Stenson, Mary

Abstract Abstract 772 The mammalian target of rapamycin (mTOR) plays a crucial role in proliferative and anti-apoptotic signals in various lymphomas. The mTORC1 inhibitors, such as the rapamycin analogs temsirolimus and everolimus, have achieved a 38% overall response rate in heavily pretreated mantle cell lymphoma (MCL); however, most responses are partial, and many patients are resistant. One mechanism for resistance to mTORC1 inhibitors is that mTORC2 remains unaffected (Blood 2009 Oct 1; 114 (14): 2926–2935). We hypothesize that combined mTORC1/mTORC2 inhibition will be more effective in MCL. We tested the dual mTORC1/mTORC2 inhibitor OSI-027 (OSI Pharmaceuticals) on MCL cell lines and patient samples that were demonstrated to have a high amount of mTORC2 (Rictor and its target p-AKTS473) and mTORC1 (Raptor and its targets pS6 and p4E-BP1) targets. Dual inhibition through OSI-027 inhibited pAKTSer473, a direct target of mTORC2 without any obvious effect on AKTTh308 in these cells. On the other hand, the mTORC1 inhibitor rapamycin had no effect on pAKTSer473. Interestingly, both drugs effectively suppressed phosphorylation of S6; only OSI-027 decreased phosphorylation of 4E-BP1Th37/46. We next assessed the effect of OSI-027 on MCL proliferation and apoptosis and showed that dual inhibition of mTORC1/mTORC2 through OSI-027 caused growth arrest and apoptosis in a dose-dependent manner in both MCL patient cells treated ex vivo and established MCL cell lines; however, we did not see any significant effect of rapamycin on apoptosis. Recent studies have shown that AKT has direct effects on some members of the apoptosis pathway such as forkhead transcription factors (FOXO), caspase 9, and BAD. OSI-027 treatment significantly inhibited p-FOXO3AT32 with some inhibitory effect on pBADS136. These data suggest that FOXO3A is an important target of AKT through which OSI-027 is exerting its apoptotic effects. There are several potential mechanisms whereby OSI-027 prevents AKTS473 phosphorylation: 1) deactivation of kinases upstream of AKT; 2) activation of phosphatases down-stream of AKT. OSI-027 treatment of MCL cells produced no change in phosphorylated PI3K p85/p55 or PDK1 protein. These data eliminate the possibility of involvement of the PI3K pathway in OSI-027 mediated AKT dephosphorylation. We did find that OSI-027 mediated deactivation of AKTS473 is, at least in part, phosphatase dependent. The PH domain leucine-rich repeat protein phosphatase (PHLPP) family of phosphatases have been shown to directly dephosphorylate and inactivate AKTS473. To determine if AKT and PHLPP1 or PHLPP2 are associated in MCL cells, we immunoprecipitated AKT from MCL cells and probed for association with endogenous PHLPP1 and PHLPP2. All the MCL lines do express endogenous PHLPP1 and PHLPP2; however, they are not complexed to AKT. Further studies using the AKT isoforms AKT1 and AKT2 found AKT2 to be associated with PHLPP1; there was no association of AKT1 or AKT2 to PHLPP2. To investigate if OSI-027 enhanced the AKT2-PHLPP1 interaction, we repeated the co-immunoprecipitation assay in the OSI-027 treated MCL cell lines and demonstrated that OSI-027 increased the interaction of AKT2-PHLPP1. Further we showed that OSI-027 exposure did not alter the expression level of PHLPP1 and PHLPP2 proteins. In summary the dual inhibition of mTORC1/mTORC2 by OSI-027 in MCL is more effective than mTORC1 inhibition by rapamycin. We present the mechanism that OSI-027 induces apoptosis in MCL cell lines by inhibition of Rictor-AKT-FOXO3A signaling, and this is mediated in part by activation of the phosphatase PHLPP1. These findings indicate that simultaneously targeting mTORC1 and mTORC2 may be effective in patients with MCL. Disclosures: Barr: OSI Pharmaceuticals: Employment.

Abstract BACKGROUND: We have demonstrated the green tea extract epigallocatechin-3-gallate (EGCG) has anticancer activity in primary CLL B-cells (Lee, Blood 2004). After dissemination of our in vitro findings by the lay press, many patients with CLL and other low grade non-Hodgkin lymphomas (NHL) began using over the counter green tea extracts as an alternative treatment strategy. We recently reported a case series of 3 patients with CLL and 1 patient with follicular lymphoma who appeared to derive objective clinical benefit from such treatment (Shanafelt, Leukemia Research 2006). Based on these findings EGCG has entered clinical testing for treatment of CLL at Mayo Clinic. Here we explore the in vitro antitumor activity of EGCG against other types of non-Hodgkin lymphoma. METHODS: Five established human B-cell lymphoma cell lines (HT, DOHH2, KARPAS, Ramos, RL) and primary lymphoma cells from 7 patients with various B-cell NHL sub-types [DLBC, FL, SMZ (2), MCL, SLL(2)] were used to evaluate the in vitro sensitivity of human lymphoma cells to EGCG. Freshly isolated primary lymphoma cells harvested in suspension from lymph nodes/spleen were obtained from patients with NHL who provided written informed consent. All patients were untreated at the time of biopsy. Lymphoma cell lines and primary lymphoma cells (n=7) were cultured with increasing doses of purified EGCG (3.12–50 ug/ml) for 24–72 hrs. Viability was assessed by using annexin/PI staining by FACS analysis. RESULTS: EGCG-induced dose dependent cell death in both established human B-cell lymphoma cell lines (average LD50 at 24 hrs between 25–50 ug/mL) and primary NHL cells (average LD50 at 24 hrs between 25–50ug/mL). In contrast, EGCG had minimal effect on purified normal B-cells (n=3) at the highest doses tested (50 ug/mL). By immunoblotting, EGCG-induced death in primary cells and cell lines was associated with PARP cleavage, suggesting the agent induced apoptotic cell death. Despite this finding, EGCG had no effect on levels of MCL-1, XIAP, or Bcl-1 by either immunoblot or FACS analysis. Based on reports that EGCG induces cell death in some cancer cell types through generation of oxidative stress (Furukawa, 2003; Nakazato, 2005), we explored this mechanism in lymphoma cells. To determine whether reactive oxygen species (ROS) generation was necessary for EGCG-induced cell death, lymphoma cell lines were cultured with or without catalase (which catalyzes the conversion of hydrogen peroxide to water and oxygen) for 30 min prior to subsequent 24 hr EGCG exposure (50 and 100 mg/ml). Pre-treatment with catalase (100 U) provided dramatic protection against cell death in both primary NHL cells and NHL cell lines suggesting that EGCG-induced cell death in lymphoma cells is dependent on ROS generation (Fig. 1 shows an example for a patient with mantle cell lymphoma and a patient with splenic marginal zone lymphoma). CONCLUSION: EGCG has in vitro anti-tumor activity against a variety of B-cell NHLs. Given its known favorable toxicity profile in vivo, EGCG is an attractive agent for clinical testing in patients with indolent NHL who otherwise are currently being managed with observation. Figure Figure

Abstract Background: Non-Hodgkin lymphomas (NHL) are increasing in incidence and are now the fifth most common tumor diagnosed each year in the United States. Most NHLs are of B-cell origin but the tumor tissue is variably infiltrated with T-cells. Our group has shown in diffuse B-cell large cell lymphoma, that a high number of intratumoral CD4+ T-cells predicts a better overall survival. It has been shown that recently-characterized CD4+CD25+ regulatory T-cells (Treg cells) played an important role in the mediation of anti-tumor immunity. However, there is no data on the role of intratumoral Treg cells in suppression of autologous infiltrating CD4+ T-cells in B-cell NHL. Goal: To investigate the effect of intratumoral Treg cells on the proliferation of tumor-infiltrating CD4+CD25- T-cells, to determine the underlying mechanism of the T-cell suppression, and evaluate the role that malignant B-cells may play in the recruitment of Treg cells to the site of B-cell NHL. Results: We identified a subset of CD4+CD25+ T-cells over-represented in biopsy specimens of B-cell NHL (these cells comprise 17% of cells in lymphoma biopsies, compared 7% of peripheral blood mononuclear cells, 12% of cells in inflammatory tonsil and 6% of cells in tumor free lymph nodes; p-value =0.001). These CD4+CD25+ T-cells are memory-like T-cells (CD45RO+ and CD45RA−) and express high levels of CTLA-4 and Foxp3 when compared to autologous tumor-infiltrating CD4+CD25- T-cells. Importantly, these CD4+CD25+ T-cells displayed the ability to suppress the proliferation and cytokine (IFN-g and IL-4) production of tumor-infiltrating CD4+CD25- T-cells in response to PHA stimulation. Treatment with anti-B7-H1 antibody or PD-1 fusion protein enhanced the proliferation of infiltrating CD4+CD25- T-cells when co-cultured with intratumoral CD4+CD25+ T-cells. Our results suggest that interaction between B7-H1 and PD-1 accounts for about 30% of intratumoral Treg cell-mediated inhibition of autologous infiltrating CD4+CD25- T-cells in tumor sites of B-cell NHL. Lastly, we found that CCL22 secreted by lymphoma B-cells is involved in the chemotaxis and migration of intratumoral CD4+CD25+ T-cells which express chemokine receptor CCR4, but not CCR8. Conclusion: Our results suggest that tumor microenvironmental CD4+CD25+ regulatory T-cells are important regulators of tumor immunity and that these cells are recruited to the area of lymphoma involvement by the malignant B-cells.

Abstract Background: Numerous studies have shown that lymphoma B-cells are resistant to CTL-mediated death, however the underlying mechanism for this resistance is not clear. In previous work, we have identified a subset of CD4+CD25+ T-cells overrepresented in the tumor sites of B-cell NHL that display a phenotype compatible with regulatory T cells (Treg cells). These cells express high levels of Foxp3 and CTLA-4, and are capable of suppressing the proliferation and cytokine production of autologous infiltrating CD4+CD25- T cells in B-cell NHL. Goal: To explore whether Treg cells exert a suppressive effect on the induction and function of autologous tumor-infiltrating CD8+ T-cells in B-cell NHL. Results: Using fresh specimens obtained from patients with B-cell lymphoma, we found that intratumoral Treg cells had a significantly suppressive effect on autologous tumor-infiltrating CD8+ T-cells in B-cell NHL. When activated CD8+ T-cells were cocultured with infiltrating CD4+CD25- T-cells, they displayed less proliferation than CD8+ T-cells cultured alone. However, we found that with a 1:4 ratio of stimulating cells (Treg cells) to responding cells (CD8+ T-cells), intratumoral Treg cells completely inhibited the proliferation of autologous tumor-infiltrating CD8+ T-cells activated with PHA. Furthermore, we have observed that 20% of infiltrating CD8+ T-cells in fresh isolated samples from B-cell NHL coexpress perforin and granzyme B. When intratumoral Treg cells were cocultured with CD8+ T-cells, the Treg cells inhibited the activation-induced production of perforin and granzyme B of autologous tumor-infiltrating CD8+ T-cells in a dose-dependent fashion. We also found that cytokine treatment (IL-2 and IL-12) or PHA activation induced the capability of tumor-infiltrating CD8+ T-cells to kill lymphoma B-cells, which was accompanied by upregulation of expression of CD107a on the surface of cytotoxic CD8+ T-cells. Intratumoral Treg cells significantly inhibit cytotoxicity of activated infiltrating CD8+ T-cells to lymphoma B-cells. Finally, we found that there was an inverse correlation of cell frequencies between intratumoral Treg cells and tumor-infiltrating CD8+ T cells in patients with B-cell NHL, suggesting that Treg cells may inhibit the migration of cytotoxic T-cells to the sites of B-cell lymphoma. Conclusion: Intratumoral Treg cells dramatically suppress the proliferation and activation-induced production of granules in infiltrating CD8+ T-cells in B-cell NHL. These Treg cells also inhibit the ability of activated tumor-infiltrating CD8+ T-cells to kill lymphoma B-cells in vitro, and may prevent the migration of CD8+ cells to the sites of lymphoma. Taken together, these data indicate an important role for intratumoral Treg cells in CTL-mediated anti-tumor immunity in B-cell NHL.

Abstract Epigenetic silencing of tumor-suppressor genes has potential therapeutic relevance if the diagnostic and prognostic utility of particular DNA methylation patterns could be established. SHP1 is a tumor-suppressor gene that can be downregulated in various neoplasms. The aims of this study are (i) whether SHP1 was lost/ suppressed in diffuse large B cell lymphoma (DLBCL) (ii) Understanding the role of various epigenetic mechanisms including CpG hypermethylation and histone modifications in SHP1 loss, and (iii) whether SHP1 in DLBCL can be re-expressed with epigenetic therapy. We initially analyzed SHP1 mRNA expression in DLBCL (n=9) patient specimens and normal B-cells by QRT-PCR. Decreased expression of SHP1 mRNA was observed in all the DLBCL patient samples as compared to normal B cells. DLBCL tumor samples (n=37) along with normal tonsils (n=10) were stained for SHP1 protein by immunohistochemistry (IHC). SHP1 expression was lost or suppressed in 53% of DLBCL tumors compared to normal controls. These data, when taken together, confirm that SHP1 is strongly expressed in normal B-cells and can be lost or suppressed in DLBCL tumors. We performed epigenetic studies of SHP1 CpG promoter 2 (named CPG1 P2) in 37 DLBCL tumors and 6 DLBCL cell lines by methylation specific (MSP1) PCR and pyrosequencing methods. Surprisingly, SHP1 CpG1 P2 hypermethylation (0/37) was not detected in patient samples or cell lines by both methods. Our data conclusively demonstrate that SHP1 CpG1 P2 hypermethylation is absent in DLBCL tumors. The finding that SHP1 was lost or silenced in many of the clinical samples without finding any CpG1 P2 hypermethylation led us to extend our methylation studies to a novel, more proximal CpG island within P2 (named CpG2) that has not been previously studied in any hematological malignancies. We designed the primers specific for these CpG2 sites and ran the pyrosequencing on the same 37 DLBCL tumors as used above for CpG1 methylation. Using a cut off of >25% (intermediate to high methylation) 57% (21/37) of patient samples were methylated. The Ly3, Ly10, and DHL2 DLBCL cell lines were also hypermethylated at CpG2. To confirm the role of CpG2 methylation in SHP1 expression, we treated the Ly3 and DHL2 cells with the DNA methyltransferase inhibitor, 5-Azacytidine (Aza). We observed increased SHP1 expression compared to untreated control cells in Ly3 and DHL2 cells followed by a progressive demethylation of SHP1 CpG2, as shown by positive U-MSP with increasing amplification intensity. This resulted in a corresponding down-regulation of activated STAT3 in a similar time dependent manner. Treatment of Ly3 and DHL2 cells with 5-Aza for 0-6 days significantly (p=0.0008 for Ly3 and p=0.03 for DHL2) reduced the cell survival (40% decrease at day 6). To further investigate epigenetic control of SHP1 in DLBCL, we proceeded to study whether histone modifications in the SHP1 promoter were involved in the regulation of SHP1 expression. Chromatin immunoprecipitation assays were performed in the Ly3, Raji cell line and normal B cells, by using antibodies to repressing marks on histones. We observed that the SHP1 promoter in the Ly3 and Raji cells was enriched for both H3K9me3 and H3K27me3 repressing marks as compared to normal B cells. To confirm the role of histone modifications in SHP1 expression, we treated Ly3 cells with the histone methyltransferase inhibitor (HMT) DZNEP and the HDAC inhibitor LBH589 (Novartis Pharmaceuticals). DZNEP has been shown to be very specific for histone suppressive marks H3K27me3 that are mediated through EZH2. Treatment with DZNEP partially reverted the H3K27me3 histone marks in the SHP1 promoter followed by increased the SHP1 expression in Ly3 cells. LBH589 treatment was not able to inhibit H3K27me3 marks, but was able to inhibit the CpG2 methylation within the SHP1 promoter 2 alone or in combination with 5- Aza. In conclusion, these data provide a comprehensive characterization of the epigenetic mechanisms leading to SHP1 deregulation in the DLBCL tumors. These findings support the notion that control of SHP1 P2 in DLBCL tumors is associated with both DNA promoter methylation and histone methylation, which explains our results that treatment with 5-Aza, DZNEP and LBH589 could reactivate SHP1 expression and inhibit DLBCL cell survival. Therefore, SHP1 may prove useful diagnostic and prognostic marker and provide a rationale to use epigenetic therapy for patients with DLBCL. Disclosures: No relevant conflicts of interest to declare.

Abstract Blood cytokine elevations have been associated with non-Hodgkin's lymphoma (NHL) such as follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Although other types of NHL have been studied, to our knowledge, cytokine deregulation in mantle cell lymphoma (MCL) has not been comprehensively studied. In this study, we studied cytokine levels in untreated and relapsed MCL patients using a multiplex assay to determine the role of dysregulated cytokines in these patients and study any prognostic relevance that could be drawn from their effect on relapse and survival. Samples from two data sets of MCL patients were used for this study. The first dataset was pre-treatment serum samples from newly-diagnosed patients with MCL (n=88) from the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource. The second dataset included pre-treatment plasma samples from relapsed MCL patients (n=20) treated on the MC0048G clinical trial studying the effect of everolimus in lymphoma patients. In addition, unrelated controls from a case-control study of lymphoma were included (n=15 serum, n=24 plasma). Thirty cytokines were assessed using a multiplex ELISA. Differences between cytokines in cases and controls were assessed using Wilcoxon rank-sum tests; associations between cytokines and event-free survival were assessed using Kaplan-Meier curves and Cox proportional hazards models. In the newly diagnosed MCL patients, IL-10 (p<0.0001), IL-1RA (p=0.001), IL-6 (p= 0.04), sIL-2Rα (p=0.005), and MIG (p=0.002) were elevated compared to normal controls. The only significantly elevated cytokine in the recurrent MCL cases compared to controls was sIL-2Rα (p= 0.0006). High levels (above median) of sIL-2Rα levels were associated with inferior event-free-survival (EFS) in both newly diagnosed (HR=2.61 (95% CI: 1.41-4.81, p= 0.0022) and relapsed MCL (HR=2.90, 95% CI: 1.08-7.80, p=0.035) patients; this remained significant after adjusting for MIPI (p=0.01 and 0.03, respectively). Moreover, in the relapsed MCL group eotaxin (p< 0.0001), IL-12 (p< 0.0001), and MCP-1 (p= 0.0002) were suppressed compared to normal controls. We hypothesized that STAT3 (pSTAT3) in MCL cells might be the possible cause behind IL-12 suppression. Indeed, 12/13 MCL samples from IL-12-suppressed relapsed MCL patients were pSTAT3+. When studying the association between cytokine levels and clinical characteristics in both untreated and relapsed MCL groups, sIL-2R was significantly associated with the MIPI score in both untreated (p<0.0001) and relapsed (p=0.007) MCL groups; and with performance status (p=0.005), lymphoma stage (p=0.004), and B symptoms (p=0.006) in the newly diagnosed MCL group.Figure 1Event-free survival of patients with untreated and relapsed MCL by sIL-2R levels.Figure 1. Event-free survival of patients with untreated and relapsed MCL by sIL-2R levels. Our study shows that cytokines are deregulated in both untreated and relapsed MCL patients. It emphasizes on the importance of sIL-2R in its association with negative clinical parameters and worse prognosis in MCL. These findings gives the rational for using sIL-2R along with MIPI and other parameters in predicting prognosis and further to monitor response in MCL. Furthermore, we showed that IL-12 is suppressed in relapsed MCL group and that pSTAT3 expression is a possible mechanism behind this suppression. Further studies are needed to investigate the effect of drugs that suppress pSTAT3 expression in IL-12 suppressed MCLs and disease progression in relapsed MCL patients. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 925 Diffuse large B-cell lymphomas (DLBCL) consists of biologically distinct subtypes: Germinal Center B-cell (GCB)–like and Activated B-cell (ABC)-like. Studies to date suggest that patients with tumors that have ABC features have an inferior survival; therefore, it is important to develop new therapies specifically for ABC-type DLBCL based on mechanistic differences. It has been shown that the ABC-DLBCL has high levels of total and phosphorylated STAT3 (pSTAT3) protein and this STAT3 activity may play a role in the survival of ABC-DLBCL cells. Indeed, when ABC and GCB cell were incubated with the novel JAK2 inhibitor TG101348 (TargeGen, San Diego, CA) we found potent cytotoxicity (LD50 2μM) in ABC DLBCL but not in GCB DLBCL. This provides further rationale to study the regulation of STAT3 in the survival of ABC type DLBCL cells. In studies of HDAC expression in ABC DLBCL cells, we found over-expression of the class 1 histone deacetylases, specifically HDAC1 and HDAC3. We then performed co-immunoprecipitation experiments to learn the functional interaction between STAT3 and HDACs. We found that STAT3 formed complexes with HDAC1 to a greater extent than HDAC3. We also showed that STAT3 did co-precipitate with HDAC1 suggesting a reciprocal relationship between these two proteins. These results suggested that HDAC1 (but not HDAC3) might be involved in the regulation of STAT3. The HDAC inhibitor LBH589 (LBH, Novartis Pharm) was more cytotoxic to ABC (LD50, 10-20nM) than GCB (LD50, 50-100nM) and was shown to dephosphorylate STAT3. These results led to experiments designed to determine the effect of LBH on STAT3 acetylation. When ABC cells were incubated with LBH, co-immunoprecipitation data showed a dose-dependent increased in acetylation of STAT3 without significant change on the levels of STAT3 immunoprecipitated. Next, we asked that how LBH is dephosphorylating STAT3. One potential mechanism is activation of a protein phosphatase. Immunoprecipitation of protein phosphatase 2A (PP2A) in the lysates of LBH-treated cells followed by western blotting of STAT3 demonstrated a positive correlation between LBH dose and amount of STAT3 co-immunoprecipitated with PP2A. Our data show that when LBH is incubated with ABC cells, the LBH increases binding of PP2A to STAT3 and the increased phosphatase activity turns off STAT3 signaling. Since both HDAC inhibitor (LBH) and the JAK2 inhibitor (TG) had selective cytotoxic effects on ABC cells, and since both agents dephosphorylate STAT3, we combined the two drugs. We found that the combination exhibited enhanced inhibitory effects on the growth of ABC cells and was able to alter the association of STAT3 with HDAC1 and subsequently dephosphorylate STAT3 to greater extent than either agent alone. Furthermore, we knocked down HDAC1 expression (through siRNA) in OCILy10 (ABC) cells and showed that inhibition of HDAC1 inhibits constitutively activated STAT3. This data suggests that inhibiting HDAC1 expression results in STAT3 dephosphorylation. In summary, we have demonstrated that a key consequence of HDAC expression in ABC cells is continuous activation of STAT3. These data indicate that HDACs activate STAT3 and that PP2A may be important in maintaining STAT3 in the activated state in the ABC cells. This study describes a new mechanism by which STAT3 mediates ABC cell survival and provides the rationale for targeting ABC type DLBCL by simultaneously inhibiting HDAC activity and STAT3 activation by pharmacologic grade inhibitors such as LBH589 and TG101348, which are currently in clinical trial. A phase 1 and 2 trial of JAK2 inhibitor, TG101348 for ABC/GCB type DLBCL is planned. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 115 Patients with diffuse large B- cell lymphoma (DLBCL) tumors that have an activated B-cell like (ABC) gene expression profile have a poorer prognosis. Understanding the mechanism(s) used by ABC tumor cells to resist the effects of common chemotherapy agents may lead to alternative approaches for the treatment of these tumors. ABC cell lines have been shown to have high levels of phosphorylated STAT3 (pSTAT3); however, the mechanisms that regulate STAT3 signaling in ABC DLBCL remain unclear. Histone deacetylases (HDACs) are enzymes that can deacetylate both non-histone and histone substrates. In this study we tested the hypothesis that HDACs in the tumor cells target a non-histone protein STAT3 in ABC DLBCL. In studies of HDAC expression in DLBCL tumors, we found over-expression of the type 1 HDACs, specifically HDAC1and HDAC3, in the pSTAT3- positive ABC tumors as compared to germinal centre B like (GCB) tumors. We then performed a co-immunoprecipitation (Co-IP) assay to learn the functional interaction between STAT3 and HDAC1. We found that STAT3 formed complexes with HDAC1 or HDAC3. Further Co-IP studies demonstrated that p300, a histone acetyltransferase (HAT), STAT3, and HDAC1 are all in the same complex. To determine whether p300 acetylates STAT3 in ABC cells, we immuno-precipitated endogenous p300 and blotted with acetylated STAT3 and showed that p300 acetylates STAT3 at lysine 685. We next tested whether HDAC inhibition could affect p300 mediated STAT3 acetylation in ABC cells. Inhibition of HDAC activity through the HDAC inhibitor LBH589 (LBH, Novartis Pharmaceuticals) increased STAT3 acetylation in a dose- dependent manner. Similar results were obtained when we used antiacetyl- lysine antibody. Furthermore HDAC1 over-expression inhibits STAT3 acetylation at lysine 685. This data implies a tight regulation of STAT3 acetylation and deacetylases in vivo in ABC lymphoma. In addition to acetylation, STAT3 can be modified by phosphorylation, thus the effect of HDAC inhibition on pSTAT3 both at serine and tyrosine residues was studied. We observed a dose-dependent decrease in pSTAT3 with some inhibitory effect on total STAT3. LBH was found to mediate STAT3 dephosphorylation by inhibiting the tyrosine phosphorylation of JAK2 and TYK2, the STAT3 upstream activators, in a dose- dependent manner. Since ABC lymphoma has higher levels of HDAC1 or HDAC3 and pSTAT3/STAT3 than GCB, we hypothesized that ABC cells will be more sensitive to HDAC inhibition than GCB. In fact, when ABC and GCB DLBCL cells were treated with LBH we observed that LBH was more cytotoxic to ABC than GCB as evidenced by annexin/PI staining and PARP cleavage. LD90 was 25 nM for ABC cells, however GCB cells required 5 times more LBH to kill 90% cells. STAT3 activation regulates genes involved in cell survival, including Bcl-2, Mcl-1, Bcl-XL, and c-Myc. LBH treatment resulted in down-regulation of Mcl-1 and c-Myc in ABC cells but has no effect in GCB cells; however, Bcl-2 and Bcl-XL levels were not decreased in both the subtype. Having established that HDAC1 physically associated with STAT3 and that LBH treatment elevated STAT3 acetylation in ABC cells, we proceeded to deplete endogenous HDAC1 with siRNA in Ly3 cells and found that HDAC1 knockdown up-regulated STAT3 acetylation indicating that HDAC1 negatively regulates the acetylation in vivo. HDAC1 inhibition also prevented phopshorylation of STAT3 and induces aopotosis in ABC cells. In summary, we have demonstrated that a key consequence of HATs and HDACs expression and activity is modulation of the STAT3 pathway in ABC lymphoma. Inhibition of this pathway with the HDAC inhibitor LBH inhibits constitutive STAT3 signaling and induces Mcl-1 mediated apoptosis. These studies provide the rationale for targeting the poorly responsive ABC-type DLBCL by inhibiting HDAC activity with epigenetic inhibitors such as LBH. We are currently testing LBH589 in relapsed DLBCL in a phase I clinical trial. Disclosures: No relevant conflicts of interest to declare.

Abstract Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and β are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3β compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3β genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3β binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3β is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3β correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.