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Abstract Background: Retinoblastoma (RB) is the most frequent pediatric retinal tumor. In the present study, to elucidate chemoresistance mechanisms and identify potential biomarkers in RB, we utilized RNA sequencing (RNAseq) technological platforms to reveal transcriptome profiles and identify any differentially expressed genes (DEGs) between an etoposide drug-resistant subline (Y79/EDR) and parental Y79 cells. Methods: To test whether Y79/EDR cells showed resistance to antineoplastic agents for RB, we treated the cells with etoposide, carboplatin and vincristine and analyzed them with a Cell Counting Kit-8 (CCK-8). Y79/EDR and parental Y79 cells were used for RNAseq and bioinformatics analysis to enable a genome-wide review of DEGs between the two lines using the DESeq R package (1.10.1). Then, DEG enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was analyzed with KOBAS software. Next, real-time quantitative reverse transcription polymerase chain reaction (real time QRT-PCR) and cytotoxicity assays were performed to experimentally and functionally validate the identified candidate biomarkers. Results: Y79/EDR cells showed resistance to etoposide, carboplatin and vincristine at different concentrations. In total, 524 transcripts were differentially expressed in Y79/EDR cells based on analysis of fragments per kilobase of transcript per million fragments mapped (FPKM); among these, 57 genes were downregulated and 467 genes were upregulated in Y79/EDR cells compared to parental Y79 cells. We selected candidate DEGs, including ARHGAP9 , HIST1H4H , RELN , DDIT4 , HK2 , STC1 and PFKFB4, for mRNA expression validation with real time QRT-PCR assays and found that the expression levels determined by real time QRT-PCR were consistent with the RNAseq data. Further studies involving downregulation of ARHGAP9 with a specific siRNA showed that ARHGAP9 altered the cellular sensitivity of Y79 cells to etoposide and carboplatin. Conclusion: Our initial findings provided a genomic view of the transcription profiles of etoposide-induced acquired resistance in RB. Follow-up studies indicated that ARHGAP9 might be a chemoresistance biomarker in RB, providing insight into potential therapeutic targets for overcoming acquired chemoresistance in RB. These findings can aid in understanding and overcoming chemoresistance during treatment of RB in the clinic.

Abstract Abstract 2688 Members of the signal transducers and activators of transcription (STAT) family of proteins function as secondary messengers mediating cellular responses to various cytokines. Aberrant activation of STAT5 protein has been implicated in the pathogenesis of hematologic malignancies due to the ability of these transcription factors to regulate genes involved in cellular proliferation and survival. The two highly homologous transcription factors collectively known as STAT5, namely STAT5A and STAT5B, display both redundant and distinct physiologic functions in non-malignant B cells. Yet, despite evidence suggesting independent functions for STAT5A and STAT5B in the pathophysiology of solid tumors, the precise roles of these isoforms in hematologic malignancies such as Waldenstrom's macroglobulinemia (WM) are not known. Initial immunohistochemical staining of WM bone marrow biopsies (n=6) revealed higher levels of STAT5 phosphorylation compared to bone marrows from normal controls (n=6). Additionally, phosphorylated STAT5 was highly expressed in CD19/CD138+ sorted cells obtained from the bone marrow of patients with WM (average ΔMFI compared to isotype control, 4.64 +/− 1.7, n=6) as determined by FACS analysis. Furthermore, western blotting demonstrated baseline STAT5 phosphorylation in both WM and IgM-secreting cell lines (MWCL-1 and BCWM-1, respectfully). The expression of other phosphorylated STAT proteins (1, 3, 4, and 6) was not detected. To better understand the overall role of STAT5 biologically in WM, MWCL-1 and BCWM.1 cells were treated with a specific inhibitor of STAT5, N′-((4-Oxo-4H-chromen-3-yl)methylene) nicotinohydrazide. Following 72 hours in culture, this inhibitor significantly decreased IgM secretion at lower doses when compared to controls (10 μM, MWCL-1: 9413 +/− 511 ng/mL vs. 6166 +/− 160 ng/mL, p<0.001; BCWM.1: 13,380 +/− 393 ng/mL +/− 10970 +/− 240 ng/mL, p<0.001) and nearly abolished IgM secretion at higher doses. Significant effects on proliferation and viability were also observed, but only at higher concentrations of drug where the specificity toward STAT5 may have diminished. To characterize the roles of the individual STAT5 isoforms in WM, we have developed doxycycline-inducible STAT5A-shRNA and STAT5B-shRNA and used these to create stable knockdowns in the MWCL-1 cell line. Addition of doxycycline to these cell lines for 72h nearly abolishes STAT5A or STAT5B protein expression, respectively, without affecting expression of the other isoform, while no change in STAT5 expression is detected in cells transduced with the empty vector (EV) control. The functional effects of isoform-specific STAT5 inhibition on WM tumor biology were assessed through proliferation assays and ELISA to determine the utility of therapeutically targeting a single STAT5 isoform. No difference in proliferation was observed in the STAT5A- or STAT5B-shRNA cells following 72h induction with doxycycline as compared to the EV control cells. However, specific knockdown of STAT5A following doxycycline induction significantly decreased IgM secretion as compared to the EV control (EV, 91.8 +/− 10.2% of baseline secretion, STAT5A, 66.7 +/− 15.1% of baseline, p<0.05), whereas IgM secretion was decreased by knockdown of STAT5B but not significantly. Similarly, IL-6 secretion was only diminished in cells lacking STAT5A as compared to EV cells (EV, 94 +/− 14.16% of baseline secretion; STAT5A, 64% +/− 7.6% of baseline, p<0.001). Overall these initial data indicate a role for isoform-specific STAT5 activation in the maintenance of WM tumors, and future studies will further our understanding of the specific roles of STAT5A and STAT5B in WM tumors. Disclosures: Ansell: Seattle Genetics, Inc.: Research Funding.

Abstract Background: Myelofibrosis (MF) is a myeloproliferative neoplasm associated with splenomegaly, debilitating symptoms, progressive bone marrow fibrosis and shortened survival. Inflammatory cytokines are associated with the disease and monitoring cytokine levels provides information about response to therapy. Momelotinib (MMB) is a JAK1/2 inhibitor under investigation for the treatment of MF that has been shown to be safe and efficacious while providing an anemia benefit to MF patients in the phase I/II studies: CCL09101 (ASH 2013) and YM387-II (EHA 2014). AIMS: To perform an exploratory analysis of the association between cytokines and anemia-related laboratories, 8 week transfusion response, spleen response by MRI at 24 weeks and total symptom score (TSS) reduction (≥50% decrease at week 24) and JAK2 mutation status, in the phase I/II, open-label YM387II trial. METHODS: 61 patients were enrolled and received MMB treatment. Whole blood samples were collected at baseline, 6 and 24 hours post-dose on day 1, and weeks 8 and 20. Plasma levels of 25 cytokines were analyzed by ELISA or MSD. Comparison of post-treatment data with baseline was carried out using Wilcoxon signed rank tests. Significant change from baseline was declared when ≥ 20% change from baseline was observed with Bonferroni adjusted p < 0.05. As this was an exploratory evaluation, comparison between treatment responders and non-responders was carried out using Wilcoxon rank sum test with no multiple testing corrections. Correlations between the cytokines and main biological parameters were investigated using Spearman correlation coefficients. RESULTS: JAK2V617F allele burden. The prevalence of JAK2V617F-positive patients was 68%. A statistically significant decline in JAK2V617F allele burden from baseline in this subgroup was observed at week 12 (median decrease 16.6%; p = 0.0063; n=21) and week 24 (median decrease 21.1%; p = 0.0019; n=18). Higher baseline IGFBP1 was associated with JAK2V617 mutation (48.2% higher in JAK2 mutated patients than in wildtype patients; p = 0.027). Cytokines and iron metabolism markers at baseline At baseline, hepcidin and IL-8 were anti-correlated with anemia-related markers such as transferrin and hemoglobin (Hb) (Spearman correlation coefficients < -0.5). IL-6, CRP and IL-10 showed strong, positive correlation (Spearman correlation coefficients > 0.5). Lower baseline IL-6 was associated with 24 week spleen response (42.3% lower in responders, p = 0.005); higher baseline thrombopoeitin (TPO) was associated with transfusion response at 8 week (3-fold higher in responders, p = 0.002). Baseline ferritin was 63.7% lower in TSS responders compared to non-responders (p = 0.015). On-treatment changes of cytokines and iron markers A significant decrease was observed in CRP, EPO, IL-6, IL-10, IL-12, TNFRII and TNFα following MMB treatment. IL-6 was reduced 30.3% as early as 6 hrs after first dose of MMB and CRP levels were reduced 27.3% after 24 hrs after treatment and continued to decline to 75% by week 4. A peak of IGFBP1 at 24h and increased TPO were observed. Decreased IL-12 and increased IL-8 at week 8 was significantly associated with 24 week spleen response (p < 0.05). Decline of CRP at week 8 was associated with TSS response (p = 0.045). Hb stayed at or above baseline for 6 months. An early peak of Hb was observed at Day 15 which was correlated with a significant increase in red blood cells and decrease in reticulocytes. CONCLUSIONS: In this exploratory analysis, a rapid and sustained reduction in inflammatory cytokines was observed with MMB treatment as expected based on the mechanism of action of MMB through JAK1/2 inhibition. Data from this study suggest an association between IGFBP1 and JAK/STAT signaling in myelofibrosis that will need to be explored in future studies with larger sample sets. Individual baseline biomarkers were correlated with MMB response, but no single biomarker was associated with all responses. Changes in IL-12 and IL-8 were significantly correlated with 24 week spleen response. The Day 15 data suggest that MMB treatment facilitates erythrocyte maturation. These observations, as well as a panel of iron metabolism markers and cellular signaling pathways, will be investigated in a planned Phase 2 translational biology study of MMB in transfusion-dependent subjects with MF (Gilead Sci: GS-US-352-1672). Disclosures Gupta: Incyte: Honoraria, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Mesa:Novartis Pharmaceuticals Corporation: Consultancy; NS Pharma: Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI Biopharma: Research Funding; Incyte Corporation: Research Funding; Genentech: Research Funding; Pfizer: Research Funding. Deininger:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zhang:Gilead Sciences: Employment. Xiao:Gilead Sciences: Employment. Collins:Gilead Science: Employment. Kwei:Gilead Sciences: Employment. Joshi:Gilead Sciences: Employment. Brachmann:Gilead Sciences: Employment.

Abstract ROR1 is a type-1 tyrosine kinase-like orphan-receptor that ordinarily is expressed during embryogenesis, but that also is found on leukemia cells of patients (pts) with chronic lymphocytic leukemia (CLL). In prior studies we found ROR1 served as a receptor for Wnt5a, which could promote survival/growth of CLL cells. We found Wnt5a in the plasma is significantly higher in pts with CLL (3.2±1.6 ng/ml (mean ±S.D.), N = 36) than in healthy adults (0.14±0.16 ng/ml, N=14, p<0.01) and also may be elaborated by accessory cells in the CLL microenvironment. On the other hand, reducing expression of ROR1 on CLL cells via siRNA adversely affected leukemia-cell survival. We hypothesized that expression of ROR1 in CLL could play a role in the pathogenesis and/or progression of this disease. Early studies failed to define differences in the expression level of ROR1 on CLL cells of different pts in a small cohort. However, upon further analyses, we observed heterogeneity in the expression levels of ROR1 among samples of different pts in larger cohort. We investigated whether the level of ROR1 expression was associated with disease progression in a cohort of 1,568 CLL pts followed by CLL Research Corium (CRC). CLL samples were uniformly examined for the expression of ROR1 using the same preparation of an Alexa-647-conjugated anti-ROR1 mAb (4A5) and a standardized procedure by the CRC Tissue Core. We used 10 CLL cases, and tested ROR1 expression at 3 different time-points for each case. We found ROR1 expression was stable over time in all CLL cases. Then, we randomly segregated pt samples into either of two cohorts, one a training set of 797, and the other a validation set of 773 cases. For the 797 cases in the training dataset, we used the profile-likelihood method in a Cox regression model of treatment-free survival (TFS) from diagnosis to determine the optimal threshold level of ROR1 expressionthat could segregate pts into 2 subgroups. In the training cohort, 294 pts were stratified into a "ROR1-Low" subgroup and 503 pts were assigned to a "ROR1-High" subgroup, defining a threshold absolute mean fluorescence intensity of CLL cells stained for ROR1 of 29. The subgroup of pts in the ROR1- High subgroup had a median TFS of 6.0 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.0 years. We applied this threshold to segregate the 773 samples of the validation cohort into ROR1-High and ROR1-Low subgroups. We found that high-level expression of ROR1retained its strong predictive adverse effect on TFS in this validation set. The subgroup of pts in the ROR1- High subgroup had a median TFS of 5.7 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.8 years. The immunoglobulin heavy-chain variable region (IGHV) mutation status was known for 593 of the 797 samples in the training set (74%) and 586 of the 773 samples in the validation set (76%). A significantly lower percentage of the ROR1-Low cases (34%, or 69 of 203) used unmutated IGHV than did the ROR1-High cases (54%, or 209 of 390) (p<0.001). Nonetheless, using a multivariate Cox regression model with either a non-delayed entry or a delayed entry, we observed that high-level expression of ROR1 had predictive value for short TFS (non-delayed entry HR 1.4, p<0.05; delayed entry HR 1.9, p<0.001) even when the IGHV mutation status was taken into consideration (non-delayed entry HR 2.8, p<0.001; delayed entry HR 2.7, p<0.001). Taken together, this study demonstrates that there is heterogeneity in the expression of ROR1 on CLL B cells. Furthermore, we find that a high level expression of ROR1 is associated with early disease progression in pts with CLL. Table 1. Number of cases with high versus low ROR1 and mutated versus unmutated IGHV in the training and validation cohort Training Dataset Validation Dataset ROR1 MFI distribution (Median) 0~475 (35.5) 0~424(34.6) ROR1Low with Mutated IGHV 139 132 ROR1Low with Unmutated IGHV 64 84 ROR1High with Mutated IGHV 181 156 ROR1High with Unmutated IGHV 209 214 Total With Known IGHV Mutation Status 593 586 Disclosures Keating: Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Other: Advisory board. Kay:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Tolero Pharma: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Byrd:Acerta Pharma BV: Research Funding. Gribben:Celgene: Consultancy, Honoraria; Janssen: Honoraria; Roche/Genentech: Honoraria; Pharmacyclics: Honoraria; Gilead: Honoraria. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.

Abstract Abstract 29 Acute lymphoblastic leukemia (ALL) cells are frequently characterized by abnormal numbers of chromosomes, a condition known as aneuploidy. Aneuploidy in ALL is not just a curiosity, but impacts on the prognosis and risk stratification in children. Additionally, aneuploidy itself contributes to the development of cancer by dysregulating gene expression, and driving the loss of heterozygosity of mutated tumor suppressor genes. The molecular underpinnings of aneuploidy in ALL are largely unknown. The mitotic checkpoint is a complex pathway that acts in mitosis to prevent the premature onset of anaphase that leads to improper segregation of chromosomes and aneuploidy. Through the concerted action of many intermediates, the mitotic checkpoint functions to inhibit the activity of a large ubiquitin E3 ligase complex known as the anaphase promoting complex/cyclosome (APC/C) that drives the separation of paired sister chromatids by effecting the ubiquitin-dependent degradation of cyclin B1 and securin. The de-ubiquitinating enzyme USP44 was recently identified as an important regulator of mitotic progression that assists in maintaining mitotic checkpoint signaling until all chromosomes are properly attached to the mitotic spindle. Microarray studies of childhood acute leukemias have observed high levels of USP44 in T-cell ALL, but the significance of this observation is unclear. Here we show that USP44 is indeed over-expressed in T-ALL in children and that its presence may contribute to aneuploidy that is frequently observed in this disease. We compared the expression of USP44 in a series of 24 samples from patients with T-ALL to that found in ten samples of peripheral T-cells isolated from healthy donors, using quantitative real-time PCR. There was a dramatic increase in the expression of USP44 in T-ALL with 18 out of 24 cases having at least 3-fold increase, with an average of 16-fold increase, in USP44 mRNA. We next studied the consequences of USP44 over-expression in primary cells using live-cell microscopy. We find that excess USP44 leads to dramatic and significant increase in chromosome missegregation with most defects involving lagging chromosomes due to improper chromosome attachment to the mitotic spindle. We observe that these mitotic errors lead to the development of aneuploidy, through a mechanism that requires USP44 catalytic activity. These effects are accompanied by reinforced mitotic checkpoint signaling that leads to delayed progression through mitosis, and a prolonged duration of mitotic arrest in cells exposed to spindle poisons such as nocodazole or paclitaxel. At the molecular level, we observe that excess USP44 leads to enhanced binding of the mitotic checkpoint effector Mad2 to the APC/C and increased levels of cyclin B1 in early mitosis. Taken together, we conclude that USP44 is a chromosome instability gene that is frequently expressed at high levels in T-ALL cells, and that this may contribute to the pathogenesis of T-ALL by leading to chromosome shuffling and aneuploidy. These data have important implications in our understanding of the pathogenesis of this disease and may contribute to understanding the therapeutic effectiveness of novel aurora inhibitors in cancer. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2958 PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. Using an antibody-based protein microarray technique, we identified Citron Rho Interacting Kinase (CRIK) as a protein that was highly expressed in primary multiple myeloma (MM) cells compared to normal plasma cell. We therefore sought to investigate the functional role of CRIK in MM cells. Methods: Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM and 4 healthy control using high-throughput proteomic analysis with antibody-based protein microarray (Clontech, CA). MM.1S, OPM2, RPMI8226, and INA6 were used to perform functional validation. Protein expression has been studied by immunoblotting. Gene expression analysis has been assessed using the Affymetrix U133A platform and qPCR. Lentivirus was used to knockdown CRIK in MM cell lines (MM.1S, RPMI8226, OPM2, and INA6). DNA synthesis, cell survival, cell cycle profiling and apoptosis were assessed by BrdU, MTT, PI and Apo2.7 staining, and flow cytometry analysis, respectively. Results: We first showed that CRIK was overexpressed in 12 patients with MM compared to normal CD138+ cells obtained from healthy controls using high-throughput protein microarray. We further confirmed CRIK expression using immunohistochemistry in the same samples of MM patients. We next correlated CRIK gene expression level (CIT) with prognosis using previously published gene expression datasets and found that CRIK correlated with poor prognosis. Knockdown of CRIK in MM cell lines led to an anti-proliferative and pro-apoptotic effect in all MM cell lines tested. Indeed, CRIK-knockdown MM cells were characterized by a reduction of 60–80% in the proliferation rate, supported by reduction of DNA synthesis and G2/M phase cell cycle arrest. Moreover, induction of cytotoxicity of caspase-3, caspase-8, caspase-9, and parp cleavage was also demonstrated in CRIK knockdown cells compared to scramble probe transfected cells. We also showed that CRIK knockdown led to cytokinesis in MM cell lines, indicating a possible mechanism of inhibition of proliferation of these cells. Conclusion: In this study, we show that MM cells express a high level of a novel protein CRIK, and that inhibition of this protein leads to significant inhibition of proliferation and survival of MM cells. CRIK could be a critical therapeutic target in MM. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Abstract It has been suggested that errors in the repair of DNA double strand breaks (DSB) can result in gross chromosomal rearrangements (GCR), including chromosomal amplifications, deletions, inversions, and translocations. To study repair of DNA DSB in vivo, we established a subclone of the U937 cell line designated F5, in which a single copy of the rare-cutting meganuclease I-SceI recognition site has been integrated into chromosome 7. We transfected an I-SceI expression vector into these cells and analyzed misrepair events. We recovered no GCR, but instead found that most DSBs were repaired by small (<50bp) indels (91.8%). We also noticed some DSBs were repaired by large (<50bp) deletions (6.4%) and rare DSBs were repaired by large (100-500 bp) insertions (1.8%). Surprisingly, the nucleotide sequence of these insertions matched distant regions of the genome. To determine if this templated sequence insertion (TSI) was peculiar to I-SceI induced DNA DSB, we repeated the experiment using TALENs to produce DNA DSB. Again, we found insertions that were derived from distant genomic regions. To determine whether this form of DNA repair was restricted to experimentally induced DNA DSB, we analyzed structural variations identified by whole genome sequence (WGS) of two myeloma cell lines (KP6, MC1286PE1), and identified 15 independent TSIs. These TSIs were verified by Sanger sequencing, and lengths of inserted fragment were 38 to 666 bp (median 189 bp). Six insertions were noted to be identical between the 2 cell lines, suggesting that these TSIs were germline polymorphic events. These TSIs were compared to a catalog of known structural variations identified by WGS of 52 healthy individuals; 13 of 15 TSI junctions were identified, suggesting that these represented polymorphic insertions in the human genome. Two TSIs were potential somatic mutations in the KP6 cell line, including one that had 85bp insertion at the junction of an acquired t(6;12) chromosomal translocation. Six TSIs showed the hallmarks of L1 retrotransposon-mediated insertions, with a 5’-TTTT/A-3’ integration site, a target site duplication (TSD), a polyA tract at the insertion site, and a polyadenylation signal. However, the inserted sequence was neither a LINE nor a SINE, but instead was a transcribed, genic region mapping to a distant genomic region. The presence of these L1 ORF1 hallmarks strongly suggests that these insertions were caused by the L1 integrase and reverse-transcriptase acting on nuclear pre-mRNA. A second class of TSI showed no preference for a 5’-TTTT/A-3’ integration site, no TSD, no poly A tract, but instead showed several bp of microhomology at the insertion junctions. This second class is similar to TSI detected at I-SceI induced DNA DSB, and we predict that these TSI were generated by reverse transcription of pre-mRNA creating a “patch” for a spontaneous DNA DSB that occurred in a germ cell. To determine if TSI at I-SceI induced DNA DSB could be repaired by RNA-templated sequences, we cotransfected murine RNA and an I-SceI expression vector into F5 cells. Approximately 10% of the TSI recovered from these experiments contained mouse sequences, indicating that they were derived from mouse RNA. To further support the contention that TSIs were derived from RNA, we again used I-SceI to induce a DNA DSB, and treated the cells with reverse transcriptase inhibitors. The TSI frequency was reduced by 70%. Finally, a subset of insertions were due to telomere repeat sequences, lending further support for the hypothesis that RNA could serve as a template for the repair of DNA DSB. TSI is novel form of potentially mutagenic DNA DSB repair, which should be considered as an alternative to a balanced chromosomal translocation in the interpretation of reciprocal interchromosomal structural variations identified by short-read deep sequencing. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Immune editing is a major mechanism used by tumors to promote their survival. We have previously reported the presence of immunosuppressive monocytes (CD14+HLA-DR low/neg) in a number of cancers. Increased presence of these cells was associated with decreased treatment response and OS. Here we report an in vitro model to examine tumor monocyte cross talk and demonstrate that these monocytes may directly promote tumor cells’ resistance to chemotherapy. Method Monocytes from healthy donors were co-cultured with lymphoma cell lines (OCI-Ly1, OCI-Ly3, OCI-Ly7, OCI-Ly10, Su-DHL2, Su-DHL6, Jeko-1, Granta-519) with or without doxorubicin (DOX). Cultured cells were assessed for phenotype, viability, proliferation, and induced protein expression. Changes in vitro were confirmed in primary tissue by gene expression profile from 48 primary diffuse large B cell (DLBCL) lymphoma tumors. Results Monocytes converted CD14+HLA-DR+ to the CD14+HLA-DRlow/neg phenotype by co-culture with 5 of the 8 B cell lymphoma cell lines. The HLA-DR suppression was not due to the known regulatory effects of IL-10 nor was the effect seen in co-culture using B cells from healthy donors. Thus, the magnitude of DR loss is mediated by a yet undetermined mechanism. DOX incubation induced apoptosis in all of the cell lines. Co-culture with monocytes improved lymphoma cell survival in 3 of the 4 lymphoma cell lines. For example, untreated OCI-Ly3 had a 1.3±0.2 fold expansion that was reduced to 0.6±0.1 when treated with DOX (p<0.0001, n=13). However, when the cells were treated with DOX and co-cultured with monocytes the survival improved to 0.8±0.2 (p<0.0001, n=13). Having previously shown that CD14+HLA-DRlow/neg were capable of suppressing patient immunity, partially through arginase I, we determined if co-culture of monocytes with tumor cells increased arginase I expression in either monocytes or tumor cells. Not only did we see arginase I expression increases during co-culture(n=10, p<0.01), but surprisingly, the greatest increases were seen during co-culture and DOX treatment (n=10, p=0.01). To determine if our in vitro observations reflect human tumors, we used gene expression data obtained from 48 DLBCL tumors and found that increased CD14 gene expression was positively correlated with increased arginase I expression (n=48, p=0.02). Conclusions We have demonstrated that certain tumor cells induced phenotype changes in monocytes in an IL-10 independent and a tumor specific way. In those cells where tumors factors mediate monocyte phenotype, the monocytes can promote tumor cell resistance to cytotoxic killing by DOX, thus demonstrating a cross-talk between monocyte and tumor function. We found that in an environment of monocyte/tumor cross talk, arginase I expression is further increased in a cytotoxic environment. Together, this data demonstrates an active cross talk between monocytes and tumors resulting in multiple mechanisms of tumor resistance to chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Abstract Multiple myeloma tumors universally target one of the three human cyclin D genes (CCND1, CCND2 or CCND3) for dysregulation (Bergsagel et al., 2005, Blood, 106:296). Using a lentivirus expressing CCND2 RNAi we first tested the effects of selective cyclin D2 knock down on My5 and H929 myeloma cell lines and found G0/G1 phase arrest, increased apoptosis and significant selective disadvantage in transfected cells. By comparison, knockout mouse models indicate that most somatic tissues can develop in the total absence of cyclin D1, D2 and D3 (Kozar et al., 2004, Cell,118:477). Targeted inhibition of specific cyclin D expression is therefore a rational therapeutic strategy in myeloma. To identify novel pharmaceutical inhibitors of CCND2 transactivation we developed an assay employing NIH 3T3 cells stably co-expressing the CCND2 transactivator c-Maf and the cyclin D2 promoter driving firefly luciferase (luc) and screened the Lopac (n=1280), Prestwick (n=1120) and Spectrum (n=2000) libraries of drugs and natural compounds. In a parallel MTS assay, the effect of each compound on 3T3 viability was determined, allowing exclusion of compounds that caused secondary suppression of CCND2 due to non-specific cytotoxicity. From the screen we identified 10 c-Maf independent putative CCND2 inhibitors. These included monensin, patulin, β-lapachone, camptothecin, dihydrogambogic acid, gentian violet, thapsigargin, brefeldin A, pristimerin and kinetin riboside. Three of the 10 compounds (gentian violet, thapsigargin and patulin) were not studied further due to toxicity cited in the literature. Subsequent validation studies using selected compounds in human myeloma cell lines (HMCL) confirmed successful suppression of both cyclin D2 and D1 proteins. Each of these compounds was then shown to be cytotoxic to a genetically diverse and standardized panel of 14 HMCL in MTT assays: monensin (10–760 nM), camptothecin (5–700nM), dihydrogambogic acid (250–800 nM), pristimerin (150–500 nM) and kinetin riboside (2.5–20μM). Cell cycle analysis confirmed induction of G0/G1 phase arrest for most compounds, consistent with cyclin D inhibition. However, camptothecin and b-lapachone induced S-phase arrest, suggesting secondary suppression of cyclin D by virtue of S-phase activity. Unsorted myeloma patient bone marrow samples demonstrated selective activity for pristimerin, dihydrogambogic acid and kinetin riboside against CD138+ myeloma cells compared with non malignant hematopoietic cells; by contrast monensin showed almost equal toxicity for normal cells. The triterpenoid, pristimerin, showed potent anti-myeloma activity and was examined in greater detail. Studies confirm that pristimerin rapidly inhibits cyclin D1, D2 and D3 expression (<6 hours) at nanomolar concentrations and induces apoptosis of primary myeloma cells characterized by caspase 9 cleavage and Annexin V binding. While pristimerin is cytotoxic to HMCL and patient myeloma cells at 0.1–0.15 mg/L, toxicity studies in vivo indicate that the drug is tolerated in mice at 2.5 mg/kg i.p. daily. In vivo activity against a xenograft model is currently being determined. Overall this targeted chemical biology screen has identified several compounds, including the triterpenoid, pristimerin, that are being further characterized for promising preclinical anti-myeloma activity.

Abstract Abstract 281 Elevated serum levels of soluble interleukin-2 receptor alpha (sIL-2Ra) have been shown to be a poor prognostic factor in various malignancies and sIL-2Ra levels have generally correlated with tumor bulk and advanced stage of disease. In a clinical trial of single agent rituximab in previously untreated follicular lymphoma (FL) patients, we found that sIL-2Ra levels were elevated compared to controls and increased sIL-2Ra levels prior to treatment were associated with a poor outcome. The mean sIL-2Ra level (+/− standard deviation) in untreated FL patients (n=32) was 2.13 ng/ml (+/− 1.81) and 1.09 ng/ml (+/− 1.53) for normal controls (n=16). The time to progression for previously untreated FL patients after 4 doses of rituximab was 12 months for patients with sIL-2Ra levels above the mean compared to 40 months for patients with low sIL-2Ra levels (p=0.003) confirming that elevated sIL-2Ra is a poor prognostic factor in FL. To explore the mechanism by which sIL-2Ra may contribute to a poor prognosis, we determined the source of sIL-2Ra and whether sIL-2Ra facilitates interleukin-2 (IL-2) signaling. Based on our previous work showing that IL-2 induces Foxp3 expression, we particularly evaluated whether sIL-2Ra may bind IL-2 and contribute to impaired anti-tumor immunity by promoting Treg cell development. We found that sIL-2Ra could be detected by ELISA in the culture medium of IL-2Ra-expressing cell lines such as SuDHL1 and Karpas299 and that activation of these cells with PMA/Ionomycin significantly increased sIL-2Ra levels in the culture medium. Production of sIL-2Ra was associated with an attenuation of surface IL-2Ra expression on SuDHL1 and Karpas299 cells suggesting that surface IL-2Ra is the source of sIL-2Ra. Next, using histo-tagged sIL-2R, we tested whether the complex between sIL-2Ra and IL-2 was able to bind to IL-2R on the malignant cell. We found that sIL-2Ra bound IL-2 and that the complex could be detected on the surface of IL-2R-expressing cells. We then compared the signaling of the sIL-2Ra/IL-2 complex to that of IL-2 alone. As expected, we found that IL-2 stimulated cell proliferation, enhanced STAT5 phosphorylation and upregulated Foxp3 expression in CD4+ T cells in a dose-dependent manner. Administration of an anti-IL-2 antibody attenuated these effects. In contrast, sIL-2Ra alone had little effect on cell proliferation, STAT5 phosphorylation and Foxp3 expression. However, the addition of sIL-2Ra to IL-2 prior to incubation with IL-2R-expressing cells enhanced IL-2-mediated phosphorylation of STAT5 and Foxp3 expression in CD4+ T cells. Compared to IL-2 alone, treatment with sIL-2Ra (25ng/ml) and IL-2 (25ng/ml) increases the number of CD4+ T cells with phosphorylation of STAT5 from 7.9% to 17.4%. Taken together, these results indicate that sIL-2R plays an active biologic role in FL by binding IL-2 and promoting IL-2 signaling rather than depleting IL-2 and blocking its function. Given the findings that IL-2 strongly induces Foxp3 expression and promotes Treg cell development and that Treg cells suppress anti-tumor immunity, we conclude that elevated serum levels of sIL-2Ra facilitate IL-2 signaling and thereby contribute to a poor prognosis in FL. Disclosures: No relevant conflicts of interest to declare.