Cultured Lymphoma Cells Research Articles

5-Azacytidine Suppresses the Expression of Tissue-Specific Oct-1 Isoform in Namalwa Burkitt’s Lymphoma Cell Culture

Overexpression of the transcription factor POU2F1 (Oct-1) increases the malignant potential of the tumor and determines the unfavorable prognosis for both solid and hematological cases of the disease in human carcinogenesis. The Oct-1 level determines the rate of development of the disease in acute myelodysplastic leukemia (AML), and a decrease in its expression significantly delays the development of leukemia in mice; however, a complete knockout of Oct-1 leads to the death of the animals. POU2F1 (Oct-1) is expressed as several isoforms transcribed from alternative promoters. They include both ubiquitous and tissue-specific isoforms. It was shown that in Burkitt’s lymphoma Namalwa cells 5-azacytidine specifically suppresses the expression of the tissue-specific isoform Oct-1L mRNA (level of Oct-1L is abnormally increased in these cells), while not causing changes in the amount of the ubiquitous isoform Oct-1A mRNA. These results show that it is possible to selectively reduce the transcription level of the Oct-1L isoform aberrantly expressed in human tumor cells.

Characterization of solvents and optimization of stability and solubility of bioactive compounds used in lymphoma cell culture treatments

Phytochemicals represent one of the rising agents in cancer research today. They are recognized as one the most abundant bio-active compounds found in many different sources, specifically in plants. Their anti-cancer effects have been frequently explored and reported in various research studies. However, the bioavailability and solubility of phytochemicals still represent the major issue in in vivo and in vitro research. This report analyses the effects of three different phytochemicals, commonly used for survival interference in malignant cancer clones, including thymoquinone (TQ), curcumin, and quercetin in a model of diffuse large B cell lymphoma (DLBCL). In order to characterize the impact of solubility of these compounds to their bioactivity in the DLBCL model, three different but highly widespread solvents were used. Determination of an optimal compound - solvent association is warranted when assessing the stability and activity of phytochemicals in cells. The results of this study indicate the dose-dependent decrease in cellular viability, including the treatments with all combinations of substances and solvents. In addition, we demonstrated that the choice of the solvent greatly influenced solubility and the overall effect of the compound in cancer cells.

Interferon-regulating activity of the CelAgrip drug and its influence on the formation of reactive oxygen species and expression of innate immunity genes in Burkitt’s lymphome cell cultures

Interferons (IFN) and IFN inducers are effective in suppressing viral reproduction and correcting of the innate immunity mechanisms. The aim of the study was to test the hypothesis of the possible involvement of the IFN inducer CelAgrip (CA) as an activator or suppressor of antiviral effects in Burkitt's lymphoma (LB) cell cultures with different ability to produce Epstein-Barr virus antigens (EBV). The kinetic analysis of the dynamics of reactive oxygen species (ROS) production and determination of gene group expression by real-time PCR in response to CA treatment were done in human cell lines LB P3HR-1 and Namalva, spontaneously producing and not producing EBV antigens. When treating CA in Namalva cells, a decrease in the ROS activation index was found; in P3HR-1 cells, an increase was observed. After treatment with CA, there was no reliable activation of the IFN-α, IFN-β and IFN-λ genes in Namalva cells, but the expression of the ISG15 and P53(TP53) genes was increased more than 1200 times and 4.5 times, respectively. When processing the CA of P3HR-1 cells, the expression of IFN-α genes increased by more than 200 times, IFN-λ - 100 times, and the ISG15 gene - 2.2 times. The relationship between IFN-inducing action of CA and the activity of ISG15 and ROS in LB cell cultures producing and not producing EBV antigens is supposed. In Namalva cells that do not produce EBV antigens the treatment of CA results in suppression of ROS generation and activation of the expression of genes ISG15 and P53 (TP53); in P3HR-1 cells producing EBV antigens, the opposite picture is observed - the formation of ROS and the expression of the IFN-α and IFN-λ genes are activated and the activity of the ISG15 and P53 (TP53) genes is suppressed.

Development of a clinically relevant chemoresistant mantle cell lymphoma cell culture model.

Mantle cell lymphoma is an aggressive subtype of non-Hodgkin’s lymphoma that claims the lives of tens of thousands of people every year. Although combination chemotherapy treatments such as CHOP have yielded promising outcomes in the clinic, the development of chemoresistance in patients has limited their long-term success. The lack of in vitro chemoresistance models has limited our ability to understand the mechanisms by which cells develop resistance, and thus our ability to develop novel therapeutics to overcome this issue. Here, we describe the development of a clinically relevant chemoresistant mantle cell lymphoma model using the JeKo-1 cell line. This was achieved through a stepwise treatment selection strategy using gradually increasing concentrations of CHOP. We show that resistant JeKo-1 cells display strong recovery and fast proliferation after treatment with an IC50 dose of CHOP. We also found that resistant JeKo-1 cells overexpress three oncogenes implicated in the development of mantle cell lymphoma—Cyclin D1, Mcl-1, and Bcl-2—compared to normal JeKo-1 cells. We anticipate that in vitro models such as this one will enable the discovery of new therapeutic strategies for overcoming chemoresistance and improve clinical outcomes in mantle cell lymphoma patients. Impact statement Mantle cell lymphoma remains one of the deadliest subtypes of non-Hodgkin’s lymphoma, in large part because patients become resistant to frontline chemotherapy. The development of strategies to treat advanced disease will be contingent upon testing in appropriate models. Most in vitro models of resistant mantle cell lymphoma are laboratory grade models that do not recapitulate the low level of chemoresistance typically observed in patients, limiting their utility. This study develops a clinically relevant in vitro model that can be used to establish the mechanisms of resistance and test new therapeutics intended to treat recurrent disease.

3,5,7-Substituted Pyrazolo[4,3- d]pyrimidine Inhibitors of Cyclin-Dependent Kinases and Their Evaluation in Lymphoma Models.

Cyclin-dependent kinases are therapeutic targets frequently deregulated in various cancers. By convenient alkylation of the 5-sulfanyl group, we synthesized 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2) H-pyrazolo[4,3- d]pyrimidines with various substitutions at position 5 with potent antiproliferative activity in non-Hodgkin lymphoma cell lines. The most potent derivative 4.35 also displayed activities across more than 60 cancer cell lines. The kinase profiling confirmed high selectivity of 4.35 toward cyclin-dependent kinases (CDKs) 2, 5, and 9, and the cocrystal with CDK2/cyclin A2 revealed its binding in the active site. Cultured lymphoma cell lines treated with 4.35 showed dephosphorylation of CDK substrates, cleavage of PARP-1, downregulation of XIAP and MCL-1, and activation of caspases, which collectively confirmed ongoing apoptosis. Moreover, 4.35 demonstrated significant activity in various cell line xenograft and patient-derived xenograft mouse models in vivo both as a monotherapy and as a combination therapy with the BCL2-targeting venetoclax. These findings support further studies of combinatorial treatment based on CDK inhibitors.

Lack of Mutagenic Activity of Sulfur Nanoparticles in Micronucleus Test on L5178Y Cell Culture

The nature and physicochemical characteristics of nanomaterials affect their biological activity. Sulfur microparticles and sulphur nanoparticles have different levels of activity against bacterial, fungal, and animal cells. The cytotoxicity and genotoxicity of sulfur nanoparticles (SNPs) of about 70 nm in size were examined on the L5178Y mouse lymphoma cell culture. The concentration of SNPs causing 50% cell death is 0.078–0.312 mg/mL. The micronucleus assay revealed no mutagenic properties of SNPs. Metabolic activation of SNPs by the rat liver microsomal fraction does not affect the toxicity. It is assumed that the mechanism of cytotoxic action is associated with the interaction of elemental sulfur with sulfhydryl groups of molecules inside the cell.

Peroxisomes protect lymphoma cells from HDAC inhibitor-mediated apoptosis

Peroxisomes are a critical rheostat of reactive oxygen species (ROS), yet their role in drug sensitivity and resistance remains unexplored. Gene expression analysis of clinical lymphoma samples suggests that peroxisomes are involved in mediating drug resistance to the histone deacetylase inhibitor (HDACi) Vorinostat (Vor), which promotes ROS-mediated apoptosis. Vor augments peroxisome numbers in cultured lymphoma cells, concomitant with increased levels of peroxisomal proteins PEX3, PEX11B, and PMP70. Genetic inhibition of peroxisomes, using PEX3 knockdown, reveals that peroxisomes protect lymphoma cells against Vor-mediated cell death. Conversely, Vor-resistant cells were tolerant to elevated ROS levels and possess upregulated levels of (1) catalase, a peroxisomal antioxidant, and (2) plasmalogens, ether glycerophospholipids that represent peroxisome function and serve as antioxidants. Catalase knockdown induces apoptosis in Vor-resistant cells and potentiates ROS-mediated apoptosis in Vor-sensitive cells. These findings highlight the role of peroxisomes in resistance to therapeutic intervention in cancer, and provide a novel modality to circumvent drug resistance.

Abstract LB-201: Preclinical toxicology of TRXE-002-1

Abstract Introduction: TRXE-002-1 is a low molecular weight super benzopyran molecule identified as having potent activity against many cancer phenotypes. It was identified from a library of analogues as being able to induce cell death in both chemoresistant CD44+/MyD88+ ovarian cancer stem cells (IC50 of ∼ 50 nM) and chemosensitive CD44-/MyD88- ovarian cancer cells, and disrupt spheroid cultures formed from cancer stem cells. In vivo studies have confirmed efficacy in a rodent intra-peritoneal model of human ovarian cancer. Here we report on pre-clinical toxicology studies conducted on TRXE-002-1. Method: We conducted a full GLP dog CV study using a 4×4 Latin Square Crossover study design where we assessed telemetric ECG recordings from 4 male dogs post IP administration at doses up to 30 mg/kg. Thirty seconds of continuous ECG tracings were collected pre-dose (Baseline) and at 0.5, 1.5, 2.5, and, 24 hours after test article or vehicle administration. In addition, TRXE-002-1 was assessed in standard assays used to identify mutagenic potential using the bacterial reverse mutation assay. The potential for TRXE-0 2-1to induce micronuclei in the bone marrow of SPF Arc(S) Swiss mice was also investigated as a measure of clastogenicity. TRXE-002-1 toxicology was evaluated in MTD, and 28-day repeat dose GLP studies in rats and dogs. Results: TRXE-002-1 was found to have no effect on cardiac parameters of blood pressure and electrocardiography in dogs. TRXE-002-1 had no reverse mutagenic potential using the bacterial reverse mutation pre-incubation method ± S9 metabolic activation (Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and the WP2 uvrA Escherichia coli strain). Similarly, TRX-E-002-1 was shown to be non-mutagenic using the gene mutation test at the TK locus in L5178Y mouse lymphoma cell culture with and without metabolic activation. High doses of TRXE-002-1 (200 and 400 mg/kg) caused a significant increase in incidence of micronuclei in male and female mice. Repeat intraperitoneal injection of TRXE-002-1 to male and female dogs and rats over 28 days was well tolerated. Adverse TRXE-002 1-related effects were limited to microscopic findings in the testes and epididymides for male rats and dogs at high doses. Macroscopic observations at necropsy were confined to sporadic distention of the cecum and colon and colon obstruction in rats only (high dose) with no observed gastrointestinal microscopic findings reported in either species. Other observations were mostly resolved after a 14-day recovery period, except microscopic findings in the testes from both species. Conclusion: These data show that TRXE-002-1 has an acceptable toxicity profile in rats and dogs, and is without genotoxicity and cardiotoxicity at clinically relevant concentrations. TRXE-002-1 has been granted orphan drug status by the U.S. Food and Drug Administration (FDA) for ovarian cancer and a first-in-human clinical trial is planned for 2016. Citation Format: Kimberley Lilischkis, Andrew Heaton, Ayesha Alvero, Gil Mor, David M. Brown. Preclinical toxicology of TRXE-002-1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-201.

(13)C MRS and LC-MS Flux Analysis of Tumor Intermediary Metabolism.

We present the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism, including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, the TCA cycle as well as its anaplerotic pathways, pyruvate–malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. The model that is called Bonded Cumomer Analysis for application to 13C magnetic resonance spectroscopy (13C MRS) data and Fragmented Cumomer Analysis for mass spectrometric data is a refined and efficient form of isotopomer analysis that can readily be expanded to incorporate glycogen, phospholipid, and other pathways thereby encompassing all the key pathways of tumor intermediary metabolism. Validation was achieved by demonstrating agreement of experimental measurements of the metabolic rates of oxygen consumption, glucose consumption, lactate production, and glutamate pool size with independent measurements of these parameters in cultured human DB-1 melanoma cells. These cumomer models have been applied to studies of DB-1 melanoma and DLCL2 human diffuse large B-cell lymphoma cells in culture and as xenografts in nude mice at 9.4 T. The latter studies demonstrate the potential translation of these methods to in situ studies of human tumor metabolism by MRS with stable 13C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T). The melanoma studies indicate that this tumor line obtains 51% of its ATP by mitochondrial metabolism and 49% by glycolytic metabolism under both euglycemic (5 mM glucose) and hyperglycemic conditions (26 mM glucose). While a high level of glutamine uptake is detected corresponding to ~50% of TCA cycle flux under hyperglycemic conditions, and ~100% of TCA cycle flux under euglycemic conditions, glutaminolysis flux and its contributions to ATP synthesis were very small. Studies of human lymphoma cells demonstrated that inhibition of mammalian target of rapamycin (mTOR) signaling produced changes in flux through the glycolytic, pentose shunt, and TCA cycle pathways that were evident within 8 h of treatment and increased at 24 and 48 h. Lactate was demonstrated to be a suitable biomarker of mTOR inhibition that could readily be monitored by 1H MRS and perhaps also by FDG-PET and hyperpolarized 13C MRS methods.

In vitro and in vivo activity of melflufen (J1)in lymphoma.

BackgroundMelphalan has been used in the treatment of various hematologic malignancies for almost 60 years. Today it is part of standard therapy for multiple myeloma and also as part of myeloablative regimens in association with autologous allogenic stem cell transplantation. Melflufen (melphalan flufenamide ethyl ester, previously called J1) is an optimized derivative of melphalan providing targeted delivery of active metabolites to cells expressing aminopeptidases. The activity of melflufen has compared favorably with that of melphalan in a series of in vitro and in vivo experiments performed preferentially on different solid tumor models and multiple myeloma. Melflufen is currently being evaluated in a clinical phase I/II trial in relapsed or relapsed and refractory multiple myeloma.MethodsCytotoxicity of melflufen was assayed in lymphoma cell lines and in primary tumor cells with the Fluorometric Microculture Cytotoxicity Assay and cell cycle analyses was performed in two of the cell lines. Melflufen was also investigated in a xenograft model with subcutaneous lymphoma cells inoculated in mice.ResultsMelflufen showed activity with cytotoxic IC50-values in the submicromolar range (0.011-0.92 μM) in the cell lines, corresponding to a mean of 49-fold superiority (p < 0.001) in potency vs. melphalan. In the primary cultures melflufen yielded slightly lower IC50-values (2.7 nM to 0.55 μM) and an increased ratio vs. melphalan (range 13–455, average 108, p < 0.001). Treated cell lines exhibited a clear accumulation in the G2/M-phase of the cell cycle. Melflufen also showed significant activity and no, or minimal side effects in the xenografted animals.ConclusionThis study confirms previous reports of a targeting related potency superiority of melflufen compared to that of melphalan. Melflufen was active in cell lines and primary cultures of lymphoma cells, as well as in a xenograft model in mice and appears to be a candidate for further evaluation in the treatment of this group of malignant diseases.

LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and ¹³C-isotopic labeling of acyl-coenzyme A thioesters.

Acyl-coenzyme A (acyl-CoA) thioesters are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoA thioesters in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoA thioesters. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoA thioesters can be labeled with either a fixed [(13)C3(15)N1] label derived from pantothenate into the CoA moiety or via variable [(13)C] labeling into the acyl chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high-resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoA thioesters by stable isotope dilution using the [(13)C3(15)N1] label and measurement of the incorporation of labeled carbon atoms derived from [(13)C6]-glucose, [(13)C5(15)N2]-glutamine, and [(13)C3]-propionate. As a proof of principle, we applied this method to human B cell lymphoma (WSU-DLCL2) cells in culture to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope-resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoA thioesters.

A mix of S and ΔS variants of STAT3 enable survival of activated B-cell-like diffuse large B-cell lymphoma cells in culture.

Activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) is characterized by increased expression and activator of signal transducer and activator of transcription 3 (STAT3). ABC DLBCL cells require STAT3 for growth in culture. In ABC DLBCL cells, eosinophils and perhaps all cells, four variant STAT3 mRNAs (Sα, ΔSα, Sβ and ΔSβ) are present as a result of two alternative splicing events, one that results in the inclusion of a 55-residue C-terminal transactivation domain (α) or a truncated C-terminal domain with 7 unique residues (β) and a second that includes (S) or excludes (ΔS) the codon for Ser-701 in the linker between the SH2 and C-terminal domains. A substantial literature indicates that both α and β variants are required for optimal STAT3 function, but nothing is known about functions of ΔS variants. We used a knockdown/re-expression strategy to explore whether survival of ABC DLBCL cells requires that the four variants be in an appropriate ratio. No single variant rescued survival as well as STAT3Sα-C, Sα with activating mutations (A661C and N663C) in the SH2 domain. Better rescue was achieved when all four variants were re-expressed or Sα and ΔSα or Sβ and ΔSβ were re-expressed in pairs. Rescue correlated with expression of STAT3-sensitive genes NFKBIA and NFKBIZ. We consider a variety of explanations why a mix of S and ΔS variants of STAT3 should enable survival of ABC DLBCL cells.

BARs (B -cell receptor a ntigens for r everse targeting): A Novel and Ultimately Specific Treatment Concept for B-Cell Neoplasms

Introduction: The major task of a B-cell receptor is the binding and internalization of its antigenic target, and its processing for antigen presentation to T-cells. Chronic antigenic stimulation has been discussed to play a role in the pathogenesis of malignant B-cell lymphomas. We therefore systematically searched for the antigenic targets of BCRs from various B-cell neoplasms.Methods: Recombinant BCRs were expressed as recombinant Fabs (rFabs) based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA lymphoma biopsies. Whenever possible, "natural" Fabs (nFabs) were also obtained by papain digestion of fresh or cultured lymphoma cells. Both nFab and rFab were used to screen for binding to proteins expressed on macroarrays derived from human cDNA expression libraries and identical binding pattern of nFabs and rFabs was demonstrated by an antigen competition assay.Results: Two antigens (paratarg-7 and sumoylated HSP-90 which are hyperphosphorylated and sumoylated, respectively, in patients compared to healthy controls) are the targets of paraproteins from (depending on ethnicity) 30-50% of all multiple myeloma patients; the BCR from 67% of patients with primary CNS lymphoma target hyperglycosylated neurabin, 26% of the BCR from ABC-type DLBCL target hypophosphorylated ARS2 and 45% of all mantle cell BCR target LRPAP1; optineurin is the BCR target of 12% follicular lymphomas and various autoantigens have been identified as the targets of roughly 30% of all CLL cases. For all autoantigens binding to its specific BCR, rapid internalization and induction of proliferation was demonstrated, indicating partial dependence on antigenic stimulation even in cell lines that had been in culture for years. Most importantly, BCR-specific cytotoxicity of recombinant pseudomonas-exotoxin conjugated ARS2 against an ABC-DLBCL cell line with BCR specific for ARS2 (OCI-Ly3) was demonstrated in vitro and in vivo after establishment of OCI-Ly3 lymphomas in SCID beige mice.Conclusions: Assuming that only a minority of BCR targets have been identified to date, the prevalence of posttranslationally modified autoantigens strongly supports a role of chronic antigenic stimulation in many B-cell neoplasms. Due to the predominance of a single or few BCR antigens in each malignant B-cell entity studied, BARs represent an attractive and novel therapeutic concept for a broad spectrum of B-cell neoplasms and are the first therapeutic approach in oncology that targets exclusively the malignant cells. BARs can be used for conjugation with toxins, radionuclides and small molecules as well as for bispecific constructs (e. g. with CD3 or CD16) and CAR T-cells, the toxicity of which should be drastically reduced due to the ultimate specificity of BARs that spares normal B-cells. Supported by Wilhelm-Sander-Stiftung DisclosuresPfreundschuh:Roche, Janssen, Celgene: Honoraria, Research Funding.

Marsdenia tenacssima extract and its functional components inhibits proliferation and induces apoptosis of human Burkitt leukemia/lymphoma cells in vitro and in vivo

Burkitt lymphoma is a fast growing non-Hodgkin lymphoma that occurs primarily in young males. The causes of Burkitt lymphoma include chromosome rearrangement and virus infection, but accurate and complete reasons remain to be discovered. The available treatment for Burkitt lymphoma is chemotherapy and radiation therapy. It is a highly aggressive B-cell neoplasm with not all patients cured, in spite of current therapies. This study evaluated the effects of traditional Chinese medicine Marsdenia tenacssima (MTE) and its component compound Tenacigenoside A (TGTA) and 11α-O-benzoyl-12β-O-acetyltenacigenin B (TGTB) on human Burkitt lymphoma growth. It was observed that MTE, TGTA or TGTB inhibited cell growth and induced apoptosis of Burkitt lymphoma cells in culture. In lymphoma bearing NOD/SCID nude mice, both TGTA and TGTB inhibited tumor growth and improved animal survival. TGTA and TGTB significantly increased tumor cell apoptosis on lymphoma bearing mice, primarily through down-regulation of BCL2 and BCL-XL and up-regulation of BID.

Abstract 1756: Preclinical activity in non-Hodgkin's lymphoma of Selinexor, a selective inhibitor of nuclear export (SINE), is enhanced through combination with standard-of-care therapies

Abstract The nuclear export protein Exportin 1 (XPO1) is overexpressed in diffuse large B-cell lymphoma (DLBCL), follicular small cell cleaved Lymphoma (FSCCL) and a wide variety of other cancers, which often correlates with poor prognosis. Selinexor is an oral SINE currently in Phase 1/2 clinical testing, which targets XPO1 to induce apoptosis across a broad spectrum of tumor types. This broad action is primarily due to forced nuclear retention and reactivation of tumor suppressor proteins (TSPs), resulting in selective tumor cell death. We have previously reported anti-lymphoma activity of selinexor in murine models of DLBCL, in spontaneous canine aggressive lymphomas and in preliminary results from B-cell lymphoma patients enrolled in a Phase 1 trial of hematological malignancies. One promising finding thus far is that efficacy of selinexor appears to be independent of the presence of the mutations in MYC and BCL2/6 found in “double-hit” DLBCL, which is typically resistant to standard treatments. Here we report combination studies involving Selinexor-Dexamethasone and Selinexor-mTOR inhibitor everolimus in Non Hodgkin's lymphoma relative to front-line standard of care. Selinexor was found to be potently cytotoxic across a broad panel of B-cell lymphoma cell lines. Furthermore, combination of selinexor with dexamethasone (Dex) or the mTOR inhibitor everolimus resulted in synergistic cytotoxicity in the WSU-DLCL2 and WSU-FSCCL cell lines. The superior cytotoxicity observed in the combination was consistent with enhanced nuclear retention of different TSPs. In WSU-DLCL2 xenografts, analogs of selinexor KPT-251 and KPT-276 showed comparable efficacy to R-CHOP and in a model of disseminated follicular lymphoma using WSU-FSCCL cells, selinexor had similar efficacy to rituximab for extending survival. The combination of selinexor and Dex or selinexor and everolimus in both xenografts are ongoing. B-cell lymphoma cells in culture are highly sensitive to selinexor. Furthermore, combination of selinexor with Dex or everolimus is synergistically toxic to DLBCL cells. This synergism is consistent with the mechanism of action of these drugs in that both selinexor and Dex antagonize NF-κB and mTOR is an XPO1 cargo protein. The comparable in vivo activity of selinexor and related SINE compounds relative standard of care is consistent with the observations of objective responses in B-cell lymphoma patients in Phase I clinical testing. Ongoing preclinical work is designed to extend comparisons of selinexor with other standard of care and broaden the characterization of combination therapies in an effort to provide a rational basis for studying specific combinations in B-cell lymphoma clinical trials. Citation Format: Asfar S. Azmi, Amro Aboukameel, Robert O. Carlson, Sivan Elloul, Sharon Shacham, Michael Kauffman, Ran Frenkel, Ramzi M. Mohammad. Preclinical activity in non-Hodgkin's lymphoma of Selinexor, a selective inhibitor of nuclear export (SINE), is enhanced through combination with standard-of-care therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1756. doi:10.1158/1538-7445.AM2015-1756

Abstract 136: Evaluation of reversible BTK inhibitors in cell lines with clinically relevant Ibrutinib resistance mutations

Abstract Background Ibrutinib, a small molecule inhibitor that covalently binds Cys481 of Bruton's Tyrosine Kinase (BTK), has recently been approved in Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). Although the majority of patients respond to this drug, eventually resistance develops. Known causes for resistance include the outgrowth of cancer cells with mutations in either the drug binding pocket of BTK or activating mutations in the downstream effector, PLCγ2. Objective To explore mechanisms of resistance to ibrutinib and evaluate the potential of next generation BTK inhibitors on ibrutinib resistant cancer, we selected for resistance in cultured lymphoma cells. We chose TMD8, a cell line model of diffuse large B-cell lymphoma (DLBCL) that has constitutive B-cell receptor signaling, and REC-1, an ibrutinib sensitive MCL line, to model resistance. Methods TMD8 and REC-1 were cultured in increasing concentrations of ibrutinib to generate resistant cell lines. Genetic changes that may account for resistance were evaluated using whole exome sequencing. Resistant cell lines were evaluated for sensitivity to a small panel of BTK inhibitors. Results We generated a TMD8 derived cell line that is 1000-fold resistant to ibrutinib. Whole exome sequencing revealed a C481Y mutation in BTK. Reversible inhibitors that are not reliant on this site may function as alternative inhibitors of C481 mutant BTK. Ibrutinib-resistant TMD8 cells were tested for response to novel, reversible BTK inhibitors. The potency was similar to or modestly reduced (3 to 10 fold) on the ibrutinib-resistant cells compared to the parental line. In addition, we generated ibrutinib-resistant cells derived from the REC-1 line. Unlike resistance generated in TMD8, REC-1 resistant cells harbor a L527W mutation in BTK and are resistant to all BTK inhibitors that we evaluated. Conclusions C481Y and L527W BTK mutations have been identified in CLL patients that have relapsed after response to ibrutinib, and C481S mutations have been identified in ibrutinib relapsed CLL and MCL patients. Our data suggest that reversible, next generation BTK inhibitors have the potential to be efficacious on ibrutinib relapsed patients that harbor mutations at C481 of BTK. Citation Format: Regina Wai-Yan Choy, Luciana Burton, Wendy Young, James J. Crawford, Elicia Penuel, Lisa D. Belmont. Evaluation of reversible BTK inhibitors in cell lines with clinically relevant Ibrutinib resistance mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 136. doi:10.1158/1538-7445.AM2015-136

Abstract B47: mTOR C1/2 activities - related protein expression and its potential prognostic/therapeutic importance in certain lymphoid malignancies

Abstract There is increasing attention for PI3K/Akt/mTOR pathway as a potential target in tumor therapy. The high activity of mTOR is a characteristic feature of many tumors including hematological malignancies. However, the pathological characterisation of mTOR activity of lymphoid malignancies - especially the activity of different complexes – is still incomplete. We studied the mTOR activity related phospho-proteins and the elements of mTOR complexes by using different techniques (ELISA, Western blot, different immunohistochemical stainings, flow cytometry) depending on the tumor sample type (fresh/fixed human tissues, isolated human cells from blood or bone marrow, cell lines). The expression and activity results were compared in order to find correlation with clinical data or to mTOR inhibitor sensitivity in vitro. The proliferation and apoptotic effects after in vitro treatments were detected by Alamar Blue assay and flow cytometry. We established the in situ measurement for the amount of mTORC1, C2 complexes by Duolink stainings to compare the effects of different mTOR inhibitors to the mTORC1/C2 complex availability in lymphoma cell cultures. Our studies described high mTOR activity in all acute lymphoid leukemia (ALL) cases and in the majority of mantle cell lymphomas, anaplastic large cell lymphomas, Burkitt lymphomas, diffuse large B cell lymphomas (DLBCL), Hodgkin lymphomas (HL) and in certain cutan T lymphomas. Further characterisation of HLs, DLBCLs and ALL cases showed that high mTOR activity especially with the presence of mTORC2 is a sign of unfavorable prognosis. mTOR activity related to p-S6 and p-4EBP1 expression (ELISA and flow cytometry) at diagnosis was significantly higher in patients with poor prognosis in ALL cases. In DLBCL patient's the results showed that high mTOR activity with Rictor overexpression significantly correlated to the worse prognosis and shorter survival. We also detected the sign of high mTOR activity in more than 90% of HLs (HLs usually with favorable prognosis), however, in these cases Rictor was not found (the mTOR activity related to mTORC1). In the different lymphoma cell lines increased mTOR activity was detected and we could describe different Rictor, Raptor and mTOR-Rictor complex expression. Moreover, the inhibitor sensitivity of the cell lines and the different expression of the elements related to mTORC1 and C2 correlated. Our results confirmed the increased mTOR activity related to mTORC1 and/or C2 complexes in certain lymphomas and ALLs. According to these analyses, the activity and expression of mTOR complexes should have potential prognostic value and high importance before mTOR inhibitor treatment, especially in the cases with high mTORC2 related protein expression. The study was supported by OTKA816624, OTKA 84262 projects and Tumor Progression Research Group of Joint Research Organization of the Hungarian Academy of Sciences and Semmelweis University Citation Format: Anna Sebestyén, Noémi Nagy, Ágnes Márk, Anna Molnár, Titanilla Dankó, Melinda Hajdu, Mónika Tóth, Botond Timár, Mónika Csóka, László Kopper. mTOR C1/2 activities - related protein expression and its potential prognostic/therapeutic importance in certain lymphoid malignancies. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B47.

IL-10 induces the development of immunosuppressive CD14(+)HLA-DR(low/-) monocytes in B-cell non-Hodgkin lymphoma.

The biological role of monocytes and macrophages in B-cell non-Hodgkin lymphoma (NHL) is not fully understood. We have previously reported that monocytes from patients with B-cell NHL have an immunosuppressive CD14+HLA-DRlow/− phenotype that correlates with a poor prognosis. However, the underlying mechanism by which CD14+HLA-DRlow/− monocytes develop in lymphoma is unknown. In the present study, we found that interleukin (IL)-10, which is increased in the serum of patients with B-cell NHL, induced the development of the CD4+HLA-DRlow/− population. Using peripheral blood samples from patients with B-cell NHL, we found that absolute numbers of CD14+ monocytic cells with an HLA-DRlow/− phenotype were higher than healthy controls and correlated with a higher International Prognostic Index score. IL-10 serum levels were elevated in lymphoma patients compared with controls and were associated with increased peripheral monocyte counts. Treatment of monocytes with IL-10 in vitro significantly decreased HLA-DR expression and resulted in the expansion of CD14+HLA-DRlow/− population. We found that lymphoma B cells produce IL-10 and supernatants from cultured lymphoma cells increased the CD14+HLA-DRlow/− population. Furthermore, we found that IL-10-induced CD14+HLA-DRlow/− monocytes inhibited the activation and proliferation of T cells. Taken together, these results suggest that elevated IL-10 serum levels contribute to increased numbers of immunosuppressive CD14+HLA-DRlow/− monocytes in B-cell NHL.

Immune independent crosstalk between lymphoma and myeloid suppressor CD14+HLA-DRlow/neg monocytes mediates chemotherapy resistance

We have previously reported a novel phenotype of myeloid suppressors in lymphoma patients characterized by a loss of HLA-DR expression on monocytes, CD14+HLA-DRlow/neg. These cells were directly immunosuppressive and were associated with poor clinical outcome. In this study, we found that lymphoma tumors could have more than 30% of their tumor occupied by CD14+ cells. This intimate spatial connection suggested substantial cell–cell communication. We examined cross talk between monocytes from healthy volunteers (normal) and lymphoma cells in co-culture to identify the mechanisms and consequences of these interactions. Normal CD14+HLA-DR+ monocytes lost their HLA-DR expression after co-culture with lymphoma cells. Lymphoma-converted CD14+HLA-DRlow/neg cells exhibited similar immunosuppressive functions as CD14+HLA-DRlow/neg monocytes from lymphoma patients. Unexpectedly monocyte additions to lymphoma cell cultures protected lymphoma from cytotoxic killing by chemotherapy drug doxorubicin (DOX). Monocyte mediated resistance to DOX killing was associated with decreased Caspase-3 activity and increased anti-apoptotic heat shock protein-27 (Hsp27) expression. Soluble Hsp27 was detected in supernatant and patient plasma. Increased Hsp27 in plasma correlated with increased proportion of CD14+HLA-DRlow/neg monocytes in patient blood and was associated with lack of clinical response to DOX. This is the first report to describe a non-immune function of CD14+HLA-DRlow/neg monocytes: enhanced lymphoma resistance to chemotherapy. It is also the first report in lymphoma of Hsp27 as a potential mediator of lymphoma and monocyte crosstalk and chemotherapy resistance. Together with previous reports of the prevalence of these myeloid suppressors in other cancers, our findings identify this pathway and these interactions as a potential novel therapeutic target.

IL-10 Contributes to the Development of Immunosuppressive CD14+HLA-DRlow/- monocytes in B-Cell Non-Hodgkin’s Lymphoma

Background - The phenotype and biological role of monocytes in B-cell non-Hodgkin lymphoma (NHL) is not fully understood, however, an increased absolute monocyte count in the peripheral blood of lymphoma patients is associated with a poor prognosis. We have previously reported that monocytes from patients with relapsed B-cell NHL displayed an immunosuppressive CD14+HLA-DRlow/- phenotype that correlated with a poor prognosis. However, the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop in patients with B-cell NHL is unknown. The goal of this study was to determine whether cytokines are responsible for the increased number and phenotype of CD14+HLA-DRlow/-monocytes present in lymphoma patients.Methods – Whole blood from patients with newly diagnosed, untreated B-cell NHL (n=20) and healthy donors (n=20) was stained with a panel of antibodies and analyzed by 10-color flow cytometry. Cytokine levels in serum and culture supernatants were measured by Luminex and ELISA, respectively. Polarization of monocytes from healthy donors was performed by incubating CD14+ cells with specific cytokines or supernatants from B-cell NHL cell lines for 24 hours. Activation and proliferation of CD4+T cells cocultured with IL-10-pretreated monocytes were measured by flow cytometry.Results – By flow cytometry, we observed that the absolute number of peripheral monocytes was increased in newly-diagnosed lymphoma patients compared to healthy donors. A significant proportion of these monocytes displayed an immunosuppressive CD14+HLA-DRlow/- phenotype and the numbers of CD14+HLA-DRlow/- cells were significantly higher in lymphoma patients than in healthy donors. To identify the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop thereby leading to increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL, we tested a panel of cytokines and found that IL-10 played a key role in the process. Firstly, treatment of monocytes with IL-10 in vitro resulted in the generation of CD14+HLA-DRlow/- cells. Second, monocytes co-cultured with IL-10 producing lymphoma B-cells, or treated with supernatants from lymphoma cell cultures, developed a CD14+HLA-DRlow/- phenotype. Thirdly, IL-10 levels were increased in the serum of DLBCL (p<0.0001) and FL patients (p=0.010) compared to healthy controls, and serum IL-10 levels correlated with increased numbers of peripheral monocytes (p=0.02). Finally, IL-10-pretreated CD14+HLA-DRlow/- monocytes were significantly immunosuppressive and inhibited the proliferation and activation of CD4+T cells in co-culture assays.Conclusions- Taken together, our results suggest that IL-10 signaling contributes to the increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL. Strategies to inhibit IL-10 production may therefore have therapeutic potential in B-cell NHL. DisclosuresNo relevant conflicts of interest to declare.