ALBERT

Description: Abstract 609 Introduction: Myelodysplastic syndromes (MDS) represent a very complex group of myeloid leukemias with diverse genetic and epigenetic characteristics. We have identified JMJD3, a histone demethylase, aberrantly upregulated in bone marrow CD34+ hematopoietic progenitor cells of MDS (Blood 2009). JMJD3 is a jmjc domain containing histone demethylase that removes methyl groups from Lys-27 of histone H3, and is also known to positively regulate the methylation on Lys-4 of histone H3. This results in activation of upstream regulators of NF-kB signaling, suggesting that JMJD3 plays a crucial role in the pathogenesis of MDS. Therefore, insight on the regulation of JMJD3 expression in MDS is of importance. Aberrant expression of miRNAs, including loss of expression, which leads to up-regulation of the expression of its target genes, has been identified in various types of malignancies including MDS. We hypothesized that alteration of regulatory miRNAs could be involved in the upregulation of JMJD3 in MDS CD34+ cells. Identification of JMJD3 regulatory miRNAs may help further dissect the molecular mechanisms underlying the pathogenesis of MDS. Methods and Results: To identify regulatory miRNAs targeting JMJD3, we performed an analysis using microRNA Genomics Resource (miRGen) and identified 40 candidate miRNAs that potentially interact with the 3′ untranslate region (3′-UTR) of JMJD3 RNA. We then characterized the interactions of these candidate miRNAs to JMJD3 by using a luciferase assay system by cloning the 3′-UTR region of JMJD3 gene into a luciferase reporter construct. By co-transfecting each of the 27 available miRNAs from the 40 candidates together with the luciferase reporter construct into human megakaryocytic blast cell line Meg-O1, we identified 7 mirRNAs (has-mir -29a, -29b, -29c, -99b, -101, -377, and -767-5p) that negatively regulate JMJD3 3′UTR associated luciferase activity. We then examined the expression for these 7 key potential regulatory miRNAs of JMJD3 in a cohort of 36 patient bone marrow CD34+ cells by microRNA Taqman probe based Q-PCR analysis. The median age of these patients was 67 years (32 to 85); 42% had low or INT-1 disease; 21% were diploid and 28% had an alteration of chromosome 5 or 7. Analysis showed that 16 of the 36 MDS CD34+ cell samples examined showed lost expression of mirRNA hsa-mir767-5p, and 7 had down-regulated expression of the same microRNA. For these 23 MDS samples with repressed mir-767-5p expression, 11 of them (~48%) had upregulated expression of JMJD3, as demonstrated by Q-PCR analysis Consistently, in miRNA transfected Meg-O1 cells, Q-PCR analysis indicates that over-expression of mir767-5p is accompanied with decrease of JMJD3 RNA by 68% based on Q-PCR analysis using 3 different probes for JMJD3. Of interest, 4 of the 5 patients with monosomy of chromosome 7 had downregulation of mir767-5p. Conclusion and Future Direction: These results support the hypothesis that mir767-5p is a key regulatory miRNA targeting JMJD3 in hematopoietic progenitor cells. Since one important mechanism for miRNA on target gene expression is through inhibiting the translation from mRNA to protein, we are now developing an antibody against endogenous JMJD3, with which we will be able to further correlate the level of mir767-5p and the expression of JMJD3 protein in both cell lines and primary MDS samples. Furthermore, correlation between alteration of mir767-5p with clinical, cytogenetic and molecular features in MDS patients will be characterized in a larger cohort of patients. These will further determine the role of mir767-5p for the regulation of JMJD3 and its role during the pathogenesis of MDS. By pursuing the studies described above, a potential link between two key epigenetic regulatory components, histone methylation regulator (JMJD3) and microRNAs, involved in pathogenesis of MDS, will be established. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2030 Poster Board II-7 Aberrant DNA methylation of multiple promoter associated CpG islands is frequent in primary ALL and predicts for poor prognosis in adult and pediatric disease. Treatment of ALL cell lines with the hypomethylating agent decitabine results in induction of global and gene specific hypomethylation, reactivation of epigenetically silenced genes and induction of apoptosis at low concentrations and prolonged exposures (Leuk Res 2005;29:739-48). Prior studies of decitabine in myeloid leukemias indicated that induction of global and gene specific hypomethylation using a 5-day schedule of decitabine is transient peaking 7 to 10 days after initiation of therapy. In view of the proliferative nature of ALL and in vitro modeling results, we designed a phase 1 study of decitabine administered daily x 5 every other week in patients with relapsed or refractory ALL. The main objective of the study is to determine the safety, activity, pharmacodynamic effects and optimal dose (based on clinical activity, toxicity profile and hypomethylating properties) of decitabine in relapsed refractory ALL. The study design follows a standard “3+3” rule with an expansion cohort of N=10 patients at the optimal dose. Patients of any age with relapsed refractory ALL are elegible regardless of performance status or organ function. Initial dose level of decitabine was 10 mg/m2 IV infused over 1 hour daily x 5 days every other week with courses of therapy repeated every 28 days. Use of steroids was allowed during the first course of therapy at the discretion of treating physician. 23 patients have been treated in 7 dose levels (10, 20, 40, 60, 80, 100 and 120 mg/m2 IV QD x 5 every other week). Cumulative doses per course ranged from 100 to 1200 mg/m2. Patient characteristics are: median age 36 years (range 8-67), median WBC 5.3 (range 0.2 to 97), median % peripheral blasts 23% (0-97), cytogenetics diploid in 4 (17%), Ph + 2 (8%), complex 17 (73%), phenotype preB/B in 15 (65%). Median number of prior therapies was 3 (range 1 to 7). No severe drug related grade 3 or 4 toxicity was observed at any dose level. Frequent toxicities included diarrhea, fatigue and liver function abnormalities that were limited and probably related to disease. Overall response rate was 23% (6 pts) including 1 CRp (complete remission with incomplete platelet recovery) and 5 complete marrow responses (blasts less than 5%). All responses lasted at least 4 weeks. Responses were observed at multiple dose leves (#1, 2, 4, 5, 7). Global and gene specific methylation was analyzed on days 0, 2,5, 14,16,19 and 28 of cycle 1. Samples were collected from 18 consenting patients. Global methylation was analyzed using the LINE bisulfite pyrosequencing assay. Median day 0 methylation was 63%, declined to 55% (p=0.01) on day 14 and increased to 61% on day 28. The most effective dose in inducing global hypomethylation was 60 mg/m2 : 61% baseline to 21% on day 28. The following genes were analyzed for gene specific methylation: p73, p15, p15, HES5, Notch3 and Jag1. Induction of hypomethylation was observed in informative patients, a process associated with gene expression reactivation. The analysis is not powered to detect association between response and hypomethylating effect. Finally, depletion of DNMT1 was measured using a Western blot assay. Depletion was only observed in 1 patient treated at 60 mg/m2 that had achieved a response. In summary, single agent decitabine is safe at higher doses than used in myeloid leukemias with clinical activity in patients with advanced refractory relapsed ALL. Of importance, hypomethylating effect is observed at cumulative doses of up to 1200 mg/m2 with a maximal effect at 600 mg/m2, doses that are considered cytotoxic in myeloid leukemias. The study continues at the expansion cohort of 60 mg/m2 IV QD x 5 every other week, the dose considered to be optimal based on toxicity, response and hypomethylating effects. Two patients have been treated but are early for assessment. A parallel study of decitabine combined with hyperCVAD is ongoing in patients that do not respond or progress after single agent decitabine. The activity of decitabine should be tested in patients in first relapse ALL. Disclosures: Off Label Use: Decitabine is not approved for treatment of ALL.

Description: Abstract 1874 Background: Cellular metabolism and oxidative stress are important in the biology and pathophysiology of hematologic malignancies. Limited studies are available regarding metabolic pathways, including production of reactive oxygen species (ROS), and biology and pathogenesis of MDS. Elevated levels of ROS have been described in other leukemias, linked to increased levels of antioxidant molecules and activation of pathways that protect cells from the oxidative stress. The Nuclear erythroid 2 related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1) - Antioxidant Response Element (ARE) complex is the main signaling pathway involved in oxidative stress. Nrf2 is a transcription factor that induces expression of antioxidant responsive genes like heme oxygenase-1, catalase, glutathione dismutates and superoxide dismutase. With low oxidative stress, Nrf2 is associated with Keap1 in the cytoplasm and is continuously degraded by the E3 ubiquitin ligase complex. Keap1 contains multiple cysteine residues that are modified by oxidation to accelerate Keap1 dissociation from Nrf2, allowing Nrf2 to enter the nucleus and bind to ARE. In MDS, high levels of ROS have been described related to iron overload. We studied expression of Nrf2 and Keap1 in newly diagnosed, untreated patients with MDS to correlate with clinical features, pathogenesis, and clinical outcomes. Methods: 24 newly diagnosed pts with MDS and 4 normal donors were evaluated for Nrf2 and Keap1. CD34+ cells were sorted from pts and normal donor bone marrow. RNA was isolated using standard protocols and cDNA was obtained by reverse transcriptase using the Invitrogen SuperScript® One-Cycle cDNA Kit. Nrf2 and Keap1 expression were analyzed by qRT-PCR using Applied Biosystems Real-Time PCR 7900 and SYBR® GreenER™ Reagent System from Invitrogen. Patient clinical and clinical-laboratory characteristics, including IPSS, blood counts, bone marrow features, serum ferritin were used for analysis. Patients were followed for time-to-event endpoints, including time to transformation to AML and survival, dated from initial sample collection. Results: The median pt age was 70 years (range 37–82); 62.5% were male. The median % bone marrow (BM) blast count was 4.5% (range 1–25). Low-risk cytogenetics by metaphase karyotype was noted in 13%, intermediate-risk in 67%, and high-risk in 21%. According to the IPSS scoring system, 25% had high-risk, 17% Int1, 37% Int2, and 21% had low-risk MDS. Bone marrow was hypercellular in 18/24 (75%) pts; 87% for males and 56% for females. The median ANC was 1579/mL (range, 90–17061); hemoglobin was 9.4 gm/dL (range, 6.4–13); and platelet was 63.5/mL (range, 9–1040). The median ferritin level was 859 ng/mL (range, 24–14564); 15/21 (71.4%) had ferritin levels higher than the normal range. The mean relative Nrf2 transcript level was 0.167 (range, .001-.69) vs. 0.004 for normal donors P= 0.147 and the mean relative Keap1 transcript level was 0.014 (range, .003-.05) vs. 0.008 for normal donors P= 0.54. Increased bone marrow (BM) blast was associated with higher Nrf2 transcript level: 7/10 (70%) pts with BM blasts 〉10% presented higher Nrf2 levels. Increased ferritin level was associated with presence of int-poor cytogenetic features(10/24). The median follow-up time is 11 months (range, 1–43); transformation to AML occurred in 5 pts and overall 18 patients have died. The median survival time is 11 months. There was a trend for shorter survival or Time To Transformation (TTT) associated with higher Nrf2 (median 6 months vs 14 months p=0.328) and higher Keap1 transcript levels (median 6 months vs 14 months p=0.45), although not statistically significant. Conclusions: CD34+ cells from bone marrow of patients with MDS have higher transcript levels of Nrf2 and Keap1 than normal donor CD34+ cells consistently with data reported in the current licterature for other hematological malignancies, such as CLL and AML. One of the reasons could be the activation and the integrity of Nrf2 signaling in MDS in response to high ROS levels. Among patients with MDS, there was a trend for shorter survival associated with higher Nrf2 and Keap1 transcript levels. These findings suggest that Nrf2/Keap1 signaling may play an important role in the pathophysiology of MDS and merit further investigation and must be confirmed in larger numbers of patients with MDS and normal donors. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Inosine 5'- monophosphate dehydrogenase (IMPDH) plays a critical role in nucleotide synthesis by serving as a rate-limiting step for the de novo production of guanine from its precursors. Overexpression of IMPDH has been observed in both solid and hematologic malignancies. FF-10501-01 is a potent new competitive IMPDH inhibitor. In this study, we systematically investigated the anti-leukemia effect of FF-10501-01 in acute myeloid leukemia (AML) cell lines, including hypomethylating agent (HMA)-resistant derivative cells. Methods Thirteen leukemia cell lines were studied, including 5 parental AML cell lines and their HMA-resistant derivatives (MOLM13, SKM1, HL60, TF1, and U937) as well as three other AML cell lines (KG1, HEL, and OCI-AML3). Cell proliferation was determined using trypan blue analysis. Flow cytometry was performed to detect drug-induced apoptosis and cell cycle status. High-performance liquid chromatography (HPLC) was performed to detect the intracellular concentration of guanine nucleotides, with mycophenolic acid (MPA) treated cells used as positive control. We also studied the effect of guanosine supplementation on FF-10501-01-treated cells. Results To understand whether FF-10501-01 was able to effectively limit AML cell proliferation, we subjected a variety of cell lines to 72 hours of FF-10501-01 treatment at various concentrations. We also included a large number of HMA-resistant cell lines in an effort to understand whether or not FF-10501-01 could be a useful secondary or complimentary treatment to HMA therapy. FF-10501-01 inhibited the proliferation of all 13 AML cell lines studied with 72 hours of treatment. The IC-50 of FF-10501-01 ranged between 4.3 and 144.5 µM. The IC50 values for HMA-resistant cells were all higher than those values in their HMA-sensitive counterparts, except SKM1, in which the HMA-sensitive line had a higher IC50 than the HMA-resistant SKM1 line. To further understand the mechanism by which FF-10501-01 effectively reduced cell numbers in these cell lines, we assessed the level of apoptosis in each line after FF-10501-01 exposure. FF-10501-01-induced apoptosis was observed in all of the studied cell lines in a dose-dependent manner except for the HMA-resistant TF-1 cell line. We then assessed whether FF-10501-01 affected cell cycle progression. This effect was highly variable. Increased numbers of cells in G1 phase and decreased numbers of cells in S phase were observed in MOLM13, SKM1, and TF-1 cell lines treated with less than 100 µM FF-10501-01. To understand the mechanistic effects of FF-10501-01, we performed rescue experiments with both HMA-resistant and HMA-sensitive MOLM13 and HL60 cells. Concurrent treatment with FF-10501-01 and guanosine in these cells partially rescued the antiproliferation effect of FF-10501-01. To further characterize the effect of FF-10501-01 on guanosine metabolism, we then performed HPLC experiments to analyze the levels of phosphoguanosine in treated MOLM13 and SKM1 cells. FF-10501-01 treatment effectively reduced the intracellular phosphoguanosine levels in both cell lines. This effect was seen for GMP, GDP, and GTP. We then sought to assess whether the combination of FF-10501-01 and HMAs could be effective in limiting MOLM13 and HL-60 cell proliferation, especially in their HMA resistant derivatives. The combination of HMA and FF-10501-01 showed little synergy beyond the effects of FF-10501-01 alone, regardless of HMA sensitivity, except in HMA-resistant HL-60 cells, in which FF-10501-01 showed moderate synergy with HMA. We further assessed the antiproliferative effect of FF-10501-01 in bone marrow blast samples taken from 3 AML patients. There was a minor dose-dependent antiproliferation effect seen in these samples, but this was not statistically significant. Notably, one patient showed a sharp increase in cell counts at the lowest concentration of FF-10501-01, but that sample's cell numbers decreased more rapidly as FF-10501-01 concentration increased. This implies that FF-10501-01 treatment response may be related to cell proliferation. Conclusions The IMPDH inhibitor FF-10501-01 can produce potent anti-proliferative and apoptotic induction effects on AML cell lines, including HMA-resistant cell lines, through inhibition of de novo guanine nucleotide synthesis. These results indicate that FF-10501-01 might be a promising new therapeutic agent for AML. Disclosures Paradiso: Strategia Therapeutics, Inc.: Employment. Iwamura:FUJIFILM Corporation: Employment.

Description: Abstract 1700 To better understand molecular bases of MDS pathogenesis, we performed a genome-wide CHIP-Seq analysis of H3K4me3, a histone mark associated with gene activation. In MDS CD34+ cells (N=4), 36 genes showed higher levels of promoter-H3K4me3 compared to controls. 10 of 11 randomly selected genes from these 36 showed increased mRNA expression in MDS CD34+ cells (N=100), supporting the positive correlation between expression and increased promoter H3K4me3. Pathway Analysis indicated that a majority of these genes are involved in innate immunity signal and NF-kB activation. This was validated by increased phospho-p65 in MDS bone marrow CD34+ specimens (N=15). Knock-down of 4 of these genes (C5AR1, FPR2, PTAFR and TYROBP) in OCI-AML3 cells resulted in reduction of p-p65. The observation of innate immunity signal activation and epigenetic deregulation led us screen key innate immunity activators, the Toll-like Receptor (TLR) family genes, and histone methylation regulators in MDS CD34+ cells. Among 8 TLRs and 24 histone methylation regulators, TLR1, 2 and 6, and Jmjc-domain histone demethylase JMJD3 were found to be significantly overexpressed in MDS CD34+ cells compared to control counterparts. This is of biological significance because TLR1 and 6 form functional hetero-dimmers with TLR2. Also, JMJD3 expression can be activated by TLR-NFkB in macrophages. To study TLR2 activation in HSC, we treated CD34+ cells with TLR2 agonists. This led to increased expression of JMJD3, supporting the biological function of TLR2 signal in HSC. We observed that JMJD3 knockdown in OCI-AML3 cells led to reduced expression of innate immunity genes (N=7), accompanied with changes of promoter H3K27me3 and H3K4me3, suggesting that JMJD3 forms a positive feedback to perpetuate innate immunity pathway. To further study the role of TLR-JMJD3 in MDS, we performed capture deep sequencing in 40 MDS bone marrow mononuclear cells (TLR1, 2, 4 and 6, JMJD3, UTX, UTY and JMJD1A). Seven different rare SNP in the coding regions of JMJD3, UTY, JMJD1A, and TLR2 were identified in 5 patients. Among them, one SNP of TLR2 causes a missense mutation, changing a conserved hydrophobic Phe217 to a hydrophilic Ser. We then analyzed the presence of TLR2 F217S in 150 MDS samples by Sanger sequencing. TLR2 F217S was observed in 17 (11%) patients. To evaluate the somatic nature of this alteration, CD3+ cells from 15 corresponding patients were sequenced and only two (13%) CD3+ cell samples carried TLR2 F217S. In the available 9 CD34+ cDNA samples, TLR2 F217S was observed in 8 (90%). We then expressed wild-type or F217S TLR2 in 293T cells, a cell line without endogenous TLR2 expression. Reporter assays indicated that in the absence of TLR2 agonist wildtype and F217S mutant TLR2 led to similar levels of NF-kB activation, whereas F217S led to an increased NF-kB activation compared to wildtype at the presence of TLR2 agonists. F217S also led to increased activation (phospho- and polyubiquitin-) of IRAK1, a key signal mediator for TLR signaling. These results suggest that TLR2 F217S led to more robust innate immunity signal activation when stimulated by agonists. We further studied the impact of TLR2 activation on hematopoietic differentiation. Colony formation of CD34+ cells indicated that TLR2 agonists led to decreased number of erythroid colonies, which was confirmed by flowcytometry that demonstrated TLR2 agonist treatments could reduce the number of CD71 high/HLADR low featured erythroid precursors. To examine the effect of targeting TLR2-JMJD3 in primary MDS cells, we transduced MDS CD34+ cells with shRNAs. In 4 CD34+ cases isolated from lower risk MDS, 3 transduced with JMJD3 shRNA and 4 transduced with TLR2 shRNA had increased ratio of erythroid colonies. In average, JMJD3 and TLR2 shRNA transduction led to a 50% increase in erythroid colonies. This was accompanied by increased expression of genes positively involved in differentiation of erythroid lineage (GYPA, GATA1 and EPOR). Finally, CD34+ cell mRNA expression levels of four genes in this study (NCF2, AQP9, MEFV and TLR1) were associated with overall survival of patients. Taken together, these studies highlight the implication of the deregulation of TLR2-JMJD3 mediated innate immunity signals in MDS pathogenesis and suggest that intervention of this pathway may have therapeutic potential in MDS. Disclosures: No relevant conflicts of interest to declare.

Description: Genomic instability is a hallmark for MDS and AML and is also important for the evolution of MDS to AML. One major cause of genomic instability is telomere dysfunction. Abnormal telomere shortening have been observed in MDS/AML and a spectrum of bone marrow failure syndromes such as dyskeratosis congenita and aplastic anemia. Studies in telomerase deficient mice also indicate that the activation of cell intrinsic checkpoints in response to telomerase dysfunction limits the repopulating capability of hematopoietic progenitor cells after serial bone marrow transplantation, eventually leading to bone marrow failure condition. POT1 is a telomere maintenance gene that encodes a telomere protection protein of the shelterin complex, and is the first member of this this structure found to be mutated in human cancer. Most recently, somatic mutations of POT1 implicated in loss of biological function have been identified in human chronic lymphocytic leukemia (CLL) (Nature Genetics 2013), indicating that POT1 dysfunction is involved in pathogenesis of hematological neoplasms. To study the role of POT1 in MDS, we first sequenced all coding exons of POT1 known to have genomic mutations in CLL. This sequencing analysis was performed via PCR-Sanger method in bone marrow mononuclear cells (BM-MNNC) of a cohort of thirty patients with MDS (15 with RAEB/RAEBT, 11 with RA/RARS/RCMD/MDS-U, 2 with CMML, and 2 with 5q- syndromes). No genetic mutation of POT1 gene was detected in this cohort. We then studied expression levels of POT1 in CD34+ bone marrow hematopoietic progenitor cells. In a cohort of sixty-five patients with MDS (partially overlapping with the patients sequenced for POT1 gene), we performed Q-RTPCR to compare POT1 RNA expression levels of patients with control CD34+ cells of healthy individuals (N=8). Results indicate that, although the overall POT1 RNA level of this patient cohort is not significantly different from controls, the subset of patients (N=13) with cytogentic deletions of chromosome 7 or 7q have a significant reduction of POT1 RNA expression (40% of controls, p

Description: Inosine 5'- monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes de novo synthesis of the guanine nucleotide and is overexpressed in both hematologic and solid tumors. FF-10501-01 is a potent new competitive IMPDH inhibitor. We investigated the anti-leukemia effect of FF-10501-01 in AML cell lines and in a Phase 1 clinical study in advanced AML and MDS, including HMA failures. Thirteen leukemia cell lines were studied, including 5 parental AML cell lines and their HMA-resistant derivatives (MOLM13, SKM1, HL60, TF1, and U937), and 3 other AML cell lines (KG1, HEL, and OCI-AML3). Cell proliferation was determined using trypan blue analysis. Flow cytometry was performed to detect drug-induced apoptosis and cell cycle analysis. High-performance liquid chromatography (HPLC) was performed to detect the intracellular concentrations of guanine nucleotides. Mycophenolic acid-treated cells were used as positive control. Effect of guanosine supplement on FF-10501-01 treatment was evaluated. Within 72 hours of treatment, FF-10501-01 inhibited proliferation of all 13 AML cell lines. The IC50 of FF-10501-01 ranged between 4.3 and 144.5 µM. MOLM13 was the most sensitive leukemia cell line, whereas the decitabine-resistant TF1 cell line was the most resistant. FF-10501-01-induced apoptosis was observed in all cell lines. Increased numbers of cells in G1 phase and decreased numbers in S phase were observed in MOLM13, SKM1 and TF1 cell lines treated with

Description: Introduction PD-1 is a negative costimulatory receptor on activated T lymphocytes which counters the activation signal provided by T cell receptor ligation. The expression of PD-1 on T lymphocytes can be dynamically modulated by DNA methylation. A recent study in solid tumors (Kleffel S et al, Cell 2015) suggested that PD-1 was not only expressed by immune cells, but also by a subpopulation of cancer cells, and the cancer intrinsic PD-1 can promote tumorigenesis. Our previous study (Yang H et al, Leukemia 2014) demonstrated an increased PD-1 mRNA expression in peripheral blood mononuclear cells from MDS patients under hypomethylating agent (HMA) treatment, but little is known about detailed PD-1 expression in MDS. PD-1 signaling has been reported to be involved in MDS pathogenesis and resistance mechanisms to HMAs (Yang H et al, Leukemia 2014). Therefore, the PD-1 pathway represents an attractive target and anti-PD-1 monoclonal antibodies are being increasingly used to study MDS. Precise understanding of PD-1 expression in MDS patients may allow for effective treatment. Methods and Human Specimens With this purpose, using multicolor flow cytometry analysis, we studied the PD-1 expression on bone marrow Lin-CD34+ cells (Lineage cells: CD2, CD3, CD4, CD7, CD10, CD11b, CD14, CD19, CD20, CD33, CD56 and CD235a) from 51 patients enrolled in a clinical trial 2014-0930. Based on 5-azacytidine treatment, these patients can be divided into two groups: group A included 29 patients (57%) (22 MDS, 5 CMML, 1 MDS/MPD and 1 AML) which were treated with 5-azacitidine in combination with ipilimumab (N=10), nivolumab (N=16) or ipilimumab + nivolumab (N=2), one patient treated with 5-azacytidine only was also included; group B included 22 patients (43%) (16 MDS, 4 CMML and 2 MDS/MPD) which did not receive 5-azacytidine treatment and were treated with ipilimumab (N=12), nivolumab (N=5) or ipilimumab + nivolumab (N=5) alone. Positive PD-1 expression was considered when the median fluorescence intensity (MFI) ratio between sample and FMO negative control was more than 2. Results In group A, 26 patients (90%) were previously untreated at the time of enrollment. PD-1 expression was not observed in 8 untreated baseline samples collected. We then performed the analysis in samples under different time points of treatment. PD-1 expression was observed in 11 patients (38%, 9 responders, 1 stable disease and 1 non-responder) in at least one time point during treatment. Interestingly, 9 (82%) of them were treated with 5-azacytidine in combination with nivolumab, the other two were treated with 5-azacytidine in combination ipilimumab and ipilimumab + nivolumab separately. PD-1 expression was observed in 56% of the patients under 5-azacytidine in combination with nivolumab treatment. Figure 1 is an example of PD-1 expression in Lin-CD34+ cells from patient treated with 5-azacytidine in combination with nivolumab. In group B, all patients except one (95%) were previously treated at the time of enrollment. PD-1 expression was observed in 2 out of 13 baseline samples collected, and the PD-1 expression was not detected on these two patients after follow-up treatment with ipilimumab. PD-1 expression was observed in two patients (9%, 1 responder, 1 stable disease) in at least one time point during treatment (1 ipilimumab, 1 ipilimumab + nivolumab). Taken together, PD-1 expression was significantly induced in patients treated with 5-azacytidine when compare to patients without 5-azacytidine treatment (38% vs 9%, p=0.023), especially in the group of patients treated with 5-azacytidine in combination with nivolumab (56% vs 10%, p=0.037, compare with 5-azacytidine in combination with ipilimumab). Induction of PD-1 expression may contribute to the response of the treatment (77% vs 46%, p=0.058). Conclusion Our study demonstrates that induced expression of PD-1 on MDS Lin-CD34+ cells may contribute to the response of MDS patients to treatment with 5-azacytidine in combination with nivolumab. PD-1 methylation as well as other biological functions related to this treatment need to be further studied. Figure 1. Figure 1. Disclosures Colla: Abbvie: Research Funding. Daver:Novartis: Research Funding; Incyte: Research Funding; Otsuka: Consultancy; Daiichi-Sankyo: Research Funding; Sunesis: Consultancy; ImmunoGen: Consultancy; Karyopharm: Consultancy; Alexion: Consultancy; Incyte: Consultancy; Kiromic: Research Funding; Pfizer: Consultancy; ARIAD: Research Funding; Pfizer: Research Funding; Novartis: Consultancy; BMS: Research Funding; Karyopharm: Research Funding; Sunesis: Research Funding.