Wellik, Linda

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 It is believed that malignant cells “condition” the microenvironment to facilitate tumor cell survival. We hypothesized that crosstalk between CLL B-cells and marrow stromal cells impacts both cell types bi-directionally and ultimately contributes to leukemic cell apoptotic resistance. To test this hypotheses, bone marrow stromal cells from core bone biopsies from CLL patients were isolated and cultured using methods we have previously described (Leuk Res 2007 31(7):899). Subsequently, we determined the impact of co-culture on CLL B-cell features including apoptosis and CD38 expression. In addition, we evaluated the release of angiogenic cytokines on co-culture and signal events in the stromal cells. Immunophenotyping demonstrated that cultured bone biopsy derived stromal cells were CD73+, CD105+, CD146+, CD14−, CD45−, CD34−, HLA-DR-, suggesting they were mesenchymal stem cells (MSC). Co-culture of these MSC with CLL B-cells protected CLL B-cells from both spontaneous apoptosis (SA) and drug-induced (fludarabine and chlorambucil) apoptosis (DA). For SA, the mean survival of CLL B-cells with or without co-culture of MSC for 5 days were 56.9 ± 10.0 and 7.7 ±3.7 (p<0.05), respectively. When CLL B cells were treated with fludarabine or chlorambucil, the fraction of CLL cells tightly adherent to MSC (TA-CLL) showed higher survival than a less adherent but viable fraction of CLL B-cells. The mean survival of TA-CLL cells treated with 10 μM of fludarabine for 48 hours in the presence of MSC were 67.5 ± 3.6 vs 29.8 ± 11.1 without MSC (P<0.05), respectively. When CLL cells with evidence for CD38 expression were co-cultured with MSC, both the percentage of CD38 positive cells and level of expression of CD38 per cell were up-regulated (mean fold change: CD38 percentage, 2.7, p<0.05; CD38 MFI, 1.9, p<0.05) after 2 weeks. In contrast, the CD38 percentage and expression were not changed in cells with minimal CD38 expression when these CLL B-cells were co-cultured with MSC. In addition, co-culture of MSC with CLL cells induced rapid ERK and AKT phosphorylation (within 30 min) in the MSC on immunoblot analysis. When CLL B cells and MSCs were cultured in transwells, the activation of ERK and AKT in MSC occurred at similar levels, indicating that activation of MSC was mediated by soluble factors. In addition, co-culture led to increased secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as well as a decrease of thrombospondin-1 (TSP-1) in the culture medium. These findings confirm that co-culture of CLL B-cells and MSC culminates in “angiogenic switch.” Taken together, these results strongly suggest interactions between MSC and CLL B cells are a bi-directional process. In leukemic cells, the interaction not only protects against spontaneous and drug induced apoptosis but also leads to an increase in CD38 expression consistent with an activated status. In MSC, the interaction leads to activation of ERK and AKT. Co-culture also facilitates angiogenic switching. These results underscore the dynamic and complex nature of the interactions between bone marrow stromal cells and CLL B-cells. Further studies are needed to dissect how crosstalk between CLL B-cells and MSC relates to disease progression, and determines whether these interactions can be targeted with therapeutic intent.

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 Background IL-10 polarized macrophages promote tumor growth and survival. The T-cell transcription factor GATA binding protein-3 (GATA3) epigenetically regulates IL10 expression. Therefore, we investigated the extent to which GATA-3 regulates IL-10 production and macrophage polarization in T-cell lymphomas. Methods Monocyte-derived macrophages were generated and polarized with T-cell lymphoma conditioned media ((TCL-CM). Immunophenotypic and functional characteristics associated with IL10-polarized macrophages were examined. GATA3-dependent cytokine production was evaluated in T-cell lymphoma lines by shRNA knockdown. Alternatively polarized macrophages and GATA3 expression were examined in a cohort of patients with peripheral T cell lymphoma, not otherwise specified (PTCL, NOS) (n=76). Comparisons among groups were evaluated using a Student's t test. Progression-free and overall survival (PFS & OS) was estimated using the Kaplan-Meier method and two-tailed log-rank test based on Cox proportional hazards model. Results To investigate the ability of T-cell lymphomas to alternatively polarize macrophages, monocytes from healthy donors were cocultured with TCL-CM. Macrophages were shown to have immunophenotypic characteristics of IL10-polarized macrophages (pSTAT3+/CD16hi/CD163hi/HLA-DR-/lo) by flow cytometry, which was abrogated by IL10, but not IL6, neutralization. TCL-CM polarized macrophages produced abundant IL-10 and were impaired in their ability to stimulate T-cell proliferation. Collectively, these results demonstrate that T-cell lymphomas promote alternative macrophage polarization in an IL-10 dependent manner, and both CD163 and pSTAT3 may aid in their identification in clinical T-cell lymphoma specimens. Therefore, CD163+/pSTAT3+ macrophages were visualized by immunofluorescence in PTCL, NOS specimens. IL-10 polarized macrophages were not identified in normal tissues, but were abundant constituents of the tumor microenvironment in 68% of the PTCL, NOS specimens examined (n=31). STAT3 phosphorylation in response to IL-10 is dependent upon the Janus kinases (JAK) JAK1 and TYK2, both of which are inhibited at nanomolar (and clinically achievable) concentrations by the “JAK2” inhibitor ruxolitinib. Therefore, we sought to determine whether ruxolitinib may prevent IL-10-induced macrophage polarization. As expected, ruxolitinib inhibited IL-10-induced STAT3 phosphorylation. More importantly, macrophages polarized in the presence of ruxolitinib lost the immunophenotype characteristics of IL-10 polarized macrophages, and were CD16-/loCD163-/loHLA-DRhi. Furthermore, IL-10 production was significantly impaired in macrophages polarized in the presence of ruxolitinib, while their ability to stimulate T-cell proliferation was significantly increased. In a separate initiative, we identified GATA3 as the key regulator of IL-10 and Th2 cytokine (IL-4, IL-13 and IL-5) expression in T-cell lymphoma lines by shRNA knockdown assays. Therefore, we examined GATA3 expression by immunohistochemistry in PTCL, NOS specimens. GATA-3 expression was observed in most of the T-cell lymphoma lines and in 47% of PTCL, NOS specimens (n=76). In the cohort of the 76 PTCL, NOS patients, the median PFS and OS observed in GATA3+ PTCL, NOS was 5 months (4-8 months, 95% CI) and 8 months (5-14 months, 95% CI), respectively. In contrast, the median PFS and OS in GATA3- PTCL, NOS was 1.3 years (0.6-1.6, 95% CI) and 1.6 years (0.7-6.9 95% CI), respectively. Conclusions Alternatively polarized macrophages are abundant in T-cell lymphomas and are generated in an IL-10- and STAT3-dependent manner. GATA-3 regulates IL-10 production in these lymphomas, and its expression identified a high-risk subset of PTCL, NOS with distinct clinicopathological features. As IL-10-dependent STAT3 phosphorylation and macrophage polarization were impaired by ruxolitinib, the JAK2 inhibitors may warrant closer scrutiny as immunomodulatory agents. Disclosures: Off Label Use: Ruxolitinib, a JAK2 inhibitor, may warrant closer scrutiny as an immunomodulatory agent in TCL therapeutics.

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 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 We have found that CLL B cells spontaneously secrete vascular endothelial growth factor (VEGF) and that a VEGF autocrine pathway can induce apoptosis resistance in these cells. Recently, we also found that hypoxia-inducible factor-1 alpha (HIF-1α) is highly expressed in CLL B cells. Since this protein is a potent transcription factor for the induction of VEGF, we were interested in further definition of HIF-1α regulation and its function in CLL B cells. CLL blood B cells overexpress HIF-1α protein but not mRNA for HIF-1α compared to normal blood and splenic B cells. Immunohistochemistry (IHC) showed that circulating blood CLL B cells and a subset of CLL marrow cells uniformly express HIF. Hypoxic conditions (i.e., 1% O2) did not increase the protein levels of HIF-1α nor mRNA for HIF-1α in CLL B cells, indicating that the high HIF-1α protein level is due to post-translation modification. Blockade of signaling pathways known to increase HIF-1α levels also did not alter the high levels of HIF-1α in CLL B cells. IHC and nuclear extraction assay demonstrated that HIF-1α was predominantly located in the CLL B cell nucleus. In addition, the nuclear extract when immunoprecipitated for HIF-1α was shown to be complexed with the co-activator p300, indicating that HIF-1α is transcriptionally active. Co-immunoprecipitation assay showed that HIF-1α from CLL B cells does not associate and form a complex with von Hippel-Landau protein tumor suppressor (pVHL), indicating that the proteasome dependent degradation pathway for HIF-1α protein in CLL B cells is dysfunctional. Using immunoblot or IHC methods, we were unable to detect pVHL protein in CLL B cells; however, we were able to use immunoprecipitation of CLL B cell lysates to demonstrate there is pVHL in CLL B cells. Prolyl hydroxylases (PHD 1, 2, and 3) are negative regulators for HIF-1α via hydroxylation of amino acid prolines in the oxygen degradation domain (ODD) which permits interaction with pVHL. RT-PCR results revealed that there is a subset of CLL patients who had ≥ 50% reduction of PHD 1 and 3 mRNA levels. However using a hydroxylation specific polyclonal antibody we found that HIF-1α from CLL B cells is indeed hydroxylated. Finally, silencing of HIF-1α by RNA interference in CLL B cells was associated with a selective decrease in VEGF mRNA levels but not VEGF-R1, Mcl-1 and prolyl hydroxylases (PHD 1–3) other downstream target genes of HIF-1α. These data show that the high endogenous HIF-1α levels in CLL B cells are due to a defect in HIF-1α degradation via the proteosomal pathway. We believe that this abnormality is linked to the autocrine VEGF pathway in CLL B cells and ultimately results in increases in their apoptotic resistance. Inhibition of HIF-1α levels may be of therapeutic benefit to CLL patients.

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 4078 Background: Cytokine stimulated signaling pathways contribute to multiple myeloma (MM) disease progression and in acquired resistance to current treatment options making MM an incurable malignancy. It is very well documented that HGF is a cytokine that is secreted by bone marrow stromal cells which has an autocrine and paracrine role in the disease progression in a myeloma setting. HGF binds to its receptor tyrosine kinase on MM cells, MET and this binding at the extracellular domain results in activation of MET which interacts with several of its target proteins resulting in increased survival, increased proliferation, cell cycle progression, motility, migration and invasion. In normal bone marrows, co-expression of MET and its ligand HGF is a rarity whereas co-expression is a common feature of MM. Elevated levels of HGF have been observed in serum and bone marrows of MM patients with a negative correlation to disease progression. In addition, increased HGF levels cause abnormal and reduced bone formation in patients. HGF gene levels in MM samples have been observed to be significantly up regulated in myeloma cells when compared to normal cells. Recently, studies have identified that HGF facilitates the MM cells to adhere to fibronectin, a bone marrow matrix protein, thereby positively impacting MM cell invasion and proliferation. Overall, HGF and its receptor mediated pathway influences tumor progression in myeloma by targeting several different aspects of the disease biology and hence is a very attractive and potentially very important target for improving treatment regimens in a myeloma setting. Methods: MK2461 was synthesized by Merck Inc. (Whitehouse Station, NJ, USA). Stock solutions were made using DMSO and working stock solutions were made using RPMI 1640 media containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In order to study the mechanism of action of the drug, immunoblotting studies were performed on lysates made from cell lines incubated with the drug for various durations. Results: MK2461 treatment led to dose and time dependent cytotoxicity in a few myeloma cell lines (OPM2, DOX40, RPMI8226 and LR5) but not in others (MM1S, MM1R, H929 and U266). The IC50 values for the sensitive lines varied from 1μ M (OPM2) to 10μ M (DOX40, RPMI8226 and LR5). However, MK2461 significantly inhibited the proliferation of MM cells at sub IC50 concentrations in all cell lines tested except MM1S. This inhibition of proliferation was observed when cells were co-cultured with stromal cells or cytokines, namely VEGF, IL6 or HGF. Culturing MM cells with increasing doses of HGF was still unable to protect them from drug induced inhibition of proliferation. MK2461 was able to induce time dependent increase in apoptosis (as measured by annexin/PI), decrease in proliferation (as measured by BrdU assay) and induction of cell cycle arrest in the drug sensitive cell lines. This effect was not observed in MM1S cells. Exploring the mechanism of action of the drug indicated that MK2461 treatment led to down regulation of pc-Met, pGab1, pAkt and pErk in both the drug sensitive (OPM2) and drug resistant (MM1S) cell lines. However, proteins down stream of Akt in the PI3K/Akt pathway, namely pGSK3β, p70S6K, Bcl2, cyclin E and cyclin D3 were down regulated only in OPM2 cells. On the contrary, we observed up-regulation of these proteins in the drug resistant cell line offering a possible explanation for the drug resistant phenotype. We have also examined combinations of MK2461 with inhibitors of PI3K/Akt pathway. Conclusion: These studies demonstrate significant in-vitro activity of MK2461 in MM. Our results suggest the presence of two populations one very sensitive to MK2461 and one insensitive. Differential effects on the signaling pathways provide important clues to the mechanisms of action of c-met inhibitors in myeloma. The results form the basis for clinical evaluation of MK2461 in MM. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.