Lymphoma Cell Lines Research Articles

Biophysical and molecular interactions of enantiomeric piperonal-derived trans β-aryl-δ-iodo-γ-lactones with cancer cell membranes, protein and DNA: Implications for anticancer activity.

Developing novel anticancer agents requires understanding their interactions with biological systems at both the cellular and molecular levels. Enantiomeric lactones have demonstrated notable cytotoxic activities against various cancer cell lines. Building on this foundation, we investigated enantiomeric piperonal-derived trans-β-aryl-δ-iodo-γ-lactones ((-)-(4S,5R,6S) and (+)-(4R,5S,6R)), focusing on their impact on cancer cells membrane (Jurkat and GL-1), model membranes, and biomacromolecules such as human serum albumin (HSA) and DNA. Also, the cytotoxicity toward red blood cells and the antitumor activity of the compounds were evaluated against a set of canine lymphoma and/or leukemia cell lines. Membrane interaction studies revealed that both enantiomers interact with the hydrophobic core of lipid bilayers, enhancing lipid acyl chain packing, with the (-)-(4S,5R,6S) isomer showing a stronger impact on membrane fluidity. Comprehensive spectroscopic and theoretical studies revealed distinct stereochemical differences in binding affinities to HSA, where the (-)-(4S,5R,6S) isomer showed higher binding affinity and significant hydrophobic interactions. Detailed biological studies demonstrated that both enantiomers exhibit antiproliferative and proapoptotic activities, with the (-)-(4S,5R,6S) enantiomer showing higher activity. This study underscores the biological activity and interactions of enantiomeric iodolactones derived from piperonal with biomacromolecules, providing comprehensive insights into their biophysical behavior and potential anticancer properties.

Two Are Better than One: The Bi-Specific Antibody Mosunetuzumab Reveals an Improved Immune Response of Vγ9Vδ2 T Cells Targeting CD20 in Malignant B Cells in Comparison to the Mono-Specific Antibody Obinutuzumab

In treating cancer, immunotherapy has been established as a later-line treatment option in clinical practice. That includes stem cell transplantation, modified or activated immune cells, and antibodies directed against aberrant cells. As an unconventional immune cell subgroup, γδ T cells have been shown to provide effects against malignant cells. They exhibit an MHC-independent activation process, which could diminish graft-versus-host disease after an adoptive transfer of allogeneic cells. Over the last years, the efficacy of therapeutic antibodies has been improved. As a bi-specific antibody, mosunetuzumab binds to both CD3 and CD20, thereby providing close proximity between effector and target cells. Here, we set out to analyze the efficiency of γδ T cells’ anti-tumor effects in combination with mosunetuzumab vs. the monoclonal anti-CD20 antibody obinutuzumab. Mosunetuzumab revealed improved responses of γδ T cells regarding their expression of IFN-γ and CD107a and their cytotoxicity towards malignant B cells from lymphoma B cell lines. In comparison to obinutuzumab, mosunetuzumab led to an equivalent or enhanced cytotoxicity against B cell lymphoma cell lines and primary patient samples, where this effect was even more prominent. In summary, we consider the combination of stimulated γδ T cells and mosunetuzumab to be a promising therapeutic approach for future clinical trials.

Epstein–Barr virus BALF0/1 subverts the Caveolin and ERAD pathways to target B cell receptor complexes for degradation

Epstein-Barr virus (EBV) establishes persistent infection, causes infectious mononucleosis, is a major trigger for multiple sclerosis and contributes to multiple cancers. Yet, knowledge remains incomplete about how the virus remodels host B cells to support lytic replication. We previously identified that EBV lytic replication results in selective depletion of plasma membrane (PM) B cell receptor (BCR) complexes, composed of immunoglobulin and the CD79A and CD79B signaling chains. Here, we used proteomic and biochemical approaches to identify that the EBV early lytic protein BALF0/1 is responsible for EBV lytic cycle BCR degradation. Mechanistically, an immunoglobulin heavy chain (HC) cytoplasmic tail KVK motif was required for ubiquitin-mediated BCR degradation, while CD79A and CD79B were dispensable. BALF0/1 subverted caveolin-mediated endocytosis to internalize PM BCR complexes and to deliver them to the endoplasmic reticulum. BALF0/1 stimulated immunoglobulin HC cytoplasmic tail ubiquitination, which together with the ATPase valosin-containing protein/p97 drove ER-associated degradation of BCR complexes by cytoplasmic proteasomes. BALF0/1 knockout reduced the viral load of secreted EBV particles from B cells that expressed a monoclonal antibody against EBV glycoprotein 350 but not a control anti-influenza hemagglutinin antibody and increased viral particle immunoglobulin incorporation. Consistent with downmodulation of PM BCR, BALF0/1 overexpression reduced viability of a diffuse large B cell lymphoma cell line whose survival is dependent upon BCR signaling. Collectively, our results suggest that EBV BALF0/1 downmodulates immunoglobulin upon lytic reactivation to block BCR signaling and support virion release, but await the development of suitable models to test its roles in EBV reactivation in vivo.

Unlocking the Therapeutic Potential of Algae-Derived Compounds in Hematological Malignancies.

Algae are a rich source of bioactive compounds that have a wide range of beneficial effects on human health and can show significant potential in the treatment of hematological malignancies such as leukemia, lymphoma, and multiple myeloma. These diseases often pose a therapeutic challenge despite recent advances in treatment (e.g., the use of immunomodulatory drugs, proteasome inhibitors, CD38 monoclonal antibodies, stem cell transplant, and targeted therapy). A considerable number of patients experience relapses or resistance to the applied therapies. Algal compounds, alone or in combination with chemotherapy or other more advanced therapies, have exhibited antitumor and immunomodulatory effects in preclinical studies that may improve disease outcomes. These include the ability to induce apoptosis, inhibit tumor growth, and improve immune responses. However, most of these studies are conducted in vitro, often without in vivo validation or clinical trials. This paper summarizes the current evidence on the in vitro effects of algae extracts and isolated compounds on leukemia, lymphoma, and myeloma cell lines. In addition, we address the current advances in the application of algae-derived compounds as targeted drug carriers and their synergistic potential against hematologic malignancies.

Combining CRISPR activation and interference capabilities using dCas9 and G-quadruplex structures.

We demonstrate that both Clustered regularly interspaced short palindromic repeats (CRISPR) interference and CRISPR activation can be achieved at RNA and protein levels by targeting the vicinity of a putative G-quadruplex (GQ)-forming sequence (PQS) in the c-Myc promoter with nuclease-dead Cas9 (dCas9). The achieved suppression and activation in Burkitt's Lymphoma cell line and in in vitro studies are at or beyond those reported with alternative approaches. When the template strand (contains the PQS) was targeted with CRISPR-dCas9, the GQ was destabilized and c-Myc mRNA and protein levels increased by 2.1- and 1.6-fold, respectively, compared to controls in the absence of CRISPR-dCas9. Targeting individual sites in the nontemplate strand (NTS) with CRISPR-dCas9 reduced both the c-Myc mRNA and protein levels (by 1.8- and 2.5-fold, respectively), while targeting two sites simultaneously further suppressed both the mRNA (by 3.6-fold) and protein (by 9.8-fold) levels. These were consistent with cell viability assays when single or dual sites in the NTS were targeted (1.7- and 4.7-fold reduction in viability, respectively). We also report extensive in vitro biophysical studies which are in quantitative agreement with these cellular studies and provide important mechanistic details about how the transcription is modulated via the interactions of RNA polymerase, CRISPR-dCas9, and the GQ.

Enhancement Of High-Dose Chemotherapy and Autologous SCT with the PARP Inhibitor Olaparib for Refractory Lymphoma.

More active high-dose chemotherapy (HDC) regimens are needed for autologous stem-cell transplantation (ASCT) for refractory lymphomas. Seeking HDC enhancement with a poly(ADP-ribose) polymerase (PARP) inhibitor, we observed marked synergy between olaparib and vorinostat/gemcitabine/busulfan/melphalan (GemBuMel) against lymphoma cell lines, mediated by inhibition of DNA damage repair. Our preclinical work led us to clinically study olaparib/vorinostat/GemBuMel with ASCT. Patients ages 15-65 with refractory lymphoma and adequate end-organ function were eligible for this phase I trial. The olaparib dose was escalated from 25 mg PO BID on days (d)-11 to -3, plus vorinostat (1,000 mg PO/d, d-10 to -3), gemcitabine (2,475 mg/m2/d IV, d-8 and -3), busulfan (target AUC 4,000 μM.min-1/d IV, d-8 to -5), melphalan (60 mg/m2/d IV, d-3 and -2), and rituximab (CD20+ tumors) (375 mg/m2, d-10), with ASCT. Fifty patients were enrolled (23 Hodgkin, 18 DLBCL, 9 T-NHL); median age 35 (range, 20-61); median 3 prior lines of therapy (range, 2-7); 17 patients had previously relapsed after CAR-T or other cellular immunotherapies; 23 patients had PET-positive tumors at HDC (9 in progression). An olaparib dose of 150 mg PO BID was identified as the recommended phase 2 dose. The main extramedullary toxicity was mucositis. The ORR/CR rates were 100%/90%. At median follow-up of 30 months (range, 12-56) months, the EFS/OS rates were 72%/82%, and 71%/88% in patients with prior CAR-T cell failure. In this first trial combining a PARP inhibitor with HDC, olaparib/vorinostat/GemBuMel was safe and showed promising activity in refractory lymphomas, including post-CAR-T relapses.

OncomiR mdv1-miR-M7-5p promotes avian lymphomatosis by modulating the BCL2/Bax mitochondrial apoptosis signaling pathway.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that infects poultry and causes fatal lymphomas in infected chickens. Notably, the mdv1-miR-M7-5p, a pivotal oncomiR encoded by MDV, is closely associated with viral replication and latency. Here, mdv1-miR-M7-5p was transfected into the chicken lymphoma cell line MSB1, which resulted in the inhibition of lymphoma cell apoptosis and an increase in lymphoma cell proliferation and migration. Additionally, the expression of the tumor suppressor genes p53 and ARRDC3 were significantly downregulated, while the MDV latency-associated genes such as ICP4 and ICP27 were significantly upregulated. The BCL2/Bax ratio was increased while the expression of genes involved in the apoptotic signaling pathway were decreased. Furthermore, our mitochondrial function experiments in MSB1 cells demonstrated that mdv1-miR-M7-5p enhanced mitochondrial ATP release and altered the mitochondrial membrane potential, thereby affecting mitochondrial function and inhibiting lymphoma cell apoptosis. Dual-luciferase assays revealed that mdv1-miR-M7-5p binds to caspase-6. For the in vivo study, a cholesterol-modified inhibitor of mdv1-miR-M7-5p was administered to chickens. Inhibition of mdv1-miR-M7-5p resulted in a lower mortality rate than that in the control groups. Furthermore, the expression levels of the cytokines interferon-gamma (IFN-γ), interleukin (IL)-4, and IL-17 in the plasma of MDV-infected chickens were significantly increased. A marked increase was observed in apoptosis in the spleen tissues, and the expression of apoptosis-related genes including caspase-3 and tumor suppressor gene PTEN in immune organs, including the spleen, bursa of Fabricius, and thymus, were markedly upregulated. In summary, the oncogenic miRNA mdv1-miR-M7-5p promotes MDV latency and may facilitate lymphoma formation by mediating the BCL2/CytC signaling pathway. This mediation enhances mitochondrial function and inhibits lymphoma cell apoptosis, thereby contributing to lymphoma development.

SP140 represses specific loci by recruiting polycomb repressive complex 2 and NuRD complex

Abstract SP140, a lymphocytic-restricted protein, is an epigenetic reader working as a corepressor of genes implicated in inflammation and orchestrating macrophage transcriptional programs to maintain cellular identity. Reduced SP140 expression is associated both to autoimmune diseases and blood cancers. However, the molecular mechanisms that link SP140 altered protein levels to detrimental effects on the immune response and cellular growth, as well as the interactors through which SP140 promotes gene silencing, remain elusive. In this work, we have applied a multi-omics approach (i.e. interactomics, ChIP-seq and proteomics) in two Burkitt lymphoma cell lines to identify both interactors and target genes of endogenous SP140. We found that SP140 interacts with the PRC2 and NuRD complexes, and we showed that these interactions are functional as SP140 directs H3K27me3 deposition and NuRD binding on a set of target genes implicated in cellular growth and leukemia progression.

Development of Hemispherical 3D Models of Human Brain and B Cell Lymphomas Using On-Chip Cell Dome System.

Lymphocytes are generally non-adherent. This makes it challenging to fabricate three-dimensional (3D) structures mimicking the three-dimensional lymphoma microenvironment in vivo. This study presents the fabrication of a hemispherical 3D lymphoma model using the on-chip Cell Dome system with a hemispherical cavity (1 mm in diameter and almost 300 µm in height). Both the human brain lymphoma cell line (TK) and human B cell lymphoma cell line (KML-1) proliferated and filled the cavities. Hypoxic regions were observed in the center of the hemispherical structures. CD19 expression did not change in either cell line, while CD20 expression was slightly upregulated in TK cells and downregulated in KML-1 cells cultured in the Cell Dome compared to those cultured in two-dimensional (2D) flasks. In addition, both TK and KML-1 cells in the hemispherical structures exhibited higher resistance to doxorubicin than those in 2D flasks. These results demonstrate the effectiveness of the on-chip Cell Dome for fabricating 3D lymphoma models and provide valuable insights into the study of lymphoma behavior and the development of new drugs for lymphoma treatment.

Novel 4-alkoxy Meriolin Congeners Potently Induce Apoptosis in Leukemia and Lymphoma Cells.

Meriolins (3-(pyrimidin-4-yl)-7-azaindoles) are synthetic hybrids of the naturally occurring alkaloids variolin and meridianin and display a strong cytotoxic potential. We have recently shown that the novel derivative meriolin16 is highly cytotoxic in several lymphoma and leukemia cell lines as well as in primary patient-derived lymphoma and leukemia cells and predominantly targets cyclin-dependent kinases (CDKs). Here, we efficiently synthesized nine novel 2-aminopyridyl meriolin congeners (3a-3i), i.e., pyrimeriolins, using a one-pot Masuda borylation-Suzuki coupling (MBSC) sequence, with eight of them bearing lipophilic alkoxy substituents of varying length, to systematically determine the influence of the alkoxy sidechain length on the biological activity. All the synthesized derivatives displayed a pronounced cytotoxic potential, with six compounds showing IC50 values in the nanomolar range. Derivatives 3b-3f strongly induced apoptosis and activated caspases with rapid kinetics within 3-4 h in Jurkat leukemia and Ramos lymphoma cells. The induction of apoptosis by the most potent derivative 3e was mediated by the intrinsic mitochondrial death pathway, as it was blocked in caspase-9 deficient and Apaf-1 knockdown Jurkat cells. However, as recently shown for meriolin16, derivative 3e was able to induce apoptosis in the Jurkat cells overexpressing the antiapoptotic protein Bcl-2. Since tumor cells often inactivate the intrinsic mitochondrial apoptosis pathway (e.g., by overexpression of Bcl-2), these meriolin congeners represent promising therapeutic agents for overcoming therapeutic resistance.

BET inhibition revealed varying MYC dependency mechanisms independent of gene alterations in aggressive B-cell lymphomas

BackgroundMYC-driven lymphomas are a subset of B-cell lymphomas characterized by genetic alterations that dysregulate the expression of the MYC oncogene. When overexpressed, typically through chromosomal translocations, amplifications, or other mechanisms, MYC can drive uncontrolled cell growth and contribute to cancer development. MYC-driven lymphomas are described as aggressive entities which require intensive treatment approaches and can be associated with poor prognosis. In the absence of direct MYC-targeting therapy, epigenetic drugs called BET inhibitors (BETi) were shown to reduce MYC levels by disrupting BRD4-dependent transcription associated with the expression of MYC, as well as other oncogenes. Here, we used BETi as molecular tools to better understand oncogenic dependencies in a panel of cell line models of MYC-driven B-cell lymphoma selected to represent their genetic heterogeneity.ResultsWe first showed that, in these models, MYC expression level does not strictly correlate to the presence of gene alterations. Our data also demonstrated that BETi induces similar growth arrest in all lymphoma cell lines independently of MYC mutational status or expression level. In contrast, BETi-induced cell death was only observed in two cell lines presenting the highest level of MYC protein. This suggests that some MYC-driven lymphoma could present a stronger dependency on MYC for their survival which cannot be predicted on the sole basis on their genetics. This hypothesis was confirmed by gene invalidation experiments, which showed that MYC loss recapitulates the effect of BETi treatment on both cell proliferation and survival, confirming MYC oncogene dependency in models sensitive to BETi cytotoxicity. In contrast, the growth arrest observed in cell lines resistant to BETi-induced apoptosis is not mediated through MYC, but rather through alternative pro-proliferative or oncogenic pathways. Gene expression profiling revealed the basal activation of a specific non-canonical WNT/Hippo pathway in cell death-resistant cell lines that could be targeted in combination therapy to restore BETi cytotoxicity.ConclusionThis work brings new insights into the complexity of MYC-dependencies and unravels a novel targetable oncogenic pathway in aggressive B-cell lymphomas.

Discovery of Carbonic Anhydrase 9 as a Novel CLEC2 Ligand in a Cellular Interactome Screen.

Membrane proteins, especially extracellular domains, are key therapeutic targets due to their role in cell communication and associations. Yet, their functions and interactions often remain unclear. This study presents a general method to discover interactions of membrane proteins with immune cells and subsequently to deorphanize their respective receptors. We developed a comprehensive recombinant protein library of extracellular domains of human transmembrane proteins and proteins found in the ER-Golgi-lysosomal systems. Using this library, we conducted a flow-cytometric screen that identified several cell surface binding events, including an interaction between carbonic anhydrase 9 (CAH9/CA9/CAIX) and CD14high cells. Further analysis revealed this interaction was indirect and mediated via platelets bound to the monocytes. CA9, best known for its diverse roles in cancer, is a promising therapeutic target. We utilized our library to develop an AlphaLISA high-throughput screening assay, identifying CLEC2 as one robust CA9 binding partner. A five-amino-acid sequence (EDLPT) in CA9, identical to a CLEC2 binding domain in Podoplanin (PDPN), was found to be essential for this interaction. Like PDPN, CA9-induced CLEC2 signaling is mediated via Syk. A Hodgkin's lymphoma cell line (HDLM-2) endogenously expressing CA9 can activate Syk-dependent CLEC2 signaling, providing enticing evidence for a novel function of CA9 in hematological cancers. In conclusion, we identified numerous interactions with monocytes and platelets and validated one, CA9, as an endogenous CLEC2 ligand. We provide a new list of other putative CA9 interaction partners and uncovered CA9-induced CLEC2 activation, providing new insights for CA9-based therapeutic strategies.

Preclinical evaluation of 64Cu/177Lu-labelled anti-CD30 monoclonal antibody for theranostics in CD30-positive lymphoma.

CD30 serves as an ideal therapeutic target for lymphoma, but its variable expression and high relapse rate pose challenges in targeted therapy. This study aims to label the anti-CD30 monoclonal antibody with 64Cu/177Lu for immuno-positron emission tomography (immuno-PET) and radioimmunotherapy (RIT). CD30 binding kinetics of anti-CD30-IgG (IMB16) were measured by Biolayer interferometry (BLI). Western blotting screened lymphoma cell lines for CD30 expression. Flow cytometry and immunofluorescence validated the specific binding of IMB16. IMB16 was conjugated to p-SCN-Bn-NOTA(NOTA) and p-SCN-Bn-DOTA(DOTA) for radiolabeling with 64Cu and 177Lu. [64Cu]Cu-NOTA-IMB16 and [177Lu]Lu-DOTA-IMB16 were used for immuno-PET and RIT in subcutaneous lymphoma NSG mouse models. IMB16 had a strong binding affinity to CD30 according to the BLI. Western blotting revealed high CD30 expression in Karpas299 cells and negative expression in Raji cells. Flow cytometry and immunofluorescence confirmed specific binding of IMB16 to CD30 on cell surface. Radiochemical purity of [64Cu]Cu-NOTA-IMB16 and [177Lu]Lu-DOTA-IMB16 exceeded 95%. In Immuno-PET imaging, CD30-positive Karpas299 tumours had a mean uptake value of 19.2 ± 0.9%ID/g (n = 3) at 24h post-injection, significantly higher than Karpas299-blocked and Raji-negative groups (P < 0.001). A high radiation dose (300µCi) of [177Lu]Lu-DOTA-IMB16 significantly inhibited tumour growth (80.2 ± 17.6% standardized tumour volume, n = 5) at 10 days post-injection, compared to controls. Ex vivo biodistribution and histological staining supported in vivo PET imaging and RIT results. Labelling IMB16 with 64Cu enabled non-invasive assessment of CD30 expression, while 177Lu labelling effectively suppressed tumour growth in CD30-positive lymphoma. CD30-targeted theranostic show promise for patient stratification and treatment enhancement, warranting further clinical evaluation.

Evaluation of Chelidonium Majus L. Alkaloid Effect on VEGF Gene Expression and Cell Apoptosis in the Burkitt Lymphoma Cell Line

Background: Burkitt lymphoma (BL) is a type of mature B-cell non-Hodgkin’s lymphoma that commonly develops in children and young adults. Vascular endothelial growth factor (VEGF) is acknowledged as a vital regulator of angiogenesis in both normal and disease states. In light of the adverse effects linked to chemical treatments, this study aimed to explore the anticancer effects of Chelidonium majus L. alkaloid on the Daudi BL cell line and to evaluate the expression of the VEGF gene. Materials and Methods: This project was an experimental study. The cytotoxic effects of the alkaloid derived from Chelidonium majus L. on the Daudi and normal cells were evaluated using MTT assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The induction of apoptosis in cells was measured utilizing Annexin V/propidium iodide (PI) flow cytometry. Additionally, the expression levels of the VEGF gene were determined via a Real-time PCR assay. Data were entered into SPSS, version 21. Student’s t-test and ANOVA test were used for comparisons of groups. Results: The 50% cytotoxic concentration (CC50) of the alkaloid from Chelidonium majus L. was found to be 56.35µg/mL in the Daudi cell. Findings from flow cytometry aligned with those from MTT assays. Real-time PCR assay results showed a significant decrease in the VEGF gene expression (P&lt;0.009). Effects observed after 48 hours of treatment across various concentrations of Chelidonium majus L. alkaloid demonstrated dose-dependency. However, in peripheral blood mononuclear cells (PBMCs) as a control, the alkaloid did not significantly affect the expression of the VEGF gene (P&gt;0.05). Conclusion: The alkaloid is derived from Chelidonium majusL. Appears to influence angiogenesis by down-regulating the VEGF gene expression, suggesting its potential as a complementary agent in chemotherapy for Burkitt lymphoma. However; further research is required to evaluate the effectiveness of the Chelidonium majusL. Extract as a definitive treatment for Burkitt lymphoma.

Targeting Non-Apoptotic Pathways with the Cell Permeable TAT-Conjugated NOTCH1 RAM Fragment for Leukemia and Lymphoma Cells.

Targeting nonapoptotic cell death offers a promising strategy for overcoming apoptosis resistance in cancer. In this study, we developed Tat-Ram13, a 25-mer peptide that fuses the NOTCH1 intracellular domain fragment RAM13 with a cell-penetrating HIV-1 TAT, for the treatment of T-cell acute lymphoblastic leukemia with aberrant NOTCH1 mutation. Tat-Ram13 significantly downregulated NOTCH1-target genes in T-ALL cell lines. Furthermore, the peptide had potent cytotoxic effects on various human leukemia and lymphoma cell lines. However, it did not affect normal lymphocytes and monocytes, some subsets of leukemia cells, or adherent tumor cells. This cell-selective cytotoxic activity was closely correlated with the peptide uptake via macropinocytosis in leukemia cells. In leukemia cells, Tat-Ram13 triggered rapid cell death. This cell death involved mitochondrial membrane depolarization and extracellular release of lactate dehydrogenase and high-mobility group box-1 protein without activation of caspase-3 or cleavage of PARP-1. These results suggest that Tat-Ram13 cell death is nonapoptotic and mediated by rapid plasma membrane rupture. Moreover, alanine scanning analysis identified four critical hydrophobic amino acids in the RAM13 domain essential for its cytotoxicity. Consequently, these results suggest that Tat-Ram13 is a tumor-selective, nonapoptotic cell death-inducing agent for treating refractory leukemia and lymphomas with apoptosis resistance.

Developmental Stage and Cellular Context Determine Oncogenic and Molecular Outcomes of Ezh2 Y641F Mutation in Hematopoiesis.

Mutations in the histone methyltransferase EZH2, particularly the Y641 hotspot mutation, have been implicated in hematologic malignancies, yet the effect of timing and cellular context on their oncogenic potential has remained unknown. In this study, we utilized a conditional allele with tissue-specific Cre drivers to investigate the effects of Ezh2 Y641F mutations at various stages of development, with a focus on the hematopoietic system. We found that ubiquitous heterozygous Ezh2 Y641F expression at birth, or conditional expression in hematopoietic or mesenchymal stem cells, led to decreased survival due to hematopoietic defects and bone marrow failure, with no evidence of malignancy. In contrast, Ezh2 Y641F expression in committed B cells drives lymphoma formation, highlighting the lineage-specific oncogenic activity of the mutation. Transcriptomic analysis of B cell progenitors revealed key pathway alterations between Cre models such as altered IL2-Stat5 signaling pathway, differential expression of E2F targets, and altered GTPase pathway expression driven by upregulation of Guanylate Binding Proteins (GBPs) in Mx1-Cre Ezh2 Y641F pro-B cells. We further found that the GBP locus is regulated by Ezh2-mediated H3K27me3, it is associated with poorer survival in Acute Myeloid Leukemia patients and has variable effects on apoptosis in human lymphoma and leukemia cell lines. These findings suggest that the Ezh2 Y641F mutation may alter immune regulatory pathways, cell differentiation and apoptosis, with potential implications for disease progression. Our results highlight the critical role of mutation timing and cellular context in EZH2-driven hematopoietic disease, resulting in distinct downstream changes that shape the oncogenic impact of EZH2.

Ubiquitin-specific peptidases in lymphoma: a path to novel therapeutics.

Ubiquitin-specific peptidases (USPs), also known as deubiquitinating enzymes (DUBs), play a crucial role in maintaining cellular homeostasis by selectively removing ubiquitin molecules from targeted proteins. This process affects protein stability, subcellular localization, and activity, thereby influencing processes such as DNA repair, cell cycle regulation, and apoptosis. Abnormal USP activities have been linked to various diseases, including cancer. Emerging evidence in lymphoma studies highlights the significance of USPs in controlling signaling pathways related to cancer initiation and progression and presents them as potential therapeutic targets. This study aimed to elucidate the multifaceted roles of USPs in lymphoma. This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Articles published in English up to May 2023 were retrieved from PubMed, Web of Science, and Scopus. The inclusion criteria focused on studies investigating the role of USPs in lymphoma cancer, involving human subjects or relevant lymphoma cell lines, exploring molecular mechanisms and signaling pathways, and assessing diagnostic or prognostic value. After the selection process, 23 studies were selected for analysis. USPs were found to affect various aspects of lymphoma development and progression. Specific USPs were identified with roles in cell-cycle regulation, apoptosis modulation, drug resistance, DNA repair, and influence of key oncogenic pathways, such as B cell receptor (BCR) signaling. This systematic review underscores the emerging role of USPs in lymphoma and their potential as therapeutic targets. Inhibitors of USPs, such as USP14 inhibitors, show promise in overcoming drug resistance. The dynamic interplay between USPs and lymphoma biology presents an exciting opportunity for future research and the development of more effective treatments for patients with lymphoma. Understanding the intricate functions of USPs in lymphoma offers new insights into potential therapeutic strategies, emphasizing the significance of these enzymes in the context of cancer biology.

Multi-Component, Time-Course screening to develop combination cancer therapies based on synergistic toxicity

Clinical trials in cancer are ideally built on a foundation of sound mechanistic rationale and well-validated drug activity in relevant disease models. The screening of approved and investigational drugs in cell-based phenotypic assays can provide evidence of drug activity, but alternative screening paradigms are needed to develop and optimize multidrug combination regimens. Here, we utilize in vitro screening outcomes across a panel of lymphoma cell lines to dissect the activity of four small-molecule drugs (Venetoclax, Ibrutinib, Prednisolone, and Lenalidomide) currently under investigation within ongoing clinical trials in lymphoma. Data from multiple concentration ranges and time points show that synergistic drug combinations promote apoptosis and cytotoxicity responses at concentrations and time points that are consistent with in vivo drug exposures. To fully map the interaction landscape of these agents in relevant cell models, we developed an in vitro assay format that facilitated time-course evaluations involving concurrent multidrug exposure which further highlighted rapid, synergistic apoptosis induction as a central engine for the activity of this multicomponent targeted therapy. In addition to several instances of exceptional drug+drug synergy, the genetically similar diffuse large B cell lymphoma models also displayed substantial heterogeneity in the degree of synergism between drug pairs. A parallel survey of chemotherapies exhibited limited combination benefit, supporting recent findings that multicomponent chemotherapy outcomes are driven by individual drug activity. Collectively, these data demonstrate how in vitro drug screening data can identify multidrug combinations that exploit drug synergy to overcome the functional diversity of human malignancies.

Combining the CRISPR Activation and Interference Capabilities Using dCas9 and G-Quadruplex Structures.

We demonstrate that both CRISPR interference and CRISPR activation can be achieved at RNA and protein levels by targeting the vicinity of a putative G-quadruplex forming sequence (PQS) in the c-Myc promoter with nuclease-dead Cas9 (dCas9). The achieved suppression and activation in Burkitťs Lymphoma cell line and in in vitro studies are at or beyond those reported with alternative approaches. When the template strand (contains the PQS) was targeted with CRISPR-dCas9, the G-quadruplex was destabilized and c-Myc mRNA and protein levels increased by 2.1-fold and 1.6-fold, respectively, compared to controls in the absence of CRISPR-dCas9. Targeting individual sites in the non-template strand with CRISPR-dCas9 reduced both the c-Myc mRNA and protein levels (by 1.8-fold and 2.5-fold, respectively), while targeting two sites simultaneously further suppressed both the mRNA (by 3.6-fold) and protein (by 9.8-fold) levels. These were consistent with cell viability assays when single or dual sites in the non-template strand were targeted (1.7-fold and 4.7-fold reduction in viability, respectively). We also report extensive in vitro biophysical studies which are in quantitative agreement with these cellular studies and provide important mechanistic details about how the transcription is modulated via the interactions of RNA polymerase, CRISPR-dCas9, and the G-quadruplex.

Cooperative participation of CagA and NFATc1 in the pathogenesis of antibiotics-responsive gastric MALT lymphoma

BackgroundThis study aimed to explore whether cytotoxin-associated gene A (CagA) can inhibit cell cycle progression by activating nuclear factor of activated T cells (NFAT) in lymphoma B cells and contribute to Helicobacter pylori eradication (HPE) responsiveness (complete remission [CR] after HPE) in gastric mucosa-associated lymphoid tissue (MALT) lymphoma.Materials and MethodsWe co-cultured three B-lymphoma cell lines (MA-1, OCI-Ly3, and OCI-Ly7) with HP strains (derived from HPE-responsive gastric MALT lymphoma) and evaluated the expression patterns of CagA, phosphorylated (p)-CagA (CagAP−Tyr), and CagA-signaling molecules, cell-cycle inhibitors, p-NFATc1 (Ser172), and NFATc1 using western blotting. Furthermore, we evaluated the association between nuclear NFATc1 expression in the tumor cells of 91 patients who received first-line HPE (59 patients with HPE responsiveness and 32 without HPE responsiveness) and HPE responsiveness and CagA expression in tumor cells.ResultsIn HP strains co-cultured with B cell lymphoma cell lines, CagA was translocated to the nucleus through tyrosine phosphorylation (CagAP−Tyr) and simultaneously dephosphorylated NFATc1, subsequently causing nuclear NFATc1 translocation and stimulating the expression of p-SHP-2/p-ERK/Bcl-xL. Activated NFATc1 causes G1 cell cycle retardation in both MA-1 and OCI-Ly3 cells by triggering p21 and p27 production. Nuclear NFATc1 localization was significantly associated with the presence of CagA in gastric MALT lymphomas (80% [41/51] vs. 33% [13/40]; p < 0.001) and with HPE responsiveness (73% [43/59] vs. 25% [8/32]; p < 0.001). Patients exhibiting both the presence of CagA and nuclear NFATc1 localization responded more rapidly to HPE than those without (median interval to CR, 4.00 vs. 6.00 months, p = 0.003).ConclusionsOur findings indicated that CagA and NFATc1 cooperatively participate in the lymphomagenesis of HPE-responsive gastric MALT lymphoma.