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Myeloid derived suppressor cells (MDSC) are a heterogeneous population of undifferentiated myeloid cells that are expanded and activated in pathological conditions and have the ability to potently suppress T-cell function and thereby contribute to immunosuppression and tumor progression. While there have been studies showing a role for MDSC in a variety of hematological malignancies, no data is available indicating that MDSCs contribute to the tumor progression in Waldenstrom's Macroglobulinemia (WM), an indolent lymphoma characterized by bone marrow (BM) infiltration of lymphoplasmacytic (LPL) cells and increased secretion of monoclonal IgM. In previous work, we have found increased GM-CSF and reduced arginine and cysteine in the BM microenvironment in WM. We hypothesized that this was due to the presence and activity of MDSCs in WM. BM aspirates from WM patients (n=17) were therefore processed to isolate LPL (CD19+/CD138+) cells from the rest of the BM cells (CD19-/CD138-). Sorted (CD19-/CD138-) cells from BM of patients with WM were studied with flow cytometry. Using a sequential gating strategy (lack of lineage markers, low levels of HLADR, CD33+, CD11b+) we identified a population of MDSCs that were then subdivided using CD14 and CD15 expression into total-, monocytic-, or granulocytic- MDSCs (m-MDSC, g-MDSC). We also analyzed unsorted BM cells using cytometry by time-of-flight (CyTOF) in order to further identify and phenotypically characterize the BM MDSC population in a group of WM patients with smoldering (asymptomatic) disease, symptomatic disease, or in remission post-treatment. BM samples from normal subjects were used as a control. Flow cytometry data showed significant higher numbers of MDSC subsets expressing PD-L1 and Arginase1 in WM patients when compared to the normal samples. BM cells from WM patients (n=18) then were compared to controls (n=11), and the absolute number of the total MDSC (p=0.05), m-MDSC (p=0.002), g-MDSC (p=0.02) was increased in WM specimens. When MDSCs from WM or normal monocytes from healthy controls were co-cultured with activated T-cells, the proliferation of activated T-cells in the presence of MDSCs from WM patients was impaired compared to controls, confirming the suppressive role of MDSCs. We then performed high dimensional analysis of the total BM MDSC cells using t-SNEand identified phenotypically distinct MDSC cell populations in the BM that were differentially present when healthy controls were compared to patients with smoldering WM or those with WM needing treatment. Specifically, WM patients needing treatment had increased numbers of a distinct MDSC population that was highly positive for CD163, and CD138. Moreover, conventional markers denoting m-MDSC and g-MDSC, such as CD14 and CD15, were highly expressed in all populations and their pattern of expression did not specifically define the MDSC subtypes, indicating that high dimensional phenotyping further details the MDSC sub-compartments beyond the conventional categorization of MDSC using conventional cytometry. In summary, we find that MDSCs are increased in the BM of WM patients compared to controls. MDSCs expressing CD163 and CD138 increase when WM patients become symptomatic and require therapy. Furthermore, MDSCs in the BM of WM patients suppress T-cell function and likely contribute to progression of the disease. MDSCs in the BM therefore present a therapeutic target that should be explored in WM patients. Disclosures Ansell: Bristol Myers Squibb: Other: research funding for clinical trials; Merck: Other: research funding for clinical trials; AI Therapeutics: Other: research funding for clinical trials; Affimed: Other: research funding for clinical trials; Takeda: Other: research funding for clinical trials; Pfizer: Other: research funding for clinical trials; Regeneron: Other: research funding for clinical trials; Seattle Genetics: Other: research funding for clinical trials.

BACKGROUND: Sézary syndrome and mycosis fungoides are two clinically distinct neoplasias of CD4-positive skin-resident T-cells that share remarkable morphologic and immunophenotypic similarities, commonly referred together as cutaneous T-cell lymphoma (CTCL). Prognostic clinical staging in CTCL requires flow cytometric quantitation of peripheral blood tumor cells (Sézary cells), based on aberrant loss of CD7 and/or CD26 within the CD4-positive T-cell compartment. Unfortunately, similar T-cell populations are frequently encountered in reactive conditions, creating uncertainty in the diagnostic interpretation. We recently reported a flow cytometric strategy to assess T-cell clonality using a single antibody (JOVI-1) against one of 2 mutually exclusive T-cell receptor beta constant (TRBC) regions randomly selected during T-cell receptor gene rearrangement. We hereby applied this strategy to a diagnostic Sézary cell flow cytometry panel, resulting in rapid, accurate and unequivocal identification and quantitation of clonal CD4-positive T-cells. METHODS: We studied 33 peripheral blood specimens from 24 patients with CTCL, and 28 specimens from patients with no clinical or laboratory evidence of T-cell malignancy. A routine Sézary cell flow cytometry panel (CD2/CD3/CD4/CD5/CD7/CD8/CD26/CD45) was modified to include a fluorescent labeled antibody against TRBC1. Gated CD4 T-cells were studied to identify discrete subsets lacking CD7, CD26 or both, in addition to other immunophenotypic abnormalities. Within each subset, clonality was defined as a dominant TRBC1-positive or TRBC1-negative population greater than 85%, or a dominant abnormal TRBC1-dim population, as previously described. RESULTS: Peripheral blood specimens from patients with CTCL showed subsets of CD4-positive T-cells lacking CD7 (median=35%; range 1.6% to 96%) or CD26 expression (median=54%; range 6.6% to 98%). Fifteen (45%) of these specimens had other identifiable phenotypic abnormalities, including decreased CD2, CD3, CD4 or CD5. Patients without a T-cell malignancy also had CD4-positive T-cells lacking CD7 (median=13%; range 0.5% to 38%) or CD26 expression (median=9.4%; range 0.7% to 30%), albeit to a lesser extent compared to CTCL patients (p<0.01 and p<0.0001 for CD7 and CD26, respectively). One febrile infant showed loss of CD2 on a small T-cell subset. Discrete CD4-positive T-cell subsets with monotypic TRBC expression were identified in 24 specimens (73%) from patients with CTCL (Figure A), ranging from 30 to12,000 cells/µL (median=632). Importantly, no TRBC-restricted CD4-positive T-cells were detected in any of the 28 patients without a T-cell malignancy. Nine specimens from CTCL patients without a TRBC-restricted subset were reviewed based on morphologic, immunophenotypic and clinical features, yielding no definitive evidence of Sézary cells. Absolute numbers of TRBC-restricted T-cells correlated with numbers of Sézary cells estimated based on lack of CD7 or CD26 expression (R=0.99), or absence of both antigens (R=0.7) (Figure B). However, all cases with no detectable clonal TRBC-restricted T-cells showed significant absolute numbers of CD4-positive T-cells lacking CD7 or CD26 (mean=208 cells/µL, range=22 to 1,078 cells/µL), resulting in common diagnostic uncertainties based on quantitation of these subsets. A narrower gating strategy on these same patients based on the combined loss of CD7 and CD26 yielded lower numbers of abnormal cells (mean=51 cells/µL, range=2 to 556 cells/µL), but grossly underestimated the number of TRBC-restricted CD4-positive T-cells by more than 500 cells/µL in 6 patients with CTCL (Figure B). CONCLUSIONS: A single anti-TRBC1 antibody added to a routine Sézary cell flow cytometry assay provides a rapid, simple and low-cost approach to query for clonality within immunophenotypically distinct CD4 T-cell subsets, eliminating the need for a separate T-cell clonality assay. Diagnostic uncertainties and quantitative biases resulting from immunophenotypic overlaps between Sézary cells and reactive T-cells are virtually resolved. Direct quantitation of clonal CD4 T-cells based on TRBC1 restriction is a biologically sound strategy for blood staging in CTCL, effectively overcoming the limitations and uncertainties of staging by immunophenotypic analysis based on current guidelines. Figure Disclosures Horna: MorphoSys AG: Research Funding.

Introduction: Most patients with newly diagnosed FL treated with rituximab (R) alone or R + chemotherapy will experience prolonged progression-free survival and overall survival (OS), but it remains unclear what factors have the greatest influence on FL-associated and other causes of death in this patient population. We utilized individual patient data from 13 first-line randomized clinical trials from the FLASH database to perform a comprehensive multistate survival analysis to examine and quantify the relationships between clinical characteristics, treatment response, and early, intermediate, and late FL outcomes. Methods: The multistate survival analysis model defined states for "Alive after beginning Induction Treatment (TX)", "Alive after beginning Maintenance TX", "Death due to FL", and "Death from Other Causes" (Figure 1). We used the Aalen-Johansen estimator, a generalization of the Kaplan-Meier estimator, to calculate the likelihood of being in each model state and estimate the course of FL over time. Making no assumptions on the probability distributions and capable of coping with censored observations, the Aalen-Johansen estimator is a convenient and reliable nonparametric estimator for clinical data. Results: Among 7,465 FL patients with median age 56 (range 18-90) years, 49.2% were female; 28.7% Stage I-III, 71.3% Stage IV, and FLIPI was 0-1 (20.0%), 2 (36.8%), ≥ 3 (43.2%). Following initiation of induction treatment, 2-, 5- and 10-year death rates were 1.7%, 3.8%, and 5.8% due to FL, and 0.7%, 2.1%, and 4.8% from other causes (Figure 2). Death rates at 2, 5, and 10 years due to FL and other causes for subgroups based on clinical characteristics and treatment response are shown in Table 1. Notably, patients > 70 years and patients with FLIPI ≥ 3 had worse outcomes and patients achieving CR at 18, 24, and 30 months experienced improved outcomes. Conclusion: This is the largest study using data from randomized trials to quantify the impact of clinical factors on early, intermediate and late mortality by cause of death. We demonstrated that age > 70 years and FLIPI ≥ 3 were linked to increased FL-associated death and response to TX distinguished patients with favorable and poor outcomes. Future analyses should quantify the impact of predictors on the rate/time of FL outcomes in multivariable models. Disclosures Salles: Roche, Janssen, Gilead, Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Amgen: Honoraria, Other: Educational events; BMS: Honoraria; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis, Servier, AbbVie, Karyopharm, Kite, MorphoSys: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Epizyme: Consultancy, Honoraria; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events. Hoster:Janssen: Research Funding; Roche Pharma AG: Other: Travel Support. Hiddemann:Bayer: Research Funding; Gilead: Consultancy, Honoraria; Vector Therapeutics: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria. Herold:Roche: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Gilead: Honoraria; Celgene: Honoraria. Morschhauser:Servier: Consultancy; Gilead: Consultancy; Janssen: Honoraria; Roche/Genentech: Consultancy; BMS: Honoraria; Celgene: Honoraria. Rummel:Roche Pharma AG: Honoraria, Research Funding; Celgene: Honoraria; Janssen: Honoraria; Sandoz: Honoraria. Kimby:AbbVie,: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Gilead: Other: educational lectures. Vitolo:Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gyan:Pfizer: Honoraria. Ladetto:Celgene: Honoraria; Roche: Honoraria; Janssen: Honoraria; Abbvie: Honoraria; Acerta: Honoraria; Sandoz: Honoraria. Nielsen:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Flowers:AstraZeneca: Consultancy; BeiGene: Consultancy, Research Funding; Eastern Cooperative Oncology Group: Research Funding; Burroughs Wellcome Fund: Research Funding; AbbVie: Consultancy, Research Funding; Optimum Rx: Consultancy; V Foundation: Research Funding; Pharmacyclics/Janssen: Consultancy, Research Funding; Bayer: Consultancy; Gilead: Consultancy, Research Funding; TG Therapeutics: Research Funding; Denovo Biopharma: Consultancy; Acerta: Research Funding; National Cancer Institute: Research Funding; Millenium/Takeda: Research Funding; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Spectrum: Consultancy;

Background: Voxelotor (GBT440) is a small molecule allosteric effector with high specificity for hemoglobin (Hb) that inhibits Hb S polymerization by increasing hemoglobin oxygen affinity. Developed as an oral, once-daily medication, it is undergoing phase 3 randomized clinical trials in sickle cell disease patients. Therapeutic monitoring of Hb quantitative fractions is routine in sickle cell disease patients to assess medication and transfusion effects and future treatment requirements. In the clinical laboratory, some therapies can alter the Hb fraction percentages or characteristic protein migration/elution patterns and can mimic complex hemoglobinopathy disorders causing misinterpretation or unnecessary additional testing. We present the effects of voxelotor on hemoglobin identification/monitoring testing in multiple testing platforms. Methods: A whole blood anticoagulated sample from a male patient who was undergoing treatment for sickle cell disease was received for routine hemoglobin testing. Five additional samples with similar features were also received from three male patients (age range of all patients was 18 to 22 years) with sickle cell disease. Hemoglobin protein analysis was performed using standard methods including cation-exchange high performance liquid chromatography (HPLC), capillary electrophoresis (CE), isoelectric focusing (IEF), intact globin chain mass spectrometry (MS), sickle solubility and hemoglobin stability studies. DNA sequencing of the alpha (HBA1/HBA2) and beta globin (HBB) genes was performed on one sample. Results: CE (initial and 50:50 mix, figures 1A and 1B, respectively) revealed a Hb S peak with a shoulder and additional small peaks at position 203 (D zone) and 234 (cusp of zones E/A2). In some cases the 234 peak interfered with instrument designation of the A2 peak (50:50 mix with normal) and shifted the Hb S peak into the E zone which risks variant misidentification (figure 1C). Our laboratory overcame this limitation by substituting the normal sample with beta thalassemia trait to enhance the Hb A2 peak after mixing. HPLC tracings (figure 1D) showed a duplex peak pattern in the D and S windows with separate peak quantification percentages (index case showed elution at 4.18 and 4.37 minutes with 14.9% and 66.4%, respectively). The dual peaks detected by HPLC partially mimicked compound Hb S/D-Punjab. IEF (figure 1E) showed additional bands at -4.7, and -11.5 (Schneider-Barwick ratios). The additional peaks identified by CE and IEF mimicked an alpha chain hemoglobin variant; however, the expected hybrid patterns were atypical prompting further investigation. Sickle solubility was positive, heat stability studies were negative and mass spectrometry did not definitively detect abnormal peaks other than βS. DNA sequencing of the alpha and beta globin genes did not reveal abnormalities other than homozygous Hb S. Similar hemoglobin patterns were identified in all samples tested. Conclusion: Voxelotor treatment in sickle cell patients results in a distinctive pattern alteration in hemoglobin testing. Methods may not give accurate variant percentage estimation and the treatment may result in shifts in variant mobility causing erroneous laboratory results. Novel therapies are increasing in the treatment of hemoglobin disorders and medication effect should be a consideration when atypical patterns are encountered in the laboratory. Knowledge of these artifacts by clinicians and laboratorians is paramount to avoid erroneous diagnosis, unnecessary additional testing and inaccurate Hb variant quantitation for monitoring purposes. Notification of current treatment regimens to testing facilities is highly recommended in these patients. Figure 1 Disclosures No relevant conflicts of interest to declare.

Abstract Introduction Myelodysplastic syndromes (MDS) are heterogeneous myeloid disorders characterized by dysplasia in one or more hematopoietic cell lines, peripheral cytopenia(s), and distinct cytogenetic abnormalities. The presence of certain cytogenetic abnormalities [e.g. 7/del(7q), -5/del(5q), 13/del(13q), i(17p)], in the setting of unexplained persistent cytopenia, has been considered presumptive evidence of MDS, despite definitive morphologic features of MDS. Conversely, chromosomal abnormalities such as isolated 20q- are not considered definitive evidence for MDS, in the absence of dysplasia. Based on our previous findings, patients with isolated 20q- without morphologic features of a myeloid neoplasm have varied clinical outcomes (Jawad MD et al. Am J Hematol.2016). In this study, we investigate the frequency of recurrent somatic gene mutations in these patients, and their relationship to progression to a myeloid neoplasm. Methods We retrospectively reviewed the bone marrow pathology reports of patients evaluated at our institution from 01/2005 to 09/2015 who had isolated 20q- in at least 2 of 20 metaphases. Patients with isolated 20q- and without myeloid neoplasms were included in the study, along with medical record review. A 35-gene next generation sequencing (NGS) panel (OncoHeme) targeting myeloid neoplasms was performed on available archived bone marrow aspirate pellets. Statistical analyses were performed using JMP Pro software version 13. Parametric (t-test) or nonparametric tests such as the Fisher-exact test, chi-square test, or Wilcoxon rank-sum test, were utilized as appropriate for test variables. Statistical significance was based on a 2-sided significance level of .05. Kaplan-Meier analysis was used for analyzing time to progression to myeloid neoplasms and progression-free survival (i.e. time to progression to myeloid neoplasms or death from any cause). Results Fifty-six (42 males, 14 females) patients with isolated 20q- were identified. The average percentage of 20q- was 38.5 % (±29.3). The median age was 68 years (range: 44-90) and the median hemoglobin (Hb), absolute neutrophil count (ANC) and platelet count (Plt) were 11.7 g/dL (range: 8.3-15.1), 2.7 x 10(9)/L (range: 0.6-9.4) and 138 x 10(9)/L (range: 16-392), respectively. The indications for bone marrow biopsies ranged from cytopenia (21), multiple myeloma (20), non-Hodgkin lymphoma (10), amyloidosis (2), CLL (2) and transplant donor evaluation (1). Twenty-three patients harbored pathogenic mutations (15 had one mutation, 1 had two mutations and 7 had three mutations). The identified pathogenic mutations included ASXL1 (8), TET2 (8), SRSF2(3),PHF6 (2), SF3B1 (2), CBL (2), U2AF1 (2), IDH1 (1), IDH2 (1), BCOR (1), JAK2 (1), PTPN11(1), TP53 (1) and RUNX1 (1). Nine patients (16.1%) progressed to myeloid neoplasms (5 MDS, 2 acute myeloid leukemia, 2 chronic myeloid neoplasm-not otherwise specified) at a median follow-up of 36.7 months (range: 1.9-57.4). Of these 9 patients, 5 had one mutation (BCOR, TET2, DNM3TA, CBL, IDH2), 1 had two mutations (SF3B1/CBL) and 3 had three mutations (ASXL1/IDH1/SRSF2,DNM3TA/PTPN11/TET2, PHF6/TET2/TET2). In contrast, only 29.8% of non-progressed cases showed at least 1 mutation. Kaplan-Meier analysis demonstrated the presence of genetic mutations in patients with isolated 20q- carried significantly higher chance of progression to myeloid neoplasms (p<.05, Figure 1A and 1B). However, the number of genetic mutations was not predictive of progression in our relatively small study cohort (p=.66). There was no significant difference between those who did and did not progress with respect to age, gender, Hb, ANC, Plt, 20q- or mutational allele burden. Conclusions Most of the patients with isolated 20q- and without morphologic features of a myeloid neoplasm did not harbor myeloid neoplasm associated gene mutations and further did not develop a myeloid neoplasm during the course of our study. Among patients with pathogenic mutations, nearly one-third showed progression to a myeloid neoplasm. Of the clinical and laboratory variables, the presence of genetic mutation is a significant risk factor for progression to a myeloid neoplasm. This study revealed the underlying molecular heterogeneity of isolated 20q- in the absence of morphologic features of a myeloid neoplasm, and underscored the prognostication value of genetic mutation analysis in these cases. Disclosures No relevant conflicts of interest to declare.

Abstract Aberrant over production of monoclonal immunoglobulin is a classic feature of the plasma cell (PC) malignancy multiple myeloma (MM). At diagnosis and more frequently upon relapse, some MM patients present with increased production of monoclonal free light chain (FLC) without a corresponding increase in intact immunoglobulin. This phenomenon is commonly referred to as light chain escape (LCE). As previously described (Walters et al. 2018, Experimental Hematology, Vol 57 p42-49), we have established a monoclonal IgA kappa MM cell line, designated as MC-B11/14, from the bone marrow (BM) aspirate of a 46 year old man diagnosed with MM. This cell line is non-hyperdiploid and possesses an 11;14 chromosomal translocation. Given our long standing interest in immunoglobulin function and secretion, we used CRISPR technology to knock out IgA heavy chain production in an effort to create a model of LCE. Successful knockout (KO) of IgA expression was demonstrated by immunofluorescence, flow cytometry, and western blot. As expected, secretion of intact IgA was undetectable in the MC-B11/14IgA- cells. Notably, no significant difference in morphology, phenotypic markers, or growth patterns was observed between the MC-B11/14WT and MC-B11/14IgA- cells. Examination of drug sensitivity between the MC-B11/14WT and MC-B11/14IgA- cells revealed that the response to pomalidomide treatment was similar between the MC-B11/14WT and MC-B11/14IgA- cells; however, the MC-B11/14IgA- cells were found to be more resistant to treatment with bortezomib. Given that increased bortezomib resistance was the only significant difference detected between the MC-B11/14WT and MC-B11/14IgA- cell lines in our initial study, we decided to take a metabolomics approach to examine whether loss of IgA heavy chain expression may alter cellular metabolism. Metabolomics or the profiling of small molecules is a powerful tool to characterize the metabolome of cells/biological systems and has proven to be useful in identifying alterations that occur in malignancy. In this regard, we employed pathway-specific targeted LC-MS/MS protocols in order to examine over 330 metabolites in 35 metabolic pathways in MC-B11/14WT and MC-B11/14IgA- methanolmetabolite extracts using an Agilent 1290 UPLC-6490 QQQ-MS liquid chromatography mass-spectrometer. Of the 330 metabolites examined, 28 were found to be significantly more abundant in the MC-B11/14IgA- cells compared to MC-B11/14 WT cells while 79 were found to be significantly less abundant. In general, the majority of significant metabolic differences between the two cell lines were found to involve metabolites from various amino acid synthesis pathways. More specifically, the MC-B11/14 WT cell line was found to possess significantly more citraconic acid, anthranilic acid, and homovanillic acid. By contrast, further analysis revealed that the MC-B11/14IgA- cell line was found to possess an 11.5 fold increase of the metabolite 2-hydroxyglutarate (2-HG). 2-HG has previously been shown to act as a competitive antagonist of α-ketoglutarate (α-KG), which results in the inhibition of α-KG-dependent dioxygenases including the JmjC domain-containing histone demethylases (KDMs) and the ten-eleven translocation (TET) family of DNA hydroxylases. Notably, these proteins are known to play a role in histone and DNA demethylation, respectively. Taken together, these data suggest that MM cells that have undergone LCE may also possess an accumulation of 2-HG which could lead to altered patterns of histone and DNA methylation. Given that MM patients who relapse with LCE typically have a poorer prognosis, further investigation of metabolomics in light-chain only expressing cell lines and patients who have relapsed with LCE is warranted. Disclosures No relevant conflicts of interest to declare.

Abstract Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of B-cell Non-Hodgkin Lymphoma (NHL), representing a third of all new cases. DLBCL is further sub-divided into various molecular sub-types based on gene expression and co-occurring genetic alterations. Gene expression based- subtypes include the germinal center B-cell (GCB) and the activated B-cell type (ABC) subtypes, with the ABC sub-type having a poorer prognosis than the GCB sub-type. Interestingly, 30-40% of all DLBCL patients harbor mutations in key epigenetic regulators, EZH2, KMT2D, CREBBP, EP300, and mutations in histone proteins themselves. Through an unbiased cell-based phenotypic screen, we discovered that inhibition of lysine demethylases, specifically KDM4C and KDM4A, represents a vulnerability across 15 different DLBCL cell lines including germinal center B-cell (GCB) and activated B-cell (ABC) type lines, with a GI50 value between 75nM to ~200nM, while sparing leukemia lines. Consistently, treatment of xenograft-based animal models of DLBCL with a low dose of KDM4A/KDM4C inhibitor delivered intra-peritoneally three times a week, results in a drastic reduction of tumor burden. Both KDM4A and KDM4C catalyze the removal of histone H3 K9 di- and tri-methylation (H3K9me2/3) and H3K36 di- and tri-methylation (H3K36me2/3). H3K9me2/3 is associated with promoter and enhancer repression, while H3K36me2/3 is present in gene bodies during transcription but also functions as a chromatin repressor Consistently, we have identified key enhancers, including those associated with IKZF1, that are "decommissioned" after inhibition of KDM4A/KDM4C. Repressed enhancer activity, through loss of H3K27 acetylation and H3K4me1, and gain of H3K9me2/3, results in a rapid transcriptional downregulation of IKZF1 and its partners including IKZF3. Given the role of IKZF1 in the transcriptional regulation of key B Cell Receptor (BCR) signaling components, we show that KDM4A/KDM4C inhibition leads to a downregulation of SYK, a proximal BCR-signaling component, which likely precedes DLBCL cell apoptosis. In addition, we observed an activation of extra-lineage transcription factors such as CEBPA and CEBPB, which are normally repressed by IKZF1 in the lymphoid lineage. A concomitant downregulation of the B-cell gene expression program and an upregulation of the myeloid (CD14+ monocytic) gene expression program is also observed, implying a "trans-differentiation" of DLBCL cells into the monocyte lineage. This lineage-switch correlates with an increased population of CD14+ expressing cells. Finally, using DLBCL patient data sets, we can show that over-expression of either KDM4A or KDM4C is associated with poor prognosis in DLBCL patients. In summary, we have discovered that KDM4A/KDM4C inhibition results in an increase of repressive histone modifications at several intra-genic "enhancers" of genes that are responsible for the survival and proliferation of DLBCL cells. The elucidation of this unique epigenetic mechanism provides a strong rationale for the development of novel targeted therapies against both multiple subtypes of DLBCL. Figure. Figure. Disclosures Shipp: Bayer: Research Funding; AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.