T-lymphoma Cell Line Research Articles

PS1322 ASTX660, A NON‐PEPTIDOMIMETIC ANTAGONIST OF CIAP1/2 AND XIAP, INDUCES APOPTOSIS IN T CELL LYMPHOMA BY ENHANCING IMMUNE MEDIATED AND DEATH RECEPTOR DEPENDENT KILLING

Background:ASTX660 is a potent, non‐peptidomimetic antagonist of the cellular and X‐linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which is currently being tested in a first in human phase I‐II study in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis and are potent anti‐tumor immune enhancers.Aims:Herein, we describe the profile of ASTX660 in a range of T cell lymphoma (TCL) cell lines and evaluate ASTX660's ability to enhance immune mediated killing of tumor cells.Methods:A panel of human and mouse T‐cell lymphoma cell lines were tested in proliferation (Alamar blue or CellTiterGlo) or apoptosis assays (activated caspase‐3 substrate assays by IncuCyte or FACS) for sensitivity to ASTX660 alone or in combination with recombinant Death Receptor ligands (TNFα, FASL or TRAIL). Additionally, we used a novel co‐culture system of tumor cell lines with anti‐CD3 activated human peripheral blood mononuclear cells (PBMC) to assess ASTX660 effects on immune mediated cell killing. Target engagement and induction of apoptosis markers were analysed by Western blotting.Results:ASTX660 antagonises IAPs in TCL cell lines, as indicated by a decrease in cIAP1 protein levels. This ASTX660‐dependent decrease in cIAP was associated with an increase in TNFα‐dependent apoptosis in the EL4 and L5178 TCL cell lines. Several T‐cell lymphoma models, including HuT‐78, HH and My‐La expressed low levels of TNFR1 and therefore did not respond to ASTX660 in the presence of TNFα. However, in these cell lines, ASTX660 conferred a significant increase in FASL or TRAIL‐dependent apoptosis, indicating that ASTX660 sensitises TCL cells to various death receptor ligands and response correlates with receptor expression levels. In addition to this direct effect on TCL cell lines, ASTX660 also enhances anti‐CD3 stimulated PBMC‐dependent killing of multiple tumour cell lines, including TCL lines, via induction of caspase activity. Additional preclinical experiments (both in vitro and in vivo) are underway to further characterise the mode of action of ASTX660 in TCL.Summary/Conclusion:The combination of both direct and indirect effects of ASTX660 on TCL lines, described here, supports the ongoing clinical testing of ASTX660 in TCL (NCT02503423). Preliminary clinical efficacy and safety data of ASTX660 in relapsed/refractory (r/r) peripheral T cell lymphoma and cutaneous T cell lymphoma is the subject of a separate abstract.

Combined Hypomethylating Agents (HMA) and Histone Deacetylase Inhibitors (HDACi) Exhibit Compelling Activity in Patients with Peripheral T-Cell Lymphoma (PTCL) with High Complete Response Rates in Angioimmunoblastic T-Cell Lymphoma (AITL)

Introduction: PTCL may represent the prototypical epigenetic malignant disorder. First, recurring mutations in important epigenetic regulators, such as TET2, IDH2, and DNMT3, have been described across several PTCL subtypes, though especially in AITL. Second, HDACi have only been approved as single agents in patients with relapsed/refractory (R/R) PTCL, and exhibit T-cell lineage-specific activity across disease subtypes. Thirdly, preclinical evidence from our group suggests marked class synergism between HDACi and HMA. In addition, across a panel of diverse T-cell lymphoma lines, the combination of HDACi and HMA induced the expression of many cancer testis antigens and genes involved in the interferon pathway/viral antigen response. Based on this collective experience, a phase 1/2 trial of romidepsin (ROMI) and oral 5-azacitidine (AZA) was launched to determine if this activity is reproduced in patients with R/R PTCL.Methods: Patients with R/R lymphoma were eligible for the phase 1, whereas the phase 2 only enrolled patients with PTCL, and included both R/R and treatment-naïve individuals. The dose-escalation portion of the study followed a 3+3 design with progressively increasing dose intensity across 7 cohorts. The phase 1 primary objectives were determination of the maximum tolerated dose and dose-limiting toxicity. Phase 2 primary objectives were overall response rate (ORR, i.e., complete response [CR] + partial response [PR]), progression free survival, and duration of response (DOR).Results: 36 patients have been enrolled to date, 26 in phase 1 and 10 in phase 2. In the intention-to-treat population the median age was 56 years [23-83] with 59% being males. Twelve patients had Hodgkin lymphoma, 8 had a B-cell lymphoma, and 16 had a T-cell lymphoma (6 enrolled in the phase 1 and 10 in phase 2). The median number of prior therapies was 6 [1-15] for the phase 1 population and 1 [0-6] for the phase 2.After a median of 2 cycles [0-16], all phase 1 patients have discontinued therapy, whereas 6 out of 10 patients in phase 2 are still on treatment after a median of 2.5 cycles [1-14]. No patient discontinued therapy due to adverse events (AE). The most frequent hematologic G3-4 AE included neutropenia (39%), lymphopenia (39%), and thrombocytopenia (28%). The most frequent non-hematologic G3-4 AE included febrile neutropenia (8%), hyponatremia (5%), and lung infection (5%). Other common G1-2 toxicities included hyperglycemia, hypoalbuminemia, nausea/vomiting, and fatigue. The recommended phase 2 dose for the combination was declared AZA 300 mg days 1-14 and ROMI 14 mg/m2 days 8, 15, and 22 on a 35-day cycle.Thirty-two patients are evaluable for response. Of these, 27 had measurable disease. The ORR and CR in the overall population are 41% and 22%, respectively. However, while only 2 (11%) patients with non-T-cell lymphoma responded, of which one was a CR, 11 of 14 (79%) patients with T-cell lymphoma responded, including 6 (43%) who attained CR (see waterfall plot in figure). Notably, all 6 evaluable patients with AITL responded, and 3 achieved a CR. Two of these 3 achieved PR before reaching CR. Responses have been durable and the median DOR has not been reached [0.2-13.1+ months]. The AUC for ROMI at 10 mg/m2 (N = 15) and 14 mg/m2 (N = 20) were 2021.5 +/- 1461.3 h*ng/mL and 1565.7 +/- 5656.2 h*ng/mL, respectively, with a median half-life of 4.8 and 4.9 hours respectively, which is comparable to single-agent values.Conclusions: The combination of AZA and ROMI is well tolerated, with cytopenias being the most common G3-4 AE. The combination appears to exhibit marked T-cell lineage-specific activity. The 100% ORR in AITL patients is unprecedented and warrants detailed follow-up. Ongoing sequencing analysis will evaluate the impact of recurring mutations on the clinical activity of the combination. The study is actively accruing (NCT01998035). [Display omitted] DisclosuresO'Connor:ADC Therapeutics: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding.

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

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

Establishment of engineered cell-based assays mediating LAG3 and PD1 immune suppression enables potency measurement of blocking antibodies and assessment of signal transduction

LAG3 is an important regulator of T cell homeostasis and studies in mouse tumor models have demonstrated that simultaneously antagonizing LAG3 and PD1 can augment tumor-specific T cell responses and induce tumor rejection. The combined use of LAG3 antagonist antibodies with established anti-PD1 therapies is currently being evaluated in human clinical trials. A functional assay for human LAG3 was developed by co-culture of a Jurkat T-cell lymphoma line overexpressing LAG3 with a Raji B-cell lymphoma line in the presence of staphylococcal enterotoxins. Reversal of LAG3 repression was measured as an increase in IL-2 production or NFAT activation in response to treatment with MK-4280, an anti-human LAG3 antagonist antibody. Changes in cytokines, chemokines, and other mRNA transcripts were in agreement with published in vitro and in vivo models for LAG3 biology which highlights the physiological relevance of the Jurkat functional assay. Additional engineering of PD1 and PDL1 components into the LAG3 assay resulted in a bi-functional assay that is capable of inducing a 10-fold response to individual antibodies blocking either PD1 or LAG3. Importantly, when MK-4280 and pembrolizumab were combined to block both pathways, a synergistic 50-fold increase in response was observed.

Cell-Penetrating CaCO₃ Nanocrystals for Improved Transport of NVP-BEZ235 across Membrane Barrier in T-Cell Lymphoma.

Owing to their nano-sized porous structure, CaCO3 nanocrystals (CaCO3NCs) hold the promise to be utilized as desired materials for encapsulating molecules which demonstrate wide promise in drug delivery. We evaluate the possibility to encapsulate and release NVP-BEZ235, a novel and potent dual PI3K/mTOR inhibitor that is currently in phase I/II clinical trials for advanced solid tumors, from the CaCO3NCs. Its chemical nature shows some intrinsic limitations which induce to administer high doses leading to toxicity; to overcome these problems, here we proposed a strategy to enhance its intracellular penetration and its biological activity. Pristine CaCO3 NCs biocompatibility, cell interactions and internalization in in vitro experiments on T-cell lymphoma line, were studied. Confocal microscopy was used to monitor NCs-cell interactions and cellular uptake. We have further investigated the interaction nature and release mechanism of drug loaded/released within/from the NCs using an alternative approach based on liquid chromatography coupled to mass spectrometry. Our approach provides a good loading efficiency, therefore this drug delivery system was validated for biological activity in T-cell lymphoma: the anti-proliferative test and western blot results are very interesting because the proposed nano-formulation has an efficiency higher than free drug at the same nominal concentration.

Antiproliferative and cytotoxic activities of furocoumarins of Ducrosia anethifolia

Context: Phytochemical and pharmacological data on Ducrosia anethifolia (DC.) Boiss. (Apiaceae), an Iranian medicinal plant, are scarce; however, furocoumarins are characteristic compounds of D. anethifolia.Objective: Our experiments identify the secondary metabolites of D. anethifolia and assess their antitumor and anti-multidrug resistance activities.Materials and methods: Pure compounds were isolated from the extract of aerial parts of the plant by chromatographic methods. Bioactivities were tested on multidrug resistant and sensitive mouse T-lymphoma cell lines. The inhibition of the cancer MDR efflux pump ABCB1 was evaluated by flow cytometry (at 2 and 20 µM). A checkerboard microplate method was applied to study the interactions of furocoumarins and doxorubicin. Toxicity was studied using normal murine NIH/3T3 fibroblasts.Results: Thirteen pure compounds were isolated, nine furocoumarins namely, pabulenol (1), (+)-oxypeucedanin hydrate (2), oxypeucedanin (3), oxypeucedanin methanolate (4), (−)-oxypeucedanin hydrate (5), imperatorin (6), isogospherol (7), heraclenin (8), heraclenol (9), along with vanillic aldehyde (10), harmine (11), 3-hydroxy-α-ionone (12) and 2-C-methyl-erythrytol (13). Oxypeucedanin showed the highest in vitro antiproliferative and cytotoxic activity against parent (IC50 = 25.98 ± 1.27, 40.33 ± 0.63 µM) and multidrug resistant cells (IC50 = 28.89 ± 0.73, 66.68 ± 0.00 µM), respectively, and exhibited slight toxicity on normal murine fibroblasts (IC50 = 57.18 ± 3.91 µM).Discussion and conclusions: Compounds 2, 3, 5, 7, 10–13 were identified for the first time from the Ducrosia genus. Here, we report a comprehensive in vitro assessment of the antitumor activities of D. anethifolia furocoumarins. Oxypeucedanin is a promising compound for further investigations for its anticancer effects.

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

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

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

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

Regulator of Calcineurin 3 Ameliorates Autoimmune Arthritis by Suppressing Th17 Cell Differentiation

Regulator of calcineurin 3 (RCAN3), an endogenous regulator of the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, inhibits the phosphatase activity of calcineurin, the nuclear translocation of NFAT, and the NFAT downstream pathway. To investigate the effects of RCAN3 on T-cell regulatory function and the development and progression of inflammatory arthritis, we studied the effects of RCAN3 transfection on regulation of Th17 cell differentiation in a murine T-lymphoma cell line and primary splenic CD4+ T cells. Overexpression of RCAN3 suppressed Th17 cell differentiation through the down-regulation of RAR receptor orphan receptor γT mRNA and up-regulation of forkhead box P3 mRNA. In mice with collagen-induced arthritis, injection of an RCAN3-overexpression vector controlled arthritis development invivo. Injection ofRCAN3 reduced the formation of osteoclasts and expression of inflammatory cytokines invivo. Antioxidants stimulated the expression of RCAN3 invitro, and combination therapy with pcDNA-RCAN3 had a synergistic suppressive effect on the development of arthritis. These data suggest that RCAN3 may be an effective treatment for rheumatoid arthritis.

Exploring Jolkinol D Derivatives To Overcome Multidrug Resistance in Cancer.

Macrocyclic monoacyl lathyrane derivatives bearing a benzoyl moiety were previously found to be strong ABCB1 modulators. To explore the effects of different substituents of the aromatic moiety, 14 new compounds (1.1-1.7, 1.10, and 2.1-2.4) were prepared from jolkinol D (1), obtained from Euphorbia piscatoria, and from jolkinodiol (2), its hydrolysis derivative. Compounds 1.8 and 1.9, having aliphatic moieties, were also obtained. The reversal of ABCB1-mediated MDR was evaluated through functional and chemosensitivity assays on the human ABCB1-gene-transfected L5178Y mouse T-lymphoma cell line. Structure-activity relationships showed that addition of electron-donating groups to the aromatic moiety improved the activity. The effects on the ATPase activity of the strongest modulator (1.3) and the inactive jolkinol D (1) were also investigated and compared. Moreover, in the chemosensitivity assay, most of the compounds interacted synergistically with doxorubicin. Compounds 1.1-1.10 and 2.1-2.4 were further assessed for their collateral sensitivity effect against the human cancer cells: EPG85-257 (gastric) and EPP85-181 (pancreatic), and the matching drug-selected cells EPG85-257RDB, EPG85-257RNOV, EPP85-181RDB, and EPP85-181RNOV. The most promising ones (1.8 and 1.10) along with compound 3, previously selected, were investigated as apoptosis inducers. The compounds were able to induce apoptosis through caspase-3 activation, with significant differences being observed between the parental and resistant cells.

Histone-Modifying Gene Mutations Are Related to Tumor Progression and Response to Histone Deacetylase Inhibitors in Patients with Peripheral T-Cell Lymphoma Not Otherwise Specified

ObjectiveHistone modifications are critically involved in pathogenesis of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS).Histonedeacetylaseinhibitors (HDACIs) exhibited potential anti-lymphoma activity through restoring normal chromatin modification patterns. Here we detected histone-modifying gene mutations in PTCL-NOS and correlated them with tumor progression and therapeutic response to different HDACIs.MethodsMutations of important lymphoma-associated histone-modifying genes (MLL2, TET2, CREBBP, EP300, SETD2 and EZH2) were detected by targeted sequencing in tumor samples of 105 newly diagnosed patients with PTCL-NOS and compared with clinical parameters. Moreover, T-lymphoma cell lines bearing representative mutations were treated with four clinically available HDACIs (valproic acid, VPA;suberoylanilidehydroxamic acid, SAHA;Romidepsin,Romi; andChidamide,Chid).ResultsA total of 65 histone-modifying gene mutations were identified in 39 of 105 PTCL patients (Figure 1A). As illustrated in Figure 1B, somatic mutations in MLL2 (encoding H3K4-specific methyltransferase) were most frequently observed (20.0%), followed by mutations of TET2 (13.3%), EP300 and CREBBP (encoding H3K18 acetyltransferase, 7.6% and 6.7%, respectively), and SETD2 (encoding H3K36 methyltransferase, 7.6%). No EZH2 mutation was detected. Clinically, mutation-positive PTCL patients presented with shorteroverall survival than patients without mutations (P=0.0038, Figure 1C).Based on the crystal structure of each gene (Figure 1D), most mutations were located on SET domain of MLL2 (p.5397-5513), HAT domain of EP300 (p.1287-1663) and SET domain of SETD2 (p.1550-1667), which could lead to loss of H3K18ac, H3K4me3 and H3K36me3 expression, respectively.Meanwhile, Jurkatcells were treated with VPA, SAHA, Romiand Chidfor 48h and the results showed that all the HDACIs increased the expression of EP300/CREBBP, as compared to the untreated group. Interestingly, VPA and SAHA specifically upregulated SETD2 expression, while Romi and Chid upregulated MLL2 expression (Figure 2A). No obvious change of TET2 expression was found during HDACI treatment. Thereafter,Jurkat cells were transfected with MLL2 mutant (p.V5389M), EP300 mutant (p.H1377R) and SETD2 mutant (p.R1598_). Compared with the WT, mutant reduced the level of H3K18ac, H3K4me3 and H3K36me3 expression, respectively. All the HDACIs restored H3K18 hypo-acetylation induced by EP300 mutant. VPA and SAHA restored H3K36me3 expression inhibited by SETD2 mutant, whileRomi andChid restored H3K4me3 expression inhibited by MLL2 mutant (Figure 2B-2D).ConclusionOur study provided the mutational spectrum of histone-modifying genes in PTCL-NOS. HDACIs targeted differently histone H3 acetylation or methylation modulated by the mutations, suggestive their distinct therapeutic efficiency in PTCL-NOS. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparison of the effect of multidrug resistance modifiers on prostate cancer, mouse lymphoma and colon cancer cells

The reason for unsuccessful tumor chemotherapy is related to multidrug resistance. An important factor is the overexpression of efflux pumps, such as P-glycoprotein. Amino- and amide-substituted steroid compounds and phenothiazine derivatives were investigated in tumor models in vitro. The inhibition of P-glycoprotein was evaluated by flow cytometry and the interaction of these compounds with doxorubicin was investigated as well. Molecular docking was used to estimate the binding energies of the compounds to P-glycoprotein. The aminosteroids showed anticancer activity on multidrug resistant mouse T-lymphoma and prostate cancer cell lines. The combination of steroids and doxorubicin potentiated its effect in hormone resistant prostate cancer cells. Among the N-hydroxyalkyl-2-aminophenothiazines, secondary amines exhibited anticancer effects on multidrug resistant colon adenocarcinoma cells. The tested phenothiazine and steroid derivatives showed potent anticancer activity, furthermore, the stereoisomerism of thioridazine did not play a role in the antitumor properties. Neither steroids nor thioridazine influenced apoptosis in hormone resistant cells. Orv. Hetil., 2016, 157(37), 1489-1495.

Triterpenoids from Momordica balsamina: Reversal of ABCB1-mediated multidrug resistance

The ability as P-glycoprotein (P-gp, ABCB1) modulators of thirty (1–30) triterpenoids of the cucurbitane-type was evaluated on human L5178 mouse T-lymphoma cell line transfected with the human MDR1 gene, through the rhodamine-123 exclusion assay. Compounds (1–26, and 29, 30) were previously obtained from the African medicinal plant Momordica balsamina, through both isolation (1–15) and molecular derivatization (16–26 and 29, 30). Compounds 27–28 are two new karavilagenin C (34) derivatives having succinic acid moieties. Apart from 4, 6, 8, 10 and 11, most of the isolated compounds (1–15) displayed strong MDR reversing activity in a dose-dependent mode, exhibiting a many-fold activity when compared with verapamil, used as positive control. At the lowest concentration tested, compounds 2 and 7 were the most active. However, a decrease of activity was found for the acyl derivatives (16–30). In a chemosensitivity assay, the MDR reversing activity of some of the most active compounds (1–3, 5, 7, 12–15) was further assessed on the same cell model. All the tested compounds, excepting 15, corroborated the results of the transport assay, revealing to synergistically interact with doxorubicin. Structure–activity relationship studies, taking into account previous results, showed that different substitution patterns, at both the tetracyclic nucleus and the side chain, play important role in ABCB1 reversal activity. An optimal lipophilicity was also recognized.

Mammalian cell cultures as models for Mycobacterium tuberculosis –human immunodeficiency virus (HIV) interaction studies: A review

Mycobacterium tuberculosis and human immunodeficiency virus (HIV) co-infections have remained a major public health concern worldwide, particularly in Southern Africa. Yet our understanding of the molecular interactions between the pathogens has remained poor due to lack of suitable preclinical models for such studies. We reviewed the use, this far, of mammalian cell culture models in HIV-MTB interaction studies. Studies have described the use of primary human cell cultures, including (1) monocyte-derived macrophage (MDM) fractions of peripheral blood mononuclear cell (PBMC), alveolar macrophages (AM), (2) cell lines such as the monocyte-derived macrophage cell line (U937), T lymphocyte cell lines (CEMx174, ESAT-6-specific CD4(+) T-cells) and an alveolar epithelial cell line (A549) and (3) special models such as stem cells, three dimensional (3D) or organoid cell models (including a blood-brain barrier cell model) in HIV-MTB interaction studies. The use of cell cultures from other mammals, including: mouse cell lines [macrophage cell lines RAW 264.7 and J774.2, fibroblast cell lines (NIH 3T3, C3H clones), embryonic fibroblast cell lines and T-lymphoma cell lines (S1A.TB, TIMI.4 and R1.1)]; rat (T cells: Rat2, RGE, XC and HH16, and alveolar cells: NR8383) and primary guinea pigs derived AMs, in HIV-MTB studies is also described. Given the spectrum of the models available, cell cultures offer great potential for host-HIV-MTB interactions studies.

Reversal of Multidrug Resistance and Computational Studies of Pistagremic Acid Isolated from Pistacia integerrima.

Pistagremic acid (PA) is a bioactive triterpenoid isolated from various parts of Pistacia integerrima plants. The aim of this research was to investigate PA for reversion of multidrug resistant (MDR) mediated by P-glycoprotein using rhodamine-123 exclusion study on a multidrug resistant human ABCB1 (ATP-binding cassette, sub-family B, member 1) gene-transfected mouse T-lymphoma cell line in vitro. Results were similar to those with verapamil as a positive control. Docking studies of PA and standard Rhodamine123 were carried out against a P-gp crystal structure which showed satisfactory results. Actually, PA cannot bind exactly where co-crystallized ligand of P-gp is already present. However, the docking study predicted that if a compound gives a lesser score then it may have some potency. The docking scores of PA and Rhodamine were similar. Therefore, we can conclude that there are certain important chemical features of PA which are responsible for the inhibiting potency of P-gp.

Isolation and Structure Elucidation, Molecular Docking Studies of Screlotiumol from Soil Borne Fungi Screlotium rolfsii and their Reversal of Multidrug Resistance in Mouse Lymphoma Cells.

A new compound namely (13-(3,3-dihydroxypropyl)-1,6-dihydroxy-3,4-dihydro-1H-isochromen-8(5H)-one (1) was isolated from an ethyl acetate extract of the borne fungi Screlotium rolfsii. Its chemical structure was elucidated by spectroscopic analysis. Screlotiumol 1 were evaluated for their effects on the reversion of multidrug resistant (MDR) mediated by P-glycoprotein (P-gp) of the soil borne fungi. The multidrug resistant P-glycoprotein is a target for chemotherapeutic drugs in cancer cells. In the present study rhodamine-123 exclusion screening test on human mdr1 gene transfected mouse gene transfected L5178 and L5178Y mouse T-cell lymphoma which showed excellent MDR reversing effect in a dose dependent manner against mouse T-lymphoma cell line. Moreover, molecular docking studies of compound-1 also showed better results as compared with the standard. Therefore the preliminary results obtained from this study suggest that screlotiumol 1 could be used as a potential agent for the treatment of cancer.

Fluorimetric Methods for Analysis of Permeability, Drug Transport Kinetics, and Inhibition of the ABCB1 Membrane Transporter.

The cell membrane P-glycoprotein (P-gp; MDR1, ABCB1) is an energy-dependent efflux pump that belongs to the ATP-binding cassette (ABC) family of transporters, and has been associated with drug resistance in eukaryotic cells. Multidrug resistance (MDR) is related to an increased expression and function of the ABCB1 (P-gp) efflux pump that often causes chemotherapeutic failure in cancer. Modulators of this efflux pump, such as the calcium channel blocker verapamil (VP) and cyclosporine A (CypA), can reverse the MDR phenotype but in vivo studies have revealed disappointing results due to adverse side effects. Currently available methods are unable to visualize and assess in a real-time basis the effectiveness of ABCB1 inhibitors on the uptake and efflux of ABCB1 substrates. However, predicting and testing ABCB1 modulation activity using living cells during drug development are crucial. The use of ABCB1-transfected mouse T-lymphoma cell line to study the uptake/efflux of fluorescent probes like ethidium bromide (EB), rhodamine 123 (Rh-123), and carbocyanine dye DiOC2, in the presence and absence of potential inhibitors, is currently used in our laboratories to evaluate the ability of a drug to inhibit ABCB1-mediated drug accumulation and efflux. Here we describe and compare three in vitro methods, which evaluate the permeability, transport kinetics of fluorescent substrates, and inhibition of the ABCB1 efflux pump by drugs of chemical synthesis or extracted from natural sources, using model cancer cell lines overexpressing this transporter, namely (1) real-time fluorimetry that assesses the accumulation of ethidium bromide, (2) flow cytometry, and (3) fluorescent microscopy using rhodamine 123 and DiOC2.

A Rare Class of New Dimeric Naphthoquinones from Diospyros lotus have Multidrug Reversal and Antiproliferative Effects.

Three new dimeric naphthoquinones, 5,4′-dihydroxy-1′-methoxy-6,6′-dimethyl-7,3′-binaphthyl-1,4,5′,8′-tetraone (1), 5′,8′-dihydroxy-5-methoxy-6,6′-dimethyl-7,3′-binaphthyl-1,4,1′,4′-tetraone (2) and 8,5′,8′-trihydroxy-6,6′-dimethyl-7,3′-binaphthyl-1,4,1′,4′-tetraone (3), were isolated from the roots of Diospyros lotus. Their structures were elucidated by spectroscopic techniques, including 1D and 2D NMR, such as HSQC, HMBS, NOESY, and J-resolved. Compounds 1–3 were evaluated for their effects on the reversion of multidrug resistance (MDR) mediated by P-glycoprotein through use of the rhodamine-123 exclusion screening test on human ABCB1 gene transfected L5178Y mouse T-cell lymphoma. Compounds 1–3 were also assessed for their antiproliferative and cytotoxic effects on L5178 and L5178Y mouse T-cell lymphoma lines. Both 1 and 2 exhibited promising antiproliferative and MDR-reversing effects in a dose-dependent manner. The effects of the tested compounds on the activity of doxorubicin were observed to vary from slight antagonism to antagonism.

Development of Masitinib for the Treatment of Peripheral T-Cell Lymphoma

Increasing resistance to chemotherapy means that approximately 30% of patients afflicted with peripheral T-cell lymphoma will develop a relapsed/refractory form and eventually succumb to the disease. To date there is no clearly established second-line therapy and investigation of new therapies is necessary to address this unmet medical need.Masitinib is an oral tyrosine kinase inhibitor that potently and selectively targets c-Kit and platelet-derived growth factor receptor (PDGFR). Studies in human T-cell lymphoma have identified aberrant expression of PDGFR-alpha and that this kinase fosters peripheral T-cell lymphoma cell proliferation via an autocrine loop. Masitinib may therefore exert an antiproliferative and pro-apoptotic action on abnormal T-cells. In addition to this direct mechanism of action, masitinib can elicit antitumor effects by acting on mast cells and macrophages. By inhibiting mast cells, masitinib reduces the release of pro-tumoral M2-polarizing cytokines as well as factors favoring metastasis and angiogenesis. Masitinib can also promote macrophage infiltration into tumors, inducing an anti-tumoral Th1 immune response.Inhibition of cell proliferation was observed following single-agent masitinib treatment in the OSW canine T-cell lymphoma line, with an IC50 of 0.005 μM, suggesting that it could be used efficiently for the treatment of T-cell lymphoma. This hypothesis was further substantiated by a case study in a companion dog with T-cell lymphoma treated with masitinib monotherapy 12 mg/kg/day, reporting a complete response following 3 weeks of treatment with increased quality of life. This preliminary observation was repeated in two separate independent studies, each comprised of 11 dogs with T-cell lymphoma. A first study reported an overall response rate (ORR) of 73%, including 3/11 dogs with complete response (CR) and 5/11 dogs with partial response (PR) after 3 months of treatment. A second study reported an ORR of 45%, including 2/11 dogs with CR and 3/11 dogs with PR after 3 months of treatment. Meta-analysis of these data (n=23) showed that masitinib treatment of canine T-cell lymphoma resulted in a CR in 6/23 dogs (26%) and in a PR 8/23 dogs (35%), resulting in an ORR of 61%. Naturally occurring tumors in dogs have more clinical and biological similarities to human cancers than any other animal cancer model, hence these data provide strong medical plausibility for masitinib in the treatment of human peripheral T-cell lymphoma.The above in vitro and in vivo data led to initiation of a multicenter, randomized, open-label, three-parallel group, phase 2 study to evaluate the combination of masitinib plus dexamethasone with or without gemcitabine in patients with relapsed or refractory peripheral T-cell lymphoma. A recent decision to accelerate to a phase 3 study was based on an observed survival benefit for masitinib treated patients when compared with the control arm, and an acceptable safety profile; passage to phase 3 was validated by the independent Data Safety Monitoring Board with data blinded to sponsor and investigator. Specifically, pooled data from all masitinib-treated patients in the phase 2 stage (n=34) estimated a median overall survival (OS) of 9.0 months, which compares favorably against the literature benchmark median OS of 5.5 months for documented chemotherapy in this indication.This phase 3 study is currently open for patient recruitment in at least 14 countries and has OS as the primary endpoint. If successful, masitinib could provide a new treatment option in relapsed or refractory peripheral T-cell lymphoma. DisclosuresOff Label Use: Masitinib is a new orally administered tyrosine kinase inhibitor that targets mast cells and macrophages, important cells for immunity, through inhibiting a limited number of kinases. Based on its unique mechanism of action, masitinib can be developed in a large number of conditions in oncology, in inflammatory diseases, and in certain diseases of the central nervous system. In oncology due to its immunotherapy effect, masitinib can have an effect on survival, alone or in combination with chemotherapy. Through its activity on mast cells and consequently the inhibition of the activation of the inflammatory process, masitinib can have an effect on the symptoms associated with some inflammatory and central nervous system diseases and the degeneration of these diseases.. Marçais:AB Science: Consultancy. Dubreuil:AB Science: Consultancy. Moussy:AB Science: Other: CEO.

Rapamycin Suppresses Tumor Growth and Alters the Metabolic Phenotype in T-Cell Lymphoma

The mTOR pathway is a master regulator of cellular growth and metabolism. The biosynthetic and energetic demand of rapidly proliferating cells such as cancer cells is met by metabolic adaptations such as an increased glycolytic rate known as the Warburg effect. Herein, we characterize the anti-tumor effect of rapamycin in a mouse model of T-cell lymphoma and examine the metabolic effects in vitro. The murine T-cell lymphoma line, MBL2, and human cutaneous T-cell lymphoma (CTCL) lines, HH and Hut78, were used in syngeneic or standard NSG mouse models to demonstrate a marked suppression of tumor growth by rapamycin accompanied by inhibition of mTORC1/2. Analysis of the metabolic phenotype showed a substantial reduction in the glycolytic rate and glucose utilization in rapamycin-treated lymphoma cells. This was associated with reduced expression of glucose transporters and glycolytic enzymes in cultured cells and xenograft tumors. As a result of the decrease in glycolytic state, rapamycin-treated cells displayed reduced sensitivity to low-glucose conditions but continued to rely on mitochondrial oxidative phosphorylation (OXPHOS) with sensitivity to inhibition of OXPHOS. Taken together, we demonstrate that rapamycin suppresses growth of T-cell lymphoma tumors and leads to a reduction in aerobic glycolysis counteracting the Warburg effect of cancer cells.