ALBERT

Description: The identification of cancer stem cells (CSCs) as initiators of carcinogenesis has revolutionized the era of cancer research and our perception for the disease treatment options. Additional CSC features, including self-renewal and migratory and invasive capabilities, have further justified these cells as putative diagnostic, prognostic, and therapeutic targets. Given the CSC plasticity, the identification of CSC-related biomarkers has been a serious burden in CSC characterization and therapeutic targeting. Over the past decades, a compelling amount of evidence has demonstrated critical regulatory functions of non-coding RNAs (ncRNAs) on the exclusive features of CSCs. We now know that ncRNAs may interfere with signaling pathways, vital for CSC phenotype maintenance, such as Notch, Wnt, and Hedgehog. Here, we discuss the multifaceted contribution of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), as representative ncRNA classes, in sustaining the CSC-like traits, as well as the underlying molecular mechanisms of their action in various CSC types. We further discuss the use of CSC-related ncRNAs as putative biomarkers of high diagnostic, prognostic, and therapeutic value.

Description: Abstract 5113 Photodynamic therapy (PDT) is a cancer therapeutic treatment that uses a compound called the “photosensitizer” and a particular type of visible light. When photosensitizers are exposed to a specific wavelength of light (600-800 nm), cytotoxic oxygen species are generated that kill cells (Dougherty, TJ et al., JNCI 90:889, 1998). Several clinical trials are currently underway to evaluate the use of PDT for a variety of cancers. A phase II study has been completed with photodynamic therapy in the treatment of patients with lymphoma or chronic lymphocytic leukemia. (NCT00054171). Recently, we have focused our attention about the properties of the photosensitizer Pheophorbide a (Pba), a chlorine, and its effects on different types of solid tumor cells (Rapozzi, V et al., Cancer Biol Ther 14:1318, 2009). The objective of the present study is to investigate the biochemical and molecular mechanisms by which PDT signals the B-NHL Raji lymphoma cell line (as model) and rendering the cells susceptible to both the cytotoxic mechanism of the tumor microenvironment in vivo or to the response to cytotoxic agents in vitro. We hypothesized that treatment of Raji cells with Pba/PDT in our in vitro system may result in the inhibition of resistance factors that regulate tumor cell responses to both chemotherapeutic and immunotherapeutic drugs. Our recent findings demonstrated that the constitutively overexpressed transcription factor Yin Yang 1 (YY1) regulates, in part, tumor cell resistance in lymphoma (Vega, MI et al., J Immun 175:2174, 2005). Accordingly, we examined whether treatment of Raji lymphoma cells with Pba/PDT will also result in the downregulation of YY1 expression and reverse resistance. The Raji cells were seeded at a cell density of 2×105/ml in Petri dishes. When the cells reached a 70% confluency, they were treated with different concentration (80-160-240 nM) of Pba for three hours in the dark and were then irradiated by an LED light source (640 nm at 12,7 mW for 9 min; 6.7 J/cm2). Following the light treatment, the cells were harvested at different times of incubation (18-36h) to assess apoptosis by the activation of caspase 3 using flow cytometry. In addition, different aliquots of cells were used to prepare slides for immunohistochemistry analyses. The results demonstrate that, indeed, treatment with Pba/PDT resulted in the inhibition of YY1 protein expression in Raji cells. By immunohistochemistry, PDT inhibited the basal nuclear and cytoplasmic expression of YY1 and resulted in weak cytoplasmic YY1 expression. The mechanism of YY1 inhibition might have been the result of PDT-mediated inhibition of NF-κB activity (Karmakar, S. et al., Neurosci lett 415: 242, 2007) since YY1 is transcriptionally regulated by NF-κB (Wang, H et al., Mol Cell Biol 67:4374, 2007). In addition, our preliminary findings demonstrate that treatment of drug-resistant tumor cells with PDT sensitizes the cells to drug-induced apoptosis. Overall, the data suggest that YY1 may be considered as a novel therapeutic target in PDT. Based on the findings here, we are currently examining the role of PDT in the dysregulation of the NF-κB/YY1/Snail/RKIP loop (Wu, K and Bonavida, B. Crit Rev Immun 29:241, 2009) that regulates cell survival and proliferation and resistance in lymphoma. (We acknowledge Doctors Oscar Stafsudd and Romaine Saxton for their assistance.) Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3152 We have reported that treatment of B-NHL cell lines with rituximab resulted in the inhibition of the constitutively activated PI3K-AKT pathway (Suzuki et al., Oncogene 26:6184, 2007). Examination of the mechanism by which rituximab inhibits the PI3K/Akt pathway revealed that it induces the expression of the PI3K/Akt inhibitor PTEN (phosphatase and tensin homolog detected on chromosome 10). Time kinetic analysis indicated that the induction of PTEN occurs as early as 6 h post-rituximab treatment. The objective of this study is to delineate the molecular mechanism by which PTEN is induced by rituximab. We hypothesized that rituximab-induced inhibition of the constitutively activated NF-κB pathway, directly and indirectly through inhibition of the PI3K/Akt pathway, may result in the inhibition downstream of the PTEN transcription factors and repressors, Snail and Yin Yang 1 (YY1). Snail has been reported to repress the transcription of PTEN (Escriva, M et al., Mol Cell Biol 28:1528, 2008). Also, YY1 has been reported to positively regulate Snail transcription and expression (Palmer, MB et al., Mol Cancer Res 7:221, 2009). In addition, the induction of PTEN by rituximab also results, in a feed-back loop, in the suppression of YY1 and Snail and potentiates the induction of PTEN (Petriella et al, Cancer Biology Therapy, 8, 1389, 2009). This hypothesis was tested using the B-NHL Ramos cells, as model, for these studies. Treatment of Ramos with rituximab (20ug/ml for 16 hours) resulted in the inhibition of NF-κB, Snail, and YY1 and induction of PTEN expression as assessed by western. The direct role of Snail and YY1 in the suppression of PTEN expression was demonstrated in cells transfected with Snail or YY1 siRNA. The treated cells demonstrated significant induction of PTEN and, concomitantly, inhibition of the PI3K/Akt pathway. We have reported that rituximab sensitizes B-NHL cells to apoptosis by various chemotherapeutic drugs and demonstrated that inhibition of the PI3K/Akt pathway by various inhibitors mimics rituximab in the sensitization of the tumor cells to apoptosis by chemotherapeutic drugs (Suzuki et al., Oncogene 26:6184, 2007). The role of PTEN induction by rituximab in the sensitization of resistanr B-NHL cells to drug-apoptosis was demonstrated in cells pre-treated with rituximab (to induce PTEN) and then transfected with PTEN siRNA. The transfected cells were resistant to drug-induced apoptosis compared to the control siRNA treated cells. Altogether, the above findings demonstrate that rituximab-induced inhibition of the PI3K/Akt pathway is due, in part, to the induction of PTEN through rituximab-induced inhibition of the PTEN repressors Snail and YY1, downstream of NF-κB. Thus, the induction of PTEN by rituximab plays a major role in the reversal of tumor cell resistance to chemotherapeutic drugs. Further, the findings reveal that the dysregulated PI3K/Akt/NF-κB/Snail/YY1/PTEN loop in B-NHL cells can be interfered by rituximab. This interference leads to the inhibition of cell survival and reversal of resistance through sensitization to drugs. We propose that the gene products in this loop are potential novel therapeutic targets in the treatment of lymphoma. Disclosures: No relevant conflicts of interest to declare.

Description: Objective and Rationale Yin-Yang 1 (YY1) is a multi-functional DNA-binding protein, which can activate, repress or initiate transcription depending on the context in which it binds. In addition, YY1 can modulate protein levels or activity through protein-protein interaction. YY1 has been identified as a potential repressor factor for several genes. We have reported that YY1 can act as a transcription repressor for both Fas and TRAIL DR5. In addition to YY1-mediated regulation of tumor cell resistance to cytotoxic immunotherapy, it also has been shown to regulate resistance to chemotherapy [Baritaki et al., J Immunol 80:6199,2008]. Overexpression of YY1 has been shown to be of prognostic significance in prostate cancer [Seligson et al., Int J Oncol 27:131,2005]. Hypothesis In this study, we have examined the expression of YY1 in MM (cell lines and patients’ bone marrow [BM]) by hypothesizing that the resistance of MM cells to various cytotoxic agents may be, in part, regulated by overexpression of YY1 and that YY1 may also be of prognostic significance. Designs and Methods MM cell lines and fresh BM samples from MM patients (n=21) were examined for YY1 expression, cytoplasmic and nuclear, by immunohistochemistry and by Western. The specificity of the anti-YY1 antibody was demonstrated by the competitive inhibition with a peptide used for immunization. Control isotype IgG did not show any staining on the cells. Results First, we found that various MM cell lines (RMPI 8226, IM-9, U266) overexpress YY1 both in the cytoplasm and the nucleus. Next, we examined the expression of YY1 in BM derived from MM patients by immunohistochemistry. BM from MM patients showed overexpression of YY1 as compared to normal bone marrow samples. Furthermore, analysis of both the intensity and frequency of cells expressing YY1 in both the cytoplasm and nucleus was shown to be significantly higher among patients with progressive disease as compared to patients with stable or responsive disease. Conclusions and Implications These findings show that the expression of YY1 among patients with MM may correlate with progression and also suggest the prognostic significance of YY1 in MM patients. Inhibition of YY1 by various agents (example: low-dose chemotherapy, proteasome inhibitors, NO donors, NF-kB inhibitors, etc.) all result in the reversal of resistance to various cytotoxic agents (eg CDDP, TRAIL) therefore, the findings also imply that agents that can inhibit YY1 expression in MM patients may be of therapeutic potential when used in combination with conventional therapeutics.

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 1970 Poster Board I-993 Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is associated with the human cancer susceptibility locus at 10q23. PTEN is one of the most frequently mutated genes in human cancer. Cells deficient in PTEN exhibit increased proliferation, reduced apoptosis and enhanced migration. PTEN primarily converts phosphatidylinositol-3,4,5,-triphosphate (PIP3) in the cytoplasm to phophatidylinositol-4,5-biphosphate (PIP2), thereby directly antagonizing the activity of PI3 kinase (PI3K). Its inactivation results in constitutive activation of the PI3K/Akt/mTOR survival pathway, normally associated with cancer development and progression. In contrast, overexpression of wild type PTEN in cancer cells induces apoptosis and blocks cell-cycle progression, colony formation and cell migration. Therefore, agents that can induce the expression of PTEN in cancer cells are needed and that can be used alone or in combination with cytotoxic drugs in the treatment of resistant cancers. We have reported that rituximab (chimeric anti-CD 20 mAb) inhibits the PI3K/Akt pathway in B-NHL cells and inhibition of this pathway contributed to rituximab-mediated sensitization of resistant tumor cells to apoptosis by chemotherapeutic drugs (Suzuki et. al., Oncogene 2007; 26:6184). It has also been reported that low levels of PTEN expression in DLBCL is a poor prognostic factor. The underlying mechanism by which the anti-CD 20 mAb inhibits the PI3K/Akt pathway is not known and is the subject of the present investigation. We hypothesized that anti-CD 20 mAb-mediated inhibition of the PI3K/Akt pathway might result from the derepression and expression of PTEN. PTEN has been reported to be inhibited by the activation of NF-ΚB and NF-ΚB has been shown to be inhibited by anti-CD 20 mAb. We also hypothesized that inhibition of NF-ΚB by anti-CD 20 mAb was due, in part, to the induction of the NF-ΚB inhibitor, Raf-1-kinase inhibitor protein (RKIP). These two hypotheses were tested using a novel anti-CD 20 mAb, namely R603 (derived from LFB). We expected that R603 treatment of B-NHL cells will result in the induction of both PTEN and RKIP expression through its inhibitory effect on NF-ΚB. Experimentally, Ramos cells were treated with various concentrations of R603 (5-40 μg/ml) for 18 hours and the lysates were prepared and examined for gene products expression by western. The findings revealed that R603 induces significantly the expression of both PTEN and RKIP and the levels of expression were a function of the antibody concentration used. The induction of PTEN and RKIP was paralleled by the inhibition of both PI3K/Akt and NF-ΚB activated pathways. Our studies in non-lymphoid cancers (prostate carcinoma and melanoma) revealed that the transcription repressor Snail, downstream of NF-ΚB, negatively regulates the transcription and expression of PTEN and RKIP and, thus, the inhibition of NF-ΚB by anti-CD 20 mAb should result in the inhibition of Snail. These findings demonstrate a novel mechanism by which R603 mediates its signaling modification in B-NHL through the induction of PTEN and RKIP and, consequently, regulates the sensitiviy of B-NHL cells to various cytotoxic drugs. 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: We have recently reported that rituximab treatment of B-NHL cell lines, like Ramos, inhibited the PI3K/AKT signaling pathway and downregulated Bcl-xL expression. The role of the AKT pathway in chemosensitization was corroborated by treating Ramos cells with the AKT inhibitor, LY294002, and resulted in sensitization of the cells to drug-induced apoptosis. We have investigated a potential underlying mechanism responsible for the rituximab-mediated inhibition of the AKT pathway. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor phosphatase and functions as a negative regulator of the PI3K pathway through its phosphatase activity. We hypothesized that rituximab may upregulate PTEN expression and thus, inhibiting the AKT pathway. Treatment of Ramos cells with rituximab (20μg/ml for 20h) resulted in significant upregulation of PTEN expression (as assessed by both Western and RT-PCR). Time kinetic analysis showed that PTEN is upregulated as early as 6–9h post rituximab treatment. In addition, since rituximab inhibits cell proliferation and cell growth, we have examined the effect of rituximab on the expression of the growth factor pleitrophin (PTN). PTN is a heparin-binding and secreted growth differentiation factor that mediates various functions such as cell motility and migration, survival, growth and differentiation. Treatment of Ramos cells with rituximab inhibited PTN expression as early as 12h post treatment. The present findings suggested that rituximab-mediated induction of PTEN expression and inhibition of both the AKT pathway and PTN epxression may be interrelated and play an important role in rituximab-mediated cell growth inhibition and chemosensitization. A recent report by Li et al., (JBC, 281:10663,2006) demonstrated that PTEN null cells exhibited upregulation of PTN expression and activation of the PI3K/AKT pathway. Further, inhibition of PTN resulted in inhibition of the AKT pathway, thus establishing a feedback mechanism. Our findings with rituximab are consistent with the Li’s findings’. The mechanism by which rituximab inhibits PTN expression is not clear. Reported studies have implicated the role of AP-1 in PTN transcription and in agreement, our findings have also demonstrated that rituximab inhibits AP-1. Overall, the present studies suggest novel targets modified by rituximab namely, PTN and PTEN, which can be considered for therapeutic intervention in the treatment of both rituximab-sensitive and rituximab-resistant tumors.

Description: There has been considerable interest in the treatment of drug-resistant tumor cells with TRAIL or agonist monoclonal antibody directed against the TRAIL receptors DR4 and DR5. TRAIL has been shown to be largely non-toxic to normal tissues and cytotoxic to transformed tumor cells. However, many tumors, including B-NHL, are resistant to TRAIL-induced apoptosis. We have reported that rituximab signals B-NHL cells and inhibits several cell survival signaling pathways leading to chemosensitization (Jazirehi and Bonavida, Oncogene;2004:2121, 2005). In addition, we have recently reported that rituximab sensitizes B-NHL cells to Fas-ligand-induced apoptosis, via inhibition of the transcription repressor Yin Yang 1 (YY1) (Vega et al. The Journal of Immunology;175:2174 2005). We have found that YY1 negatively regulates DR5 transcription and expression and thus, we hypothesized that rituximab-mediated inhibition of YY1 may sensitize TRAIL-resistant B-NHL cells lines to TRAIL-induced apoptosis. The B-NHL cells lines, Ramos and Daudi, were treated with rituximab (20μg/ml for 6h) and were then exposed to various concentrations of recombinant TRAIL (2.5–10 ng/ml for 24h). Following incubation, the cells were examined for apoptosis by assessing activation of caspase-3 and by Annexin V/PI. The findings revealed that the cell lines were relatively resistant to TRAIL but following treatment with rituximab significant potentiation of apoptosis and synergy were achieved. Optimal apoptosis was observed with a concentration of TRAIL of 10ng/ml. The rituximab-treated cells showed a 2 fold upregulation of cell surface DR5 expression as compared to untreated cells. In addition, rituximab treated cells showed significant inhibition of YY1 expression as determined by Western and EMSA. We have also examined the expression of YY1 in tissue arrays containing formalin fixed, paraffin embedded sections from AIDS lymphoma, obtained from the Aids and Cancer Specimen Resource of the NCI. These arrays consisted of 21 Burkitt, 29 Large Cell Lymphoma and 6 Small Cell Lymphoma and were examined for YY1 by immunhistochemistry. The findings revealed that YY1 was overexpressed as compared to normal tissues. Currently, we are examining the effect of rituximab-mediated sensitization of patients derived B-NHL cells to TRAIL-induced apoptosis. The present findings demonstrate that drug-resistant and TRAIL-resistant B-NHL cells can be sensitized by rituximab to TRAIL-induced apoptosis. Further, the studies revealed a potential novel mechanism of rituximab-mediated effect in vivo by recruiting host cells expressing/secreting TRAIL to exert a cytotoxic activity on the rituximab-treated cells. The findings also suggest the potential therapeutic efficacy, in vivo, of combination of rituximab and either recombinant TRAIL or agonist monoclonal antibodies against DR4 or DR5 in the treatment of resistant cells. We propose that inhibitors of YY1 can serve as a sensitizing agent for TRAIL-induced apoptosis in rituximab-resistant B-NHL cells.