ALBERT

Description: The leukocyte activation marker CD69 is a novel regulator of the immune response, modulating the production of cytokines including transforming growth factor-β (TGF-β). We have generated an antimurine CD69 monoclonal antibody (mAb), CD69.2.2, which down-regulates CD69 expression in vivo but does not deplete CD69-expressing cells. Therapeutic administration of CD69.2.2 to wild-type mice induces significant natural killer (NK) cell–dependent antitumor responses to major histocompatibility complex (MHC) class I low RMA-S lymphomas and to RM-1 prostatic carcinoma lung metastases. These in vivo antitumor responses are comparable to those seen in CD69-/- mice. Enhanced host NK cytotoxic activity correlates with a reduction in NK-cell TGF-β production and is independent of tumor priming. In vitro studies demonstrate the novel ability of anti-CD69 mAbs to activate resting NK cells in an Fc receptor–independent manner, resulting in a substantial increase in both NK-cell cytolytic activity and interferon γ (IFNγ) production. Modulation of the innate immune system with monoclonal antibodies to host CD69 thus provides a novel means to antagonize tumor growth and metastasis.

Description: Trib1, Trib2, and Trib3 are mammalian homologues of the Tribbles protein family, an evolutionarily conserved group of proteins that can mediate proteasome-dependent degradation. Evidence suggests that these proteins function as adapters, where they recruit E3 ligases and enhance ubiquination of the target protein in order to promote its degradation. To date, increased Trib1 and Trib2 mRNA expression has been shown to correlate with acute myelogenous leukemia (AML) in humans and induces AML in mice; whereas Trib3 has not been associated with AML. In order to understand the effects of Trib family members in hematopoietic cells, we reconstituted mice with hematopoietic progenitors retrovirally expressing Trib1, Trib2, or Trib3. Trib1 and Trib2 mice developed AML whereas Trib3 mice did not. Our previous data suggested that Trib2-mediated degradation of the transcription factor, C/EBPα, is important for leukemogenesis. We now show that Trib1, like Trib2, strongly binds C/EBPα and induces its degradation. In contrast, Trib3 weakly binds C/EBPα and fails to induce its degradation. Consistent with the ability to strongly bind and degrade C/EBPα, Trib1 and Trib2, but not Trib3, block differentiation of myeloid cells. We are currently mapping the domains that account for the differences between Trib1/Trib2 and Trib3 in leukemogenesis. Together, our results strengthen the correlation between Trib-induced C/EBPα degradation and induction of AML. Furthermore, our data show that different Tribbles family members have distinct targets and understanding this specificity may provide opportunities to therapeutically target Tribbles.

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: Background. Different types of BCR-ABL fusion mRNAs can be found in pts with CML due to different genomic breakpoints and alternative splicing. The two major forms join ABL exon 2 with exons 13 or 14 of BCR, resulting in two main transcripts, b2a2 and b3a2, respectively, that codify for a p210 protein present in the majority of CML patients. b3a2 is more frequent in newly diagnosed patients than the b2a2 transcript, and occasionally both transcripts may be present. The clinical significance of the specific transcript among CML pts treated with imatinib has not been clearly established, and some have suggested that b2a2 may be associated with better outcome. (Blood2006108: Abstract 4780) Genet.Mol. Res.4(4): 803–811 (2005)Journal of Clinical Oncology, 2007 Vol 25, No 18S (June 20 Supplement), 2007: 7043. Purpose: To determine if there is a difference in outcome after imatinib therapy in CML pts according to their BCR-ABL transcript. Methods: We analyzed 480 pts with CP CML treated with imatinib, 251 receiving imatinib as frontline therapy and 229 after interferon (IFN) failure. Molecular response was evaluated using RT PCR every 3 months (mo). Results: The median follow-up was 62 mo (range 1–92 mo) Median age was 51 years (range 15–84). Overall, 187 of 480 (39%) pts expressed b2a2, 234 (49%) b3a2, 55 (11%) expressed both, and 4 (1%) expressed e1a2. The rates of major and complete cytogenetic response were similar for pts with b3a2 and b2a2, both in the newly diagnosed and the post-IFN failure: CCyR 91% for b3a2 and 89% for b2a2 in frontline, and 72% and 78%, respectively in the post-IFN failure group. However, among 433 pts evaluable for molecular response, transcript levels were significantly lower at 3, 6 and 12 months from start of therapy for pts with b3a2 compared to those with b2a2 (Table 1). Table 1. Median BCR-ABL transcript levels over time by transcript type 3 months 6 months 9 months overall b2a2 1.8284 0.318 0.186 0.0464 b3a2 0.5175 0.0553 0.0352 0.0033 p-value 0.001

Description: Galiximab is a primatized anti-CD80 antibody that is being investigated as a treatment for non-Hodgkin’s lymphoma. Results to date from clinical trials indicate that galiximab is well-tolerated and suggest clinical activity both as monotherapy and in combination with rituximab (anti-CD20). CD80 (B7.1), expressed not only by malignant B cells but also by normal B cells, monocytes/macrophages, dendritic cells as well as T cells, plays an important role in the complex and dynamic regulation of adaptive immunity by interacting with either co-stimulatory (e.g. CD28) or co-inhibitory (e.g. CTLA-4, PD-L1) receptors expressed by T lymphocytes. Inhibition of anti-lymphoma immunity that exists within the tumor microenvironment may reflect selection of inhibitory immune regulation mediated through CD80. Galiximab may thus function by directly targeting malignant and possibly non-malignant CD80-expressing cells for antibody-dependent cytotoxicity, or by blocking CD80 interactions involved in suppressing anti-tumor immunity. In preclinical xenograft models, which do not assess the effect of galiximab on non-malignant cells given a lack of cross-reactivity with murine CD80, treatment with galiximab exhibited enhanced tumor growth inhibition when used in combination with either rituximab or with the chemotherapeutic drugs fludarabine and doxorubicin (Hariharan et al., abstract 3040, ASCO, 2007). However, the mechanisms by which galiximab enhances the cytotoxic effects of chemotherapy remain unclear. Rituximab has been shown by us to chemosensitize tumor cells by inhibiting intracellular survival pathways. Thus, we hypothesized that treatment of B lymphoma cells with galiximab facilitates the cytotoxic activity of chemotherapeutic drugs by modifying intracellular anti-apoptotic signaling pathways, resulting in the alteration of pro and anti-apoptotic gene products. The above hypothesis was examined using Raji (Burkitt lymphoma) and IM-9 (multiple myeloma) cell lines as models. Treatment with galiximab resulted in significant inhibition of cell growth and proliferation and modest apoptosis with high concentration. Both Raji and IM-9 are resistant to chemotherapeutic drugs; however, pretreatment with galiximab significantly sensitized the tumor cells to apoptosis induced by CDDP. The synergy achieved was found with subtoxic concentrations of CDDP (5–10 ug/mL) and galiximab. Analysis of the survival pathways and gene products regulating apoptosis following treatment with galiximab, CDDP, and combination will be presented. The present findings demonstrate that galiximab can synergistically enhance cytotoxicity mediated by chemotherapeutic drugs. These findings suggest that in vivo galiximab may have a chemosensitizing effect on resistant B cell malignancies, in addition to its direct cytotoxic effect via ADCC and CDC. The clinincal relevance of these findings will be discussed.

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: 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: 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.