Apoptotic Lymphoma Cells Evoke a Pro-Senescent Stromal Signal That Limits Lymphoma Development.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2392-2392
Author(s):  
Maurice Reimann ◽  
Soyoung Lee ◽  
Christoph Loddenkemper ◽  
Jan Dörr ◽  
Vedrana Tabor ◽  
...  
Keyword(s):  
B Cell ◽  
Tumor Cells ◽  
Transgenic Mouse ◽  
Cellular Senescence ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Anti Cancer ◽  
Mouse Lymphoma

Abstract Abstract 2392 Poster Board II-369 Introduction: Cancer entities frequently exhibiting constitutive Myc expression, such as aggressive B-cell lymphomas, typically display significant amounts of apoptotic cell death. So far, cellular senescence as another cell-autonomous oncogene-inducible safeguard program has been recognized in RAS/BRAF-driven scenarios but not as a bona fide Myc-evoked anti-cancer mechanism. Understanding how oncogenic Myc may provoke not only apoptosis but cellular senescence as a failsafe mechanism to counter tumor development has broad implications for the clinical presentation and therapeutic strategies in frequently Myc-driven lymphoma entities such as Burkitt's lymphoma and diffuse large B-cell lymphoma (DLBCL). Results: Using the Burkitt's like Eμ-myc transgenic mouse lymphoma model, we show here that cellular senescence serves as another crucial anti-neoplastic barrier during Myc-driven tumorigenesis in addition to apoptosis. Eμ-myc lymphomas harbor a substantial fraction of senescent tumor cells, that stain positive for histone H3 lysine 9 (H3K9)-trimethylation. Lymphomas lacking the H3K9 methyltransferase Suv39h1 display no senescence and develop significantly faster, although apoptosis is not affected by Suv39h1 deficiency. While Myc, unlike other Ras-type oncogenes, shows rather modest pro-senescent activity in vitro, we identified the cytostatic cytokine TGF-β as the main paracrine senescence trigger in vivo. When neutralizing TGF-β action during Myc-driven lymphomagenesis utilizing a secretable TGF-β receptor II ecto-domain, senescence is completely blunted and tumor latency is significantly shortened. We identify macrophages, but not lymphoma cells, as the main source of exogenous TGF-β, that is secreted upon phagocytosis of apoptotic lymphoma cells. Lymphomas harboring a Bcl2-mediated apoptotic block presented with a much lower frequency of both infiltrating macrophages and senescent cells in vivo, suggesting that there is a functional link between cell-autonomous Myc-triggered apoptosis and non-cell-autonomous, macrophage-induced senescence. Both pharmacological suppression of TGF-β production in macrophages via the angiotensin-converting enzyme (ACE) inhibitor lisinopril and depletion of macrophages in Eμ-myc lymphoma-harboring mice by systemic exposure to clodronate resulted in a profound reduction of senescence, thereby underscoring the important role for tumor-infiltrating macrophages in TGF-β-mediated senescence in vivo. We recapitulated components of such a mechanism in human aggressive B-cell lymphomas, a frequently Myc-activated entity where TGF-β1 signaling has previously been identified as a component of the prognostically favourable “stromal-1” signature (Lenz-G et al., NEJM, 2008). A panel of 30 DLBCL samples was sub-divided based on Ki67 immunoreactivity into a very high proliferation (Ki67hi; ≥80% Ki67-positive cells) and a lower proliferation (Ki67lo; <80% Ki67-positive cells) group. Ki67lo samples exhibited a higher frequency of H3K9me3-positive cells, indicative of cellular senescence. Importantly, the Ki67lo group also presented with a higher fraction of apoptotic cells, more lymphoma-infiltrating macrophages, and a stronger reactivity for the TGF-β signaling mediator Smad3-P, thereby representing a subgroup in DLBCL that displays features highly reminiscent of the macrophage-derived mechanism of senescence induction. Conclusions: Our study expands the relevance of oncogene-induced senescence to Myc-driven cancers and demonstrates that different tumor suppressor programs - such as apoptosis and senescence - are enforced in an interdependent fashion between the tumor- and non-malignant stroma cells during lymphomagenesis. Utilizing the Eμ-myc transgenic mouse lymphoma model and furthermore supported by evidence from human aggressive B-cell lymphoma samples, this study establishes a novel network of heterotypic cell-cell interactions within a tumor in which apoptotic tumor cells induce a paracrine response in non-malignant bystander cells that limits lymphomagenesis by cellular senescence. Given the anti-cancer relevance of senescence and the demonstrated inducibility of senescence by a non-DNA damaging cytokine, such as TGF-β, these findings open the exciting perspective to utilize Suv39h1/H3K9me3-mimicking approaches for future cancer therapies. Disclosures: No relevant conflicts of interest to declare.

In Vitro and In Vivo Effects of CT-011, a Humanized Anti–PD-1 Monoclonal Antibody, in Combination with Rituximab against Human B-Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 724-724
Author(s):  
Fuliang Chu ◽  
Myriam Foglietta ◽  
Hong Qin ◽  
Rakesh Sharma ◽  
Qing Yi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
In Vivo Effects ◽  
Human B Cell

Abstract Abstract 724 Background: Programmed death (PD)–1 is an inhibitory receptor that impairs the function of activated T-cells and natural killer (NK) cells when engaged by its ligands PD-L1 or PD-L2. We have previously demonstrated that PD-1 is markedly up-regulated in intratumoral and peripheral blood CD4+ and CD8+ T cells in patients with follicular lymphoma (FL), a finding associated with impaired T-cell function, suggesting that PD-1 blockade may improve FL immune control. CT-011, a humanized anti PD-1 monoclonal antibody, was previously studied in a phase I clinical trial in patients with advanced hematological malignancies. CT-011 was well tolerated and induced sustained elevations of CD4+ T cells in the peripheral blood. More importantly, apparent clinical benefit was observed in six patients, including one patient with FL who had large tumor masses that achieved a durable complete remission lasting >14 months. Here, we studied the in vitro and in vivo effects of CT-011 on T-cell and/or NK-cell immune responses against human B-cell lymphoma and the hypothesis that CT-011 may improve tumor control when combined with rituximab, a chimeric anti-CD20 monoclonal antibody for the treatment of human FL. Materials and Methods: To determine the effects of CT-011 on antitumor T cells, intratumoral T cells were isolated from primary FL tumor samples, and cultured with or without autologous tumor cells in the presence or absence of CT-011 or isotype control antibody (50 μg/ml each) for 5 days, and tested for proliferation by 3H thymidine incorporation assay. To determine the effects of CT-011 on NK cells, peripheral blood mononuclear cells (PBMCs) derived from normal donors or patients with FL were cultured in the presence or absence of CT-011 (50 μg/ml) with or without IL-2 for 96 hours and analyzed for expression of various activating receptors including CD16, CD32, CD64, Fas ligand, NKG2D, NKp30, NKp44, and NKp46. The in vivo effects of CT-011 were tested in two B-cell lymphoma xenograft models. Ramos and RL lymphoma tumor cells were injected subcutaneously into nude and SCID mice, respectively, and CT-011 (10 μg/mouse) was injected weekly with or without rituximab starting approximately 7–10 days after tumor inoculation. Results: We observed that CT-011 significantly increased the proliferation of intratumoral T cells in response to autologous tumor cells compared with isotype control antibody. Treatment with CT-011 enhanced the expression of Fas ligand, CD32, CD64, and NKp30 on human NK cells in the presence of IL-2 as compared with PBMCs treated with IL-2 alone or media control. In the RL lymphoma xenograft model in SCID mice, treatment with CT-011 significantly delayed tumor growth (P≤0.05) and improved survival (P≤0.01) compared with control mice injected with saline. In a Ramos lymphoma xenograft model in nude mice, treatment with CT-011 and rituximab eradicated established tumors in a significant proportion of mice (P≤0.05) and markedly improved survival compared with rituximab alone or saline. Conclusions: Taken together, these studies suggest that blockade of PD-1 with CT-011 enhances the function of anti-tumor T-cells and augments the expression of activating receptors on NK cells. Treatment with CT-011 led to improved tumor control against human B-cell lymphoma in xenograft models and the combined use of CT-011 and rituximab was more effective that rituximab alone. These results provide the rationale to test the combination of CT-011 with rituximab in patients with B-cell lymphoma, given that the combination is likely to be complementary and may even be synergistic, leading to enhanced clinical efficacy without increasing toxicity. The development of such approaches that activate both the innate (NK-cells) and adaptive (T-cells) immune systems is likely to minimize the emergence of immune escape variants and improve clinical outcome in patients with lymphoma. A clinical trial evaluating CT-011 in combination with rituximab is planned in patients with relapsed FL. Disclosures: Rodionov: Cure Tech Ltd.: Employment. Rotem-Yehudar:Cure Tech Ltd.: Employment.

scholarly journals Deoxypodophyllotoxin Exerts Anti-Cancer Effects on Colorectal Cancer Cells Through Induction of Apoptosis and Suppression of Tumorigenesis

2019 ◽  
Vol 20 (11) ◽  
pp. 2612 ◽  
Author(s):  
Chathurika D. B. Gamage ◽  
So-Yeon Park ◽  
Yi Yang ◽  
Rui Zhou ◽  
İsa Taş ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Apoptotic Pathway ◽  
Anti Cancer ◽  
Cervical Tumors ◽  
Skin Xenograft

Deoxypodophyllotoxin (DPT) is a cyclolignan compound that exerts anti-cancer effects against various types of cancers. DPT induces apoptosis and inhibits the growth of breast, brain, prostate, gastric, lung, and cervical tumors. In this study, we sought to determine the effect of DPT on cell proliferation, apoptosis, motility, and tumorigenesis of three colorectal cancer (CRC) cell lines: HT29, DLD1, and Caco2. DPT inhibited the proliferation of these cells. Specifically, the compound-induced mitotic arrest in CRC cells by destabilizing microtubules and activating the mitochondrial apoptotic pathway via regulation of B-cell lymphoma 2 (Bcl-2) family proteins (increasing Bcl-2 associated X (BAX) and decreasing B-cell lymphoma-extra-large (Bcl-xL)) ultimately led to caspase-mediated apoptosis. In addition, DPT inhibited tumorigenesis in vitro, and in vivo skin xenograft experiments revealed that DPT significantly decreased tumor size and tumor weight. Taken together, our results suggest DPT to be a potent compound that is suitable for further exploration as a novel chemotherapeutic for human CRC.

Apoptotic and Non-Apoptotic Cellular Pathways Executed by Bortezomib (BTZ) in Rituximab-Resistant Lymphomas

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4967-4967
Author(s):  
Juan Gu ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Cory Mavis ◽  
Natalie M Czuczman ◽  
Karen E Thudium ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Tumor Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mitotic Catastrophe ◽  
M Cell ◽  
Lymphoma Cells

Abstract Abstract 4967 Rituximab-chemotherapy relapsed/refractory (r/r) B-cell lymphomas represent an emerging clinical challenge that underlies the need to develop alternative therapeutic strategies. A better understanding of the mechanism(s)-of-action of BTZ and other proteasome inhibitors (PI) is likely to aid in the identification of biomarkers that can be used to determine clinical responsiveness and/or help in the rational development of novel PI-based therapeutic combinations (e.g. incorporating biologics, small molecules and/or chemotherapy) in r/r B-cell lymphoma. Previously we demonstrated that rituximab resistance was associated with increased proteasome activity leading to a de-regulation in the apoptotic threshold of lymphoma cells to multiple chemotherapy agents. Pharmacological and genetic (e.g. siRNA silencing of BAK/BAX) inhibition of apoptosis partially affected BTZ activity in rituximab-resistant (RSCL) but not in rituximab-sensitive cell lines (RSCL) suggesting the existence of alternative pathways of cell death associated with PI exposure. To this end we evaluated the contribution of cellular senescence, cell cycle inhibition, or mitotic catastrophe to the anti-tumor activity of BTZ as a single agent or in combination with chemotherapeutic agents in RSCL, RRCL and in primary tumor cells. Lymphoma cells were exposed to BTZ (10-25nM) for 24–48 hrs. Cell senescence was determined by SA-β-gal staining using a senescence assay kit and inverted phase-contrast microscopy was performed. Changes in cell cycle were analyzed by the FACScan DNA method and changes in cell cycle regulatory proteins (i.e. cdc2, cyclinA/B, p21, CDK2/4/6) were analyzed by Western blotting. Mitotic index was determined by Wright-Giemsa stain and positive cells were counted under a Nikon microscope. Mitotic catastrophe was determined by confocal microscopy by staining with α-tubulin antibody. Finally, changes in ATP content was determined by the Cell Titer Glo assay. Baseline differences were observed between RSCL and RRCL in terms of cell morphology, proliferation rate and senescence. RRCL (Raji2R and Raji4RH) were considerably larger in size, had a slower proliferation rate and an exhibited a 3-fold increase the number of cells in senescence than RSCL. In vitro exposure of RSCL and RRCL to BTZ attenuated the number of cells in senescence by 50–75%. Cell cycle analysis demonstrated that RRCL had more cells in S phase when compared to RSCL. In vitro exposure to BTZ-induced G2/M arrest in RRCL, but not in RSCL. Overexpression of G2/M cell cycle regulatory proteins cyclin B and cdc2 were observed in RRCL and in tumor cells isolated from r/r B-cell lymphoma patients. Mitotic catastrophe with multi-nucleated cells were only detected in RRCLs exposed to BTZ. In vitro and ex vivo exposure of RSCL and RRCL to BTZ potentiated the cytotoxic effects of paclitaxel and overcame the acquired resistance to chemotherapy drugs in RRCL and primary tumor cells isolated from r/r lymphoma patients in a dose-dependent manner. Our results suggested that BTZ activates several death pathways in B-cell lymphoma pre-clinical models. In addition to apoptosis, BTZ is capable in triggering mitotic catastrophe in rituximab-chemotherapy lymphoma cells with decreased levels of pro-apoptotic proteins. Moreover, sensitization of RRCL to drug therapy involves interplay between cellular senescence attenuation, G2/M cell cycle regulation, and mitotic catastrophe. Hence, proteasome inhibition may provide a novel therapeutic approach for treating apoptosis-resistant B-cell lymphoma. Research, supported in part as a subproject of NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute. Disclosures: Hernandez-Ilizaliturri: Genmab: Research Funding; Amgen: Research Funding; Celgene: Consultancy. Czuczman:Millennium: Honoraria, Research Funding.

Cross-Species Investigations Reveal Role, Mechanism and Predictive Power of Paracrine, Secondary Senescence in B-Cell Lymphoma Therapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3715-3715
Author(s):  
Jan R. Dörr ◽  
Maja Milanovic ◽  
Yong Yu ◽  
Julia Kase ◽  
Dido Lenze ◽  
...  
Keyword(s):  
B Cell ◽  
Predictive Power ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Brdu Incorporation ◽  
Lymphoma Cells ◽  
Long Term Outcome

Abstract Abstract 3715 Apoptosis and cellular senescence operate as anti-tumor safeguard mechanisms. Unlike apoptotic cells, senescent cells remain viable, and, hence, may crosstalk to other cells in their vicinity over extended periods of time. In fact, cells that entered oncogene-induced senescence or anticancer therapy-induced senescence (TIS) present with a senescence-associated secretory phenotype (SASP), a massive production of secretable factors, which reportedly reinforces senescence through an intracellular mechanism. Utilizing the Eμ-myc transgenic mouse lymphoma model, we provide evidence for an outcome-relevant paracrine, DNA damage-independent secondary senescence program (SecS) in vitro and in vivo. Apoptosis-blocked (bcl2-infected) lymphoma cells from different genetic backgrounds were treated with the DNA-damaging anticancer agent adriamycin in vitro or the alkylating agent cyclophosphamide upon lymphoma formation in mice in vivo. TIS and SecS was detected based on senescence-associated b-galactosidase activity (SA-b-gal), Ki67 staining and BrdU incorporation. The secretome of senescent cells was analyzed by proteomics, gene expression and protein arrays. Overall and progression free survival in mice and patients was assessed by Kaplan-Meier analysis. Transcriptome and secretome analyses followed by functional studies found extracellular matrix proteins, especially small leucine-rich proteoglycans (SLRP), but not NF-kB-dependent cytokines and chemokines, to induce SecS in proliferating lymphoma cells in a paracrine fashion, and linked a “high secretor” status to stronger SecS induction. Dissecting senescence-mediating pathways in recipient cells by biochemical, genetic and pharmacological means unveiled an essential role for the LDL receptor-related protein 1 (LRP1), a receptor for SLRP and other SASP components, through the cell-cycle inhibitor p21CIP1 in SecS. Accordingly, mice harboring TIS-capable but genetically SecS-defective lymphomas (e.g. lacking LRP1 or p21CIP1 expression) experienced inferior long-term outcome to therapy. Not only the recipient cell-based LRP1 status but also the genetically and biologically distinct donor cell-based secretor gene signature stratified outcome in mice. Strikingly, humanized versions of both classifiers were predictive in a large cohort of diffuse large B-cell lymphoma (DLBCL) patients, where they identified – although composed of different gene sets – largely overlapping patient subgroups with superior prognosis, again suggesting SecS as the critical underlying treatment effector principle. Our study highlights the predictive power of senescence for treatment outcome in DLBCL, and provides functional examples (which will be discussed at the meeting) for SASP-related non-genotoxic pro-senescent therapies. Disclosures: No relevant conflicts of interest to declare.

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.

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.

Macrophage Depletion Can Enhance Combined Radiation and Anti-CD40 Monoclonal Antibody Therapy of B-Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2501-2501
Author(s):  
Jamie Honeychurch ◽  
Tim M. Illidge
Keyword(s):  
T Cell ◽  
Radiation Dose ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Low Dose Radiation ◽  
Antigen Presenting ◽  
Dose Radiation

Abstract Treatment of B-cell lymphoma with combined radiation and anti-CD40 monoclonal antibody (mAb) can result in long-term CD8+ T-cell mediated tumor protection. Survival correlates closely with radiation dose, such that for combinations of 5 Gy and anti-CD40 mAb over 80% of animals survive beyond 100 days, but at 2 Gy maximum survival time is only a week over controls. We hypothesized that this reflected a greater initial cell kill with the higher dose, thus de-bulking tumor and slowing down disease progression, and the fact that there is a larger amount of dying tumor cells available for uptake by antigen presenting cells (APC) thus providing a greater source of antigen for subsequent T-cell priming. However, the fate of irradiated tumor cells (ITCs) in vivo following combination therapy, and the precise interaction and influence of host immune response to tumor remains unknown. It is likely that the nature of the APC involved in the clearance of ITCs may vary depending on the amount of cell death occurring and that this in turn may impact upon the quality of the ensuing immune response. An increased understanding of these events would enable the design of strategies to optimise host immune responses after anti-cancer therapies. In an attempt to address some of these issues, we have examined the influence of macrophages (Mø), one of the primary antigen presenting cell populations, on therapeutic outcome following combined radiation and anti-CD40 therapy. Using syngeneic murine B-cell lymphoma models, we have shown that tumor cells undergo apoptosis (as assessed by flow cytometry measuring DNA content with propidium iodide) and importantly, express surface phosphatidylserine (as measured by Annexin V binding) in a radiation dose-dependent fashion. Microscopy using fluorescently labeled tumor cells reveals that most ITCs dying in vivo are taken up by Mø, with the degree of phagocytosis again correlating with radiation-dose and amount of cell death (>40% clearance post-2Gy; >70% clearance post 5Gy). Using clodronate-liposomes, we have been able to deplete Mø in a manner that is both selective and complete (>95% depletion in spleen [primary site of tumor growth] and peritoneum). Following depletion of Mø, clearance of ITCs in vivo is reduced with <20% uptake post 5Gy. To examine the role of Mø on therapeutic outcome we have conducted combination therapies in mice given clodronate-liposomes or control, PBS-liposomes. Whilst the degree of survival after 5 Gy irradiation plus anti-CD40 mAb was unchanged in Mø depleted animals compared to controls, therapy involving low-dose radiation (2Gy) was greatly enhanced in Mø depleted cohorts, with a median survival time of >50 days compared to less than 5 days in control cohorts. In conclusion, depleting Mø and thus reducing the clearance of ITCs in vivo results in enhanced survival after combination therapy with low-dose radiation plus anti-CD40 mAb in lymphoma. We suggest that Mø depletion increases availability of ITCs for uptake and presentation by other APC (eg. dendritic cells), which may promote T-cell responses more effectively and improve therapeutic results and that these new insights can be readily translated to the clinic.

B-Cell Lymphoma Cells Overexpressing BMI-1 Are Correlated with Drug Resistance through Enhanced Expression of Survivin and Are Effectively Eliminated by T Cells with Anti-CD38 Chimeric Receptor

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3613-3613
Author(s):  
Joyeeta Bhattacharyya ◽  
Keichiro Mihara ◽  
Motoaki Ohtsubo ◽  
Shin'ichiro Yasunaga ◽  
Yoshihiro Takihara ◽  
...  
Keyword(s):  
Drug Resistance ◽  
T Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
Survivin Expression ◽  
B Cell Lymphoma ◽  
Drug Resistant ◽  
Lymphoma Cells ◽  
Anti Cancer

Abstract Abstract 3613 BMI-1 is essential for the self-renewal and proliferation of leukemic and hematopoietic stem and progenitor cells. Increased expression of BMI-1 is known to be an indicator for a poor prognosis in cancer patients. Analysis of the expression of BMI-1 and survivin in 6 patients with B-cell lymphoma (3 drug-resistant and 3 sensitive cases) showed that in the drug-resistant patients, high levels of BMI-1 and survivin were maintained even after drug administration in vitro. However, there observed was a down-regulation of both BMI-1 and survivin expression in the drug-sensitive patients. BMI-1 transduction induced the drug-resistance of two B-cell lymphoma cell lines, HT and RL, to the anti-cancer drugs etoposide and oxaliplatin, but not to irinotecan. The expression of survivin was clearly augmented in the cells transduced with BMI-1. Moreover, we detected sustained expression of survivin level in the presence of etoposide in the BMI-1-overexpressing cells. By contrast, the mock-transduced cells succumbed in the medium with anti-cancer drugs with an accompanying decrease in the expression of survivin as well as BMI-1. Survivin has been reportedly implicated in resistance to chemotherapeutic agents. Intriguingly, survivin mRNA levels in BMI-1-overexpressing cells were consistent with those in controls. Also, the level of survivin was enhanced by treatment with a proteasomal inhibitor, MG132, suggesting that overexpression of BMI-1 stabilized survivin expression post-translationally. We further showed that sh RNA-mediated knockdown of BMI-1 or survivin restored sensitivity to etoposide in the HT cells overexpressing BMI-1. Our findings suggest survivin as a potential target for BMI-1. Thus BMI-1, by acting as an upstream regulator, may control the expression of survivin, facilitating drug resistance in B-cell lymphoma. Next, we examined whether B-cell lymphoma cells overexpressing BMI-1 are abrogated by immunotherapy with T cells containing anti-CD38 chimeric receptor in vitro. Interestingly, these B-cell lymphoma cells were effectively eliminated by specific T cells against B-cell lymphoma cells bearing CD38. These results suggest that the immunotherapy is useful for treatment of patients with B-cell lymphoma cells overexpressing BMI-1, which are refractory to chemotherapeutic reagents. BMI-1 may be an important prognostic marker as well as a future therapeutic target in the treatment of drug-resistant lymphomas. Disclosures: No relevant conflicts of interest to declare.

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