ALBERT

Description: We have identified a novel regulatory erythroid kinase (REDK) that is homologous to a family of dual-specificity kinases. The yeast homolog of REDK negatively regulates cell division, suggesting a similar function for REDK, which is primarily localized in the nucleus. REDK is present in hematopoietic tissues, such as bone marrow and fetal liver, but the RNA is expressed at significant levels only in erythroid or erythropoietin (EPO)-responsive cells. Two novel forms of cDNA (long and short) for REDK have been isolated that appear to be alternative splice products and imply the presence of polypeptides with differing amino termini. The ratio of short-to-long forms of REDK increases dramatically in CD34+ cells cultured with EPO, suggesting differing regulation and function for each form. REDK is predominantly found in nuclear, rather than cytoplasmic, protein extracts, and immunoprecipitated REDK is active in phosphorylating histones H2b, H3, myelin basic protein, and other coimmunoprecipitated proteins. Antisense REDK oligonucleotides promote erythroid colony formation by human bone marrow cells, without affecting colony-forming unit (CFU)-GM, CFU-G, or CFU-GEMM numbers. Maximal numbers of CFU-E and burst-forming unit–erythroid were increased, and CFU-E displayed increased sensitivity to suboptimal EPO concentrations. The data indicate that REDK acts as a brake to retard erythropoiesis.

Description: Thrombotic events are life-threatening complications of human hemolytic anemias such as paroxysmal nocturnal hemoglobinuria, sickle cell disease, and thalassemia. It is not clear whether these events are solely influenced by aberrant hematopoietic cells or also involve aberrant nonhematopoietic cells. Spherocytosis mutant (Spna1sph/Spna1sph; for simplicity referred to as sph/sph) mice develop a severe hemolytic anemia postnatally due to deficiencies in -spectrin in erythroid and other as yet incompletely defined nonerythroid tissues. Thrombotic lesions occur in all adult sph/sph mice, thus providing a hematopoietically stressed model in which to assess putative causes of thrombus formation. To determine whether hematopoietic cells fromsph/sph mice are sufficient to initiate thrombi, bone marrow from sph/sph or +/+ mice was transplanted into mice with no hemolytic anemia. One set of recipients was lethally irradiated; the other set was genetically stem cell deficient. All mice implanted withsph/sph marrow, but not +/+ marrow, developed severe anemia and histopathology typical of sph/sph mice. Histological analyses of marrow recipients showed that thrombi were present in the recipients of sph/sph marrow, but not +/+ marrow. The results indicate that the -spectrin–deficient hematopoietic cells of sph/sph mice are the primary causative agents of the thrombotic events.

Description: Introduction: The revised 2016 WHO classification of MDS has highlighted the value of morphologic evaluation and mutation analysis of bone marrow (BM)/ peripheral blood (PB) to further refine prognostication. These highlights include: (1) increased emphasis on lineage dysplasia compared with cytopenias; (2) objective enumeration of blast % for reproducibility; (3) accurate quantification of ring sideroblasts (RS); and (4) mutation analysis for SF3B1 in cases showing RS 〉5% and TP53 in MDS with isolated del(5q). Most of the proposed changes are within the categories of low-grade MDS. In this study, we evaluated 264 cases of MDS with diploid karyotype using the 2016 WHO system. Methods: We selected consecutive cases of MDS with diploid karyotype with BM morphological evidence of dysplasia and reclassified using the 2016 WHO system. Mutation analysis for SF3B1 (exons 14 and 15), SRSF2 (exon 1) and U2AF1 (exons 2 and 6) was performed using Sanger sequencing. Patient data were collected from the medical record. The Kaplan-Meier method was used to estimate OS and time-to-AML transformation. The associations between outcome and clinical and pathological parameters were determined using univariate and multivariate Cox proportional hazards regression models. Results: The study group included 264 MDS patients: 168 (64%) men and 96 (36%) women with a median age of 66.9 years (range, 28.3 - 89.1). The median hemoglobin, absolute neutrophil count (ANC), platelet count, and white blood cell (WBC) count were 10.0 g/dL, 1.9 x 109/L, 114.5 x 109/L, and 3.5 x 109/L, respectively. The median BM blast percentage was 2.5; 74% of the patients had 〈 5% BM blasts. MDS sub-classification according to the 2008 WHO classification was: RCUD, n=5 (2%); RA, n=9 (3%); RARS, n=16 (6%); RCMD, n=152 (58%); RAEB-1, n=56 (21%); RAEB-2, n=20 (8%), and MDS-U, n=6 (2%). Reclassification using the 2016 WHO classification: MDS with single lineage dysplasia (MDS-SLD, n=14, 5%), MDS with multi-lineage dysplasia (MDS-MLD, n=112, 42%), MDS with RS (including single lineage and multi-lineage dysplasia, MDS-RS, n=56, 21%); MDS-EB1, n=56 (21%), MDS-EB2, n=20 (8%) and MDS-U, n=6 (2%). Grading of fibrosis using reticulin/ trichrome stains showed absent-minimal fibrosis (grades 0-1) in 56/85 (66%) and moderate-severe fibrosis (2-3) in 29/85 (34%) cases. Mutation analysis for splicing factors was performed on 15 cases. Ten cases with 0-5% RS showed 2 cases each with SRSF2 and U2AF1 mutations. No cases had SF3B1 mutation. 5 cases with 〉5% RS showed SF3B1 mutations in 4 cases and 1 case each with SRSF2 and U2AF1 mutations. Over a median follow-up duration of 22.4 months (range, 0-156.8), 128 (48%) patients died. The median OS was 46.1 months (95% CI: 32.3, 58.4). Patients categorized as MDS-SLD by 2016 WHO had the best OS (156.8 months), followed by MDS-RS (58.7 months), MDS-MLD (46.3 months) and MDS-EB (21.2 months) (p1% RS. Figure Figure. Disclosures Jabbour: ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy.

Description: Abstract 1305 Cancer cells exist in a stressed environment, mainly due to lack of nutrients and oxygen, particularly during chemotherapy, and rely on metabolic homeostatic regulatory mechanisms for protection against these lethal challenges. Increasing glucose metabolism and continuous reactive oxygen species (ROS) production is one strategy of metabolic adaptation utilized by tumor cells to relieve this stress. Thioredoxin interacting protein (TXNIP) is a negative regulator for both redox thioredoxin (ROS production) and cellular glucose uptake, not well understood but found to be repressed in various cancers, including diffuse large B-cell lymphomas (DLBCL), the most common form of non-Hodgkin lymphoma that continues increasing in incidence and remains incurable in many cases, primarily due to development of chemo-resistance. The molecular mechanisms by which TXNIP expression is down-regulated during cancer progression and chemo-resistance development have not been completely elucidated. Since key gene silencing events have now been identified in the pathogenesis of DLBCL, recent therapeutic interest has focused on dysregulated histone modifications as potentially important therapeutic targets, for developing strategies that can reactivate silenced tumor suppressor genes. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), is a highly conserved histone methyltransferase that targets lysine-27 of histone H3 (H3K27). Studies in human tumors show that EZH2 is frequently over-expressed in a wide variety of tumors, including lymphomas. More importantly, recent studies using whole-genome sequencing in primary DLBCL, identified frequent mutations in the EZH2 gene that leads to critical gene silencing in DLBCL pathophysiology. Our study showed that EZH2 is either over-expressed or mutated in representative DLBCL cell lines and primary DLBCL cells, and that down-regulation of EZH2 with siRNA leads to the reactivation of TXNIP, with subsequent inhibition of tumor cell growth and survival mediated through both thioredoxin and glucose metabolism in DLBCL. We also found that histone deacetylation (HDAC) is also involved in EZH2-mediated silencing of TXNIP in DLBCL. Pharmacologic agents aimed at reactivating TXNIP genes include histone methylation inhibitor 3-Deazaneplanocin A (DZNep) that targets EZH2, as well as HDAC inhibitor Vorinostat. DZNep is currently the only histone methylation inhibitor that is commercially available. Our data indicated that DZNep is highly effective in inhibiting cell growth in various DLBCL cell lines, particularly in chemo-resistant DLBCL cell lines. Vorinostat, on the other hand, has been a good drug and is currently in clinical trial for relapsed DLBCL and has been FDA approved for treating cutaneous T-cell lymphoma patients. Our data showed synergistic activity of DZNep and Vorinostat in reactivating TXNIP gene expression and inhibiting DLBCL cell growth and survival. We also discovered that EZH2 controls constitutive NF-κB activity through both, the canonical and alternative NF-κB pathways in DLBCL. This function of EZH2 is independent of its histone methyltransferase activity. These findings reveal that EZH2 and NF-κB, the two oncogenic factors display functional crosstalk in DLBCL cells. Our findings have indicated that deregulated EZH2 leads to constitutive NF-kB activation and to epigenetic silencing of TXNIP, resulting in uncontrolled tumor cell growth and survival mediated through both thioredoxin and glucose metabolism in DLBCL, and that targeting this pathway represents a novel, rational, and effective therapeutic approach to selectively reverse chemoresistance in DLBCL patients, particularly relapsed/refractory patients. Disclosures: No relevant conflicts of interest to declare.

Description: Background: The prognosis of children with relapsed/refractory B-cell (CD20+) non-Hodgkin lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemo-radiotherapy resistance (Cairo et al, JCO, 2012, Cairo et al, BJH, 2018). Rituximab has been widely used in frontline treatment of B-NHL, however, some patients retreated with rituximab will relapse which limits patient treatment options (Goldman/Cairo, Leukemia, 2013). Several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering of NK cells with chimeric antigen receptors (Chu/Cairo et al, Can Imm Res 2015), as well as second-generation engineered anti-CD20 antibodies (Tiwari/Cairo et al BJH 2015). To enhance NK based therapy, our group has successfully expanded the functionally activated peripheral blood NK cells (exPBNK) with irradiated feeder cells (Chu/Cairo, et al, Can Imm Res 2015). To enhance NK cell-based targeted therapy, we had successfully engineered expanded NK cells with chimeric antigen receptors (Chu/Cairo, et al, Can Imm Res 2015) and combined expanded NK with an anti-CD20 targeted IL-15 fusion protein N-820 (2B8T2M) targeting rituximab-sensitive and -resistant BL (Chu/Cairo, et al, ASH 2017). N-820 was generated by fusing ALT-803 to four single-chain antibody domains of rituximab (Liu/Wong, et al, JBC, 2016). ALT-803 is a superagonist of an IL-15 variant bound to an IL-15RαSu-Fc fusion with enhanced IL-15 biological activity (Zhu et al. 2009 J Immunol), longer half-life and increased potency (Han, et al. Cytokine. 2011). N-820 displayed tri-specific binding activity through its recognition of CD20 on tumor cells, activated NK cells to enhance antibody-dependent cellular cytotoxicity (ADCC), and induced apoptosis of B-lymphoma cells (Liu/Wong, et al, JBC, 2016). Objective: To investigate how N-820 modulates the crosstalk between BL tumor cells and expanded NK cells by monitoring cytokines, chemokines and growth factors released and the anti-tumor effects of N-820 on NK cells in BL xenografts. Method: PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells and isolated as we have previously described with K562-mbIL15-41BBL (Chu/Cairo, et al, Can Imm Res 2015). ALT-803 and N-820 were generously provided by Altor Bioscience. Equal doses of N-820, Rituximab (R), ALT-803, R+ALT-803, obinutuzumab (obinu, generously provided by Christian Klein, PhDfrom Roche) were used for comparison. IgG was used as control. Rituximab-sensitive Raji and -resistant BL cells Raji-2R and Raji-4RH were used as target cells. NK cells were sorted by Beckman Coulter MoFlo XDP high-speed sorter and NK purity was confirmed by flow cytometry. Cytokines, chemokines and growth factors mRNA levels in NK cells were monitored by real-time PCR. Secreted cytokines, chemokines and growth factors were examined by standard ELISAs. Raji-2R-Luc cells were injected into NSG mice. ExPBNK+N-820 and controls were injected to the xenografted NSG mice. The tumor burden was measured with the IVIS-200 system. Results: We found that ALT-803 and N-820 at equal doses significantly enhanced the expression of NK activating receptors such as NKG2D, NKp30, NKp44, and CD16 on exPBNK compared to IgG controls. Additionally, N-820 significantly enhanced exPBNK cytotoxicity against Raji, Raji-2R and Raji-4RH cells compared to the controls IgG, R, ALT-803, R+ALT-803, or obinu (p

Description: Background Despite some robust initial responses, anti-CD19 Chimeric Antigen Receptor (CAR) T-cell therapy can be associated with significant short-term (cytokine release syndrome) and long-term (B-cell deficiency) toxicities. CAR-engineered natural killer (NK) cells potentially provide a safer alternative while maintaining efficacy. Activated Natural Killer (aNKTM) cells are a clinical grade cell line derived from the NK-92R cell line that has demonstrated potent cytotoxicity towards a broad spectrum of malignant cell lines as well as safety and efficacy in phase I trials. Variants of the aNKcell line are currently in Phase I/II clinical trials: a CAR-expressing aNK cell line and the haNKTM cell line, which have been engineered to carry a high-affinity version of the CD16/FcγRIII receptor to allow for combination therapy with monoclonal antibodies. haNK cells have also been genetically modified to express an endoplasmic reticulum-retained version of IL-2 (ERIL-2), which provides IL-2 independence and limits IL-2 secretion to sub-physiological, safe levels. A key factor for the efficacy of cellular immunotherapies against a given target is biodistribution, which affects the local effector to target ratio. Inability to reach the tumor cells, either by lack of homing or by the accumulation of extracellular matrix (ECM) surrounding a tumor, can be responsible for the clinical failure of even the most effective CAR. The chemokines CCL19 and CCL21 drive recruitment of CCR7-expressing immune cells to secondary lymphoid organs. Engineering aNK cells to express the CCR7 receptor is likely to improve their efficacy by increasing their targeted migration to lymphoma tumor sites. Methods and Results Clinical grade aNK cells were electroporated with a non-viral vector containing the CCR7 receptor, an anti-CD19 CAR, and a high affinity CD16 receptor. To assay the migration of these engineered cell lines, a modified Boyden Chamber assay was performed using Matrigel coated Transwells. K562 cells or modified K562 cells engineered to express CCL19 (K-19) were placed in the destination chamber and CFSE-stained effector cells were placed in the top well. After 24 hours, cells in the bottom well were analyzed by flow cytometry to measure the number of effectors which had migrated through the Matrigel (Fig 1a). The excellent activity of the CAR in stably transfected cells was confirmed against SUP-B15 cells (aNK-resistant), while the ADCC activity was tested against a SUP-B15 variant expressing CD20, but engineered to lack the CD19 antigen (Sup-B15 CD19-, CD20+). Migration towards human lymph node chemokine CCL19 was also tested in vivo in NSG mice with bilateral subcutaneous tumors - with parental K562 in one flank and K-19 tumors on the contralateral flank. CFSE-stained effector cells were delivered via tail vein injection once average tumor size reached 100mm3 and following randomization. Tumors were then harvested at multiple time points, dissociated, and the number of infiltrating effectors in each tumor compared by flow cytometric analysis. (Fig 1b). In testing, monoclonal cell lines expressing all components of the polycistronic system displayed preferential migration towards CCR7 chemokines both in vitro and in vivo, as well as robust cytotoxicity vs. K562 (92.4% +/- 2.4% at 5:1 E:T), Sup-B15(97% +/- 0.6% at 5:1 E:T), and Sup-B15(CD19-, CD20+) when pre-incubated with Rituximab(83.2% +/- 2.8% at 5:1 E:T) but not with control antibody Trastuzumab (22.3% +/- 1.1% at 5:1 E:T) in standard cytotoxicity and ADCC assays. Conclusion We show here that the incorporation of a CCR7 receptor into an off the shelf CAR engineered NK cell line improves their homing towards lymph node chemokines both in vitro and in vivo. This improved homing should result in a greater ratio of effector to target in lymphoid tissue, and maximize the immunogenic cell death. Disclosures Schomer: NantKwest, Inc.: Employment, Equity Ownership. Boissel:NantKwest, Inc.: Employment, Equity Ownership. Jiang:NantKwest, Inc.: Employment, Equity Ownership. Klingemann:NantKwest, INc.: Employment, Equity Ownership, Patents & Royalties. Lee:NantKwest, Inc.: Employment, Equity Ownership. Soon-Shiong:NantKwest, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Description: Thrombotic events are life-threatening complications of human hemolytic anemias such as paroxysmal nocturnal hemoglobinuria, sickle cell disease, and thalassemia. It is not clear whether these events are solely influenced by aberrant hematopoietic cells or also involve aberrant nonhematopoietic cells. Spherocytosis mutant (Spna1sph/Spna1sph; for simplicity referred to as sph/sph) mice develop a severe hemolytic anemia postnatally due to deficiencies in -spectrin in erythroid and other as yet incompletely defined nonerythroid tissues. Thrombotic lesions occur in all adult sph/sph mice, thus providing a hematopoietically stressed model in which to assess putative causes of thrombus formation. To determine whether hematopoietic cells fromsph/sph mice are sufficient to initiate thrombi, bone marrow from sph/sph or +/+ mice was transplanted into mice with no hemolytic anemia. One set of recipients was lethally irradiated; the other set was genetically stem cell deficient. All mice implanted withsph/sph marrow, but not +/+ marrow, developed severe anemia and histopathology typical of sph/sph mice. Histological analyses of marrow recipients showed that thrombi were present in the recipients of sph/sph marrow, but not +/+ marrow. The results indicate that the -spectrin–deficient hematopoietic cells of sph/sph mice are the primary causative agents of the thrombotic events.

Description: Deregulation of pro-inflammation signals plays an important role in MDS and AML. We have previously demonstrated that the histone H3 demethylase JMJD3 (KDM6B), a key regulator of inflammatory genes, is significantly overexpressed in the bone marrow progenitor cell population of patients with MDS (Wei et al, 2013). GSK-J4, a novel selective inhibitor against JMJD3, has been shown to modulate pro-inflammatory signals and affect cell growth in T-ALL and glioma. We therefore sought to evaluate the effect of GSK-J4 in MDS and AML. First, we observed that GSK-J4 significantly reduces the survival of MDS and AML cell lines (MDS-L and TF1) by 70% at concentrations 1-3 uM. These doses have no obvious cytotoxic effect on primary normal peripheral blood cells. The repression of survival was associated with induction of apoptosis, cell cycle blockade, and inhibition of p38MAPK activation. Second, we evaluated the impact of GSK-J4 on histone H3 methylation and found that the compound did not affect overall histone H3K4 and K27 methylation, which is consistent with previous findings (Ntziachristos et al, 2014). To pinpoint functionally relevant targets of JMJD3 inhibition in AML/MDS, we sought to identify disease-relevant inflammatory cytokines because JMJD3 binds and modulates promoters of many pro-inflammatory genes, particularly cytokines. We analyzed 38 inflammatory cytokines in bone marrow plasma specimens of patients with MDS (N=37) using a Luminex-based cytokine array, which revealed that levels of the cytokines CCL2 (also called MCP-1) and CCL11 (also called eotaxin-1) are significantly elevated in patients compared to healthy controls by 700 and 330 fold respectively (p=0.002 and 0.04, respectively). Both CCL2 and 11 are c-c motif chemokines whose encoding genes are located in the human chromosome 17q cytokine gene cluster. Furthermore, compared to pre-treatment samples, CCL2 was significantly elevated in paired plasma samples that were collected after hypomethylating agent (HMA) treatment (2.5 fold increase, N=11 pairs, p=0.04). Luminex results were confirmed by cytokine-specific ELISA. Previous studies have shown that JMJD3 binds to CCL2 gene promoter in mouse macrophage cells (De Santa et al, 2009). Together these results implicate that CCL2 may be a relevant pro-inflammatory signal modulated by JMJD3 in MDS/ AML. We therefore sought to evaluate impact of JMJD3 inhibition on CCL2 production by MDS/AML cells. In MDS-L and TF1 cells, GSK-J4 significantly repressed RNA expression and cytokine release of CCL2. Furthermore, in HMA-resistant MDS-L and TF1 derivative cell lines, productions of CCL2 are elevated by 1.8 and 2.5 fold respectively compared to their parental lines. Treatment of HMA-resistant lines with GSK-J4 improved the anti-leukemia effect of decitabine and had a synergistic effect with HMA. In summary, we have demonstrated that pharmacological inhibition of JMJD3 by GSK-J4 has anti-leukemia effects in MDS/AML cells and has the potential to improve hypomethylation-based therapy in this disease. A possible mechanism for this effect is through the modulation of the inflammatory cytokine CCL2. To further evaluate this hypothesis, we will characterize the effect of JMJD3 and its inhibition on histone methylation of the CCL2 promoter in future work. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1428 Acquired chemo-resistance (ACR) is currently the most important cause of treatment failure and early mortality in DLBCL, arguably the most important unmet need in lymphoma therapy today. Diffuse Large B cell Lymphoma (DLBCL), the most common human lymphoma, comprises a genetically and clinically diverse group of aggressive B cell non-Hodgkin lymphomas (NHL-B), among a small group of important human cancers increasing in incidence in the US over the last four decades. NHL-B are the fifth most common cancers in the USA (〉62,000 new cases/20,000 deaths) expected in 2011. The molecular biologic and genetic basis of the patho-physiology of these important lymphoid tumors is still mostly unresolved. This is due primarily to the lack of valid patho-biologic experimental models allowing for identification of the key patho-physiologic molecular/genetic mechanisms involved in chemo-resistance, resulting in mostly unsuccessful empiric new drug salvage trials, rather than efficient drug-targeting key growth/survival/chemo-resistance (GSC) pathways essential for effective salvage therapies. We have been developing such novel translational experimental DLBCL systems (〉25 DLBCL cell lines derived from relapsed DLBCL patients) and novel agents as the conceptual basis of this model. We have distinguished a set of cell lines that are more resistant to chemo-therapy and identified that the transcription factor p52 component of the alternative NF-kB pathway is highly expressed in DLBCL cell lines that show the highest chemo-resistance characteristics. Down-regulation of p52 sensitizes resistant cells to chemotherapy. This is of particular interest since previous studies have not as yet established definitive role(s) for the alternative NF-kB pathway, particularly p52, in chemo-resistance development. We have discovered that the second generation proteasome inhibitor, Carfilzomib can target the alternative NF-kB-p52 pathway by down-regulating the TNF-receptor family BAFF-R, resulting in lymphoma cell growth inhibition and apoptosis induction. NFATc1, another important multifunctional regulatory molecule (transcription factor (TF), chromatin remodeler, etc), that we have shown to be intrinsically involved with NF-kBs in most DLBCL, and whose involvement in DLBCL is becoming increasingly important on multiple levels, that was recently confirmed genetically, identifying NFATc1 expression as a candidate oncogene in ABC DLBCLs. We have also discovered that GSK3b, a key upstream natural inhibitor of NFATc1, is constitutively phosphorylated in DLBCL cells and can negatively regulate NFATc1 activation. The PKC beta II inhibitor Enzastaurin, affectively inhibits pGSK3b, leading to NFATc1 inactivation and inhibiting cell growth/survival in a broad range of DLBCL cell lines, both GCB and ABC subtypes, with IC: 50 values in the low uM ranges. Enzastaurin strongly synergizes with Carfilzomib to inhibit DLBCL cell growth and induce apoptosis, particularly in chemo-resistant DLBCL cells. Carfilzomib alone enhances pGSK3b and NFATc1 activation, while Enzastaurin abolishes CFZ-induced pGSK3b and NFATc1, suggesting a mechanism for the synergistic activity of the drugs. Novel drug combinations with agents that target multiple growth, survival, and chemo-resistance pathways, such as Carfilzomib and Enzastaurin, represent promising, emerging therapeutic options for reversing chemo-resistance in relapsed/refractory DLBCL patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2717 The biological significance of the ubiquitin-proteasome system in the control of cellular processes has been well-recognized; however, the pathophysiological importance of the immunoproteasome, the inducible form of the proteasome, has not been well-appreciated in cancer cells, particularly in this common diffuse large B-cell lymphoma (DLBCL), a clinically challenging, aggressive B-cell non-Hodgkin's lymphoma (NHL-B). The primary function of the immunoproteasome was originally believed to be only in immune cells to improve MHC-I antigen presentation efficiency in adaptive immune responses. It has now becomes evident that the immunoproteasome possesses broader biological functions, and is associated with various types of cancer. Using the Oncomine database to analyze the mRNA expression levels of immunoproteasome subunits in DLBCL, we found that the subunits MECL-1 and LMP-2 are highly expressed in comparison to normal B lymphocytes. We then examined the clinical significance of MECL-1 and LMP-2 mRNA expression in primary DLBCL, and found that increased MECL-1 mRNA expression is significantly associated with decreased cumulative overall survival rate (P=0.019). We then analyzed the protein expressions of MECL-1 and LMP-2 in various (20) DLBCL cell lines, and discovered that most of the DLBCL cell lines highly expressed both MECL-1 and LMP-2 but there is a subset of cell lines that did not express MECL-1 and LMP-2. Further analysis indicated that MECL-1 and LMP-2 subunits of the immunoproteasome are not associated with constitutive NF-kB activation in DLBCL since MECL-1 and LMP-2-negative DLBCL cell lines also express constitutive NF-kB activation. RNA interference-mediated knock-down of MECL-1 or LMP-2 leads to cell growth inhibition in DLBCL cell lines in vitro. These results strongly suggest that the immunoproteasome has important biological function in controlling growth and survival mechanisms in DLBCL and thus selective targeting of the immunoproteasome may offer therapeutic opportunity for this deadly disease. Bortezomib (BZ) is the first in the class of proteasome inhibitor (PSI) and represents a major advance in NHL, particularly mantle cell lymphoma. However, with the emergence of a new class of PSIs, such as Carfilzomib (CFZ), we are presented with opportunities to improve patient care in relapsed/refractory NHL-B. To elucidate the role of proteasome inhibitors in DLBCL, we analyzed the effect of BZ and CFZ in our representative DLBCL cell lines. BZ and CFZ treatments in DLBCL cell lines (20) have shown strong responses, with IC50s in the low nM ranges (2–50 nM). We have shown that DLBCL cell lines lacking both MECL-1 and LMP-2 are more resistant to CFZ than DLBCL cell lines that have both MECL-1 and LMP-2. To investigate a potential CFZ resistance mechanism(s) in these cell lines, we measured the 20S proteasome activity and compared this activity to the CFZ sensitive DLBCL cell lines. The results indicated that DLBCL cells that are more sensitive to CFZ show higher immunoproteasomal activity. The immunoproteasome activity in the resistant DLBCL cell lines is comparable to the proteasome activity found in normal B cells. These results suggest that the immunoproteasome is deregulated in DLBCL and represents a potential target for therapy in personalized medicine. Our studies emphasize understanding the mechanisms responsible for abnormal proteasomal function in DLBCL, that are critical for establishing an etiologic link to chemo-resistance and the development of new specific therapies for DLBCL targeting defective proteolysis through the immunoproteasome. Disclosures: No relevant conflicts of interest to declare.