ALBERT

Description: Transgenic coexpression of a class I–restricted tumor antigen–specific T cell receptor (TCR) and CD8αβ (TCR8) redirects antigen specificity of CD4+ T cells. Reinforcement of biophysical properties and early TCR signaling explain how redirected CD4+ T cells recognize target cells, but the transcriptional basis for their acquired antitumor function remains elusive. We, therefore, interrogated redirected human CD4+ and CD8+ T cells by single-cell RNA sequencing and characterized them experimentally in bulk and single-cell assays and a mouse xenograft model. TCR8 expression enhanced CD8+ T cell function and preserved less differentiated CD4+ and CD8+ T cells after tumor challenge. TCR8+CD4+ T cells were most potent by activating multiple transcriptional programs associated with enhanced antitumor function. We found sustained activation of cytotoxicity, costimulation, oxidative phosphorylation– and proliferation-related genes, and simultaneously reduced differentiation and exhaustion. Our study identifies molecular features of TCR8 expression that can guide the development of enhanced immunotherapies.

Description: Rituximab (chimeric anti-CD20 mAb) has been used for the treatment of Non Hodgkin B cell lymphomas (B-NHL), alone or in combination with CHOP. However, a subset of patients does not respond to treatment or develops refractoriness to further treatments. Therefore, there is an urgent need to develop new alternatives to treat these patients. We have reported that treatment of B-NHL cell lines with rituximab inhibits anti-apoptotic survival pathways and down regulates the expression of anti-apoptotic Bcl-2 family proteins (i.e. Bcl-2/Bcl-xl) resulting in sensitization to chemotherapeutic drugs. Further, rituximab-resistant clones showed over-expression of anti-apoptotic gene products (Jazirehi et al., Cancer Research1:1270–81, 2007). Therefore, we hypothesize that inhibitors of anti-apoptotic Bcl-2 family may reverse the resistance to apoptotic stimuli. We examined chemical inhibitors that mimic natural ligands of the anti-apoptotic BH3-only proteins. GX15–070 (Gemin X Biotechnologies, Inc., Canada) inhibits Bcl-2 protein-protein interactions resulting in Bak and Bax oligomerization, release of cytochrome C, and activation of caspases (Shore and Viallet, Hematology, 2005; ASH, 226–230). Treatment of B-NHL cell lines (Raji, Ramos, 2F7, DHL-4) with subtoxic concentrations of GX15–070 (20–50 uM) resulted in inhibition of cell proliferation and subsequently induction of apoptosis as determined by TUNEL. There was a time and concentration-dependent effect of GX15–070 on cytostasis and apoptosis. Analysis of cells treated with GX15–070 by western blotting revealed that Bcl-2, Bcl-xl, and Mcl-1 protein expressions were significantly inhibited as compared to controls. The protein inhibition by GX15–070 was not expected and needs further investigation. We also examined the effect of combination treatment of GX15–070 with the chemotherapeutic drug CDDP and there was an additive or synergistic cytotoxic effect. Treatment of rituximab-resistant clones (generated from 2F7, Raji and Ramos) treated with GX15–070 resulted in significant inhibition of cell growth and apoptosis. The cytotoxicity of GX15–070 in the B-NHL cell lines was tumor specific, because treatment of human peripheral blood leukocytes from different donors did not show any cytotoxic effect. Likewise, treatment of nude mice with different concentrations of GX15–070 did not show any detectable toxicity. These findings demonstrate that GX15–070 is cytotoxic to various drug/rituximab-resistant B-NHL cell lines and is not toxic to normal human leukocytes. This study suggests that combination of GX15–070 with subtoxic concentrations of chemotherapeutic drugs may have additive/synergistic effects. The present findings support the potential therapeutic application of GX15–070 in the treatment of patients with B-NHL that are resistant to current therapies.

Description: There have been significant advances in the treatment of patients with B-NHL using combination of rituximab and CHOP. However, a subset of patients does not initially respond or develop resistance to further treatments; hence, the need for alternative therapies to overcome resistance. TRAIL and agonist DR4/DR5 monoclonal antibodies have been examined clinically against a variety of tumors in Phase I/II. However, the majority of B-NHL derived from patients and cell lines are resistant to TRAIL-induced apoptosis. Recent findings demonstrated that treatment of TRAIL-resistant-B-NHL with rituximab sensitizes the tumor cells to TRAIL apoptosis. The underlying mechanism of rituximab-induced sensitization to TRAIL, however, is not clear. We have recently reported that treatment of tumor cells with sensitizing agents (example CDDP, proteasome inhibitors) resulted in the reversal of resistance to TRAIL via induction of Raf-1 kinase inhibitor protein (RKIP) and demonstrated the pivotal role of RKIP in the regulation of tumor cell sensitivity to TRAIL. Hence, since rituximab induces the expression of RKIP in B-NHL, we determined the role of RKIP induction by rituximab in the sensitization of B-NHL to TRAIL apoptosis. Various B-NHL cell lines were used as models for study. Treatment of B-NHL cells with rituximab (20 ng/ml) and TRAIL (5–10 ng/ml) resulted in significant potentiation of apoptosis and synergy was achieved. Rituximab induced the expression of RKIP as determined by RT-PCR and western concomitantly with inhibition of NF-kB. The inhibition of NF-kB resulted in upregulation of RKIP expression and was mediated, in large part, by inhibition of the transcription repressor Snail (downstream of NF-kB). Further, RKIP-induced inhibition of NF-kB by rituximab resulted in downstream inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) and concomitantly with the upregulation of DR5 expression. The role of RKIP induction by rituximab in the upregulation of DR5 and sensitization to TRAIL apoptosis was corroborated by the use of cells over expressing RKIP which were sensitive to TRAIL apoptosis in the absence of rituximab. Our findings reveal a novel mechanism of rituximab-induced sensitization of B-NHL to TRAIL apoptosis via inhibition of NF-kB and Snail and upregulation of RKIP and DR-5. The combination of rituximab and TRAIL may be effective in the treatment of B-NHL. Further, our studies suggest that agents other than rituximab that can induce RKIP can reverse resistance to TRAIL in B-NHL that are unresponsive to rituximab treatment.

Description: Abstract 2887 Galiximab (anti-CD80 mAb) is a primatized mAb (human IgG1 constant region and Cynomogous macaque variable region) that binds CD80 on lymphoma cells. It has been shown in vitro that Galiximab inhibits tumor cell proliferation and mediates ADCC. Galiximab is currently in clinical trials for a variety of cancers. Our preliminary findings demonstrated that Galiximab treatment of B-NHL cell lines, like Raji, triggers the cells and inhibits the constitutively activated NF-κB pathway. We hypothesized that Galiximab-induced inhibition of NF-κB may result in the inhibition downstream of several anti-apoptotic gene products and sensitizes cells to drug-induced apoptosis. Raji cells were treated with Galiximab (20-100 μg/ml) for 18h and followed by treatment with the chemotherapeutic drug CDDP (5-10 μg/ml) for 24h and apoptosis was determined by flow for activation of caspase 3. The findings demonstrated that the cells treated with Galiximab were sensitized to CDDP-induced apoptosis. Analysis of the apoptotic pathway following treatment with Galiximab revealed the inhibition of anti-apoptotic gene products such as Bcl-2 and Bclxl. We have also found that Galiximab, like rituximab, inhibits the Fas and DR5 transcription repressor Yin Yang 1 (YY1) and the direct inhibition of YY1 resulted in tumor cell sensitization to both Fas-L and TRAIL. We examined whether inhibition of YY1 by Galiximab was also involved in the sensitization to CDDP apoptosis. Raji cells were treated with YY1 siRNA and, unlike control siRNA or non-treated siRNA cells, the tumor cells were sensitized to CDDP apoptosis. The inhibition of YY1 by siRNA correlated with the inhibition of Bcl-2 and Bclxl. The direct role of Bcl-2 and Bclxl in the regulation of resistance was corroborated by treatment of cells with the Bcl-2 family inhibitor, 2MMA3, and such cells mimicked Galiximab and were sensitive to CDDP-induced apoptosis. The mechanism by which treatment with YY1 siRNA resulted in the inhibition of Bcl-2 and Bclxl and the reversal of resistance is not clear. We suggest that YY1 inhibition, following Galiximab-induced inhibition of NF-κB, will result in the inhibition of Snail transcription (Palmer, MB et al., Mol cancer Res 7:221, 2009). Inhibition of the RKIP (Raf kinase inhibitor protein) repressor Snail will result in the induction of RKIP (Wu, K and Bonavida, B Crit Rev immu 29:241, 2009) and, in turn, RKIP will inhibit NF-κB and resulting downstream in the inhibition of Bcl-2 and Bclxl. In addition, it has been reported that YY1 negatively regulates p53 (Sui, G et al., Cell 117:889, 2004) and YY1 inhibition by Galiximab will upregulate p53 and which will result in the inhibition of Bcl-2 and Bclxl (see scheme below). The present findings demonstrate that Galiximab sensitizes drug-resistant B-NHL cells to drug-induced apoptosis via modulation of the NF-κB/YY1/Snail/RKIP/p53 loop. Current studies are validating the present findings with freshly-derived B-NHL cells and also examining the molecular mechanism by which YY1 regulates Bcl-2/Bclxl expression and the reversal of resistance. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4931 Conventional treatments of non-Hodgkin's lymphoma (B-NHL) consist primarily of chemotherapy. Currently, rituximab is used alone or in combination with chemotherapy. However, there are subsets of patients who do not respond initially or develop resistance to further treatment. Therefore, there is an urgent need to develop other immunotherapies with less toxicities. At present, both TRAIL and agonist antibodies directed against TRAIL-R1 and -R2 have been explored for various cancer treatments in various phase 1 and phase 2 clinical trials. We have recently demonstrated that rituximab sensitizes TRAIL-resistant B-NHL cells to TRAIL-induced apoptosis. Sensitization was the result of rituximab-induced inhibition of the constitutively activated NF-κB pathway and downstream the DR5 transcription repressor Yin Yang 1 (YY1). The direct role of YY1 in the regulation of resistance to TRAIL was demonstrated in cells transfected with YY1 siRNA and that became sensitive to TRAIL- apoptosis. Treatment with rituximab did not have any observed effects on the expression of DR4. Based on these findings, it was possible that rituximab-mediated sensitization to TRAIL may invoke either TRAIL-R1 (DR4) or TRAIL-R2 (DR5), or both; thus, this possibility is currently being examined by the use of either neutralizing antibodies against each death receptor or by the use of silencing RNA. Currently, clinical trials are being conducted with both mapatumumab (anti-TRAIL-R1,) and lexatumumab (anti-TRAIL-R2) against a variety of cancers. These agonist antibodies have been evaluated clinically as single agents and in combination with standard therapy in solid and hematologic malignancies. It is not clear whether tumors can develop resistance to agonism of either one or both death receptors and thus, may not respond to monotherapy alone. Combination therapies may be required and we have hypothesized that the combination treatment of rituximab and agonist antibodies may be complementary or synergistic. This hypothesis was based on our findings that rituximab inhibits survival pathways and downregulates anti-apoptotic gene products and, thus, significantly reducing the threshold of resistance. Thus, this rituximab-mediated effect will facilitate the direct cytotoxicity of the agonist death receptor antibodies. The present study investigated whether rituximab can sensitize TRAIL-resistant tumor cells by either agonist TRAIL-R1 or TRAIL-R2 antibodies To address this question, we have examined the effect of agonist antibodies directed against either TRAIL-R1 (mapatumumab) or TRAIL-R2 (lexatumamab). Treatment of the TRAIL-resistant Ramos B-NHL cells with rituximab for 24h and followed with treatment with non-toxic concentrations of mapatumumab (12 μg/ml) or lexatumumab (12 μg/ml) for 18h resulted in significant sensitization to apoptosis as assessed by activation of caspase 3. The mechanism of the sensitization by rituximab for each antibody was also examined. These findings demonstrated that rituximab sensitizes tumor cells to apoptosis by activation of either DR4 or DR5. Although there is heterogeneous expression of TRAIL-R1 and TRAIL-R2 in B-NHL cells, such cells may still be sensitive to rituximab-mediated sensitization to apoptosis by the corresponding agonist death receptor antibody. Recent findings demonstrated that some tumors expressing both DR4 and DR5 were shown to respond to TRAIL by preferential activation of DR4 and not DR5. Therefore, preclinical findings obtained with the use of TRAIL may not be predictive of outcome compared to the use of TRAIL-receptor specific agonist antibodies; mapatumumab or lexatumumab. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4919 Rituximab, a chimeric anti-CD20 mAb, has being used, alone or in combination with chemotherapy, in the treatment of patients with B-NHL and rheumatoid arthritis. It is also being tested clinically in the treatment of other B cell malignancies. The mechanisms by which the antibody depletes the B cells have been shown to be mediated via ADCC, CDC, and apoptosis. In addition, the antibody also signals the cells and modifies various survival pathways and sensitizes the resistant tumor cells to various apoptotic stimuli (Jazirehi and Bonavida, Oncogene 24:2121, 2005). The role of the host innate cytotoxic cells, such as NK cells, in cooperation with rituximab in the depletion of B-NHL cells has been poorly explored. Studies by us and others have reported that rituximab sensitizes resistant B-NHL tumor cells to both Fas ligand and TRAIL-induced apoptosis (Bonavida, Oncogene 26:3629, 2007; Daniel, D. et al., Blood 110:4037, 2007). Since NK cells express on the surface TRAIL, we hypothesized that rituximab may also sensitize the TRAIL-resistant tumor cells to NK-mediated cytotoxicity. Accordingly, we have examined various TRAIL-resistant B-NHL cell lines and used peripheral blood-derived purified human NK cells. Treatment of various B-NHL cell lines with rituximab sensitized the cells to TRAIL-induced apoptosis. The mechanism of TRAIL-induced cytotoxicity was found to be the result of TRAIL-induced inhibition of NF-κB and downstream inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) as well as inhibition of anti-apoptotic gene products such as Bclxl. Treatment of various B-NHL cell lines with rituximab, unlike treatment with control IgG1, resulted in significant cytotoxicity in the presence of purified NK cells. The extent of the cytotoxic activity was a function of the E:T ratios used. We then examined the contribution of TRAIL expressed on the NK cell surface for its role in NK-mediated cytotoxicity of rituximab-pretreated B-NHL cells. We used a neutralizing TRAIL antibody that was added in the reaction mixture and demonstrated that the NK cytotoxic activity was significantly reduced compared to controls. These studies with rituximab were also confirmed with other CD20 mAbs. We are currently examining the sensitization of freshly-derived B-NHL and CLL cells that are treated with rituximab and other anti-CD20 mAbs to NK-mediated cytotoxicity for validation of the findings with cell lines. The present findings suggest that, in vivo, patients who are treated with rituximab may recruit NK and other effector cells to mediate, independently of ADCC, cytotoxicity via the TNF-family ligands (e.g. TNF-α, Fas-L, TRAIL). The studies also suggest that this B cell-depletion mechanism by NK cells may contribute to the mechanism of rituximab- mediated depletion of B-NHL cells in vivo. Noteworthy, the proposed host cytotoxic mechanism may not be functional if the therapeutic treatment consists of the combination of rituximab and immunosuppressive chemotherapeutic drugs that may lead to depletion or inactivation of host cytotoxic cells. Disclosures: No relevant conflicts of interest to declare.

Description: Treatment of B-NHL cell lines with rituximab inhibited the p38 MAPK and NF-κB pathways, resulting in chemosensitization to drug-induced apoptosis (Vega et al., 2004 Oncogene, 23:3530–40; Jazirehi et al., 2005 Cancer Res., 65:264–76). Chemosensitization was the direct result of inhibition by these pathways of the anti-apoptotic gene products Bcl-2/Bcl-xL. Cell signaling by rituximab in B-NHL patients has not been investigated and thus, we have examined an in vivo model bearing a tumor xenograft for validation. Balb/c nude mice were transplanted s.c. with the B-NHL cell line Raji; a group of mice was left untreated and another group was treated with rituximab (1000 μg) at days 5 and 10 following implantation of 8x106 tumor cells. The animals were monitored for tumor cell growth and sacrificed at day 30 and tumors were obtained for further analysis. Treatment with rituximab resulted in significant inhibition of tumor cell growth compared to control. Tumor tissues were examined by immunohistochemistry for phospho and non-phospho p38 MAPK and NF-κB (p50), Bcl-2 and Bcl-xL expression. Analysis of tumor-derived tissues from control mice revealed overexpression of phospho-p38 MAPK and NF-κB (p50) and strong nuclear localization of phospho-p50. In addition, there was overexpression of Bcl-2 and Bcl-xL. In contrast, tumor tissues derived from rituximab-treated mice demonstrated significant inhibition of phospho-p38 MAPK, phospho-p50-NF-κB and reduced nuclear localization of phospho-p50. In addition, Bcl-2 and Bcl-xL expression was also significantly reduced. These findings established for the first time, in a pre-clinical model, rituximab-mediated inhibition of the cell survival pathways mediated by p38 MAPK and NF-κB. In addition, the study also corroborates the role of Bcl-2 and Bcl-xL expression in resistance and their inhibition by rituximab. In a separate abstract, tumor tissues derived from many untreated B-NHL patients were analyzed by immunohistochemistry for the activation of both phospho-p38 MAPK and NF-κB and compared to biopsies derived from control individuals. Overall, this study suggests that the p38 MAPK and NF-κB survival pathways are targets for rituximab-mediated effects and also suggests that such targets can be used for intervention in cases of rituximab resistance.

Description: The CD80 antigen, also called B7.1, is the natural ligand for the T cell receptor CD28 and which maintains T cell and B cell adhesion. Galiximab (anti-CD80 mAb) is a primatized (human IgG1 constant regions and Cynomolgous macaque variable regions) mAb that binds CD80 on lymphoma cells and has been shown in vitro to inhibit tumor cell proliferation, upregulate apoptosis and induce ADCC. A phase I/II trial as single agent Galiximab with dose escalation demonstrated that it is well tolerated and produced modest clinical activity. Also, a phase I/II trial evaluated the combination of Rituximab and Galiximab in patients with relapse refractory follicular NHL. The combination produced an overall response rate of 66% with a median PFS of 12.4 months. We have reported that Galiximab sensitized Raji and IM-9 cells to drug-induced apoptosis. The present study extends these findings and examines the underlying molecular mechanism by which Galiximab sensitizes NHL cells to apoptosis by cytotoxic drugs. We hypothesized that Galiximab inhibits intracellularly cell survival anti-apoptotic pathways such as constitutively activated NF-kB, leading to sensitization to drug-induced apoptosis. We have used CD80-expressing Raji cells as a model for our studies. We demonstrate that following treatment of Raji with Galiximab (25–50 μg/ml) for 24 hours, cell lysates were assessed for various gene products of the NF-kB pathway by Western. There were significant downregulation of both p65 and phospho-p65, both IkB-α and phospho-IkB-α and downstream inhibition of Bcl-2 and BclXL and induction of Bak. In addition, there was a strong induction of the metastasis suppressor and immune surveillance cancer gene product Raf-1 kinase inhibitor protein (RKIP) and downregulation of the inactive and phosphorylated form of RKIP. The induction of RKIP by Galiximab was, in part, the result of NF-kB-induced inhibition downstream of the metastasis inducer and RKIP transcription repressor Snail. Galiximab also inhibited downstream both the Fas and DR5 transcription repressor Yin-Yang 1 (YY1) concomitantly with upregulation of Fas and DR5. These findings establish a molecular mechanism by which Galiximab sensitizes tumor cells to drug/immune-induced apoptosis via inhibition of NF-kB and Snail and induction of RKIP expression. We have previously reported that Rituximab modifies intracellular pathways including NF-κB and sensitizes B-NHL to apoptosis (Jazirehi and Bonavida, Oncogene, 24:2121, 2005). Thus, the combination treatment with Rituximab and Galiximab, through common and complementary mechanisms, may result in the reversal of CD20+/CD80+ B-NHL tumor cell resistance. The studies also suggest the potential combination treatment of Galiximab and non-toxic chemotherapeutic drug or immunotherapeutic drug (example: TRAIL) in the treatment of refractory CD80+ B cell malignancies.

Description: Treatment of patients with relapsed or refractory low-grade follicular B-NHL lymphoma with rituximab has resulted in ~50% response rate. The mechanism underlying the failure of rituximab to affect the remaining 50% of the patients is not clear, though their tumors express CD20. The in vivo effector functions of rituximab include ADCC, CDC, and seldom apoptosis. In addition, we have reported that rituximab signals the cells and inhibits several intracellular cell survival pathways that are responsible for the immune and chemo-sensitizing effects of rituximab on resistant B-NHL cell lines (see review Jazirehi and Bonavida, Oncogene24:2121, 2005). The objective of this study was to develop novel and fully humanized anti-CD20 monoclonal antibodies with enhanced effector functions and molecular signaling that may potentiate their therapeutic efficacy. Novel humanized anti-CD20 monoclonal antibodies were derived from a chimerized form of murine anti-CD20 1K11791, shown to exert more important ADCC, CDC, and apoptotic activities as compared to rituximab (Nishida et al., Int J Oncol., 31:29, 2007). A representative humanized monoclonal antibody, BM-ca (Biomedics Inc., Tokyo, Japan) was used to examine its molecular cell signaling on a representative Ramos B-NHL cell line. The studies were also performed in parallel with rituximab treatment. Ramos cells were treated with various concentrations of BM-ca monoclonal antibody and it was found to inhibit cell proliferation in a concentration-dependent manner. The optimal concentration of BM-ca mAb (20–40 μg/ml) was used for further molecular analyses. Following treatment of Ramos with BM-ca mAb for 24 hours, cell lysates were assessed for the expression of various gene products by Western. Compared to untreated cells, treatment with BM-ca inhibited constitutively activated NF-kB as assessed by inhibition of phospho-p65, phospho-IkB-α, but not unphosphorylated forms. In addition, the NF-kB upstream kinase phospho-p38 MAPK was also inhibited. Inhibition of NF-kB and phospho-p38 MAPK resulted in downstream inhibition of the anti-apoptotic gene product Mcl-1 and induction of the proapototic gene products Bax and Bak. BM-ca significantly induced the expression of the metastasis suppressor and immune surveillance cancer gene product, Raf-1 kinase inhibitor protein (RKIP). RKIP has been shown to inhibition NF-kB activity and inhibition of NF-kB results downstream in the inhibition of the metastasis-inducer gene product Snail. Snail is a transcription factor that has been shown recently to negatively regulate the expression of RKIP (Beach et al, Oncogene27:2243, 2008). BM-ca inhibited Snail expression in Ramos cells. In comparison with rituximab, BM-ca showed qualitative and quantitative differences in the regulation of gene expression. These findings demonstrate that BM-ca triggers CD20-expressing B-NHL cells and results in significant alteration of several gene products that regulate chemo and immune-resistance and metastasis.

Description: Motivation Automated profiling of cell–cell interactions from high-throughput time-lapse imaging microscopy data of cells in nanowell grids (TIMING) has led to fundamental insights into cell–cell interactions in immunotherapy. This application note aims to enable widespread adoption of TIMING by (i) enabling the computations to occur on a desktop computer with a graphical processing unit instead of a server; (ii) enabling image acquisition and analysis to occur in the laboratory avoiding network data transfers to/from a server and (iii) providing a comprehensive graphical user interface. Results On a desktop computer, TIMING 2.0 takes 5 s/block/image frame, four times faster than our previous method on the same computer, and twice as fast as our previous method (TIMING) running on a Dell PowerEdge server. The cell segmentation accuracy (f-number = 0.993) is superior to our previous method (f-number = 0.821). A graphical user interface provides the ability to inspect the video analysis results, make corrective edits efficiently (one-click editing of an entire nanowell video sequence in 5–10 s) and display a summary of the cell killing efficacy measurements. Availability and implementation Open source Python software (GPL v3 license), instruction manual, sample data and sample results are included with the Supplement (https://github.com/RoysamLab/TIMING2). Supplementary information Supplementary data are available at Bioinformatics online.