ALBERT

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: 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 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 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: Introduction: We have recently reported that the expression of Krüppel-like factor 4 (KLF4) is upregulated in pediatric Burkitt lymphoma and it was suggested to be a prognostic biomarker for survival (Valencia-Hipolito et al., 2014)*. In addition, preliminary findings have demonstrated that KLF4 is also a resistant factor for drug-induced apoptosis. Rationale: Analysis of the role of KLF4 in resistance may have prognostic and therapeutic implications. Objective: To investigate the molecular mechanism underlying KLF4-induced resistance. Hypothesis: We have reported that the transcription factor Yin Yang 1 (YY1) is overexpressed in hematological malignancies and regulates both drug resistance and immune resistance. In Non-Hodgkin lymphoma (NHL), KLF4 is overexpressed and is under the transcriptional regulation of YY1. Hence, we hypothesized that KLF4 may also regulate drug/immune resistance and its inhibition would reverse the resistance of tumor cells to drug-induced apoptosis. Methods: We have used the NHL cell lines Ramos and DHL4 as models. The expression of KLF4 was assessed by Real Time-PCR and Western. KLF4 inhibition was achieved using the inhibitor Kenpaullone and the inhibition of YY1 and KLF4 was induced by transfection with corresponding siRNAs. Apoptosis was determined by activation of caspase 3 by flow cytometry. Tumor biopsies from pediatric NHL patients were analyzed by IHC. Results: Several NHL human tumor cell lines showed overexpression of KLF4, although at different levels. Treatment with the KLF4 inhibitor, Kenpaullone, resulted in the inhibition of KLF4 expression and the cells were sensitized to Doxorubicin-induced apoptosis. Treatment of tumor cells with YY1 siRNA inhibited both YY1 and KLF4 expressions and the cells were sensitized to drugs-induced apoptosis. Tumor biopsies from patients were divided into two groups, namely, one group with high KLF4 and YY1 and the other group with low levels of KLF4 and YY1. Kaplan Meier analysis revealed that patients with low expression of KLF4 and YY1 responded subsequently to CHOP, whereas patients with high KLF4 and YY1 did not respond to CHOP. Conclusions: The findings revealed the followings: (1) KLF4 overexpression is under the transcriptional regulation of YY1 (2) Inhibition of KLF4 by the chemical inhibitor Kenpaullone, or by YY1 siRNA resulted in the sensitization of tumor cells to drug-induced apoptosis and (3) Patients with NHL whose tumors overexpressed KLF4 and YY1 did not respond to CHOP treatment. Implications: We suggest that the overexpression of KLF4 in NHL may be a novel prognostic biomarker for response to chemotherapy and may also be a therapeutic target. In addition, a recent report by Farrugia M et al (2015)** reported that KLF4 is associated with both BclxL and Mcl-1 and, thus, drug resistance is primarily under the influence of the overexpression of BclxL and Mcl-1. Consequently, since KLF4 is regulated by YY1 and YY1 is in turn, regulated by NF-κB, we suggest the presence of an NF-κB/YY1/KLF4/BclxL/Mcl-1 resistant axis in NHL and gene products in this axis maybe potential novel prognostic biomarkers and therapeutic targets. References: * (Valencia-Hipolito et al 2014, Lek & Lympho 55:1806-1814 ** Farrugia M et al (2015_cell death and disease 19;6:e1699. 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.