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.

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.

B Cell Lymphomas Are Resistant towards Bone Morphogenetic Protein (BMP) Induced Growth Inhibition.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2381-2381
Author(s):  
Kanutte Huse ◽  
Marianne B. Eide ◽  
Christian Kersten ◽  
Erlend B. Smeland ◽  
June H. Myklebust
Keyword(s):  
Growth Inhibition ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Lymphoma Cells ◽  
Bmp Receptors ◽  
Induced Apoptosis

Abstract Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily, and mediate their effects mainly through the Smad signalling pathway. Whereas TGF-β is well established as one of the most potent negative regulators in hematopoietic cells, the role of BMPs remains more elusive. We have previously shown that BMP-6 inhibits the growth of naïve and memory human B cells. As high BMP-6 mRNA expression is associated with poor outcome in diffuse large B cell lymphoma (DLBCL; Rosenwald et al, N Engl J Med 2002), we hypothesized that resistance towards BMP-induced growth inhibition is a possible mechanism for lymphomagenesis. In the current study, 7 B cell lymphoma cell lines (representing Burkitt lymphoma (BL) and DLBCL) and tumour material from lymphoma patients were investigated to unravel the role of BMPs in lymphomas. We analyzed the expression of BMP receptors by FACS analysis, and found variable expression of the BMP receptor type I (Alk2, Alk3 and Alk6) and type II (BMP RII, Activin RIIA and RIIB) among the cell lines and in primary lymphoma cells, suggesting variable binding of BMPs. We next investigated the effect of BMP-2, BMP-4, BMP-6 and BMP-7 on proliferation and survival of B lymphoma cell lines, and found 2 of 7 cell lines to be resistant towards BMP-2 and BMP-4 induced growth inhibition. In contrast, 4 of 7 and 7 of 7 cell lines were resistant to BMP-6 and BMP-7 induced growth inhibition, respectively. In Sudhl6 cells that were highly sensitive to BMP-2 and BMP-6 induced apoptosis and inhibition of proliferation, we demonstrated that the cytokines IL-10, CD40 Ligand and BLyS were able to counteract the negative effects induced by BMPs, while IL-2 and IL-4 were not. On the contrary, both BMP-2 and BMP-6 greatly increased anti-IgM activation induced apoptosis. In resistant lymphoma cells, the BMPs were not able to induce detectable levels or induced low levels of phosphorylated SMAD1/5/8 compared to sensitive cell lines. Low or no increase in phosphorylation of SMAD1/5/8 induced by BMPs could only partly be explained by low/ undetectable expression of BMP receptors. Hence, upregulation of inhibitory Smads (Smad6, Smad7) or mutations in receptors or Smads represent other possible mechanisms for resistance to BMPs in lymphomas, and this is currently under investigation. We also investigated if the lymphoma cells produced BMPs themselves and found that 5 of 7 cell lines and 3 of 5 primary lymphomas produced significant amounts of BMP-7. Some lymphoma cells also had detectable levels of BMP-4 and BMP-6. Our findings that lymphoma cells are resistant towards BMP-7 and to some degree BMP-6 induced growth inhibition, whereas they produce these cytokines, suggest that resistance towards BMP induced signalling in B cell lymphomas can contribute to increased tumour growth.

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.

Targeting DKK1 for the Immunotherapy of B-Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 465-465
Author(s):  
Jianfei Qian ◽  
Sungyoul Hong ◽  
Liang Zhang ◽  
Yuhuan Zheng ◽  
Haiyan Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Specific Ctls

Abstract Abstract 465 Immunotherapy may complement the current treatments for lymphomas. The lack of suitable shared lymphoma-associated antigens limits its applicability. Therefore, identification and utilization of novel and more potent tumor-associated antigens, particularly those shared among patients, are urgently needed to improve the efficacy of immunotherapy in the diseases. Recent studies have shown that Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by myeloma and other tumor cells, and is absent from normal tissues and organs except placenta and prostate. In the present study we demonstrated that DKK1 is also overexpressed in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Using DKK1 peptide-pulsed dendritic cells (DCs), we successfully generated HLA-A*0201+ DKK1-specific CTL lines and clones in vitro. These CTLs effectively lysed DKK1+/HLA-A*0201+ lymphoma cell lines Jeko-1 and Granta 519 cells, but not DKK1-/HLA-A*0201+ BJAB, RL and Mino cells nor DKK1+/HLA-A*020- CA46 and Daudi cells. Furthermore, the T-cell clones efficiently killed DKK1+/HLA-A*0201+ primary B-cell lymphoma cells from patients but not lymphoma cells from DKK1–/HLA-A*0201+ patients. HLA-ABC or HLA-A*0201 blocking mAbs significantly inhibited T cell-mediated cytotoxicity against peptide-pulsed T2 cells (P < .01, compared with medium control). No inhibitory effect was observed with mAb against HLA-DR and isotype control IgG. The results indicate that the cytotoxicity was attributed to MHC class I and more specifically, HLA-A*0201-restricted CD8+ CTLs. The CTLs did not kill DKK1–/HLA-A*0201+ DCs, B cells, or PBMCs, These results suggest that the CTLs recognized DKK1 peptides that are naturally processed and presented in the context of HLA-A*0201 molecules on lymphoma cells. To determine the in vivo antitumor activity, NOD-SCID and SCID-hu mice were used for lymphoma cell lines and primary lymphoma cells, respectively. Mice were treated with DKK1-specific CTLs after tumor established in NOD-SCID and SCID-hu mice. Control mice were treated with naïve CD8+ T cells or PBS alone. Tumor burden was measured according to levels of circulating human B2M, and survival rates were determined. Low levels (< 50 ng/ml) of circulating human B2M were detected in group treated DKK1-specific CTLs, while high levels (≥ 150 ng/ml) of circulating human B2M were detected in control mice. In SCID-hu model, X-ray examination showed that established tumors were eradicated in 60% mice treated with DKK1-specific CTLs, while large tumor burdens were found in all control mice. In NOD-SCID model, 40% of mice survived with the treatment of DKK1-specific CTLs. TUNEL assay further confirmed that tumor cells were lysed by DKK1-specific CTLs not naïve CD8+ T cells. These results indicate that DKK1-specific CTLs are able to eradicate established, patient-derived primary B- cell lymphoma in the hosts and adoptive transfer of DKK1-specific CTLs may be used for B-cell lymphoma therapy. Disclosures: No relevant conflicts of interest to declare.

Combination of NVP-BEZ235 and Enzastaurin on B-Cell Lymphoma Cell Lines: An Effective Therapeutic Strategy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2719-2719 ◽  
Author(s):  
Monica Civallero ◽  
Maria Cosenza ◽  
Samantha Pozzi ◽  
Stefano Sacchi
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoid Cells ◽  
Lymphoma Cell ◽  
Lymphoma Cells ◽  
Apoptosis Pathways ◽  
Extrinsic Apoptosis

Abstract Abstract 2719 Non-Hodgkin's lymphoma is the most common hematologic neoplasm in adults. Chemotherapy combined with CD-20 monoclonal antibodies has improved survival in both indolent and aggressive B-NHL. However, a substantial subset of patients does not achieve a cure or long disease remission. This has promoted the identification of new targeted treatments and new agents that have shown promising efficacy for future B-NHL therapies. The phosphatidylinositol 3-kinase (PI3K) mammalian target of rapamicin (mTOR) pathway mediates proliferation, survival and drug resistance in lymphoma cells. NVP-BEZ235 (BEZ235) is a new, orally bio available inhibitor of PI3K and mTOR and a representative of a new class of anti-tumour agents. In the current study, the efficacy of the combination of two orally available inhibitor to PI3K/mTOR (BEZ235) and PKCbeta/AKT (enzastaurin) was evaluated in B-cell lymphoma cell lines (RL, WSH-NHL, Jeko and Granta). First, we tested the anti-lymphoma activity of BEZ235 alone and in combination with enzastaurin, everolimus and perifosine. Results using MTT assay were expressed as fraction of cells killed by the individual drug or the combination in the drug-treated versus untreated cells. The interaction between drugs was analyzed by isobologram analysis using the STACorp8.2 software program based upon the Chou-Talalay method to determine if the combination were additive or synergistic. We found that enzastaurin, everolimus and perifosine enhanced the cytotoxicity triggered by BEZ235; a clear synergistic interaction (CI<1) appeared after 48 hours using low concentrations of the all compounds. We examined the functional effects of BEZ235 alone and in combination on apoptosis in lymphoma cells. We demonstrated that BEZ235 (20nM) alone after 24 hours induces an increase of 8–10% of apoptotic cells versus untreated, instead BEZ235 (20nM) in combination with enzastaurin (5microM) after 24 hours induces an increase of 25%. We next defined mechanisms whereby BEZ235 alone and in combination induce apoptosis in lymphoid cells. In particular, BEZ235 combined with enzastaurin induces both intrinsic and extrinsic apoptosis pathways with caspase 3, caspase 9, caspase 8 cleavage. We also showed that the combination of BEZ235 and enzastaurin decreases viability and induce apoptosis in B-cell lymphoma cell lines and peripheral blood mononuclear cells (PBMCs) from lymphoma patients. The combination has no effect on normal PBMCs and suppresses cell prolipheration of B-cell lymphoma cell lines (RL and Jeko) when co-cultured with bone marrow stromal cells in a system that mimics the bone marrow microenvironment. BEZ235, enzastaurin, everolimus and perifosine are inhibitors of intracellular pathways, thought we investigated effects of BEZ235 alone and in combinations with the other compounds in targeting p-AKT, p-mTOR, p-GSK3beta, p-p70, p-p90, p-MAPK, p-4EBP1 and cyclin D1 pathways by Western Blot. In addition, we demonstrated that BEZ235 plus enzastaurin resulted in increased expression of pro-apoptotic Bim, and in decrease expression of anti-apoptotic Bcl-2, which could not be abrogated by BEZ235 alone. In conclusion, our data suggest that in B cell lymphoma cell lines, BEZ235 in combination with enzastaurin elicits its antitumor effect better that combinated with perifosine and everolimus. Our data reveals that the drug combination targets phosphorilation of PI3K/Akt/mTOR pathways and induces both intrinsic and extrinsic apoptosis pathways. Furthermore, inhibition of Bcl-2 anti-apoptosis family members may, in part, explain the efficacy of signalling blockade in lymphoma cells and suggests an additional therapeutic targeting strategy. Therefore, these preclinical data support the potential use of BEZ235 in patients with NHL, and in particular provide rationale for combination with enzastaurin. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Prosurvival and Chemoprotective Effects of Tumor-Associated Macrophages Reversed By Anti-CSF-1R Monoclonal Antibody in B-Cell Lymphomas

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 498-498
Author(s):  
Anupama Gopisetty ◽  
Myriam Foglietta ◽  
Min Zhang ◽  
Zhiqiang Wang ◽  
Nathan Fowler ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
B Cell Lymphomas ◽  
Lymphoma Cells ◽  
Tumor Survival

Abstract The results of gene expression profiling (GEP) and immunohistochemical studies indicate that survival is worsened by macrophages (MΦ) in the tumor microenvironment of various B-cell lymphomas including follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Tumor-associated macrophages (TAMs) are known to be different from other types of MΦ, but the effects of TAMs that worsen prognosis in B-cell lymphoma are essentially unknown, as are the mechanisms of these effects. Here, we determined the phenotype and effects of TAMs on tumor survival, proliferation, and drug resistance in B-cell lymphomas and evaluated strategies to reverse their effects. As compared to peripheral blood monocytes (Mo) from normal donors (ND), Mo from FL patients were differentiated less into M1 MΦ (defined as CD68+CD163loCD206loCD86hi) by culture with CSF-1 for 5 days followed by IFN-g + LPS for 2 days more. In contrast, Mo from FL patients and ND were differentiated similarly into M2 MΦ (defined as CD68+CD163hiCD206hiCD86lo) by culture with CSF-1 followed by IL-4. Consistent with this, MΦ gene signatures from FL tumors were more similar to previously-described signatures of M2 rather than M1 MΦ (Martinez et al, J Immunol, 2006, 177(10):7303-11). In co-culture, primary FL tumor cells and lymphoma cell lines (including RL, a transformed FL cell line; Granta 519, a mantle cell lymphoma (MCL) cell line; and Raji, a Burkitt lymphoma cell line) induced differentiation of Mo into MΦ. Differentiation could be prevented by CS4 monoclonal antibody (mAb), a fully human IgG1 anti-human CSF-1R mAb (ImClone/Eli Lilly), but not isotype control Ab. Elevated levels of CSF-1 in culture supernatants after addition of CS4 mAb and real-time PCR of tumor cells suggested secretion of CSF-1 by lymphoma cells. Spontaneous apoptosis of primary FL and MCL tumor cells, determined by Annexin V and propidium iodide staining, was significantly reduced by co-culture with ND Mo (p<0.01), whether pre-differentiated into MΦ with CSF-1 or not, but this protection could be reversed by CS4 mAb. Mo and/or pre-differentiated MΦ protected primary FL and MCL tumor cells from cytotoxic effects of doxorubicin and/or bendamustine (p<0.01), but CS4 mAb reversed this effect. To assess effects of MΦ on proliferation, lymphoma cell lines (RL, Granta 519, and Raji) were CFSE-labeled prior to co-culture with Mo and doxorubicin, and proliferation assessed by CFSE dilution by flow cytometry in the presence or absence of CS4 or isotype control mAbs. MΦ promoted proliferation of all three cell lines, but this effect could be reversed by CS4 mAb. To further understand the mechanism by which MΦ promote tumor survival and growth, we performed phosflow analysis and found increased phosphorylation of STAT3 in co-cultured lymphoma cells. Consistent with this, we observed a correlation between an 11-gene STAT3 activation signature, described by Huang et al in DLBCL tumors (J Clin Oncol, 2013, 52.8414), and a MΦ gene signature in whole genome GEP studies of 191 FL tumors (Pearson correlation co-efficient=0.396, p<0.001). In conclusion, our results suggest that Mo from FL patients are predisposed to differentiate into an M2-like MΦ state. The interaction between lymphoma cells and Mo/MΦ is reciprocal: a change in Mo (MΦ differentiation) induced by interaction with lymphoma tumor cells leads to a change in the tumor cells (promotion of survival, proliferation, and chemoresistance). More importantly, our results demonstrate that targeting TAMs using CS4, an anti-CSF-1R mAb, can be an effective strategy to overcome the adverse effects of TAMs and reverse chemoresistance. Further studies are needed to determine whether STAT3 activation contributes to the protumor effects of TAMs. This may provide novel insights into the molecular mechanisms related to TAMs and lymphoma cells and offers additional targets for therapeutic development. In the long term, strategies targeting TAMs is especially appealing, as they should be able to be combined with existing therapies including chemotherapy, other immunotherapy, and targeted therapy, potentially improving their efficacy without increasing toxicity for FL, DLBCL, and other B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

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