Human Lymphoma Cell Research Articles

The T/Tn-Specific Helix pomatia Lectin Induces Cell Death in Lymphoma Cells Negative for T/Tn Antigens.

Simple SummaryChanges in glycosylation, such as incomplete synthesis and higher density of O-glycans on the cell surface, are frequently observed in cancer cells. Several types of truncated O-glycan structures, e.g., T/Tn antigens, are suspected to disrupt molecular interactions between tumor microenvironment and immune cells, for instance, facilitating cancer immune-escape. Therefore, numerous exogenous lectins targeting aberrant O-glycans are interesting tools for cancer diagnosis, prognosis, and therapy. However, the ability of exolectins to detect subtle alterations in the glycome of tumor cells and to interfere in tumor/healthy cell interactions remains largely unknown. The present article reports for the first time that the Helix pomatia (HPA) lectin, a well-known T/Tn-specific lectin, currently used as a tool in cancer diagnostics, kills Tn-positive leukemia cells and Tn-negative lymphoma cells but does not affect healthy lymphocytes. Thus, HPA could be used to discriminate between tumor and healthy cells, and detect subtle alterations in the glycosylation profile.Morniga G is a T/Tn-specific lectin, inducing cell death in Tn-positive leukemias but not in healthy lymphocytes. Helix pomatia lectin (HPA) is another T/Tn-specific lectin, currently used as tool for cancer diagnostics. The HPA-mediated tumor cell death was evaluated on human leukemia and mouse lymphoma cells, and compared to the effect of Morniga G. Both lectins induced an equivalent percentage of cell death in Tn-positive Jurkat human leukemia. In contrast, EL4 mouse lymphoma resisted Morniga G-mediated cytotoxicity but were killed by HPA at concentrations of 2.5 μg/mL (0.032 nM) and higher. In both malignant cells, HPA-mediated cell death showed features compatible with apoptosis (annexin-externalization, caspase-activation, mitochondrial membrane depolarization, and ROS production). Cytometry analysis indicated that EL4 cells are T/Tn-negative. Because previous results showed a high amount of N-acetylgalactosamine (GalNAc, sugar present in Tn antigen) on EL4 cell surface, this GalNAc could be involved in the formation of truncated O-glycans other than the T/Tn residues. When compared to Morniga G, bioinformatic analysis suggested that HPA benefits from an extended carbohydrate-binding site, better adapted than Morniga G to the accommodation of more complex branched and truncated O-glycans (such as core 2). Finally, HPA killed EL4 cells but not healthy lymphocytes in a mixture of lymphoma cells + lymphocytes, suggesting that HPA selectively triggers tumor cell death.

Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt's lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sμ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.

Downregulation of the Tumor Suppressor SAMHD1 in Classical Hodgkin Lymphoma

Introduction: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which has been initially described to restrict human immunodeficiency virus type 1 (HIV-1) in certain cell types through depletion of intracellular dNTP substrates for HIV-1 reverse transcription. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS) and have been identified as putative drivers of chronic lymphocytic leukemia resulting in decreased SAMHD1 mRNA and protein levels. Therefore, a line of evidence suggests that SAMHD1 may play a role in oncogenesis as a tumour suppressor. In addition, SAMHD1 may confer resistance to nucleoside-based chemotherapies by hydrolysing their active triphosphate metabolites, and thus, it may lead to resistance to certain chemotherapeutic agents with cytarabine in acute myeloid leukemia being a recent example (Herold et al, Nat Med 2017; 23(2):256-263). The expression patterns and the potential role of SAMHD1 in the pathogenesis of classical Hodgkin lymphoma (HL) are not yet known.Methods: SAMHD1 protein levels were assessed by Western blot analysis in 20 human lymphoma and leukemia cell lines including 5 classical HL (L1236, L428, HDML2, HDMyZ, MDA-V) and 3 diffuse large B-cell lymphoma (DLBCL) (RC-K8, OCI-Ly3, U2932) cell lines. As our findings from in silico analysis revealed potential binding sites for MYC on the SAMHD1 gene promoter, HL cell lines were treated with selective MYC inhibitors to assess possible MYC-associated regulation of SAMHD1 expression. The patient cohort included 212 cases of classical HL diagnosed and treated at the University of Texas MD Anderson Cancer Center (USA), University of Athens (Greece) and University of Crete (Greece). A control group of 4 reactive lymph nodes and 76 B-cell non-Hodgkin lymphomas (58 DLBCLs) was included in the study for comparison. All tissue samples were obtained prior to therapy. SAMHD1 expression was assessed by immunohistochemistry performed on TMAs with duplicate tumor cores from each case or full tissue sections using a monoclonal antibody (Bethyl Laboratories, San Antonio, TX). An arbitrary 10% cutoff was used to define SAMHD1 positivity in the neoplastic cells. Overall survival (OS) was defined as time from diagnosis to death or last follow-up. Event-free survival (EFS) was defined as time from diagnosis to relapse, death, or last follow-up. Survival analyses were performed using the Kaplan-Meier method (log-rank test) and Cox regression models.Results: In 4 out of 5 HL cell lines, SAMHD1 protein levels were dramatically reduced as compared to all 3 DLBCL and other lymphoma and leukemia cell lines tested. Inhibition of MYC activity resulted in a substantial increase in the SAMHD1 protein level suggesting a possible MYC-associated gene regulation. Similarly, the neoplastic Hodgkin-Reed Sternberg (HRS) cells of classical HL were SAMHD1-negative in 132 of 212 (62.3%) patients. Among the SAMHD1-positive cases, the percentage of positive HRS cells ranged from 10 to 95% with a median 40%. By contrast, SAMHD1 was negative in only 15% of the control group of DLBCLs. In addition, most of the germinal center (GC) cells of the reactive lymph nodes were SAMHD1-positive with consistently higher intensity of immunostaining as compared to HRS cells of classical HL. No significant associations between SAMHD1 levels and clinicopathologic parameters were observed in the HL cohort. In a subset of 56 HL patients with long follow up period (median 195 months), FFS at 10 years was 79% vs. 71% for patients with SAMHD1-negative vs. SAMHD1-positive HL, respectively. Similarly, OS at 15 years was 71% vs. 63% for SAMHD1-negative vs. SAMHD1-positive HL, respectively. These differences did not reach statistical significance (p>0.05, log-rank) likely because of the relatively small number of patients included in this preliminary survival analysis.Conclusions: SAMHD1 is shown for the first time to be downregulated in HRS cells of classical HL further supporting its role as a tumor suppressor and its regulation may involve MYC. Preliminary survival analysis shows a statistical trend towards inferior FFS and OS for patients with SAMHD1-positive HRS cells, however, analysis of the entire cohort of HL patients is underway to draw definite conclusions. DisclosuresKhoury:Pfizer: Research Funding; Angle: Research Funding; Kiromics: Research Funding; Stemline Therapeutics: Research Funding.

Fascin Inhibition As a Novel Therapeeutic Strategy in Diffuse Large B-Cell Lymphoma

Fascin is an actin bundling protein used to form cellular projections (e.g. filipodia) involved in cell motility and invasion. Cancer cells often upregulate fascin, and in many cancer types this is associated with poor prognosis, likely due to increased invasive and metastatic potential. We have developed fascin inhibitors that prevent actin bundling and block breast cancer metastasis in mice. Here we explored the potential role of fascin inhibition in lymphoma. We have shown that fascin is upregulated in 44% of canine B-cell (n=62) and 57% of T-cell lymphoma cases (n=28), and that increased fascin expression is correlated with worse overall survival (p=0.03). B-cell lymphoma cases had a median survival time of 388 days while that of T-cell cases was 263 days. We have further shown that a fascin inhibitor, NP-G2-044, kills human and canine diffuse large B-cell lymphoma (DLBCL) cells in vitro, and in vivo in mice bearing canine DLBCL xenografts, with GI50s ranging from 5 to 200uM. Animal toxicology studies (performed by Charles River Laboratories) show no hematologic or other toxicity in dogs and rats at concentrations up to 1,000 mg/kg/day for 28 days; these doses are much higher than the doses needed to achieve concentrations that kill lymphoma in vivo . Therefore, we are planning clinical studies in pet dogs with lymphoma to establish proof of principle prior to a human clinical trial. Collectively, our results demonstrate that fascin is a potential new therapeutic target in DLBCL. DisclosuresHuang:Novita: Equity Ownership, Patents & Royalties: Cornell University and Novita Pharmaceutical has patient on the fascin described here. .

The Effect of the MK0339 Neutrophil Elastase Inhibitor on Proliferation and Maturation of iPSC Derived from Patients with ELANE, TCIRG1 and Whim Syndrome Associated Neutropenia

Genetic neutropenias are heterogeneous group of hematological diseases characterized by impaired survival and maturation of myeloid precursors in bone marrow. G-CSF and HSCT are currently the only effective treatment options. MK-0339 is a potent, cell permeable, orally absorbed inhibitor of neutrophil elastase (NE), previously investigated in preclinical and clinical studies by Merck/DuPont as a potential anti-inflammatory drug. We have previously reported that MK-0339 increases neutrophil proliferation in cellular models of ELANE associated neutropenia (2016 [ASH Annual Meeting Abstracts] Blood. 2016;128:406; 2017 J Leuk Biol [in press]). To examine the specificity of these responses, we investigated the effects of MK-0339 on iPSCs from healthy volunteers and compared its effects on differentiation of iPSC derived from patients with ELANE, TCIRG-1 and CXCR4 associated neutropenia.We generated induced pluripotent stem cells (iPSC) from dermal fibroblasts from healthy volunteers and neutropenic patients harboring ELANE, TCIRG1 and CXCR4 mutations and developed a feeder free, reproducible and efficient protocol for myeloid differentiation.In our initial experiments, we observed that iPSC lines derived from patients with ELANE mutations (P139L and G214R) consistently grew much slower and formed much smaller colonies than the controls using both mTeSR1/matrigel and DEF-CS feeder free culture medium systems. We observed more than 3-fold growth reduction of patient derived lines compared to healthy volunteers.We next tested the effects of MK-0339 under the same culture conditions. Growth of control and ELANE patient cell lines was enhanced, and the cells appeared more robust, after addition of 1uM MK-0339. We were surprised to see a similar proliferative effect (~40% increase) with Ramos (human Burkitt's lymphoma cell line) cells cultured in the presence of 1uM MK-0339, although neither the Ramos cells nor the ELANE iPSCs had detectable levels of ELANE expression. The enhanced growth of control and patient iPSCs and the results with Ramos cells suggested that the effects of MK-0339 are not solely mediated via inhibition of NE.We next compared myeloid differentiation and CD34+ cell proliferation of iPSC from patients with ELANE, TCIRG1 and CXCR4 associated neutropenia in HPC expansion medium, using granulocytic differentiation markers and flow cytometry. Myeloid maturation was impaired and inefficient for both ELANE (P139L and G214R) patient derived cell lines. Myeloid differentiation of the TCIRG1 patient derived was also impaired. Introduction of MK-0339 restored the impaired myeloid differentiation capacity of both ELANE and TCIRG1 positive patient derived cell lines. An iPSC line derived from a WHIM (CXCR4 mutation) patient appeared to have normal maturation and proliferation. Western blot analysis revealed elevated levels of endoplasmic reticulum chaperone GRP78/BiP in ELANE and TCIRG1 mutant cell lines which was corrected by MK-0339 treatment.These studies demonstrate heretofore unreported proliferative effects of MK-0339 on cell lines not expressing neutrophil elastase. They suggest important mechanisms beyond enzyme inhibition to account for these findings. Although preliminary, we believe that this work suggests that MK-0339 may be a novel, oral therapy for both ELANE and non-ELANE associated neutropenia. DisclosuresDale:University of Washington: Employment, Research Funding; National Institutes of Health: Research Funding; WedMD/Medscape: Membership on an entity's Board of Directors or advisory committees; Wolter Kluwer: Other: Editor, Current Opinions in Hematology; Philips: Research Funding; X4Pharma: Research Funding; Shire: Other: Independent Review Board; Omeros: Other: DSMB; Coherus: Consultancy; Boheringer-Ingelheim: Consultancy; Prolong: Consultancy; Hospira: Consultancy; Genkyotex: Other: DSMB (work completed 6/2015); Genzyme (now owned by Sanofi-Aventis): Consultancy, Patents & Royalties, Research Funding; Cellerant: Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Consultancy, Editor, Current Opinions in Hematology, Honoraria; Amgen: Consultancy, Research Funding; American College of Physicians: Other: Editor and author; GlaxoSmithKline: Equity Ownership; Johnson&Johnson: Equity Ownership.

Preclinical Activity of Novel BET Protein Proteolysis Targeting Chimeras (BETP-PROTACs) and Their Combination with Venetoclax or CDK4/6 Inhibitor Against Human Mantle Cell Lymphoma (MCL) Cells

Among the genetic alterations described in Mantle Cell Lymphoma (MCL) are those involving cyclin D1, deletion of INK4a/ARF, as well as copy number gains of MYC, CDK4 and BCL2 genes. Activated B-cell receptor (BCR) signaling, and the ensuing downstream pro-growth and pro-survival NFkB activity, is also a notable feature of MCL. Collectively, the genetic alterations and the ensuing deregulated signaling and activity of transcription factors, including c-MYC and NFkB, creates the MCL-specific ‘transcriptome’ that promotes growth and survival of MCL cells. Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase (BTK), exhibits unprecedented single-agent activity in relapsed/refractory MCL, however approximately 40% of patients demonstrate primary resistance and secondary resistance is common, with a poor one-year survival rate of only 22%. Mutations in CARD11/IKBKB/TRAF2/BIRC3/NIK or the C481S mutation in BTK, sustain alternative or classical NFkB signaling despite ibrutinib treatment and confer resistance to ibrutinib in MCL. We hypothesized that the resistance to ibrutinib in MCL cells would be governed by a dysregulated transcriptome dependent on the activity of the chromatin reader BET proteins (BETPs), including BRD4. Therefore, we determined the lethal preclinical activity of BETP PROTACs (proteolysis targeting chimeras) ARV-825, and its pharmacologically superior version ARV-771, that proteasomally degrade BETPs, against the following cellular models of ibrutinib resistance (MCL-IR): in vitro generated cultured MCL Mino-persister/resistant (Mino-P/R) cells (~4-fold resistant compared to parental Mino cells), endogenously resistant cultured Z138 and Maver-1 cells, as well as patient-derived xenograft (PDX) PDX1 and PDX2 cells that had exhibited in vivo resistance to ibrutinib. Protein expression evaluations by RPPA and/or Western analyses demonstrated that MCL-IR cells expressed relatively high levels of p-AKT over AKT, p-S6, p-4EBP1, MYC, BCL2, BCl-xL, XIAP and MCL1. Treatment with BETP-PROTAC dose-dependently (20 to 200 nM) induced significantly more growth inhibition and apoptosis than the BETP inhibitor BETi OTX015 against the cellular models of MCL-IR (p < 0.001), which was associated with marked depletion of BRD4, BRD2, MYC, BCL2, BCL-xL, cyclin D1, CDK4 and pRB (S807/811) levels, as well as of NFkB target gene expressions, including cIAP2, XIAP, cFLIP, TNFAIP3, BCl-xL, IL10, TNFa and BTK. Concomitantly, BETP-PROTAC increased expression of HEXIM1 and p21 levels in the MCL-IR cells. Treatment with the BCL2 inhibitor venetoclax or the CDK4/6 inhibitor abemaciclib dose-dependently induced apoptosis of MCL-IR cell types (p< 0.01). Notably, co-treatment with BETP-PROTAC ARV-771 and venetoclax or abemaciclib synergistically induced apoptosis of Mino-P/R, Maver-1 and PDX1 and PDX2 cells (combination indices < 1.0). Additionally, compared to treatment with vehicle control, treatment of luciferase-transduced PDX2 cells infused by the tail vein and engrafted in immune-depleted NSG mice with ARV-771 (30 mg/kg SQ daily) and venetoclax (15 mg/kg PO daily) for 3 weeks, significantly reduced the MCL burden (at 2 weeks of treatment) and improved survival of the mice. These findings demonstrate that BETP-PROTACs deplete BETPs and important MCL-relevant oncogene levels, including MYC, cyclin D1, CDK4, BCL2, Bcl-xL, as well as NFkB target gene expressions, leading to synergistic lethality in vitro in combination with venetoclax or abemaciclib, as well as exert in vivo activity in combination with venetoclax against PDX model of MCL-IR cells. DisclosuresRaina:4Arvinas, LLC. New Haven, CT: Employment. Qian:4Arvinas, LLC. New Haven, CT: Employment. Crew:Arvinas, Inc: Employment.

A Novel Antibody-Cell Conjugation Method to Enhance Cellular Therapies for the Treatment of Hematological Malignancies

IntroductionCellular therapies including γδ T cells, NK-92 cells, and cytokine-induced killer (CIK) cells have shown modest anti-tumor activity against multiple hematological and solid malignancies in preclinical and phase I/II clinical trials. We investigate whether the antitumor activity of these cellular therapies can be augmented by using a novel method that rapidly affixes tumor-targeting antibodies directly to cell surface proteins.MethodsMonoclonal antibodies including rituximab and daratumumab were conjugated to the cell surface proteins using a three step approach. First, we conjugate single-stranded DNAs (ssDNAs) to the therapeutic monoclonal antibodies, as previously described (ref below). Next, we conjugate complementary ssDNAs to surface proteins of cells being used for cellular therapy. Finally, the modified antibodies are attached to the modified cells via hybridization of the complementary DNA strands.ResultsWe demonstrate that multiple anti-neoplastic monoclonal antibodies including rituximab and daratumumab can be conjugated to the surface proteins on a variety of cell including γδ T cells, NK-92 cells, CIK cells, and primary human T cells. Then we evaluated the killing activity of these conjugated cells in vitro, in vivo, and ex vivo . In vitro, Rituximab-armed γδ T cells and NK-92 cells demonstrated increased cytokine production (IFN-γ and granzyme B) and enhanced cytotoxic killing against of multiple human B-cell non-Hodgkin lymphoma (NHL) and multiple myeloma cell lines. Similar enhanced anti-tumor activity was seen with rituximab-armed or daratumumab-armed CIK cells, as well as increased CD107a expression (a marker of cytotoxicity activity) by the antibody-armed CIK cells. Rituximab-armed primary T cells also show enhanced cytotoxic killing against Raji cells, although not as pronounced as autologous rituximab-armed CIK cells. We next evaluated the therapeutic potential of antibody-armed cell therapies in vivo in a disseminated xenograft mouse model using SCID-beige mice. Rituximab-armed γδ T cells exhibited potent anti-lymphoma activity against Raji cells, which lead to improved overall survival; whereas unarmed γδ T cells alone or with rituximab co-infusion demonstrated limited anti-lymphoma activity. We also evaluated the therapeutic activity of antibody-armed cell therapies against a primary lymphoma target ex vivo. Rituximab-arm CIK cells demonstrated enhanced cytolytic activity against primary follicular lymphoma freshly collected from a patient via fine needle aspiration biopsy.DiscussionWe have demonstrated a novel approach to create tumor-specific cellular immunotherapies without the need for genetic engineering. The antibody to cell conjugation method is straight forward. The modified antibodies can be made and stored until needed. It takes approximately 1 hour to attaching these modified antibodies to the cell surface and is performed in standard aqueous buffers, such as phosphate-buffered saline (PBS). This method is also flexible being able to conjugate multiple different antibodies to the surface proteins of a variety of different cell types. We also see enhanced cell killing across multiple hematological targets using multiple cell populations including γδ T cells, NK-92 cells, CIK cells, and primary human T cells. Overall, our data demonstrate existing monoclonal antibodies can be used to “antibody program” cell therapies for the treatment of hematological malignancies.RefHsiao et al Langmuir 2009,25(12), 6985-6991 DisclosuresHsiao:Acepodia, Inc: Employment.

Single Agent and Synergistic Combinatorial Efficacy of First-in-Class Small Molecule Imipridone ONC201 in Hematological Malignancies In Vitro and In Vivo through Apoptosis Via the Integrated Stress Response Pathway

▪ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies.ONC201 was evaluated in 20 human leukemia cell lines (Figure 1): 13 acute myeloid leukemia (AML), 5 acute lymphoblastic leukemia (ALL) and 2 chronic myelogenous leukemia (CML) cell lines. We observed a dose-dependent decrease in cell viability in AML, ALL and CML cells (GI50 1-8 µM). ONC201 also decreased cell viability in 2 primary chronic lymphocytic leukemia (CLL) patient samples with 48-72h treatment. Dose- and time-dependent induction of apoptosis (caspase-3 cleavage), inhibition of Akt and Foxo3a phosphorylation was observed in AML cells treated with ONC201 for 24-48 hr. Sub-G1 analysis confirmed that ONC201 induced apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. ONC201-mediated apoptosis in ALL cells involved PARP cleavage and caspase-9 activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim was upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells, which are known to confer a poor clinical prognosis.Low micromolar concentrations of ONC201 also reduced multiple myeloma (MM) cell viability in a time- and dose-dependent manner. ONC201 reduced cell viability and induced apoptosis in dexamethasone-resistant MM.1R cells with efficacy comparable to dexamethasone-sensitive MM.1S cells (Figure 2). ONC201 caused activation of the integrated stress response (ISR) pathway, as observed with induction of ATF/CHOP in MM cells. XIAP is highly up-regulated in MM cells and sownregulation of IAP family proteins induces apoptosis. ONC201 treatment of MM cells resulted in a dose-dependent decrease in the anti-apoptotic proteins XIAP, cIAP1 and cIAP2.Next, ONC201 was evaluated in 33 human lymphoma cell lines: 3 anaplastic large cell lymphoma (ALCL), 7 Burkitt's lymphoma, 2 cutaneous T-cell lymphoma (CTCL), 8 diffuse large B-cell lymphoma (DLBCL), 1 MCL, 2 nodular sclerosis (NS) and 10 other lymphoma cell lines. ONC201 demonstrated low micromolar range efficacy (GI50 1-6 µM) across all lymphoma subtypes. Low micromolar concentrations of ONC201 reduced cell viability of MCL and DLBCL cell lines and primary cells in a time- and dose-dependent manner (Figure 3). ONC201 caused activation of the ISR pathway, as observed with induction of ATF4 in Pfeiffer DLBCL cells. In patient-derived MCL cells, ONC201 reduced cyclin D1 expression, a diagnostic feature of MCL.We then tested the efficacy of ONC201 in combination with approved therapies in hematological malignancies in vitro and in vivo. ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine confirmed by combination indices in AML cell lines CMK, THP-1, HL60 and ALL Kasumi-2 cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model exhibited superior tumor growth inhibition compared to either agent alone (Figure 4). ONC201 demonstrated synergistic reduction in cell viability in combination with 5-azacytidine that was confirmed by combination indices in AML cell lines HL-60, MOLM14 and MV411. ONC201 synergistically reduced cell viability in combination with dexamethasone or ixazomib in RPMI-8226 MM cells. ONC201 in combination with bortezomib synergistically reduced cell viability in KARPAS 299 ALCL cells and RPMI-8226 MM cells and improved apoptosis induction in UPN2 MCL cells. ONC201 combined with bortezomib was evaluated in a Burkitt's lymphoma xenograft model. Histological evaluation of tumor xenografts revealed a reduced tumor cell density at 3-4 weeks and enhanced CHOP induction at 48 hours with combination treatment compared to either agent alone. The combination treatment did not result in body weight loss.These results serve as a rationale for ongoing ONC201 single agent trials in relapsed/refractory acute leukemia (NCT02392572), non-Hodgkin's lymphoma (NCT02420795), MM (NCT02609230) and combination trial with dexamethasone in MM (NCT02863991). Our findings provide pharmacodynamic biomarkers and identify synergistic combinatorial regimens that are well tolerated and can be explored in the clinic. [Display omitted] DisclosuresPrabhu:Oncoceutics: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Oster:Oncoceutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Allen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc: Equity Ownership, Patents & Royalties.

Evaluation and Optimization of High-Field Asymmetric Waveform Ion-Mobility Spectrometry for Multiplexed Quantitative Site-Specific N-Glycoproteomics.

The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has been shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified immunoglobulin M (IgM) protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size, and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 methods provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass spectrometry-based qualitative and quantitative N-glycoproteomics.

Cell-Penetrating Peptides: Applications in Tumor Diagnosis and Therapeutics.

Since their identification over twenty-five years ago, the plethora of cell-penetrating peptides (CPP) and their applications has skyrocketed. These 5 to 30 amino acid in length peptides have the unique property of breaching the cell membrane barrier while carrying cargoes larger than themselves into cells in an intact, functional form. CPPs can be conjugated to fluorophores, activatable probes, radioisotopes or contrast agents for imaging tissues, such as tumors. There is no singular mechanism for translocation of CPPs into a cell, and therefore, many CPPs are taken up by a multitude of cell types, creating the challenge of tumor-specific translocation and hindering clinical effectiveness. Varying strategies have been developed to combat this issue and enhance their diagnostic potential by derivatizing CPPs for better targeting by constructing specific cell-activated forms. These methods are currently being used to image integrin-expressing tumors, breast cancer cells, human histiocytic lymphoma and protease-secreting fibrosarcoma cells, to name a few. Additionally, identifying safe, effective therapeutics for malignant tumors has long been an active area of research. CPPs can circumvent many of the complications found in treating cancer with conventional therapeutics by targeted delivery of drugs into tumors, thereby decreasing off-target side effects, a feat not achievable by currently employed conventional chemotherapeutics. Myriad types of chemotherapeutics such as tyrosine kinase inhibitors, antitumor antibodies and nanoparticles can be functionally attached to these peptides, leading to the possibility of delivering established and novel cancer therapeutics directly to tumor tissue. While much research is needed to overcome potential issues with these peptides, they offer a significant advancement over current mechanisms to treat cancer. In this review, we present a brief overview of the research, leading to identification of CPPs with a comprehensive state-of-the-art review on the role of these novel peptides in both cancer diagnostics as well as therapeutics.

Retraction.

Retraction: "Knockdown of long noncoding RNA HOTAIR inhibits cell growth of human lymphoma cells by upregulation of miR-148b," by Xianxian Zhao, Xiaoyan Tian, J Cell Biochem. 2019; 12348-12359: The above article, published online on 08 March 2019 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/abs/10.1002/jcb.28500), has been retracted by agreement between the the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. Several flaws and inconsistencies between results presented and experimental methods described were found, the editors consider the conclusions of this article to be invalid. The authors collaborated in the investigation initially, but were not available for a final confirmation of the retraction.

RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production.

Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.

Enhanced electrochemiluminescence ratiometric cytosensing based on surface plasmon resonance of Au nanoparticles and nanosucculent films

Ramos cells are human Burkitt's lymphoma cells, which are a kind of cancer cells to facilitate the monitoring of the relevant biological processes of cancers. Sensitive and accurate detection of Ramos cells using emerging ratiometric ECL biosensing technology shows increasing importance, however, the target analytes of current ratiometric ECL biosensors are mainly limited to DNA/RNA or proteins. In this study, we proposed a dual-potential ratiometric sensing strategy for the electrochemiluminescence detection of Ramos cells based on two types of electrochemiluminescence (ECL)-responding molecular. Au nanosucculent films (AuNFs) were electrodeposited on the fluorine doped tin oxide (FTO) electrode to increase the effective area of the electrode for more efficient assembly of DNA and effectively improving the conductivity of the sensing interfaces. In the presence of Ramos cells, aptamers capped with Au@luminol would conjugate with Ramos cells and then remove from AuNFs, accompanying the decrease of ECL signal from Au@luminol. Then, Au–DNA was captured and alternately hybridized with DNA-modified CdS nanocrystals (NCs) on the surface of AuNFs with the formation of a super reticulate structure. The reticulate structure not only raised another identified ECL signal from CdS NCs but also greatly promoted its ECL intensity from the surface plasmon resonance originating from Au NPs. The value of log (ECLCdS/ECLluminol) and the logarithm of the number of cells exhibit considerable linear relation ranging from 80 to 8 × 105 cells mL−1 with a low detection limit of 20 cells mL−1 (S/N = 3). The selectivity and specificity of this dual-potential ECL sensor showed good performance and indicated considerable promise in avoiding false-positive results in detection.

Fomitopsis cajanderi extracts have differential tumoricidal actions on human MCF7 and U-937 cancer cells and inhibit TNF secretion by U-937 monocytes

Abstract This study aimed to determine if hot ethanol and aqueous extracts from the Fomitopsis cajanderi mushroom species are cytotoxic to human cancer cell lines and modulate TNF secretion by human monocytes. The hot ethanol extract was made via a 2 hour Soxhlet extraction of F. cajanderi fruiting body in ethanol using a cold finger condenser. The hot aqueous extract was made via a 2 hour decoction in water at 100°C. Human MCF7 mammary adenocarcinoma and U-937 histiocytic lymphoma cells were treated with increasing concentrations of extract or solvent controls and were assessed for cytotoxicity by XTT colorimetric assay. Effects of the aqueous extract on TNF secretion by PMA-differentiated, LPS-stimulated U-937 cells were determined by TNF ELISA. Glycosyl content and linkage analysis of the aqueous extract were assessed by the Complex Carbohydrate Research Center (Athens, Georgia, USA). The hot ethanol extract induced concentration-dependent cytotoxicity of MCF7 and U-937 cells, with stronger U-937 tumoricidal effects. The hot aqueous extract was not cytotoxic to MCF7 cells, but had cytotoxic effects on undifferentiated U-937 cells at high concentrations. The aqueous extract, found to contain beta glucans, inhibited TNF secretion by LPS-stimulated U-937 cells in a concentration-dependent manner. Hot ethanol and aqueous F. cajanderi extracts showed differential tumoricidal actions on human cancer cells. The ethanol extract was cytotoxic to the human cancer cells tested. The aqueous extract contained beta glucans reported to be immunomodulatory, and inhibited U-937 TNF secretion. These results warrant further research of F. cajanderi extracts as novel adjunctive anti-cancer and anti-inflammatory agents.

Identification of Differentially Expressed Human Endogenous Retrovirus Families in Human Leukemia and Lymphoma Cell Lines and Stem Cells.

Endogenous retroviruses (ERVs) are becoming more and more relevant in cancer research and might be potential targets. The oncogenic potential of human ERVs (HERVs) has been recognized and includes immunosuppression, cell fusion, antigenicity of viral proteins, and regulation of neighboring genes. To decipher the role of HERVs in human cancers, we used a bioinformatics approach and analyzed RNA sequencing data from the LL-100 panel, covering 22 entities of hematopoietic neoplasias including T cell, B cell and myeloid malignancies. We compared HERV expression in this panel with hematopoietic stem cells (HSCs), embryonic stem cells (ESCs) and normal blood cells. RNA sequencing data were mapped against a comprehensive synthetic viral metagenome with 116 HERV sequences from 14 different HERV families. Of these, 13 HERV families and elements were differently expressed in malignant hematopoietic cells and stem cells. We found transcriptional upregulation of HERVE family in acute megakaryocytic and erythroid leukemia and of HERVFc family in multiple myeloma/plasma cell leukemia (PCL). The HERVFc member HERVFc-1 was found transcriptionally active in the multiple myeloma cell line OPM-2 and also in the Hodgkin lymphoma cell line L-428. The expression of HERVFc-1 in L-428 cells was validated by qRT-PCR. We also confirm transcriptional downregulation of ERV3 in acute megakaryocytic and erythroid leukemia, and HERVK in acute monocytic and myelocytic leukemia and a depression of HERVF in all malignant entities. Most of the higher expressed HERV families could be detected in stem cells including HERVK (HML-2), HERV-like, HERVV, HERVT, ERV9, HERVW, HERVF, HERVMER, ERV3, HERVH and HERVPABLB.

The Effect of a New Glucose-Methotrexate Conjugate on Acute Lymphoblastic Leukemia and Non-Hodgkin's Lymphoma Cell Lines.

Patients with hematologic malignancies require intensive therapies, including high-dose chemotherapy. Antimetabolite–methotrexate (MTX) has been used for many years in the treatment of leukemia and in lymphoma patients. However, the lack of MTX specificity causes a significant risk of morbidity, mortality, and severe side effects that impairs the quality of patients’ life. Therefore, novel targeted therapies based on the malignant cells’ common traits have become an essential treatment strategy. Glucose transporters have been found to be overexpressed in neoplastic cells, including hematologic malignancies. In this study, we biologically evaluated a novel glucose–methotrexate conjugate (Glu–MTX) in comparison to a free MTX. The research aimed to assess the effectiveness of Glu–MTX on chosen human lymphoma and leukemia cell lines. Cell cytotoxicity was verified by MTT viability test and flow cytometry. Moreover, the cell cycle and cellular uptake of Glu–MTX were evaluated. Our study reveals that conjugation of methotrexate with glucose significantly increases drug uptake and results in similar cytotoxicity of the synthesized compound. Although the finding has been confined to in vitro studies, our observations shed light on a potential therapeutic approach that increases the selectivity of chemotherapeutics and can improve leukemia and lymphoma patients’ outcomes.

Antioxidant-modulated cytotoxicity of silver nanoparticles.

The properties of silver nanoparticles (AgNPs) synthesized using compounds exhibiting biological activity seem to constitute an interesting issue worthy of examination. In these studies, two types of AgNPs were synthesized by a chemical reduction method using well-known antioxidants: gallic acid (GA) and ascorbic acid (AA). Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed that the AgNPs were spherical. The average size was equal to 26 ± 6 nm and 20 ± 7 nm in the case of ascorbic acid-silver nanoparticles (AAgNPs) and gallic acid-silver nanoparticles (GAAgNPs), respectively. Surface-enhanced Raman spectroscopy (SERS) confirmed that the AgNPs were not stabilized by pure forms of applied antioxidants. Changes in mitochondrial activity and secretion of inflammatory and apoptosis mediators after the exposure of human promyelocytic (HL-60) and histiocytic lymphoma (U-937) cells to the AgNPs were studied to determine the impact of stabilizing layers on nanoparticle toxicity. The GAAgNPs were found to be more toxic for the cells than the AAgNPs. Their toxicity was manifested by a strong reduction in mitochondrial activity and induction of the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and caspase-9. The addition of pure antioxidants to the AgNP suspensions was found to influence their toxicity. There was a significant positive effect in the case of the mixture of AA with AAgNPs and GA with GAAgNPs. The results obtained suggest that the presence of stabilizing agents adsorbed on the surface of AgNPs is the main factor in shaping their toxicity. Nevertheless, the toxic effect can be also tuned by the introduction of free antioxidant molecules to the AgNP suspensions.

NRF1-regulated CircNSUN2 promotes lymphoma progression through activating Wnt signaling pathway via stabilizing HMGA1

ABSTRACT Lymphoma is the malignant tumor in the lymphatic system. Circular RNAs (circRNAs) are non-coding RNAs with closed structure, which have been reported to perform critical functions in various tumor progressions. However, the role of circNSUN2 in lymphoma has not been well explored. Quantitative reverse transcription real-time polymerase chain reaction (RT-qPCR) assay was performed to test the expression of circNSUN2 in malignant lymphoma tissues and normal lymph tissues, as well as in human peripheral blood lymphocyte cell line and malignant lymphoma cell lines. Cell counting kit-8 (CCK-8) assay and Transwell assays were used to evaluate the function of circNSUN2 on lymphoma cell proliferation, migration and invasion. DNA pull-down assay, chromatin immunoprecipitation (ChIP) and luciferase reporter assay were employed to test the interaction between circNSUN2 and NRF1. TOP/FOP flash reporter assay was performed to detect influence of circNSUN2 on Wnt pathway. Luciferase reporter assay and RNA pull-down assay were performed to explore interaction between HMGA1 and circNSUN2 through Wnt pathway. CircNSUN2 expression was abnormally high in malignant lymphoma tissues and cell lines. CircNSUN2 inhibition could reduce proliferation and invasion of lymphoma. Bioinformatic analysis, DNA pull-down, ChIP and luciferase reporter experiments confirmed that circNSUN2 could be modulated by transcription factor NRF1. Through RT-qPCR, western blot and luciferase reporter assays, circNSUN2 was proved to influence Wnt pathway by modulating HMGA1. CircNSUN2 regulated by transcription factor NRF1 could promote lymphoma progression through activating Wnt pathway via stabilizing HMGA1.

An Integrated Genomic, Proteomic, and Immunopeptidomic Approach to Discover Treatment-Induced Neoantigens.

All nucleated mammalian cells express major histocompatibility complex (MHC) proteins that present peptides on cell surfaces for immune surveillance. These MHC-presented peptides (pMHC) are necessary for directing T-cell responses against cells harboring non-self antigens derived from pathogens or from somatic mutations. Alterations in tumor-specific antigen repertoires — particularly novel MHC presentation of mutation-bearing peptides (neoantigens) — can be potent targets of anti-tumor immune responses. Here we employed an integrated genomic and proteomic antigen discovery strategy aimed at measuring how interferon gamma (IFN-γ) alters antigen presentation, using a human lymphoma cell line, GRANTA-519. IFN-γ treatment resulted in 126 differentially expressed proteins (2% of all quantified proteins), which included components of antigen presentation machinery and interferon signaling pathways, and MHC molecules themselves. In addition, several proteasome subunits were found to be modulated, consistent with previous reports of immunoproteasome induction by IFN-γ exposure. This finding suggests that a modest proteomic response to IFN-γ could create larger alteration to cells’ antigen/epitope repertoires. Accordingly, MHC immunoprecipitation followed by mass spectrometric analysis of eluted peptide repertoires revealed exclusive signatures of IFN-γ induction, with 951 unique peptides reproducibly presented by MHC-I and 582 presented by MHC-II. Furthermore, an additional set of pMHCs including several candidate neoantigens, distinguished control and the IFN-γ samples by their altered relative abundances. Accordingly, we developed a classification system to distinguish peptides which are differentially presented due to altered expression from novel peptides resulting from changes in antigen processing. Taken together, these data demonstrate that IFN-γ can re-shape antigen repertoires by identity and by abundance. Extending this approach to models with greater clinical relevance could help develop strategies by which immunopeptide repertoires are intentionally reshaped to improve endogenous or vaccine-induced anti-tumor immune responses and potentially anti-viral immune responses.

Effect of Autophagy to Drug Resistance of B-cell Lymphoma Cells

To investigate the effect of autophagy on the drug resistance of different human lymphoma cells. Human Burkitt's lymphoma cell Daudi, human B lymphoma cell SUDHL-4, and human mantle cell lymphoma cell JeKo-1 were taken as the research subjects. The expression of Atg5 was inhibited by the treatments of autophagy inhibitors or stable interference via lentivirus infection. The autophagy activity of B lymphoma cell was changed, and the changes of lymphoma cells to the drug resistance of ADR and VCR was observed. JeKo-1 cells showed the strongest resistance to ADR and VCR, followed by SUDHL-4, and Daudi cells showed the weakest resistance to ADR and VCR. At the same time, JeKo-1 cells showed the strongest autophagy activity, followed by SUDHL-4, and Daudi cells showed the weakest autophagy activity. After the treatments of autophagy inhibitors or stable Atg5 interference, the resistance of lymphoma cells to ADR and VCR was significantly weakened, and there was the positive correlation at the drug resistance and the autophagy activity of B lymphoma cell. The higher autophagy activity in lymphoma cells, the lower chemotherapy resistance of the lymphoma cells after autophagy was inhibited.