Combination of Enzastaurin and HDAC Inhibitors Synergically Induce Apoptosis in Lymphoma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4783-4783
Author(s):  
Juraj Bodo ◽  
Jan Sedlak ◽  
Jaroslaw P. Maciejewski ◽  
Eric D. Hsi
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Lymphoma Cell ◽  
Parp Cleavage ◽  
Primary Lymphoma ◽  
Low Concentrations

Abstract Abstract 4783 Introduction Histone deacetylase inhibitors (HDACis) are approved for use in the setting of cutaneous T-cell lymphoma with modest benefit. Enzastaurin is an investigational PKCβ inhibitor that has growth inhibitory and pro-apoptotic effects in both B and T-cell lymphoma. Specifically, enzastaurin-induced inhibition of PKC leads to rapid accumulation of β-catenin that triggers c-Jun dependent induction of p73, followed by apoptosis. We investigated the cytotoxicity and mechanisms of cell death of combination enzastaurin and low concentrations of HDACis in B-cell lymphoma and T-cell lymphoma cell lines and primary lymphoma/leukemia cells. Experimental design Apoptosis was measured by flow cytometry and PARP cleavage. Phospho-GSK3β (S9), pS6, phospho-c-jun (S73) and β-catenin were analyzed by Western blot or quantum-dot immunoflourescence as measures of PKCβ inhibition. Cytotoxicity was determined by WST-1 proliferation assay and colony forming cell (CFC) assays. Results As expected, enzastaurin induced dephosphorylation of GSK3β and S6RP associated with increased β-catenin expression followed by phosphorylation of c-jun (S73) and PARP cleavage in SU-DHL-6 (diffuse large B-cell lymphoma line) cells. Treatment with low concentrations of suberoylanilide hydroxamic acid (SAHA) showed slight or no changes in studied proteins. Combined enzastaurin/SAHA treatment resulted in strong synergistic apoptosis in two treated germinal center B-cell-like and two activated B-cell-like lymphoma cell lines, two T-cell lymphoma cell lines and four different primary lymphoma/leukemia samples. Similarly, combined enzastaurin/ valproic acid treatment induced synergistic apoptosis in SU-DHL-6 cell line, suggesting the synergy is generalizable to other HDACis. In comparison to the single agent treatment, combined enzastaurin/ SAHA treatment resulted in activation of proapoptotic MAPK, c-jun N-terminal kinase, further increase of phospho c-jun (S73) levels, increased FasL levels, and amplification of PARP cleavage. Quantitative immunofluorescence assay showed a more rapid increase of β-catenin levels with the combination than either agent alone. Furthermore, compared to the low dose SAHA treatment alone, hyperacetylation of histone H3 was detected in samples when enzastaurin was added in combination with low dose SAHA, likely the consequence of displacement of HDAC by β-catenin. In addition, no change in CFC output in normal bone marrow exposed to this combination was observed. Conclusion Enzastaurin/ HDACi therapy can synergistically inhibit growth and induce apoptosis in lymphoid malignancy through increased biochemical effects attributed to each agent. These data support further investigation of addition of PKCβ inhibitors to HDACi in order to increase their anti-lymphoma effects. Disclosures: No relevant conflicts of interest to declare.

Anti-CD45 Mediated Cytolysis of Human T-Cell Lymphoma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4637-4637
Author(s):  
Gerald G. Wulf ◽  
Anita Boehnke ◽  
Bertram Glass ◽  
Lorenz Truemper
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cytolytic Activity ◽  
T Cell Lymphoma ◽  
Lymphoma Cell ◽  
Lymphoma Cells ◽  
Human T Cell

Abstract Anti-CD45 mediated cytoreduction is an effective means for T-cell depletion in rodents and humans. In man, the CD45-specific rat monoclonal antibodies YTH24 and YTH54 are IgG2b subclass, exert a predominantly complement-dependent cytolytic activity against normal T-lymphocytes, and have been safely given to patients as part of conditioning therapies for allogeneic stem cell transplantation. The efficacy of such antibodies against human lymphoma is unknown. Therefore, we evaluated the cytolytic activity of YTH24 and YTH54 by complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), as well as by direct apoptotic and antiproliferative effects, against a panel of Hodgkin disease (HD) and non-Hodgkin lymphoma (NHL) cell lines, and against primary specimens. Significant CDC activity (>50% cytolysis) of the antibodies YTH54 and YTH24 was observed against three of five T-cell lymphoma lines, but against only one of nine B-cell lymphoma lines and none of four HD cell lines. The combination of YTH54 and YTH24 induced ADCC in all T-cell lymphoma cell lines and three primary leukemic T-cell lymphoma specimens, but were ineffective in B-cell lymphoma and HD cell lines.There were only minor effects of either antibody or the combination on lymphoma cell apoptosis or cell cycle arrest. In summary, anti-CD45 mediated CDC and ADCC via the antibodies YTH24 and YTH54 are primarily effective against lymphoma cells with T-cell phenotype, and may be an immunotherapeutic tool for the treatment of human T-cell lymphoma.

scholarly journals Identification of Anti-Lymphoma Biomarkers of Response to the Anti-CD37 Antibody Drug Conjugate (ADC) IMGN529

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4187-4187 ◽  
Author(s):  
Eugenio Gaudio ◽  
Chiara Tarantelli ◽  
Alberto Arribas ◽  
Luciano Cascione ◽  
Ivo Kwee ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Lymphoma Cell ◽  
Cell Of Origin ◽  
Antibody Drug Conjugate ◽  
Induced Apoptosis

Abstract Background IMGN529 is an antibody drug conjugate (ADC) consisting of an anti-CD37 antibody with direct anti-tumor activity conjugated via a thioether linker to the cytotoxic maytansinoid antimicrotubule agent DM1. IMGN529 has shown pre-clinical (Deckert et al, Blood 2013) and clinical activity in lymphoma (Stathis et al, ASH 2014; NCT01534715). Here, we assessed the anti-tumor activity of IMGN529 on a large panel of B cell and T cell human lymphomas to identify potential biomarkers of response. Methods Fifty-four lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), n.=27; mantle cell lymphoma (MCL), n.=10; anaplastic large T-cell lymphoma, n.=5; marginal zone lymphomas, n=6, others, n=6] were exposed to increasing doses of IMGN529 or to the unconjugated DM1 for 72h. Cell proliferation was measured using the MTT. Apoptosis induction was defined by at least 1.5-fold increase in caspase 3/7 signal activation with respect to controls using the Promega ApoTox-Glo Triplex Assay. CD37 surface expression was assessed by cytofluorimetry. Gene expression profiling (GEP) was done with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > |2|, P<0.05, FDR<0.25) and limma t-test (FC> |1.2|; P< 0.05; top 200 up and top 200 down). Results. The IMGN529 median IC50 in the 54 cell lines was 780pM (95%C.I., 263pm-11.45nM). Activity was stronger (P<0.001) in B cell lymphoma cell lines (n= 46; median IC50=450pM; 95%C.I., 150-800pM) than in T cell lymphoma cell lines (n=8; median IC50=22.5nM; 95%C.I., 14-40nM). The median IC50 for DM1 was 30pM (C.I.95%, 20-40pM) with no differences between B and T cell lymphoma origin. IMGN529 induced apoptosis in 33/54 (61%) lymphoma cell lines. Surface CD37 expression was higher in cell lines derived from B than from T cells (P< 0.0001): IMGN529 IC50 values, but not of DM1, were negatively correlated with surface CD37 expression across all cell lines (R=-0.39; P= 0.018), but not within the individual B or T cell subgroups. Among B cell lines, DLBCL cell of origin, TP53 status or the presence of BCL2 translocation did not affect the sensitivity to IMGN529, while IC50s were higher in the presence of MYC translocation (P= 0.043). No association was seen between IMGN529-induced apoptosis or the sensitivity to DM1 with DLBCL cell of origin, TP53 status or the presence of BCL2 or MYC translocations. We then compared the baseline gene expression profiling of DLBCL cell lines that were highly sensitive to IMGN529 (IC50< 800pM; "S") versus less sensitive/resistant DLBCL cell lines (IC50>10nM, "R"), separately for germinal center B cell type (GCB) (S, n=11; R, n=8) and for activated B cell like (ABC) (S, n=4; R, n=3). In both DLBCL groups, MYC targets, genes involved in unfolded protein response, glycolysis and DNA repair were enriched in transcripts more expressed in R than S cell lines. Transcripts associated with low sensitivity included CD44, VIM, ANXA2, BCL2, ANXA2P1, HSP90B1, NFKBIZ, CDK6, BIRC5 in GCB and HSPA1B, HSP90AA1, CADM1, CD86, TUBB2A, TUBG1, NOTCH1 in ABC cell lines. HEBP1, PHB, PSME3, RNU6-15, RPL13 were more expressed in both GCB and ABC R. Genes involved in PI3K/AKT/mTOR, hypoxia, INF-gamma, TNFA signaling via NFKB and in complement were more expressed in S than in R cell lines. Genes associated with sensitivity to IMGN529 comprised: CD37 (IMGN529 target), CD79A, CHI3L2, FAM117B, LPAR5, NFATC1, PTPN22, RBM38, SGPP1, SLC6A16 in both GCB and ABC cell lines; BASP1, CXCR5, BIK, LY86, TLR10, CD86, LCK, CD22, PTPN22, BCL6, PIK3IP1, CDKN2A in GCB; AFF3, PIM1, MGMT, PDE4B, NFKBIE, SYK, FOXO1in ABC. Conclusions. IMGN529 showed a very strong anti-tumoral activity in pre-clinical lymphoma models. High expression of CD37 and mostly genes involved in BCR signalling were associated with sensitivity to IMGN529. Conversely, the presence of MYC translocation, a high expression of MYC targets and of genes known to be involved in drug resistance (BCL2, BIRC5, CDK6, heat-shock proteins, annexins, proteasome and tubulin components) appeared to negatively affect the response to the ADC but also represent therapeutic targets for novel combinations to be explored. Disclosures Rossi: Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Sloss:Immunogen Inc: Employment.

The bidirectional increased risk of B-cell lymphoma and T-cell lymphoma

Blood ◽  
2021 ◽  
Author(s):  
Dai Chihara ◽  
Graça Dores ◽  
Christopher Flowers ◽  
Lindsay M Morton
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Population Based ◽  
Lymphocytic Leukemia ◽  
T Cell Lymphoma ◽  
Primary Lymphoma ◽  
Second Primary ◽  
Increased Risk

Lymphoma survivors have a significantly higher risk of developing second primary lymphoma than the general population; however, bidirectional risks of developing B- and T-cell lymphomas (BCL; TCL) specifically are less well understood. We used population-based cancer registry data to estimate the subtype-specific risks of second primary lymphoma among patients with first BCL (n=288,478) or TCL (n=23,747). We observed nearly five-fold increased bidirectional risk between BCL and TCL overall (TCL following BCL: standardized incidence ratio [SIR]=4.7, 95% confidence interval [CI]=4.2-5.2; BCL following TCL: SIR=4.7, 95%CI=4.1-5.2), but the risk varied substantially by lymphoma subtype. The highest SIRs were observed between Hodgkin lymphoma (HL) and peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS) (PTCL-NOS following HL: SIR=27.5, 95%CI=18.4-39.4; HL following PTCL-NOS: SIR=31.6, 95%CI=17.3-53.0). Strikingly elevated risks also were notable for angioimmunoblastic T-cell lymphoma (AITL) and diffuse large B-cell lymphoma (DLBCL) (AITL following DLBCL: SIR=9.7, 95%CI=5.7-15.5; DLBCL following AITL: SIR=15.3, 95%CI=9.1-24.2). These increased risks were strongest within the first year following diagnosis but remained persistently elevated even at ≥5 years. In contrast, SIRs were &lt;5 for all associations of TCL with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and follicular lymphoma (FL). These patterns support etiologic heterogeneity among lymphoma subtypes and provide further insights into lymphomagenesis.

scholarly journals Deletion of Murine Rhoh induces More Aggressive Diffuse Large B Cell Lymphoma (DLBCL) Via Interaction with Kaiso and Regulation of BCL-6 Expression

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1574-1574
Author(s):  
Hiroto Horiguchi ◽  
Marioara Felicia Ciuculescu ◽  
Anja Troeger ◽  
Haiming Xu ◽  
Christian Brendel ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Nuclear Localization ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Germinal Center Formation

Abstract RHOH encodes a GTPase-deficient, hematopoietic-specific small GTPase first identified as a hypermutable gene in DLBCL (Pasqualucci et al. 2001). RhoH is critical for T cell receptor signaling and Rhoh-deficient (RhohKO) mice have T cell lymphopenia (Gu et al., 2006) and loss of function mutations of RHOH are associated with Epidermodysplasia Verruciformis (Crequer et al., 2012). However, the role of RhoH in the biology of DLBCL is still unknown and its role in B lymphoid development is incompletely studied. We investigated the role of RhoH in normal germinal center formation and in a murine model of DLBCL by crossing RhohKO mice with Iµ-HABcl-6 transgenic (Bcl-6Tg) mice (Cattoretti G, et al., 2005). In young RhohKOmice, deficient development of CXCR5+ follicular T helper (Tfh) cells results in defective germinal center (GC) formation and impaired immunoglobulin switching in vivo. In spite of this defect in GC formation, RhohKO; Bcl-6Tg (KOTg) mouse demonstrated accelerated lymphoma progression associated with larger spleens and significantly earlier death (Log-rank test p<0.01, Figure 1). Immunohistochemistry data suggested increased expression of IRF-4 and enhanced expression of BCL-6 in KOTg mice, findings confirmed by immunoblot and consistent with an activated B-cell (ABC)-DLBCL phenotype. To analyze the mechanism underlying these results, B cell lymphoma cell lines from KOTg lymphoma mice were established. Multiple attempts to establish RhohWT lymphoma cell lines failed, although we also successfully established a lymphoma cell line from RhohKO; Bcl-6(ntg) (KONtg) mice. Re-expression of RhoH in these lines via retrovirus mediated gene transfer led to significantly decreased proliferation (5.9x106±9.6x105 cells vs 8.6x106±9.6x105 cells after 5-days culture; KOTg vs KOTg-RhoH, mean±SEM, p<0.05) that was associated with clear reduction in BCL-6 expression. These data suggest that BCL-6 is a direct or an indirect transcriptional target of RhoH. Our laboratory previously reported that KAISO, a dual-specific, Broad complex, Trantrak, Bric-a-brac/Pox virus, Zinc finger (POZ-ZF) transcription factor interacts and colocalizes with RhoH in the nucleus, whereas knockdown of RhoH inhibits the nuclear localization of KAISO in Jurkat cells (Mino A, et al., 2016). In addition, Kaiso has been shown to be a key regulator of spleen germinal center formation by repressing Bcl-6 expression in splenocytes (Koh D, et al., 2013). We hypothesized that the deletion of Rhoh may lead to the decreased nuclear localization of KAISO and result in increased the expression of Bcl-6. We first confirmed that RhoH bound KAISO in RhoH-transduced KO lymphoma cells by co-immunoprecipitation. Further immunoblot analysis and quantitative PCR (qPCR) demonstrated decreased BCL-6 expression in lymphoma cells in which RhoH was re-expressed (KOTg-RhoH and KONtg-RhoH) compared with empty vector-transduced lymphoma cell lines. Interestingly, p53 a BCL-6 target was increased in RhoH-transduced lymphoma cell lines. These data indicate that RhoH affects BCL-6 expression in B cell lymphoma cell lines and suggest that RhoH may be involved in DLBCL development by co-regulating BCL-6 expression affecting downstream targets via interaction with KAISO. Figure. Figure. Disclosures Williams: Bluebird Bio: Research Funding.

scholarly journals Asparagine Synthetase Expression and L-Asparaginase Sensitivity in Aggressive Lymphomas

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5494-5494 ◽  
Author(s):  
Willy Berlier ◽  
Karine Aguera ◽  
Anne-Marie Chevrier ◽  
Fanny Gallix ◽  
Alexandra Traverse-Glehen ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
T Cell Lymphomas ◽  
Large B Cell

Abstract L-asparaginase (L-ASPA) displays a strong clinical benefit in the treatment of acute lymphoblastic leukemia (ALL), where it is included in most of current chemotherapy regimen. L-ASPA depletes plasmatic asparagine (ASN), an amino acid essential for the proliferation of leukemic cells. Since these cells are deficient in asparagine synthetase (ASNS), they rely on external (plasmatic) source of ASN and can be starved to death by L-ASP treatment. Several studies evidenced the potential of ASN depletion to treat lymphomas. Indeed, many animal and human lymphoma cell lines have been shown to be sensitive to L-ASPA in vitro. In veterinary medicine, L-ASPA is routinely administered to treat effectively both feline and canine lymphomas (Wypig et al., 2013). L-ASPA regained attention in the treatment of human lymphomas since its adjunction in current chemotherapy regimens significantly improved the outcome of patients with NK/T cell lymphoma (Zou et al., 2014). Some studies also evidenced its benefit in combined chemo or monotherapy for the treatment of B-cell and T-cell lymphomas (Sun et al., 2006; Takahashi et al., 2010). In this study, we assessed the in vitro sensitivity to L-ASPA of 6 lymphoma cell lines and we analyzed ASNS expression in biopsies from 166 cases of lymphomas (130 B-cell lymphomas and 17 T-cell lymphomas). Sensitivity to L-ASPA (expressed as an IC50) was assessed in vitro by measuring the cell viability in the presence of various concentrations of E.coliL-ASPA. ASNS expression in biopsies (TMA, USBiomax, Rockville, MD) was assessed with a validated immunohistochemistry (IHC) method attributing a score to each tumor based on ASNS labeling intensity from 0 (no expression) to 3 (strong expression). Tumors expressing no/low ASNS (scores 0 and 1) were considered potentially sensitive to asparagine depletion. As shown in the following table, all cell lines were proved to be sensitive to L-ASPA. Their in vitrosensitivity exceeded cell lines MOLT-4 (ALL) and HL-60 (AML). Table 1Cell lineSensitivity to L-ASPA (IC50 in IU/mL)HuT-78 (Peripheral T-cell lymphoma,PTCL)0.11 ± 0.02Toledo (Diffuse large B-cell lymphoma, DLBCL)0.19 ± 0.03SU-DHL-8(Diffuse large B-cell lymphoma, DLBCL)0.10 ± 0.04SU-DHL-10(Diffuse large B-cell lymphoma, DLBCL)0.10 ± 0.01REC-1 (Mantle cell lymphoma, MCL)0.15 ± 0.03KHYG-1 (NK/T-cell lymphoma)0.16 ± 0.06MOLT-4 (acute lymphoid leukemia, ALL)0.19 ± 0.07HL-60 (acute myeloid leukemia, AML)0.23 ± 0.02 As shown in the following table, ASNS expression was null/low in 85% in the entire population of patients with B-cell lymphomas. Considering DLBCL, 63% of patients displayed no ASNS expression at all. ASNS expression was also null/low in 88% of patients with T-cell lymphomas (n=17). Table 2ASNS expression (IHC score)Type of lymphoma(% of cases)DLBCL (n=110)Others BCL (n=20)PTCL (n=3)Others TCL (n=14)MCL(n=3)Hodgkin (n=16)Negative (0)62,770,00,057,133,343,8Low positive (1)21,825,066,635,766,656,3Positive (2)7,35,033,37,10,00,0Highly positive (3)8,20,00,00,00,00,0 Globally, these results suggest that L-ASPA is potentially effective for the treatment of several lymphomas. Indeed, B-cell as well as T-cell lymphoma cell lines are sensitive to L-ASP in vitroand the majority of lymphoma tissues express no/low ASNS. Based on our results on ASNS expression in lymphoma biopsies, L-ASPA therapy may be beneficial for up to 85% of patients with DLBCL. Up to 90% of patients with other B-cell lymphomas or T-cell lymphomas may be sensitive to L-ASPA treatment as well. However, L-ASPA has only been used scarcely in the treatment of lymphomas despite promising clinical responses. Its well known serious side-effects (hypersensitivity, coagulation disorders, pancreatitis, and liver failure) render its use hazardous, particularly in older or frail patients. Therefore, the development of a new formulation of L-ASPA with safer profile has to be considered in order to allow the clinical development of L-ASPA in the treatment of aggressive lymphomas. Disclosures Berlier: ERYTECH: Employment, Equity Ownership. Aguera:ERYTECH: Employment. Chevrier:ERYTECH: Employment. Gallix:ERYTECH: Employment. Godfrin:ERYTECH Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Biological Effects of Rabbit Anti-Thymocyte Immunoglobulin (rATG, Thymoglobulin®) in T-Cell Non-Hodgkin’s Lymphoma Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4628-4628
Author(s):  
Francisco J. Hernandez ◽  
Nishita Reddy ◽  
Sujatha Nallapareddy ◽  
Myron S. Czuczman
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Biological Effects ◽  
T Cell Lymphoma ◽  
Lymphoma Cell ◽  
T Cell Lymphomas ◽  
Mantle Cell ◽  
T Cell Lines

Abstract Monoclonal antibodies (mAbs) have emerged as powerful adjuncts in the treatment of patients with B-cell lymphoproliferative disorders. While the treatment of B-cell lymphomas has incorporated mAbs and other biological agents into standard chemotherapy regimens, the treatment options for patients with T-cell lymphomas remain relatively limited. There exists a dire need to develop targeted therapies for T-cell lymphomas. Thymoglobulin® (rATG) is a rabbit polyclonal antibody targeting various receptors present on T-cell lymphocytes. When administered at high doses, rATG is known to deplete various subsets of T-cell lymphocytes and induce tolerance in solid organ or bone marrow transplant settings. Using several pre-clinical models, we evaluated the biological effects of rATG against various T-cell lymphoma cell lines. Experiments were conducted in HH, H9, Loucy and HT102 cell lines. A B-cell mantle cell lymphoma cell line was used as a control (MJ). rATG-induced cell-growth inhibition was measured by [3H]-Thymide incorporation assays and measured at 24 and 48 hours. Induction of apoptosis in T-cell lines following rATG exposure was determined by annexin-V/propidium iodine staining and quantified by flow cytometric analysis. Standard functional assays for ADCC/CMC were performed using rATG (5 or 25mg/ml) in 51Cr-labeled T-cells. We found that rATG inhibited DNA synthesis in all the T-cell lines tested. No biological effect was observed in the B-cell mantle cell lymphoma line. Treatment with rATG at either 5 or 25mg/ml resulted in a 30 to 50% growth inhibition when compared to isotype or vehicle controls (P&lt;0.05). Induction of apoptosis was demonstrated in 30 to 40% of T-cell lymphoma cells 24 hrs following exposure to ATG. Biological effects of rATG were dose-dependent. In addition, rATG induced significant ADCC and CMC in T-cell lymphoma cell lines. In conclusion, our data demonstrate that rATG is active against a variety of T-cell lymphoma cell lines in vitro. Anti-tumor effects of rATG are mediated by induction of direct signaling and via the activation of the innate immune system. Additional in vivo studies using T-cell lymphoma are underway and will be presented at the annual meeting.

scholarly journals Schedule-Dependent Synergy Between Belinostat and Pralatrexate in T-and B-Cell Lymphoma Cells in Vitro

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5128-5128
Author(s):  
Godefridus Peters ◽  
FHA van Gemert ◽  
I Kathmann ◽  
Saskia AGM Cillessen ◽  
G Jansen ◽  
...  
Keyword(s):  
Folic Acid ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Drug Exposure ◽  
Cell Lymphoma ◽  
Fixed Ratio ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Substrate Affinity

Abstract Pralatrexate (Folotyn; FOL) and belinostat (Beleodaq; BEL) are two new agents that have recently been registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and have also shown promising activity in other types of lymphoma. FOL is a folate analogue and a potent inhibitor of dihydrofolate reductase (DHFR), designed to accumulate in cancer cells preferentially via the reduced folate carrier (RFC) and retained therein via efficient polyglutamylation. Inhibition of DHFR leads to an imbalance of deoxynucleotides (e.g. depletion of dTTP and an increase in dUTP) resulting in DNA strand breaks and inhibition of DNA synthesis. BEL is a hydroxamic acid-based pan-histone deacetylase (HDAC) inhibitor that broadly inhibits all of the zinc-dependent HDAC enzymes, with high affinity for the Class I, II and IV isozymes. HDAC inhibition results in an alteration in the degree of histone and non-histone protein acetylation, which in turn affects transcription of genes essential in cellular proliferation, cell cycle and DNA repair. We investigated whether folate transporters other than RFC, i.e. folate receptor α (FRα) and the proton-coupled folate transporter (PCFT) could contribute to the efficacy of FOL. Moreover, we explored whether the toxicity of FOL can be controlled by levo-leucovorin (Fusilev), the natural stereoisomer of leucovorin, and whether in combination experiments BEL had the ability to potentiate the cytotoxicity of FOL. A panel of lymphoma cell lines was used for the combination studies including: the B-cell lymphoma cell lines SU-DHL-4, SU-DHL-5, HT, Jeko-1 and T-cell lymphoma cell lines Karpas-299 and Hut-78. RFC-mediated uptake efficiency of FOL was determined in competition uptake experiments with [3H] Methotrexate (MTX), revealing a 6-fold better RFC substrate affinity for FOL, and 2-fold better than levo-leucovorin. FOL displayed very poor substrate binding affinity for FRα (>100-fold lower than folic acid and > 10 lower than levo-leucovorin). FOL had a low substrate affinity for PCFT (>10-fold lower than folic acid and levo-leucovorin in [3H] leucovorin uptake competition experiments). Levo-leucovorin could completely protect toxicity by FOL, but had no effect on BEL toxicity. Sensitivity of lymphoma cell lines (IC50 concentrations after 72 hrs drug exposure) to FOL drug varied from 2.8 nM (Hut-78), 5.5 nM (SU-DHL4 and 5), 7.4 nM (HT) to 20 nM (Karpas-299 and Jeko-1) while IC50 values for BEL were in the range of 100 nM (SU-DLH-4 and 5, Jeko-1 and Hut-78) to 200 nM (Karpas-299 and HT). The interaction between BEL and FOL was studied using the median-drug effect analysis with Calcusyn software. At a fixed ratio between the drugs based on the IC50 concentration the average combination index (CI) for all the lymphoma cell lines revealed an additive effect (CI: all around 1.0). In two selected cell lines (SU-DHL-4 and HT) sequential exposure to the drugs (24 hr pretreatment with either BEL or FOL) followed by 48 hr to the combination, did not improve the results with CI values varying between 0.9 and 1.4. As an alternative approach a non-fixed ratio was used by exposing SU-DHL-4 and HT cells to IC25 concentrations of either BEL or FOL in combination with the other drug. Exposure to IC25 concentrations of FOL did not decrease the IC50 for BEL (CI around 1.2), but exposure to IC25 concentrations of BEL markedly increased the sensitivity to FOL as reflected by the low CIs varying from 0.40 to 0.66. Mechanistic studies focused on induction of apoptosis, showed cleavage of caspase 8 and 9 in HT and SU-DHL-4 cells for both drugs at their IC50s, being similar in the combination setting. Moreover, at these concentrations, the drugs were shown to confer an S-phase arrest. In conclusion, the combination of FOL and BEL showed additive activity in various lymphoma cell lines, while a schedule-dependent synergism was observed. Based on these data, proficient inhibition of HDAC activity by BEL holds promise in sensitization of tumor cells to FOL. Furthermore, toxicity of FOL could be completely protected by levo-leucovorin. Disclosures Reddy: spectrum: Employment, Equity Ownership.

BTK inhibitor PCI-32765 targets endogenous and microenvironment-mediated B-cell receptor signaling to suppress lymphoma cell growth and is highly synergistic with Bortezmib against non-Hodgkin B-cell lymphomas

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4721-4721
Author(s):  
Bing Xia ◽  
Xiaowu Li ◽  
Le Zhang ◽  
Qing Guo ◽  
Xin Jin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Lymphoma Cell ◽  
Primary Lymphoma ◽  
Lymphoma Cells

Abstract Abstract 4721 Background: B-cell receptor (BCR) signaling is a critical pathway in the pathogenesis of B-cell malignancies and Bruton tyrosine kinase(Btk) is essential for BCR signaling and function. PCI-32765, a specific and irreversible small-molecule Btk inhibitor, has recently been reported to display a significant clinical activity against non-Hodgkin B-cell lymphomas (NHL) especially chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL). In this study we set to explore 1) the role of Btk in NHL cell apoptosis and proliferation, 2) the role of BtK in bone marrow strom-mediated lymphoma cell survival and 3) to test if PCI-32765 as a therapeutic agent in single or in combination with Bortezomib for NHL therapy. Methods: B-cell lymphoma cell lines including mantle cell lymphoma lines (Jeko-1 and HBL-2), Burkitt lymphoma cell line (Raji) and transformed large cell lymphoma cell line (SUDHL-10) as well as primary lymphoma cells from various NHL samples were used for the experiments. These cells were cultured in the presence or absence of bone marrow mesenchymal stromal cells (MSC). The endogenous and MSC-induced Btk and its signaling activation such as BtK, ERK1/2 and AKT expression and phosphorylation status as well as its inhibition by were examined PCI-32765 by Western blot. The effects of PCI-32765 on lymphoma cell growth and appotosis were analyzed by using MTT, DAPI stain and flow cytometric annexin V/PI staining. Furthermore, the combined effect of PCI-32765 and Bortezomib on lymphoma cell growth and apoptosis was analyzed using the CalcuSyn software program in search for a synergistic or additive effect. Results: We found constitutive expression and activation of Btk and its downstream signaling in most of these cell lines and primary lymphoma cells. Furthermore, co-culture with MSC cells further enhanced the phosphoration of Btk and AKT in these cells. Incubation of Jeko-1, Raji, HBL-2 and SUDHL-10 cell lines with PCI-32765 induced cell growth inhibitory effects. We found that PCI-32765 exhibited a significant dose-dependent induction of cytotoxicity in these cells at various time points as measured by MTT. We also found significant apoptosis in these cells treated with PCI-32765. In addition, PCI-32765 significantly inhibited phpsphorylation of AKT and Btk, confirming the block of BCK signal pathways in these cells. Finally, MTT assays indicated that combined PCI-32765 with Bortezomib induced a synergistic cytotoxicity against these NHL cells (CI<1). Discussion: Our studies therefore highlight the biological significance of Btk in B-cell lymphoma cell growth and survival. PCI-32765 effectively antagonizes B-cell survival provided by bone marrow stromal cells and synergistically in combination with Bortezomib eliminates lymphoma cells. This study provide rational for targeting BCR and Btk as a novel therapeutic approach for NHL. Disclosures: No relevant conflicts of interest to declare.

Motexafin Gadolinium Enhances Rituximab-Induced Cytotoxicity Against B-Cell Lymphoma Cell Lines: Role of Elevated Intracellular Calcium.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4829-4829
Author(s):  
Jun Chen ◽  
Jason Ramos ◽  
Mint Sirisawad ◽  
Richard A. Miller ◽  
Louie Naumovski
Keyword(s):  
B Cell ◽  
Intracellular Calcium ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Parp Cleavage ◽  
Ionophore A23187 ◽  
Motexafin Gadolinium ◽  
Synergistic Cytotoxicity

Abstract Motexafin gadolinium (MGd, Xcytrin®) is a tumor selective redox active drug that is directly cytotoxic to some hematolymphoid cell lines and chronic lymphocytic leukemia (CLL) patient samples. MGd has shown single agent activity in early phase II studies of lymphoma and CLL patients. Rituximab, an anti-CD20 antibody, is used widely in the treatment of B-cell malignancies. We evaluated the effects of MGd, rituximab and the combination in HF-1, a follicular lymphoma-derived cell line. Data analysis with CalcuSyn software revealed that the combination of MGd and rituximab showed synergistic growth inhibition and cytotoxicity compared to either agent used alone. MGd/rituximab activated a caspase-dependent apoptotic pathway as demonstrated by loss of mitochondrial membrane potential and PARP cleavage. Similar results were obtained with the combination of MGd and rituximab in DHL-4 and Ramos lymphoma cell lines. Since intracellular calcium ([Ca2+]i) levels have been implicated in rituximab cytotoxicity, we explored [Ca2+]i in rituximab and MGd/rituximab treated cells. Rituximab and MGd/rituximab treated cells have increased levels of [Ca2+]i. Elevation of [Ca2+]i with thapsigargin, an agent that releases calcium from internal stores or the ionophore A23187 that transports calcium into cells, results in synergistic cytotoxicity with rituximab or the MGd/rituximab combination. These in vitro findings suggest a role for [Ca2+]i in MGd/rituximab-induced cell death and support the combined use of MGd and rituximab in the treatment of B-cell lymphoma.

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