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Background: Accurate characterization of tumor stiffness and adhesion is crucial when surgically resecting meningiomas. Currently, such determinations are difficult to ascertain through standard preoperative imaging diagnostics and clinical experience. Herein, we explored magnetic resonance elastography (MRE) as a means of assessing these parameters and thereby improving neurosurgical resections. Methods: This prospective study of 193 patients with meningiomas involved three distinct patient subsets (cohort 1, 88 [2017–2021]; cohort 2, 55 [2022–2023]; cohort 3, 50 [2023–2024]. We analyzed preoperatively obtained MRE data on meningioma stiffness (cohorts 1 and 2) and adhesion (cohort 2) for accuracy, while matching subjects with or without MRE studies (cohort 3, 1:1 ratio) to define the role of preoperative MRE in improving operative outcomes. Results: Areas under the curve (AUCs) for MRE in gauging meningioma stiffness and adhesion were 0.87 and 0.89, respectively. Both parameters significantly surpassed empiric predictions of stiffness and adhesion rendered by surgeons and radiologists, based on routine magnetic resonance imaging (MRI). MRE-determined stiffness measures independently correlated with prolonged resection duration, whereas MRE-assessed adhesion showed independent associations with increased blood loss, longer duration of tumor resection, and higher risk of pial injury. Ultimately, results based on cohort 3 demonstrated that MRE has great potential in this setting to improve neurosurgical procedures by significantly reducing intraoperative bleeding, resection duration, and risks of pial injury. Conclusion: MRE is a promising, noninvasive tool for accurately assessing meningioma stiffness and adhesion, standing to improve the precision and safety of needed surgical intervention.

Abstract Abstract 960 Diffuse large B cell lymphoma (DLBCL) is an aggressive lymphoma whose survival depends on various signaling pathways one of which is Signal transducer and activator of transcription 3 (STAT3)/Janus kinase 2 (JAK2). We have demonstrated that JAK2 is constitutively activated at the auto-phosphorylation site in many cases of DLBCL. The most common mechanism causing abnormal JAK2 activation is through dysregulated cytokine signaling. Cytokines are deregulated in several hematological malignancies and may play a role in tumor cell growth through receptor-mediated and ligand dependent activation of the JAK2 kinase. In this study we investigated the role of cytokine signaling in JAK2 activation in DLBCL and use of a novel JAK2 inhibitor (JAK2i) to inhibit IL-10 induced JAK/STAT signaling. We studied serum cytokines from 70 patients with new untreated DLBCL who participated in a clinical trial in the North Central Cancer Treatment Group (Micallef, IN et al Blood 2011). Compared to control sera, patients with DLBCL had higher levels of several JAK2 kinase related cytokines (IL-2, IL-6, IL-10, and EGF). IL-10 and IL-6 were significantly higher in DLBCL patients vs controls with p values >0.03 and >0.001, respectively. We next examined whether DLBCL cell lines produced IL-10. The phospho-JAK2 (pJAK2) positive DLBCL cell lines Ly10, DHL2 and HBL1 produced more IL-10 (40–80 pg/ml) than the pJAK2 negative cell lines Ly1, Ly19 and DHL6 (0–5 pg/ml). Analysis of the surface expression of IL-10 receptors (IL-10R) determined that most p-JAK2 positive DLBCL cells express either IL-10Ra or IL-10Rb or both. IL-10 had no effect on DLBCL cell survival in vitro; however, it did promote their proliferation. Only IL-10 (but not the other elevated cytokines) was specifically able to activate JAK2 and STAT3 phosphorylation in a subgroup of DLBCL cell lines. IL-10 was not able to activate JAK1, STAT1 and STAT5, which suggests a specific role of IL-10 in the JAK2 pathway. Moreover, neutralizing antibody to IL-10 inhibited IL-10-induced JAK2 and STAT3 tyrosine phosphorylation. We studied the effect of SAR302503 (SAR503, Sanofi, Cambridge, MA), a selective JAK2i currently in clinical trial for myelofibrosis on constitutive JAK2 signaling in DLBCL cells. SAR503 was able to inhibit JAK2 and STAT3 phosphorylation in a dose and time dependent manner in a variety of DLBCL cell lines and patient samples. JAK2 inhibition with SAR2503 caused a dose-dependent inhibitory effect on survival of pJAK2 positive DLBCL cells not observed in pJAK2 negative cells. Activation of STAT3 signaling up-regulates the expression of various genes involved in cell survival and proliferation such as bcl-xl, bcl-2, mcl-1 and c-myc. We observed a dose-dependent decrease in c-myc protein and mRNA levels in Ly3 and DHL2 cells with SAR503 treatment; however, SAR503 did not effect expression of bcl2, mcl-1 and bcl-xl proteins in DLBCL cells. Interestingly, JAK2 inhibition with SAR503 inhibited the autocrine secretion of IL-10 in a dose-dependent manner. Next, we determined if higher pre-treatment serum IL-10 correlates with the overall survival of 81 DLBCL patients. IL-10 by ELISA demonstrated that IL-10 levels were high in 51% (41/81) of patients (median, 57.7pg/ml; range, 26.1–503.7), and low in 49% (40/81) (median, 15.9 pg/ml; range, 0–25.9). Using a cut-off value of 26.1pg/ml, patients with a high serum IL-10 level had a significantly worse event-free survival (p=0.01). Clinical correlation of serum IL-10 with disease parameters showed that the IL-10 level was correlated with elevated serum LDH (p= 0.0085) and IPI score (p=0.01); there was no correlation with the number of extranodal sites, age, or B symptoms. DLBCL tumors from 40 patients were classified into germinal center B-cell type (GCB) and non-GCB type using the Hans algorithm. There was a clear trend towards a higher serum IL-10 in non-GCB DLBCL patients (p <0.06). In summary, our data identifies mechanisms of JAK2 kinase activation in DLBCL and supports the hypothesis that the IL-10-JAK2-cmyc pathway is activated in patients with DLBCL. Finally our data provide a mechanistic basis of selecting and targeting DLBCL tumor cells with high IL-10 levels and/or constitutive JAK2 activity with potent and novel JAK2 inhibitors such as SAR503. Disclosures: No relevant conflicts of interest to declare.

Abstract The mammalian target of rapamycin (mTOR) exists in two complexes, mTORC1 and mTORC2. Most mTOR inhibitors used for hematologic diseases such as rapamycin, everolimus, and temsirolimus are considered mTORC1 inhibitors and do not target mTORC2. The central role of the mTOR kinase in controlling cell growth has sparked interest in discovering mTOR inhibitors that bind to the ATP active site and therefore target both mTORC2 and mTORC1. mTOR also regulates protein synthesis by phosphorylating the P70S6-Kinase and the eIF4E-binding-protein, 4EBP1. The effect of active site dual mTOR inhibitors on cap dependent translation has not been investigated in lymphomas. In this study we moved beyond the effect of the dual mTOR inhibitor CC214-1 (Celgene Pharmceuticals) on the mTORC2/AKT pathway to focus on its effects on cap dependent protein translation. Cap dependent translation is regulated by the translation initiation complex eIF4F (eIF4E-eIF4G-eIF4A). First, we assessed the formation of the active eIF4F (m7GTP-eIF4E-eIF4G) translation complex by a pull down assay using agarose-immobilized m7GTP cap analog to capture eIF4E and its binding partner in lymphoma cell lines (Jeko, Mino, Granta and JVM2, Ly3, Ly1, DHL6) and in primary cells (n=5). Our data demonstrate that the cap bound fraction from normal B cells contain very little eIF4G and eIF4A; however, lymphoma cells were enriched with eIF4G, eIF4E and eIF4A. Increased mRNA of eIF4E and eIF4G was observed in all lymphoma cell lines tested as compared to normal B-cells. 150 untreated diffuse large B cell lymphoma samples were stained for eIF4E by immunohistochemistry and 76% (135/150) were found eIF4E positive. Taken together, these data suggest that lymphoma cells exist in a translational activated state compared to normal B-cells. Next, we examined the effect of the dual mTOR inhibitor CC214-1 on the integrity of the eIF4F complex. In the CC214-1 treated cells the quantity of the eIF4E bound to 4EBP1 was increased whereas the amount of the eIF4E bound to eIF4G was decreased in the CC214-1 treated lymphoma cells but not in those treated with a selective mTORC1 agent (everolimus). We also found that the phosphorylation of 4EBP1 that controls protein synthesis is partially resistant to everolimus/rapamycin but fully suppressed with CC214-1. CC214-1 had a dose-dependent inhibitory effect on lymphoma cell survival; however, everolimus and rapamycin were only cytostatic. Moreover, CC-214 was much more potent than rapamycin and everolimus in blocking protein translation measured by the leucine incorporation assay. To elucidate the role of the eIF4E complex in the response to CC214-1, we transiently expressed WT-eIF4E plasmid in Jeko cells and then treated with CC214-1. Ectopic expression of wt-eIF4E led to a loss in the CC-214-1 dependent decrease in the eIF4E bound eIF4G as compared with the control plasmid. Moreover, overexpression of eIF4E also protected against CC214-1-induced apoptosis. To evaluate the efficiency of cap-dependent translation in the presence of CC214-1 and rapamycin, we used the bicistronic reporter assay where translation initiation of the first cistron is dependent on the 5′ cap, whereas initiation of the second cistron depends on a viral internal ribosome entry site. Transfection of this plasmid in CC214-1 treated cells caused a significant decrease in cap-dependent, but not IRES-dependent, translation. We next looked the effect of CC214-1 on the eIF4E regulated translationally active mRNA targets. We isolated eIF4E known mRNA targets (Myc, Cylin D1, Cyclin D2 and MCL-1) by immunoprecipitation assays using eIF4E antibody (mRNA-IP). CC214-1 treated RNA-IP data showed decreased expression of Myc but not the other mRNA targets. Myc is highly expressed in the DLBCL tumors and would be a good prognostic or predictive biomarker for CC214-1 therapy. Translation deregulation is an important mechanism that causes aberrant cell growth in lymphoma cells. Currently, there is a need to identify additional agents that target this pathway. These studies provide a mechanistic basis of targeting cap dependent translation in lymphoma through next generation dual mTOR inhibitors. This study also suggests eIF4E (indicative of activated eIF4F complex) as a potential biomarker for the selection of patients for next generation mTOR inhibitors clinical trials in lymphoma patients. Disclosures: No relevant conflicts of interest to declare.

Abstract T cell lymphoma (TCL) is a heterogeneous group of malignant lymphomas and is subdivided in three major subgroups, (i) cutaneous (mycosis fungoides (MF), sezary syndrome (SS) and primary CTCL), (ii) nodal (peripheral T-cell lymphoma, PTCL NOS, anaplastic large cell lymphoma (ALCL), angioimmunoblastic (AITL)) (iii) and extranodal. Because of this broad morphological spectrum and immunophenotypic variation the pathogenesis of most of the TCL is poorly understood. Constitutive STAT3 signaling provides growth-promoting signals in many forms of malignancy; however in-vivo activation of phosphorylated STAT3 and its clinical correlation in subtypes of TCLs has not been extensively studied. In this study we examined constitutive STAT3 activation in various subtypes of TCL tumors and performed pre-clinical testing to determine whether STAT3 inhibitors will be effective. First, we assessed the STAT3 activation (pSTAT3Tyr705) by immunohistochemistry (IHC) on a tissue microarray (TMA) from 169 untreated TCL patient samples. Using a cutoff of ≥30% pSTAT3 nuclear staining, 38% (64/169) of TCL tumors were pSTAT3+. Interestingly, pSTAT3 was differentially expressed among the various TCL subgroups. 27% (16/59) PTCL NOS, 29% (11/38) AITL, 93% (14/15) ALK+ ALCL, 57% (13/28) ALK- ALCL, 50% (6/12) extranodal NK/T cell tumors and 17% (1/6) primary cutaneous showed pSTAT3 positivity. Using either a 30% or 80% cut-off, pSTAT3 positivity was associated with inferior event free survival for AITL, PTCL NOS and ALK neg ALCLs TCLs. Due to small sample size within each group statistical significance (p=0.03) was met only for ALK neg ALCL (80% cutoff). Further analysis in the human TCL cell lines and normal control T-cells also revealed differential expression of pSTATs. ALCL cell lines, Karpas 299 and SR786 were pSTAT3 positive but pSTAT5 negative. Both the CTCL (SS) cell lines HuT78 and SeAx were pSTAT3 negative, however MyLa a cell line of MF origin was strongly pSTAT3+ and pSTAT5+. The normal control CD4 T-cells was pSTAT3- and pSTAT5+. These data clearly suggest that pSTAT3 is differentially expressed among the TCL tumors and is a potential therapeutic target. Potential mechanisms for STAT3 activation could be aberrant secretion of cytokines/chemokines produced by the tumor itself or the tumor microenvironment. We compared the cytokine profile of the culture supernatant from pSTAT3 positive SUDHL1 and pSTAT3 negative HuT78 TCL cell lines using a 30-plex ELISA. The most significantly different cytokines among these 2 cell lines were interleukin -10 (IL-10) and soluble interleukin-2 receptor alpha (sIL-2Ra). The SUDHL1 was found to secrete a very high level (1700 pg/ml) of IL-10, in contrast to only 167 pg/mL in the HuT78 supernatant. Moreover, SUDHL1 secretes 3.5 times more sIL-2Ra (6600 pg/ml) than the pSTAT3 negative cell line HuT78 (1800 pg/ml). In-vitro exposure to IL-10 but not sIL-2Ra was able to further upregulate pSTAT3 in SUDHL1 and Karpas 299. Overall, these data suggest that autocrine secretion of IL-10 but not sIL-2Ra is important for STAT3 activation in ALCL TCLs. Next we compared the cytokine profile of 2 pSTAT3 positive cells from different origin; SUDHL1 (ALCL) and MyLa (MF). In contrast to SUDHL1 (which produce high level of IL-10), MyLa was producing very little IL-10 (18pg/ml). The other most significantly different cytokines between these cells was RANTES with MyLa producing a very high level (28897 pg/ml) and SUDHL1 a very low level (8pg/ml). We also noticed differential level of IL-13 between these cells; MyLa producing 113pg/ml while SUDHL1 only 13 pg/ml. SAR302 (Sanofi, Cambridge, MA), a selective JAK2 inhibitor currently in clinical trial for myeloproliferative neoplasms was able to inhibit autocrine secretion of IL-10 but not sIL-2Ra in a dose-dependent manner followed by STAT3 dephosphorylation and decreased lymphoma cell survival. In summary, our data provide first evidence of in-vivo STAT3 activation in AITL, PTCL NOS, ALK negative ALCL TCLs using TMA from patient samples. Comprehensive cytokine analysis determined IL-10 as an autocrine factor driving STAT3 activation in ALCLTCLs, while RANTES and IL-13 might be important drivers for STAT3 activation in MF TCLs. Finally our data provide a mechanistic basis of selecting and targeting TCL tumor cells with high IL-10 levels and/or constitutive STAT3 activity with potent and novel JAK2 inhibitors such as SAR302 or other STAT3 inhibitors. Disclosures: No relevant conflicts of interest to declare.

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 Abstract 2655 Diffuse large B cell lymphoma (DLBCL) has been classified into two distinct molecular subtypes: germinal center B cell–like (GCB), non-germinal centre-like (non-GCB). To improve outcomes for DLBCL patients, it is necessary to identify additional novel targets within GCB and non-GCB classifications to further stratify patients for prognosis and assist in choosing therapy for the individual patient. We have recently demonstrated that STAT3 activation is frequent in the DLBCL tumors, however the exact molecular mechanism(s) of STAT3 activation in DLBCL tumors are not known. Molecular mechanisms such as epigenetic silencing of negative regulators of the STAT3 pathway such as protein tyrosine phosphatase 6 (PTPN6) may contribute to STAT3 activation. We aimed to learn whether PTPN6 was expressed in GCB and non-GCB DLBCL, and if so, how that expression correlated with STAT3 activation and prognosis. We first performed epigenetic studies of PTPN6 in 38 DLBCL tumors and 6 DLBCL cell lines by methylation specific (MSP) PCR and high resolution melting array (HRM) methods. Surprisingly, PTPN6 promoter hypermethylation (0/38) was not detected in patient sample and cell lines by both the methods. Since the MSP PCR technique yields qualitative rather than quantitative data, we next performed pyrosequencing on the 38 DLBCL samples, and found results consistent with the MSP PCR analysis. Our data conclusively demonstrate that PTPN6 hypermethylation is absent in DLBCL tumors. We next sequenced the 600 bp upstream of the transcription initiation site of PTPN6 promoter 2 and 1 in 10 DLBCL tumors. We did not detect any point mutations in the promoter 2 and 1 core regions. Since PTPN6 promoter hypermethylation and mutations were absent in DLBCL tumors, we determined the expression level of the PTPN6 protein in 40 DLBCL tumors by molecular subtype. Formalin fixed paraffin-embedded DLBCL tumor samples were stained by immunohistochemistry (IHC) for the determination of molecular subtype using the Hans algorithm and the detection of PTPN6 expression. Using a threshold of ≥30%, 75% (30/40) of cases were PTPN6 positive with various levels of expression: 11 cases had 30–80% of tumor cells staining positive and 19 had >80% of cells PTPN6 positive. PTPN6 expression by IHC was only correlated with higher IPI scores and a trend towards a shorter event free survival (EFS) (p=0.07). Within the 29 GCB tumors 69% (20/29) were PTPN6 positive; 100% (10/10) of non-GCB cases were PTPN6 positive. These data clearly suggest that PTPN6 expression is found in both GCB and non-GCB with the latter being uniformly positive (p=0.03) and PTPN6 negative cases being uniformly GCB. PTPN6 mRNA and protein was detected in all three ABC lines (LY3, Ly10, DHL2). Within the GCB lines (DHL6, Ly1 and Ly19) DHL6 was weakly positive and Ly1 and Ly19 were negative for PTPN6 mRNA and protein. Furthermore, within the GCB group PTPN6 positive cases had inferior EFS as compared to PTPN6 negative cases. In the non-GCB group all cases were PTPN6 positive with an EFS similar to PTPN6 positive GCB cases. The role of PTPN6 in the persistent activation of the STAT3 pathway in DLBCL patients has not been investigated. We hypothesized that tumors with activated STAT3 would have loss of PTPN6. Interestingly, this hypothesis was disproven. Within the 15-pSTAT3 positive cases 12 (80%) were PTPN6 positive. Conversely, 26% (6/23) of the pSTAT3 negative cases were PTPN6 negative. The distribution of pSTAT3 and PTPN6 by IHC in samples was evaluated in both GCB and non-GCB groups. Within the PTPN6+/pSTAT3+ group out of the 12 cases most were non-GCB (8/12; 66%). However, within the PTPN6-/pSTAT3- group all the 5 cases were GCB (5/5; 100%). Survival analysis revealed that the groups with the best EFS were those with PTPN6-/pSTAT3- tumors (n=5); those with PTPN6+/pSTAT3+ group (n=12) had the shortest EFS; and those with PTPN6+/pSTAT3- tumors (n=17) being intermediate between the other groups. In summary, we have demonstrated for the first time that PTPN6 is highly expressed in DLBCL tumors, and a common abnormality in non-GCB subtypes, which is positively correlated with activated STAT3. PTPN6 expression in the DLBCLs is not regulated through SHP1 promoter hypermethylation or point mutations. The finding that SHP1 loss was found only in GCB cases and was especially favorable should be explored further and may provide an important new stratification factor for future DLBCL studies. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1575 We recently demonstrated in a phase II study that the anti-CD22 monoclonal antibody epratuzumab added to RCHOP improved DLBCL survival parameters. Despite these advances, 30–40% of DLBCL patients still relapse and die of disease. Recent advances have led to the identification of a variety of intracellular oncogenic pathways as potential targets for lymphoma therapy. Specifically, many studies have found that activation of the Signal transducer and activator of transcription 3 (STAT3) pathway promotes tumor cell survival in various types of cancers. STAT3 is a transcription factor and exerts its anti-apoptototic effect through several downstream targets such as MYC. MYC protein can be expressed in lymphoma cells with or without the presence of MYC translocation. The frequency of phosphorylated STAT3 (pSTAT3) and MYC expression and their prognostic relevance are unknown within diffuse large B-cell lymphoma (DLBCL), germinal center B-cell (GCB) and non-GCB subtypes. This study studied the tumor cell expression of pSTAT3 and MYC by IHC paired with serum cytokine levels in a DLBCL patient population uniformly treated on N0489. DLBCL tumor samples (n=38) were stained for detection of nuclear pSTAT3 expression. Using a threshold of ≥30% of tumor nuclei staining positive, 35 % (14/40) of tumors were pSTAT3+. An additional 17% (7/40) had between 10–30% pSTAT3+ cells. Non-malignant tonsil tissues (n=10) were positive for tSTAT3 in all cases but all but one case were pSTAT3 negative. Twenty-four of the same DLBCL tumors used for pSTAT3 expression were stained for MYC and 50% (12/24) were MYC positive (all nuclear) as defined by the criteria of ≥30% of cells staining positive. In cases (n=23) where both MYC and pSTAT3 IHC were performed, a positive pSTAT3 was more likely to have MYC expression whereas a positive MYC stain did not inform pSTAT3. By using a break apart probe for the MYC gene, MYC translocations in the major breakpoint regions were found in 10% (3/29) of cases. When MYC FISH was correlated with MYC IHC in the 24 DLBCL cases that had both techniques performed, all 3 MYC translocation cases were GCB by IHC, two were strongly positive for MYC by IHC, and 1 was negative. Among the 21 MYC FISH negative cases, 10 were MYC positive by IHC. These data suggest that MYC expression in lymphoma is not only controlled by genetic events such as translocations but also by other signaling pathways such as STAT3. pSTAT3 expression was correlated with an elevated serum LDH (p=0.0007). Neither MYC or pSTAT3 tumor expression correlated with other clinical or pathological features. Survival analysis revealed a trend toward shorter EFS for DLBCL patients whose tumors expressed MYC protein by IHC (p=0.2) or had a MYC translocation (p=0.09) by FISH; pSTAT3 expression status did not predict EFS (p=0.9). Within the GCB group 10 cases were MYC negative (10/17; 59%) and 7 cases were MYC positive (7/17; 41%). Among the non-GCB group 83% (5/6) were MYC positive. These data clearly suggest a trend of higher MYC positivity in the non-GCB DLBCL group (p=0.07). MYC positive cases had a clear trend of inferior EFS in both GCB (p=0.2) and in non-GCB (p=0.5) groups. The distribution of pSTAT3 (n=38) expression was also evaluated in the GCB (n=26) and non-GCB (n=12) DLBCL. In the GCB group 27% (7/26) were pSTAT3 positive compared to 58% (7/12) in the non-GCB group. Thus, there was a clear trend toward higher pSTAT3 positivity in non-GCB DLBCL tumors (p=0.06); however, pSTAT3 status did not correlate with EFS in either GCB or non-GCB DLBCL patients. In the present study we correlated pretreatment serum cytokines levels of these patients with pSTAT3 and MYC tumor cell expression. Out of the 30 cytokines tested, the only pre-treatment cytokines that were significantly correlated with pSTAT3 expression were IL-10 (p=0.05), G-CSF (p=0.03) and TNFa (p=0.04); none were correlated with MYC expression. Overall, our data provide evidence that over-expression of pSTAT3 and MYC is common in DLBCL. These biomarkers have potential as prognostic factors in the case of MYC and as a tool for selecting therapy for pSTAT3. MYC may be especially useful to further identify an adverse group of DLBCL patients within the otherwise favorable GCB tumor group. The availability of JAK/STAT and STAT-specific inhibitors provides the rationale to incorporate pSTAT3 staining in tumors from patients who are participating in these trials to learn if this biomarker can predict response Disclosures: No relevant conflicts of interest to declare.

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 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.