html). The expression levels and pairwise correlations of STAT5B, DCAF13, p53 and xCT in MCL patients were detected, respectively. STAT5B was silenced to confirm their criticality in MCL ferroptosis. the effects of blocking necrosis, apoptosis and ferroptosis on the anti-MCL effects of STAT5B were examined. Cells with STAT5B overexpression and/or DCAF13 silencing were constructed to confirm the involvement of DCAF13 in the STAT5B-regulated p53/xCT pathway. The regulation of p53 ubiquitination was confirmed by DCAF13 overexpression and MG132. The effects of silencing DCAF13 and MG132 on STAT5B overexpression on MCL was clarified by a tumor-bearing nude mouse model. DCAF13 was overexpressed in MCL and positively correlated with STAT5B, negatively correlated with p53, and positively correlated with xCT. Inhibition of ferroptosis alleviated the inhibitory effects of siSTAT5B on MCL, while inhibition of necrosis and apoptosis had few effects. Silencing of DCAF13 led to the blocking of STAT5B regulation of p53/xCT and ferroptosis. The changes in DCAF13 and the addition of MG132 did not have statistically significant effects on p53 mRNA. Elevation of DCAF13 resulted in downregulation of p53 protein levels, and this inhibition was reversed by MG132. In animal models, the promotion of MCL and the inhibition of ferroptosis by STAT5B. Silencing of DCAF13 blocked STAT5B inhibition of p53 and induction of xCT, GPX4, and GSH. STAT5B suppresses ferroptosis by promoting DCAF13 transcription to regulate p53/xCT pathway to promote MCL progression.

Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression

Whole-genome sequencing of longitudinal tumor pairs representing transformation of follicular lymphoma to high-grade B cell lymphoma with MYC and BCL2 rearrangements (double-hit lymphoma) identified coding and noncoding genomic alterations acquired during lymphoma progression. Many of these transformation-associated alterations recurrently and focally occur at topologically associating domain resident regulatory DNA elements, including H3K4me3 promoter marks located within H3K27ac super-enhancer clusters in B cell non-Hodgkin lymphoma. One region found to undergo recurrent alteration upon transformation overlaps a super-enhancer affecting the expression of the PAX5/ZCCHC7 gene pair. ZCCHC7 encodes a subunit of the Trf4/5-Air1/2-Mtr4 polyadenylation-like complex and demonstrated copy number gain, chromosomal translocation and enhancer retargeting-mediated transcriptional upregulation upon lymphoma transformation. Consequently, lymphoma cells demonstrate nucleolar dysregulation via altered noncoding 5.8S ribosomal RNA processing. We find that a noncoding mutation acquired during lymphoma progression affects noncoding rRNA processing, thereby rewiring protein synthesis leading to oncogenic changes in the lymphoma proteome.

MiR-525-5p inhibits diffuse large B cell lymphoma progression via the Myd88/NF-κB signaling pathway.

Diffuse large B-cell lymphoma (DLBCL) is a B-cell lymphoma with a high degree of aggressiveness. Recently, evidence has shown that miR-525-5p is decreased in DLBCL, suggesting its possible involvement in tumor progression. In this study, miR-525-5p suppressed proliferation, invasion and clonogenicity, and increased apoptosis of U2932 cells, whereas miR-525-5p silencing enhanced tumor cell growth. Next, miR-525-5p targets the 3'-UTR of Myd88, and Myd88 protein was increased in lymphoma tissues. Similar to the miR-525-5p mimic, Myd88 siRNA suppressed proliferation, invasion, and clonogenicity, and enhanced apoptosis of U2932 cells. We observed that Myd88 reversed the inhibitory effect of miR-525-5p on tumor cell growth by transfecting cells with miR-525-5p mimics alone or together with Myd88 overexpression vector. In addition, in vivo studies have shown that compared to the control group, U2932 cells with upregulated miR-525-5p expression have a reduced ability to induce tumor formation. In conclusion, our results demonstrate that miR-525-5p inhibits the progression of DLBCL through the Myd88/NF-κB pathway, which largely fills the gap of previous studies, and our results may provide a new reference for the targeted treatment of DLBCL.

Abstract 2226: Modeling the role of Tet-deficiency and restoration in Bcl2-driven B cell lymphoma progression

Abstract Loss of function in DNA methylation regulators such as the Ten-eleven translocation (TET1-3) proteins, have been implicated in the progression of lymphoma. TET enzymes constitute a family of iron and αKG-dependent dioxygenases that oxidize 5-methylcytosine in DNA to promote demethylation and the reversal of gene silencing. The TET enzymes require vitamin C for their optimal catalytic activity, however, many patients with hematological malignancies are vitamin C deficient. Enhancing and restoring TET function with high-dose vitamin C has been shown to block the progression of solid tumors and blood malignancies. A combination of genetic and epigenetic diversity in lymphoma can result in the aberrant overexpression of oncogenes, loss of tumor suppressor function, and genomic instability. Our research models Bcl2-driven B cell lymphoma with Tet-deficiency using transgenic RNAi for the knockdown of Tet1 or Tet2. In vitro, our results have shown that this model shows increased self-renewal capacity of early B cell progenitors and precursors that can be suppressed by genetic restoration of Tet expression or upon treatment with vitamin C. In vivo, the B cell compartment of mice with Bcl2 overexpression and Tet1 or Tet2 deficiency exhibit a block in B cell maturation during a long period of pre-malignancy, and a decreased frequency of follicular B cells in the spleen from 3 months of age. Our aim is to determine mechanistically the effects of Tet-deficiency on B cell lymphoma progression and whether vitamin C can restore Tet activity to suppress lymphoma progression. Citation Format: Minh Quang Lam, Luisa Cimmino. Modeling the role of Tet-deficiency and restoration in Bcl2-driven B cell lymphoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2226.

P1307: TESTING THE IMPORTANCE OF ANTIOXIDANTS ON THE PATHOGENESIS OF B CELL LYMPHOMA

Background: Our group found that antioxidant supplementation of the diet – in doses that reduce reactive oxygen species (ROS) levels – markedly accelerates progression and metastasis of malignant melanoma and lung cancer in mice. These findings can help explain the puzzling outcome of clinical trials with antioxidants and led to a heated debate about the safety of supplements for cancer patients. The findings, combined with those of dozens of clinical antioxidant trials, suggest that cancer patients and survivors should avoid antioxidant supplements. Although multiple other cancer types including breast, colon, and pancreatic cancer has been found to respond similarly to antioxidants, little is known about the effects in hematologic malignancies. In the latter, high doses of VitC – that increase oxidative stress – have shown therapeutic effects in some studies, including in B cell lymphoma. However, other studies have raised doubts about high-dose VitC therapy. Thus, many questions remain about the impact of antioxidant administration for B cell lymphoma progression, in particular in ROS-lowering doses. Aims: Define the impact of antioxidant administration on B cell lymphoma cell proliferation and viability in vitro and on the progression and survival of lymphoma in vivo. Methods: For in vitro experiment, we performed analyses of cell proliferation, cell cycle, apoptosis, and ROS levels; RNA from cells following interventions was isolated for RNAseq analysis; real-time PCR was performed to confirm candidate genes; and ChIP-qPCR to study protein-DNA binding. For in vivo experiments, we used xenograft models by subcutaneous injection of human B lymphoma cell lines, and used λMYC mice as a model of endogenous B cell lymphoma (these mice harbor a mutant human MYC gene controlled by a reconstructed Igλ locus). In vivo antioxidants were administered both orally in the drinking water, and was injected in different doses and combinations. Results: The antioxidants N-acetylcysteine (NAC), vitamin C (VitC), L-Cystein, and glutathione in doses that reduce ROS levels markedly reduced the viability of human B cell lymphoma cell lines in a MYC-dependent fashion. Intraperitoneal injections of NAC and VitC dose-dependently reduced tumor growth in xenograft experiments. Knockdown of MYC restored tumor growth in the presence of antioxidants and switching-on MYC expression in B cells rendered them sensitive to NAC and VitC. Molecular analyses revealed that antioxidants increased the binding of EGR1 to MYC and stimulated EGR1/MYC binding to apoptotic gene promotors—thus stimulating apoptosis. Intraperitoneal injections or life-long oral antioxidant administration reduced tumor growth and improved survival in λMYC transgenic mice. Summary/Conclusion: High MYC expression renders B lymphoma cells sensitive to therapy with common soluble antioxidants and indicates that this strategy might be useful—alone or in combination with lower doses of standard chemotherapy—in patients with MYC-driven lymphomas such as the diffuse large B cell variant.

Targeting lysophosphatidic acid receptor with Ki16425 impedes T cell lymphoma progression through apoptosis induction, glycolysis inhibition, and activation of antitumor immune response.

Lysophosphatidic acid (LPA) is a small phospholipid that acts as an extracellular lipid mediator. It promotes cancer progression by altering a wide array of cellular processes, including apoptosis, survival, angiogenesis, invasion, and migration through binding with its cognate receptors. Intriguingly, our previous study showed that in vitro treatment of LPA induced survival of T lymphoma cells. Hence, the present investigation was designed to investigate the antitumor potential of Ki16425, an antagonist of LPA receptors, against T cell lymphoma. Our in vitro results showed inhibition of LPA-mediated survival and metabolic activity of T lymphoma cells by Ki16425. Further, in vivo experimental findings indicated the tumor retarding potential of Ki16425 against T cell lymphoma through apoptosis induction, glycolysis inhibition, and immunoactivation. The administration of Ki16425 triggered apoptosis by down-regulating the expression of Bcl2 and up-regulating p53, Bax, cleaved caspase-3, and Cyt c expression. Further, Ki16425 suppressed glycolytic activity with concomitantly decreased expression of GLUT3 and MCT1. Moreover, we also noticed an elevated level of NO and iNOS in tumor cells after Ki16425 administration which might also be responsible for apoptosis induction and suppressed glycolysis. Additionally, we observed an increased population of total leukocytes, lymphocytes, and monocytes along with increased thymocytes count and IL-2 and IFN-γ levels. Besides, we observed amelioration of tumor-induced kidney and liver damages by Ki16425. Taken together, this is the first study that demonstrates that LPA receptors could be potential future therapeutic targets for designing promising therapeutic strategies against T cell lymphoma.

Nimbolide retards T cell lymphoma progression by altering apoptosis, glucose metabolism, pH regulation, and ROS homeostasis.

Nimbolide is reported as one of the potential anticancer candidates of the neem tree (Azadirachta indica A. Juss). The cytotoxic action of nimbolide has been well reported against a wide number of malignancies, including breast, prostate, lung, liver, and cervix cancers. Interestingly, only a few in vivo studies conducted on B cell lymphoma, glioblastoma, pancreatic cancer, and buccal pouch carcinoma have shown the in vivo antitumor efficacy of nimbolide. Therefore, it is highly needed to examine the in vivo antineoplastic activity of nimbolide on a wide variety of cancers to establish nimbolide as a promising anticancer drug. In the present study, we investigated the tumor retarding action of nimbolide in a murine model of T cell lymphoma. We noticed significantly augmented apoptosis in nimbolide- administered tumor-bearing mice, possibly due to down-regulated expression of Bcl2 and up-regulated expression of p53, cleaved caspase-3, Cyt c, and ROS. The nimbolide treatment-induced ROS production by suppressing the expression of antioxidant regulatory enzymes, namely superoxide dismutase and catalase. In addition, nimbolide administration impaired glycolysis and pH homeostasis with concomitant inhibition of crucial glycolysis and pH regulatory molecules such as GLUT3, LDHA, MCT1, and V-ATPase, CAIX and NHE1, respectively. Taken together, the present investigation provides novel insights into molecular mechanisms of nimbolide inhibited T cell lymphoma progression and directs the utility of nimbolide as a potential anticancer therapeutic drug for the treatment of T cell lymphoma.

LncRNA SNHG5 can Regulate the Proliferation and Migration of Diffuse Large B Cell Lymphoma Progression via Targeting miR-181-5p/XIAP.

Background: It has been verified that long noncoding RNAs (lncRNAs) may participate in the pathogenesis of various human diseases. This study aims to investigate the roles of lncRNA SNHG5 in diffuse large B cell lymphoma (DLBC), especially the impacts of lncRNA SNHG5 on proliferation and migration of human diffuse large B cell lymphoma cells and the related mechanism.Methods: qRT-PCR analysis was carried out to examine the expression pattern of SNHG5 in DLBC tissue and adjacent normal tissue. The effect of SNHG5 on the proliferation, apoptosis, migration, and invasion of DLBC cells was detected by MTT, flow cytometry analysis, wound healing assay and transwell assay. The correlation between SNHG5, miR-181-5p and XIAP were certified by bioinformatics analysis, and dual-luciferase reporter assay. Furthermore, rescue assays were performed to analyze the effects of SNHG5-miR-181-5p-XIAP axis on the biological behaviors of diffuse large B cell lymphoma cells. Finally, the effects of SNHG5 axis on the growth of DLBC tumor was examined by in vivo analysis.Results: SNHG5 was significantly upregulated in diffuse large B cell lymphoma tissues. Knockdown of SNHG5 inhibited the proliferation, migration, and invasion of diffuse large B cell lymphoma cells in vitro and in vivo. LncRNA SNHG5 acted as a ceRNA through binding with miR-181-5p in DLBC cells.Conclusion: LncRNA SNHG5 may promote proliferation and migration of diffuse large B cell lymphoma cells via targeting miR-181-5p/XIAP.

High Expression of IKZF2 in Malignant T Cells Promotes Disease Progression in Cutaneous T Cell Lymphoma.

Cutaneous T cell lymphoma is a generally indolent disease derived from skin-homing mature T cells. However, in advanced stages, cutaneous T cell lymphoma may manifest aggressive clinical behaviour and lead to a poor prognosis. The mechanism of disease progression in cutaneous T cell lymphoma remains unknown. This study, based on a large clinical cohort, found that IKZF2, an essential transcription factor during T cell development and differentiation, showed stage-dependent overexpression in the malignant T cells in mycosis fungoides lesions. IKZF2 is specifically over-expressed in advanced-stage mycosis fungoides lesions, and correlates with poor prognosis. Mechanistically, overexpression of IKZF2 promotes cutaneous T cell lymphoma progression via inhibiting malignant cell apoptosis and may contribute to tumour immune escape by downregulating major histocompatibility complex II molecules and up-regulating the production of anti-inflammatory cytokine interleukin-10 by malignant T cells. These results demonstrate the important role of IKZF2 in high-risk cutaneous T cell lymphoma and pave the way for future targeted therapy.

Suppression of latent transforming growth factor-β (TGF-β)-binding protein 1 (LTBP1) inhibits natural killer/ T cell lymphoma progression by inactivating the TGF-β/Smad and p38MAPK pathways

BackgroundNatural killer/T cell lymphoma (NKTCL) is a distinct subtype of Non-Hodgkin's lymphoma with highly aggressive clinical behavior. We aim to investigate the function of Latent transforming growth factor β binding protein 1 (LTBP1) and transforming growth factor beta1 (TGF-β1) and complex molecular pathogenesis of this disease. MethodsNKTCL patients and reactive lymph nodes patients were recruited in this study. The expression of LTBP1 and TGF-β1 was examined using qRT-PCR, Western blot, IHC and ELISA analyses in biopsied tissues and serum from participants and NKTCL cell lines. Cell proliferation was determined using CFSE. Cell cycle and apoptosis were evaluated using flow cytometric analyses. The expression of Ki-67, CDK4 and cyclinD1 proteins was measured using Western blot analyses. The roles of LTBP-1/TGF-β1 in EMT program were determined by measuring E-cadherin, N-cadherin and Vimentin using Western blot analyses. The effects of LTBP-1 and TGF-β1 on tumor progression in vivo were determined by animal experiments. ResultsLTBP-1 and TGF-β1 levels were elevated in NKTCL tissues and serum. The expression of LTBP-1 was positively correlated with the expression of TGF-β1 in NKTCL tissues. LTBP-1 was overexpressed in NKTCL cells. Knockdown of LTBP-1 suppressed cell proliferation and cell cycle progression, induced cell apoptosis, and suppressed EMT program in NKTCL cells. These effects of LTBP-1 knockdown were attenuated after TGF-β1 stimulation. Knockdown of LTBP-1 inhibited NKTCL tumor weight and volume in vivo. Also, stimulation of TGF-β1 attenuated the suppressive effects on tumor growth from sh-LTBP-1. Silencing of LTBP-1 lowered cellular TGF-β1, phosphorylated-Smad2, phosphorlyatd-Smad3, and phosphorylated-p38 and the suppressive effects were reversed after stimulation of TGF-β1. ConclusionOur findings suggested that inhibition of LTBP-1/TGF-β1 suppressed the malignant phenotypes of NKTCL cells and tumor growth via inactivating the canonical TGF-β/Smad signaling and p38MAPK signaling.

Neutrophils Extracellular Traps Induced By Lymphoma-Derived IL-8 Could Promote Diffuse Large B Cell Lymphoma Progression

Background: Neutrophils extracellular traps (NETs) are extracellular strands of decondensed DNA in complex with citrullinated histones (H3cit) and granule proteins including myeloperoxidase (MPO) (Sørensen O E, et al. 2016). Although this structure was initially described as an antimicrobial defense mechanism (Brinkmann V, et al. 2007), recent evidence has linked NETs to tumor metastases, and a previous study has demonstrated that the interaction of NETs and CD5+ B cells favored the transition from autoimmunity to lymphoma (Sangaletti S, et al. 2014). But it remains unclear whether NETs promote diffuse large B cell lymphoma (DLBCL) progression and can be a therapeutic candidate. Thus, our objectives were to detect NETs in the DLBCL samples, define the role of NETs in progression of DLBCL and reveal the underlying mechanism.Methods: We collected 123 tissue samples from DLBCL patients. NETs in tissues were detected by immunofluroscent staining. In cocultured model, we tested the effect of NETs on cell proliferation and migration of DLBCL cell lines. In vivo, murine A20 cells were inoculated in BALB/C mice (n = 5 mice/group), tumor growth and axillary lymph nodes metastasis were monitored by computer tomography. Tumor tissues and plasma were collected and NETs were detected by immunofluroscence and ELISA.Results: We detected more NETs structures in advanced stage samples than early stage samples (P= 0.035). Higher NETs expression was significantly associated with more B symptoms, extranodal involvement, higher IPI, elevated LDH level and lower overall response rate. In a univariate analysis, we found high NETs expression was associated with significant inferior survival (OS, HR: 3.250, 95%CI: 1.451-7.278, P= 0.002; progression free survival, HR: 2.678, 95%CI: 1.271-5.642, P= 0.007). In a multivariate analysis, high NETs expression was also found to be an independent predictor of OS and PFS (OS, HR: 2.824, 95%CI: 1.242-6.423, P= 0.013; PFS, HR: 2.398, 95%CI: 1.125-5.113, P= 0.024).Next, we wanted to determine whether DLBCL could induce NETs formation and how tumor-induced NETs were formed. As previous study proved that IL-8 could prime neutrophils for NETosis (Alfaro C, et al.2016), we detected IL-8 expression in the same tissue samples by immunostaining, and found advanced stage presented with high IL-8 expression and IL-8 also predicted inferior OS, suggesting that IL-8 may interact with NETs. Using a transwell chamber assay, we found only the media of human Su-dhl2 and murine A20, not human Su-dhl4 or Su-dhl6, potentiated species-matched neutrophils to generate extensive NETs. The level of IL-8 was highest in the supernatant of Su-dhl2 (3181.00 ± 386.60 pg/ml), nearly 10.63 and 8.57 folds higher than Su-dhl4 and Su-dhl6 (P= 0.002); and blocking IL-8 significantly reduced the ability of Su-dhl2 to induce NETs. CXCL1 and CXCL2, IL-8 murine homologues, were also high in the media of A20 cells, and NETosis induced by A20 cells was attenuated by murine CXCL1 or/and CXCL2 neutralizing antibody. We also found IL-8 mainly bound to CXCR2, not CXCR1, to induce NETs via the activation of MAPK and Src pathway.Then we sought to investigate whether NETs formation affected the growth and dissemination of DLBCL. In vitro, we treated Su-dhl2 cells and A20 cells with PMA-stimulating neutrophils media, and we observed that the treatment of NETs increased tumor proliferation at 72 hours and promoted cell migration, which were attenuated by administration of DNase I, a known inhibitor of NETs formation. In vivo, we found that NETs formed in the tumor tissues, which was associated with significantly increased serum MPO-DNA complexes in untreated mice. However, the systemic administration of DNase I or CXCR2 neutralizing antibody significantly decreased NETs formation, and retarded heterotopic tumor growth and axillary lymph nodes metastases compared with untreated group.Conclusion: In the present study, we firstly demonstrated the value of NETs to predict DLBCL inferior survival, and provided clear evidence that tumor-derived IL-8 bound to its receptor CXCR2, not CXCR1, to induce NETs formation via the activation of Src and MAPK pathway. And we also proved that this structure could promote DLBCL progression. Most importantly, our findings provided preliminary evidence that the use of DNase I or CXCR2 neutralizing antibody could reduce DLBCL progression and will hopefully pave the way for future clinical trials. DisclosuresNo relevant conflicts of interest to declare.

A novel lncRNA TCLlnc1 promotes peripheral T cell lymphoma progression through acting as a modular scaffold of HNRNPD and YBX1 complexes

Long noncoding RNAs (lncRNAs) play an essential role in tumor progression. Few researches focused on the clinical and biological relevance of lncRNAs in peripheral T cell lymphoma (PTCL). In this research, a novel lncRNA (ENST00000503502) was identified overexpressed in the main subtypes of PTCL, and designated as T cell lymphoma-associated lncRNA1 (TCLlnc1). Serum TCLlnc1 was associated with extranodal involvement, high-risk International Prognostic Index, and poor prognosis of the patients. Both in vitro and in vivo, overexpression of TCLlnc1 promoted T-lymphoma cell proliferation and migration, both of which were counteracted by the knockdown of TCLlnc1 using small interfering RNAs. As the mechanism of action, TCLlnc1 directly interacted with transcription activator heterogeneous nuclear ribonucleoprotein D (HNRNPD) and Y-box binding protein-1 (YBX1) by acting as a modular scaffold. TCLlnc1/HNRNPD/YBX1 complex upregulated transcription of TGFB2 and TGFBR1 genes, activated the tumor growth factor-β signaling pathway, resulting in lymphoma progression, and might be a potential target in PTCL.

LncRNA SNHG14/miR-5590-3p/ZEB1 positive feedback loop promoted diffuse large B cell lymphoma progression and immune evasion through regulating PD-1/PD-L1 checkpoint

Diffuse large B cell lymphoma (DLBCL) is the commonest disorder derived from the B-lymphocytes. Inhibiting the immune checkpoint through naturalizing programmed death-1 (PD-1) and programmed death ligand 1 (PD-L1) is proved to be a successful therapeutic regime for lymphoma. Long non-coding RNAs (lncRNAs) are unceasingly reported to be promising biological targets for the cancer therapies. This study planned to explore the regulation of small nucleolar RNA host gene 14 (SNHG14) on DLBCL. SNHG14 level in DLBCL samples and cell lines was analyzed by GEPIA bioinformatics tool and RT-qPCR. Biological functions of SNHG14 in DLBCL were detected by CCK-8, colony formation, and transwell invasion assays. Molecular interaction was determined by RNA immunoprecipitation (RIP) and luciferase reporter assays. MiR-5590-3p-related pathway was identified through KEGG pathway analysis applying DAVID6.8 online bioinformatics tool. Effect of SNHG14 on CD8+ T cells was detected by flow cytometry. Results depicted that SNHG14 was upregulated in DLBCL and its depletion retarded proliferation, migration and epithelial-to-mesenchymal transition (EMT). Mechanistically, SNHG14 sponged miR-5590-3p to upregulate Zinc finger E-box binding homeobox 1 (ZEB1), and ZEB1 transcriptionally activated SNHG14 and PD-L1 to promote the immune evasion of DLBCL cells. In conclusion, we firstly showed that SNHG14/miR-5590-3p/ZEB1 positive feedback loop promoted diffuse large B cell lymphoma progression and immune evasion through regulating PD-1/PD-L1 checkpoint, indicating that targeting SNHG14 was a potential approach to improve the efficacy of immunotherapy in DLBCL.

PO-507 Deregulated mechanisms downstream of PLCG1 promoting cutaneous T cell lymphomaprogression

IntroductionCutaneous T Cell Lymphoma (CTCL) is a heterogeneous group of non-Hodgkin lymphomas with unfavourable prognosis at advanced stages. The malignant network that control the development of CTCL is now starting to be characterised. NGS-derived studies have identified multiple molecular alterations in specific genes and signalling pathways including PLCG1 and JAK-STAT. It has been shown that up to 50% of CTCL cases develop deregulated PLCG1-downstream signalling as shown by activating PLCG1 mutations and PRKCQ amplifications. Also, frequent mutations in JAK1 and JAK3 and in members of the STAT family have been identified, thus revealing a potentially important role of these signalling pathways in CTCL. We aimed at exploring the interactions between deregulated PLCG1 and JAK-STAT activity in CTCL using pre-clinical models and in samples from CTCL patients at different stages of the disease.Material and methodsHEK-BlueTM IL-6 and CTCL cell lines were used to study PLCG1 downstream signalling towards NFAT and STAT3 activation. Immunohistochemistry (IHC) of NFAT, pSTAT1, pSTAT3, pSTAT5, p50, p52, p65 and RelB wasperformed in 71 patient samples. The status of PRKCQ was analysed in 45 samples by fluorescent in situhybridization (FISH).Results and discussionsDeregulated PLCG1 activity elicited NFAT and STAT3 activation ex vivo. Downstream of PLCG1, pharmacological and genetic inhibition of PRKCQ negatively affected NFAT and STAT3 activation whereas a constitutively active PRKCQ mutant (A148E) was sufficient to trigger their transcriptional activity. Our data shows that downstream of PLCG1, PRKCQ can activate transcription mediated by both NFAT and STAT proteins. We also analysed nuclear accumulation of NFAT, STAT and NF-κB family proteins by IHC in patient samples. Interestingly positive P-STAT3 staining correlated with advanced diseases. Moreover, most CTCL-tumour stage cases studied were positive for PRKCQ amplification and/or 10q23 polysomy.ConclusionOur results support PLCG1 playing a mayor role at controlling CTCL progression towards advanced stages. Downstream of PLCG1 or due to genetic amplification, PRKCQ can mediate the activation of NFAT and STAT3. Thus, mutations in PLCG1, PRKCQ amplifications and/or nuclear NFAT and STAT3 accumulation can serve as diagnostic markers for CTCL. Moreover, these can also provide rational to develop specific therapies targeting PRKCQ alone or in combination with calcineurin or JAK-STAT inhibitors.

Targeting the PI3K/Akt/mTOR signaling pathway by pterostilbene attenuates mantle cell lymphoma progression.

Mantle cell lymphoma (MCL) is an aggressive and mostly incurable B-cell malignancy with frequent relapses after an initial response to standard chemotherapy. Therefore, novel therapies are urgently required to improve MCL clinical outcomes. In this study, MCL cell lines were treated with pterostilbene (PTE), a non-toxic natural phenolic compound primarily found in blueberries. The antitumor activity of PTE was examined by using the Cell Counting Kit-8, apoptosis assays, cell cycle analysis, JC-1 mitochondrial membrane potential assay, western blot analysis, and tumor xenograft models. PTE treatment induced a dose-dependent inhibition of cell proliferation, including the induction of cell apoptosis and cell cycle arrest at the G0/G1 phase. Moreover, the PI3K/Akt/mTOR pathway was downregulated after PTE treatment, which might account for the anti-MCL effects of PTE. Synergistic cytotoxicity was also observed, both in MCL cells and in xenograft mouse models, when PTE was administered in combination with bortezomib (BTZ). The antitumor effects of PTE shown in our study provide an innovative option for MCL patients with poor responses to standardized therapy. It is noteworthy that the treatment combining PTE with BTZ warrants clinical investigation, which may offer an alternative and effective MCL treatment in the future.

B cell lymphoma progression promotes the accumulation of circulating Ly6Clo monocytes with immunosuppressive activity

ABSTRACT Monocytosis is considered a poor prognostic factor for many cancers, including B cell lymphomas. The mechanisms by which different monocyte subsets support the growth of lymphoma is poorly understood. Using a pre-clinical mouse model of B cell non-Hodgkin's lymphoma (B-NHL), we investigated the impact of tumor progression on circulating monocyte levels, subset distribution and their activity, with a focus on immune suppression. B-NHL development corresponded with significant expansion initially of classical (Ly6Chi) and non-classical (Ly6Clo) monocytes, with accumulation and eventual predominance of Ly6Clo cells. The lymphoma environment promoted the conversion, preferential survival and immune suppressive activity of Ly6Clo monocytes. Ly6Clo monocytes expressed higher levels of immunosuppressive genes including PD-L1/2, Arg1, IDO1 and CD163, compared to Ly6Chi monocytes. Both monocyte subsets suppressed CD8 T cell proliferation and IFN-γ production in vitro, but via different mechanisms. Ly6Chi monocyte suppression was contact dependent, while Ly6Clo monocytes suppressed via soluble mediators, including IDO and arginase. Ly6Clo monocytes could be selectively depleted in tumor-bearing hosts by liposomal doxorubicin treatment, further enhanced by co-administration of anti-4-1BB monoclonal antibody. This treatment led to a reduction in tumor growth, but failed to improve overall survival. Analogous immunosuppressive monocytes were observed in peripheral blood of diffuse large B cell lymphoma patients and actively suppressed human CD8 T cell proliferation. This study highlights a potential immune evasion strategy deployed by B cell lymphoma involving accumulation of circulating non-classical monocytes with immunosuppressive activity.

The Expression and Effect of B7-H3 in Mantle Cell Lymphoma

▪Objective: To investigate the effects of B7-H3 (CD276) on oncogenesis and chemosensitivity in mantle cell lymphoma (MCL).Methods: The B7-H3 expression was detected by flow cytometry in cell lines, 20 patients with MCL and 20 volunteers. B7-H3 knockdown was performed using lentivirus transduction in the Maver and Z138 mantle cell lymphoma cell lines, respectively. The effects of B7-H3 on cell proliferation, cycle, migration and invasion were investigated by CCK-8 assay, methyl cellulose colony forming assay, PI staining, and Transwell assays in vitro. By establishing Maver and Z138 xenograft models, the effects of B7-H3 on tumourigenicity were observed, and Ki-67 and PCNA was detected through immunohistochemical. Moreover, the impacts of B7-H3 RNAi on the anti-tumor effect of chemotherapy drugs were determined with CCK-8, Annexin V-FITC/PI and Hoechst 33342 staining assays in vitro and with xenograft models in vivo.Results: The frequency of B7-H3positive expression cases was 65.0% (13/20) in MCL patients and 10.0% (2/20) in volunteers. The down-regulation of B7-H3 significantly decreased tumor proliferation in MCL in vitro and in vivo. In the B7-H3 knockdown groups of Maver and Z138 xenograft models, the mean inhibition rate of tumor growth was 59.1% and 65.0% (p = 0.010 and 0.003), and the expression of both Ki-67 and PCNA were significantly lower, respectively. After B7-H3 silencing, the cell cycles of Maver and Z138 were both arrested at G0/G1 phase, and the expression of cell cycle-related proteins Cyclin D1 and CDK4 was lower. The cell migration rates and invasion capacity were decreased, and the rates of distant metastasis in B7-H3 knockdown both Maver and Z138 xenografts were significantly declined as well. The expression of invasion-related proteins MMP-2 and MMP-9 was lower in B7-H3 knockdown cells and xenografts. The silencing of B7-H3 increased the sensitivity of Maver and Z138 cells to Rituximab and Bendamustine and enhanced the drug-induced apoptosis, respectively. The activity of caspase-3 in vitro and the expression of caspase-3 in both Maver and Z138 xenografts was significantly increased in the B7-H3 shRNA combined with chemotherapy drugs groups.Conclusions: B7-H3 levels in MCL patients were signifiantly higher than that in volunteers. Our study demonstrates for the first time that B7-H3 promotes mantle cell lymphoma progression and B7-H3 knockdown significantly enhances the chemosensitivity. This may provide a new therapeutic approach to mantle cell lymphoma. DisclosuresNo relevant conflicts of interest to declare.

B7-H3 silencing inhibits tumor progression of mantle cell lymphoma and enhances chemosensitivity.

B7-H3 (CD276), known as a member of B7 immunoregulatory family, is a type I transmembrane glycoprotein aberrantly expressed in numerous types of cancer and associated with poor prognosis. However, the role of B7-H3 in oncogenesis and chemosensitivity of mantle cell lymphoma (MCL) remains unknown. We determined the effects of downregulating B7-H3 expression on tumor progression and the sensitivity of chemotherapeutic drug in mantle cell lymphoma. B7-H3 knockdown was performed using lentivirus transduction in the Maver and Z138 mantle cell lymphoma cell lines, respectively. The effects of B7-H3 on cell proliferation, cycle, migration and invasion were investigated by CCK-8 assay, methyl cellulose colony forming assay, PI staining, and Transwell assays in vitro. By establishing Maver and Z138 xenograft models, the effects of B7-H3 on tumorigenicity were observed, and Ki-67 and PCNA was detected by immunohistochemistry. The downregulation of B7-H3 significantly decreased tumor proliferation in MCL in vitro and in vivo. In the B7-H3 knockdown groups of Maver and Z138 xenograft models, the mean inhibition rate of tumor growth was 59.1 and 65.0% (p=0.010 and 0.003), and the expression of both Ki-67 and PCNA were significantly lower, respectively. After B7-H3 silencing, the cell cycles of Maver and Z138 were both arrested at G0/G1 phase, and the cell migration rates and invasion capacity were decreased as well. Moreover, the impacts of B7-H3 RNAi on the antitumor effect of chemotherapy drugs were determined with CCK-8 and Annexin V-FITC/PI assays in vitro and with xenograft models in vivo. The silencing of B7-H3 increased the sensitivity of Maver and Z138 cells to rituximab and bendamustine and enhanced the drug-induced apoptosis, respectively. Our study demonstrates for the first time that B7-H3 promotes mantle cell lymphoma progression and B7-H3 knockdown significantly enhances the chemosensitivity. This may provide a new therapeutic approach to mantle cell lymphoma.