Gupta, Mamta

Abstract Long noncoding RNAs (lncRNAs) are defined as RNA-like transcripts that are over 200 nucleotides and lack significant open reading frames. Some lncRNAs such as HOTAIR, MALAT1 and H19 have been found to be associated with clinical prognosis and are potential drivers of cancer progression in cancers of the breast, lung, and liver respectively. The role of lncRNAs in lymphoma is unknown. Dysregulation of eIF4E (a key component of the translation initiation complex eIF4F) influences global protein translation, especially the translation of “weak” mRNAs that can be malignancy-related. We and others have found that eIF4E is dysregulated in B-cell lymphoma. The aim of this study is to identify eIF4E-associated lncRNAs through next generation RNA-Sequencing (NGS RNA-Seq) and delineate their role in protein translation in lymphoma. RNA-immunoprecipitation (RNA-IP) was used to pull down eIF4E-bound lncRNA in lymphoma cells. eIF4E-bound lncRNAs were immunoprecipitated with eIF4E antibody or IgG control in Jeko, a mantle cell lymphoma (MCL) cell line and sent for microarray analysis and NGS-RNA-Seq for identification of lncRNAs. The microarray analysis showed that several lncRNAs were enriched with eIF4E antibody compared to IgG control. These included SNHG4 (13.6 fold), SNHG12 (4.8 fold), NCRNA00171 (4.8 fold) and IPW (4.6 fold), GNASAS (3.5 fold), SNHG7 (3.3 fold), NCRNA00182 (2.7 fold), NCRNA00094 (2.6 fold), NCRNA00188 (2.4 fold) and NCRNA00201 (2.1 fold). The binding of these lncRNAs to eIF4E was further confirmed by RT-PCR in Jeko, Mino and Granta MCL cell lines. Next, we looked the expression of these lncRNAs by qRT- PCR in the MCL cell lines and normal controls. We found SNHG4 and IPW to be overexpressed in all the MCL cell lines, while SNHG12 and NCRNA00201 were overexpressed in the selected cell lines. No significant difference was found for the expression of NCRNA00171 and NCRNA00182 in any of the MCL cell lines compared to controls. Overall, these data suggest that several lncRNA have altered expression in malignant B-cells. Considering that the microarray assay only covered a limited number of lncRNAs, we further confirmed eIF4E bound lncRNA by NGS RNA-Seq in Jeko MCL and normal control. The binding of 10/13 lncRNA mentioned above with eIF4E were found upregulated by NGS-RNA-Seq. In addition several novel lncRNAs such as SNHG1 (161.6), AC091814.2 (98.8) and RP11-304L19.5 (64.2) showed up in NGS-RNA-Seq data. These data suggest that lncRNAs, such as SNHG12, SNHG4, and SNHG1 bind to eIF4E with high affinity in malignant B-cells and might play a role in protein translation. We knocked down the expression of SNHG4 through siRNA and demonstrated that cell proliferation and global protein translation was inhibited in lymphoma cells. To further confirm the role of SNHG4 in translation regulation, a plasmid, which contains a renilla luciferase driven by SV40 promoter, was co-transfected with SNHG4 siRNA into Mino cells. The luciferase signal, decreased compared with the cells transfected with nontargeting siRNA. These data suggest that SNHG4 is involved in the regulation of protein translation. In order to clarify the mechanism of lncRNAs bound to eIF4E we searched for RNA binding sites or motifs in eIF4E protein using the web-based tools, BindN and PPRInt. Interestingly two RNA binding motifs, KNKRGGRWLITLNKQQRRS and SHADTATKSGSTTKNR, were found in eIF4E based on the prediction. To examine whether lncRNAs bind with eIF4E through these RNA binding motifs, an eIF4E mutant plasmid with both RNA binding motifs deleted (eIF4EDel), was constructed and transfected transiently into HEK-293T cells along with eIF4EWT plasmid. RNA-IP data showed that the lncRNAs SNHG12, SNHG4 and SNHG1 were not able to bind with eIF4E in eIF4EDel-transfected cells compared with that of eIF4EWT, suggesting that these lncRNAs complex with eIF4E through RNA-binding motifs within the eIF4E. Overall, our results show that the lncRNAs, SNHG1 and SNHG4 are able to bind with eIF4E and regulate protein translation. Since lncRNAs had been found to play roles in the regulation of gene expression, including transcription, splicing and mRNA stability, our results may broaden the view of the functional role of lncRNAs in translation in lymphoma cells and in other cancers. Furthermore, our results also suggested that SNHG4 lncRNAs might be served as potential biomarkers for MCL and other B cell lymphomas for translation therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3678 The JAK/STAT pathway is becoming a relevant target for lymphoma therapy and inhibitors of the pathway are now in clinical trial. It would be ideal to identify patients for trial by assessment of pathway activation. We analyzed the primary tumors from 50 patients with diffuse large B cell lymphoma (DLBCL) using antibody against phosphorylated tyrosine signal transducer and activator of transcription 3 (STAT3) revealed that 52% (21/40) of DLBCL tumors express tyrosine phosphorylated STAT3 (pSTAT3). There are several potential mechanisms of activation of the JAK/STAT pathway. Protein tyrosine phosphatases such as SHP1 are key negative regulators of this pathway. SHP-1 is also considered a tumor suppressor gene because of its ability to antagonize the action of tyrosine kinases. Persistent activation of STAT3 seems to play a critical role in the pathogenesis of several malignancies. In an attempt to determine mechanism of STAT3 phosphorylation, we analyzed SHP1 expression and its correlation to STAT3 phosphorylation in DLBCL tumors. We analyzed SHP1 expression by immunohistochemistry (IHC) in a tissue microarray composed of 27 DLBCL tumor samples. SHP1 was found silenced in 33% (7/27) of DLBCL cases. To confirm our IHC data SHP1 mRNA expression was also analyzed in DLBCL patients (n=7) and CD19+ normal B cells (n=3) using reverse transcriptase polymerase chain reaction (RT-PCR). SHP1 expression was found silenced in 57% (4/7) of cases, however normal B cells expressed a significant amount of SHP1 mRNA transcripts. We next sought to determine the mechanism of SHP1 silencing in these patients. DNA methylation is the most common mechanism of gene silencing and gene inactivation in human cancers and methylation of a cluster of CPG sites near the proximal SHP1 promoter 2 has been shown in myeloma and T cell lymphoma cells. We analyzed the frequency of SHP1 DNA methylation in 40 DLBCL patients and in DLBCL cell lines by analyzing the proximal promoter regions for SHP1 with methylation specific (MSP) and unmethylation specific (UMSP) PCR primers. We found SHP1 methylation in 2.5% (1/40) DLBCL tumors and none of the DLBCL cell lines tested (OCILy3, OCILy10, SUDHL2, SUDHL6, OCILy19 and OCILy1) were positive for methylation. Furthermore, bisulphite sequencing of the MSP product confirmed methylated cytosines in the one MSP-positive DLBCL patient sample and in the positive control (methylated DNA). We next confirmed our MSP methylation results in the 40 DLBCL tumors DNA with a high resolution melting (HRM) methylation array for accurate detection of changes in the CpG methylation. The primers were designed to flank CpG islands, not to specifically amplify (converted) methylated or unmethylated DNA (as in MSP PCR). Data were normalized to an unmethylated control. Based on the melting profile, there was no methylation in the controls but detectable methylation (>5%) in the SHP1 promoter region was found in 10% (4/40) of DLBCL samples. The one sample that was positive with MSP PCR was also >50% methylated by HRM. Next we analyzed whether there is any correlation between SHP1 expression and tyrosine phoshorylated STAT3 (pSTAT3). In the 27 cases of DLBCL studied, 16 were pSTAT3 negative and 11 were pSTAT3 positive by IHC. 50% (8/16) of the pSTAT3 negative cases had very high expression of SHP1. Similarly, 45 % (5/11) of pSTAT3 positive cases were SHP1 negative consistent with loss of SHP1 gene expression. These data suggest that loss of SHP1 expression is one of several potential mechanisms that can contribute to increased tyrosine STAT3 activation in DLBCL. In addition, these data suggest that CPG island promoter hypermethylation in the SHP1 promoter is not associated with SHP1 gene silencing in DLBCL tumors. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 970 The mammalian target of rapamycin, mTOR, is a highly conserved serine/threonine kinase known to play a role in regulating mRNA translation, cell cycle progression, cell proliferation and apoptosis. As a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, mTOR is a component of two distinct complexes, TORC1 and TORC2. While TORC1 facilitates cell cycle progression from G1 into S phase by phosphorylating p70S6 kinase and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), TORC2 catalyzes the activating phosphorylation of Akt on Ser473, providing a feedback loop for further activation of mTOR. Phase II trials have shown activity of the TORC1-selective inhibitor rapamycin and its analogs in a wide range of lymphoma subtypes. The purpose of this study was to evaluate the anti-proliferative and pro-apoptotic effects of the dual TORC1/TORC2 inhibitor OSI-027 in human neoplastic lymphoid cells in vitro. MTS assays demonstrated that OSI-027 inhibited proliferation in a wide range of lymphoid lines, including SeAx (Sezary syndrome), DoHH2 (large cell lymphoma), RL (follicular lymphoma) and Jurkat (T cell ALL), as well as clinical lymphoma and T cell ALL samples, with IC50 values ranging from 0.078 to 10 μM. Propidium iodide staining followed by flow cytometry for subdiploid cells revealed induction of apoptosis within 48 h of treatment with OSI-027 (but not rapamycin) in SeAx, DoHH2, and Jurkat cells. Examination of Jurkat variants with alterations in key proteins involved in the death receptor versus mitochondrial pathway revealed diminished apoptotic responses to OSI-027 when Bcl-2 was overexpressed or caspase 9 was silenced, indicating involvement of the mitochondrial pathway. Immunoblotting for Bcl-2 family members revealed upregulation of Bim and Puma after a 48-hour exposure to OSI-027 but not rapamycin. This upregulation was also seen at the mRNA level, with a 12- to 20-fold increase in Puma mRNA and 4- to 12-fold induction of Bim mRNA. Small interfering RNA (siRNA)-mediated knockdown of Bim and Puma significantly diminished the apoptotic response to OSI-027. Because the Foxo3a transcription factor has been implicated in Bim and Puma expression and is known to be activated when Akt is inhibited, we next examined whether Bim and Puma induction was Foxo3a-dependent. Luciferase reporter assays showed that OSI-027 activated the full-length Puma and Bim promoters and that this activation was diminished when the Foxo3a binding sites were deleted or mutated. In addition, OSI-027 induced nuclear translocation of Foxo3a, while Foxo3a siRNA diminished OSI-027-induced apoptosis in Jurkat cells. Collectively, these results indicate that OSI-027 inhibits proliferation and induces apoptosis in a wide range of neoplastic lymphoid cells through a process that involves Foxo3a-mediated upregulation of Bim and Puma. These results also suggest that dual inhibition of TORC1 and TORC2 may be an effective treatment strategy in lymphoid malignancy. Disclosures: Barr: OSI Pharmaceuticals: Employment. Witzig:Novartis and Celgene: Patents & Royalties, Research Funding, Served on advisory boards with Novartis and Celgene – both uncompensated with compensation to Mayo Clinic.

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 1591 Background: The serum free light chain (FLC) assay quantitates free kappa and free lambda immunoglobulin light chains. We previously showed elevated FLC are present in approximately 1 in 3 diffuse large B cell lymphoma (DLBCL) patients from both a phase II cooperative group clinical trial and a large clinic based epidemiology cohort. Approximately 10% of DLBCL patients had elevated FLC that was monoclonal in these series. Patients with monoclonal elevated FLC have very poor outcome with almost 50% of patients progressing in the first 15 months following initial treatment with immunochemotherapy. Unlike patients with polyclonal FLC elevation, where a variety of host effects may cause a rise in both kappa and lambda, it is likely the excess FLC in monoclonal patients is directly related to the tumor. DLBCL are a heterogenous group of tumors that can be classified into germinal center (GCB) or activated B cell (ABC) groupings using immunohistochemistry (IHC) staining algorithms. Here we examine the association of monoclonal free light chains with ABC vs. GCB DLBCL tumors. Methods: Newly diagnosed DLBCL patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER) or NCCTG clinical trial N0489. Serum FLC was quantitated from enrollment research serum draws using the Freelite FLC assay (The Binding Site, Ltd., Birmingham, UK). Monoclonal elevated FLC was defined as an elevated kappa or lambda with a corresponding abnormal FLC ratio. Immunohistochemistry (IHC) staining was performed on paraffin tissue from research tissue microarrays (TMAs). Results: IHC tissue staining was available from 12 patients with monoclonal elevated FLC unrelated to renal failure. Germinal center markers were predominantly negative: 2 of 11 were LMO2 positive (18%), 1 of 12 was CD10 positive (8%), and zero cases out of 11 were GCET1 positive. FoxP1 was positive in 5 of 9 cases (56%) and MUM1 was positive in 4 of 12 cases (33%). By the Tally algorithm, 10 of 11 cases (91%) were ABC type DLBCL The percentage of ABC patients by the Tally algorithm in the corresponding DLBCL patients without monoclonal FLC was 45% (N=108, p=0.004). Conclusions: Approximately 10% of DLBCL patients have elevated monoclonal FLC and these patients have poor prognosis. Elevated monoclonal FLC may be a serum marker for ABC-type lymphoma, as the tumors from patients are predominantly negative for germinal center stains and ABC by the Tally algorithm. The secretion of light chains by the tumor suggests an activated B cell clone and the poor prognosis is characteristic of ABC type DLBCL and. Given the likelihood of the excess monoclonal FLC being tumor related, the potential exists for tracking of tumor response and progression via serum FLC in these patients. Replication of these findings in an additional cohort of DLBCL patients is planned. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1570 B-cell non-Hodgkin lymphoma encompasses a heterogeneous group of B-lymphocyte-derived malignancies that are characterized by chromosomal translocations involving the immunoglobulin (IG) gene loci and specific histological subtypes of disease are associated with a different spectrum of IG translocations. Marginal zone derived B cell lymphomas encompass three distinct entities: extranodal marginal zone B lymphoma (MZL) of mucosa associated lymphoid tissue (MALT), nodal MZL (NMZL), and splenic MZL (SMZL). MALT lymphoma is genetically unique and four mutually exclusive chromosomal translocations have been identified in this disease thus far. However, the known translocations are only present in a subset of cases suggesting that additional uncharacterized translocations or other genetic events may exist that contribute to disease development. In previous studies we characterized the novel t(X;14)(p11.4;q32) translocation in a patient with MALT lymphoma and found that GPR34, an orphan G-protein coupled receptor (GPCR), was highly expressed due to its juxtaposition to the IGHSA2 switch region. We then measured GPR34 mRNA expression in tissue biopsies from a panel of MALT, NMZL, SMZL, lymphoplasmacytic (LPL), diffuse large B cell (DLBCL), follicular (FL), and mantle cell (MCL) lymphomas. Expression of GPR34 was detected in all tissues examined, but was significantly increased in MALT (37 fold), LPL (23 fold), NMZL (18 fold), and SMZL (21 fold) compared to normal CD19+ B cells. GPR34 mRNA expression was also analyzed by gene expression profiling (GEP) of a panel of lymphomas or control lymphoid tissue and we confirmed elevated expression of GPR34 in the t(X;14) case and detected elevated GPR34 mRNA expression in MALT, LPL, NMZL, SMZL, and ABC DLBCL. When we grouped specimens by high and low expression of GPR34, a clear molecular subtype of SMZL could be identified and suggests that GPR34 high expressing tumors have a unique GEP. To confirm the qPCR and GEP analysis of GPR34 expression, we analyzed surface expression of GPR34 by flow cytometry of normal and malignant tissues and found that expression of GPR34 was detected on all MALT and SMZL B cells with an average DMFI of 2.08 (DMFI range 1.26–3.18, n=12) and on the JeKo-1 lymphoma B cell line (DMFI= 4.75). Analysis of CD19+ cells from normal controls (n=3) revealed a low level of GPR34 on (average DMFI =1.12). Little is known about the function of GPR34 and to further characterize the impact of GPR34 overexpression on cell signaling, OCI-LY19 lymphoma B cells and HeLa cells were transduced with GPR34 or a vector control. To determine if MAPK activity, a pathway regulated by GPCRs, was upregulated, the phosphorylation status of ERK1/2 in GPR34 or control cells was analyzed and we found that ERK1/2 was constitutively phosphorylated at significantly higher levels in GRP34 expressing cells. To further explore which signaling pathways were affected by GPR34 overexpression, GPR34 or vector control cells were transfected with an AP-1, CRE, NF-kB, E2F, SRE, NFAT, or ISRE- luciferase reporter plasmid and GPR34 expressing cells had significantly increased luciferase activity driven by AP-1 (5.35-fold), CRE (4.7-fold), NF-kB (2.6-fold), and E2F (2.1) when compared to vector control cells. To determine the biologic impact of GRP34 overexpression, the proliferation rates of vector control and GPR34 cells were compared and we found that proliferation of GPR34 WT cells was significantly higher than that seen in the control. We next tested the effect of the MEK inhibitor on proliferation and saw a dose dependent decrease in proliferation of GPR34 expressing cells. We then tested the ability of GPR34 cells to generate colony formation in a soft-agar colony formation assay. After 3 weeks of culture, the GPR34 cells had formed significantly more colonies than the vector control suggesting that overexpression of GPR34 can result in increased anchorage-independent cell growth. Taken together, our results identify a novel translocation in MALT lymphoma, emphasize a novel role for GPR34 in regulation of gene expression and tumor cell growth, and suggest that MEK inhibitors may be useful in a subset of translocation–harboring or GRP34 high-expressing tumors. Disclosures: No relevant conflicts of interest to declare.

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 4136 Background: The serum free light chain (FLC) assay quantitates free kappa and free lambda immunoglobulin light chains. This assay has prognostic value in MGUS, multiple myeloma (MM), solitary plasmacytoma, and amyloid light chain (AL) amyloidosis, and has been incorporated in response criteria for both MM and AL amyloidosis. We previously showed elevated FLC was associated with poor outcome in diffuse large B-cell lymphoma (DLBCL) patients from a phase II cooperative group clinical trial (Blood (ASH Annual Meeting Abstracts) 2009 November 20, 2009; 114(22): 136). Here we replicate those findings in a large clinic based epidemiology cohort, as well as examine FLC abnormalities in other lymphoma subtypes. Methods: Newly diagnosed lymphoma patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). Serum FLC was quantitated from enrollment research serum draws within 9 months of diagnosis using the Freelite FLC assay (The Binding Site, Ltd., Birmingham, UK). Elevated FLC was defined as either kappa or lambda above the published reference range. Monoclonal elevated FLC was defined as elevated FLC with an abnormal FLC ratio; polyclonal elevated FLC was defined as elevated FLC with an FLC ratio in the normal reference range. Clinical data was abstracted using a standard protocol for the MER and patients were followed systematically for event-free (progression, retreatment, or death due to any cause) and overall survival (EFS and OS, respectively). Cox proportional hazards models were used to evaluate the unadjusted association between FLC and outcome. Results: 1118 lymphoma patients diagnosed between September 2002 and February 2008 with available serum were assayed. At a medium follow-up of 49 months (range 1–94), 484 patients (43%) had an event and 249 patients died (22%). Elevated FLC was present in 27% of lymphomas, with prevalence ranging from 13% in follicular lymphoma (FL) to 64% for mantle cell lymphoma (MCL) and 77% for lymphoplasmacytic lymphoma. Elevated FLC was present in 27% of DLBCL and associated with poor EFS (monoclonal hazard ratio (HR)=2.56, polyclonal HR=1.99, all p<0.001) and OS (monoclonal HR=3.22, polyclonal HR=2.91, all p<0.0001), replicating our previous findings. Elevated FLC was generally polyclonal (FLC ratio normal) across lymphoma subtypes, though monoclonal elevated FLC (FLC ratio abnormal) was common in MCL and the post-follicular lymphomas. Elevated FLC was associated with inferior EFS in all lymphomas combined (monoclonal HR=1.92, 95% CI:1.44-5.55, polyclonal HR=1.38, 95% CI:1.09-1.74); this association remained consistent in most subtypes (see table). In addition, elevated FLC was associated with poor EFS in MCL (monoclonal) and Hodgkin lymphoma (HL; polyclonal). Elevated FLC was also significantly associated with OS in HL (polyclonal HR=6.24, 95% CI 2.44–15.9), FL (polyclonal HR=5.85, 95% CI 2.62–13.0), post follicular lymphomas (polyclonal HR=4.16, 95% CI 1.17–14.8), as well as for all lymphomas combined (monoclonal HR=2.24, 95% CI 1.52–3.29, polyclonal HR=2.33, 95% CI 1.74–3.11). Conclusions: A significant percentage of lymphoma patients have elevated FLC. Our previous discovery of elevated FLC predicting poor outcome in DLBCL was confirmed in a large, independent dataset. Prevalence and type of elevated FLC (monoclonal vs. polyclonal) varies by lymphoma subtype. Elevated monoclonal and polyclonal FLC is associated with poor outcome broadly in lymphoma, as well as specifically for a number of lymphoma subtypes, supporting FLC as a potentially useful prognostic biomarker in many lymphoma subtypes. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2690 Diffuse large B cell lymphoma (DLBCL) is the most common type of adult non-Hodgkin lymphoma. Aberrant gene expression and mutations are involved in the pathogenesis of DLBCL and may have prognostic and therapeutic relevance. We have shown that STAT3 (by immunohistochemistry) is constitutively activated in approximately 50% of new untreated DLBCL cases. To investigate whether genetic mutations play a role in the activation of JAK2/STAT3 signaling pathway in DLBCL, we sequenced the coding regions of the JAK2 gene and found no novel missense mutations. Since we did not find any missense mutations in the JAK2 gene, we proceeded to bi-directionally sequence all the 24 exons of the STAT3 gene in the same DLBCL patient tumors (n=40). We discovered mutations in 2 patients, one in exon 7 (T>A) and the other at the stop codon in exon 24 (G>A). While the G to A mutation in exon 24 was a silent mutation, the T to A mutation in exon 7 led to methionine substitution by lysine at codon 206 (M206K). Codon 206 is located in the coil-coiled domain of STAT3, which is a conserved amino acid in STAT3 from many different species. In order to delineate the functional significance of this STAT3 M206K mutation we generated STAT3 expression plasmids with the lentiviral expression vector pLEX-MCS. STAT3 constructs carrying the M206K mutation were generated by site directed mutagenesis of wild type STAT3. Since there are two STAT3 isoforms in humans, full-length STAT3α and the splicing variant STAT3β (which lacks the C-terminal transactivation domain), we constructed stable cell lines containing mutant and wild type gene of both STAT3α and STAT3β in HEK-293T cells. The mutation was found to increase the phosphorylation of Tyr705 in STAT3α mutant cell lines with no effect on STAT3β mutant cell line. Moreover the mutant STAT3α cell line was more sensitive to IL-10 in the activation of Tyr705 compared to its wild type while STAT3β mutant cell line was insensitive to IL-10. Both the STAT3 mutants (STAT3α and STAT3β) were insensitive to IL-6, which suggests that the mutant can sense the signal specifically from IL-10. Furthermore JAK2 inhibition through the pharmacological inhibitor TG 101348 dephosphorylated STAT3 in WT cells; however, had minimal effect in both the STAT3 mutants. Immunofluorescence staining showed that the M206K mutation did not affect the nuclear translocation of both STAT3α and STAT3β, which was confirmed by immuno-blotting on the cytosol and nucleus of the cells. Interestingly, the mutation activated the transcriptional activation activity of both STAT3α and STAT3β by luciferase reporter assay. Overall these results suggest that the M206K STAT3 gene mutation is rare (2.5%) in DLBCL tumors, leads to STAT3 activation, and enhances STAT3 transcriptional activation activity. Thus, STAT3 mutations in addition to cytokines can produce STAT3 pathway activation in DLBCL. Our data suggest that clinical trials of novel STAT3 inhibitors need not be restricted to tumors with STAT3 mutations but rather should focus on tumors with demonstrated aberrant STAT3 activation. Disclosures: No relevant conflicts of interest to declare.