ALBERT

Description: Abstract 291 Although cytogenetic abnormalities are common in MDS, search for genetic alterations has been less informative with few prevalent abnormalities thus far known. To identify genes aberrantly activated in MDS, we developed a novel approach based on chromatin immuno-precipitation combined with massive parallel sequencing (CHIP-Seq) using the Solexa 1G sequencing technology. To our knowledge this is the first example of the use of this technology in primary human samples. For CHIP analysis we used an antibody against H3K4me3 (histone-H3-lysine 4-trimethylation). H3K4me3 is a chromatin mark of gene activation that localizes to active gene promoter regions. CHIP-Seq was performed in CD34+, CD34 neg cells and whole bone marrow (WBM) from 6 patients with MDS and 4 normal controls. In total 30 samples were sequenced. Patients samples were obtained at the time of initial referral at MDACC and were sorted immediately using standard separation procedures. When compared to normal controls for each cellular compartment, we identified 36, 156 and 32 potential active gene promoters associated with H3K4me3 in CD34+, CD34 neg cells and WBM respectively. Of importance, gene promoter regions identified did not overlap among the different cellular compartments analyzed (differences were observed comparing normal vs MDS but also among different MDS compartments), indicating that chromatin structure and gene expression profiles are aberrant and distinct in non-CD34+ cells that may also contribute to the pathobiology of MDS. Here we focus on H3K4me3-associated gene promoters in CD34+ cells. To confirm the results obtained with the CHIP-seq approach, we studied the expression levels of the top 9 CHIP-Seq identified genes in an independent cohort of in CD34+ cells obtained from 54 MDS at the time of initial diagnosis. Patient characteristics have been previously reported (Leukemia, in press): 11 (20%) low risk, 20 (37%) int-1, 15 (27%) int-2 and 8 (14%) high risk by IPSS. We confirmed gene expression up-regulation of 7 (C5AR1, FPR1, FPR2, AQ9, FYB, FCAR, IL8RA) of 9 genes detected by CHIP-Seq. Using Ingenuity Pathway Analysis of the 36 genes identified in CD34+ cells revealed NF-κB as central activated knot in CD34+ cells. This was confirmed by phospho-p65 immuno-staining in primary cells. Furthermore up-regulation of all 10 NF-κB activation associated genes was confirmed in MDS CD34+ cells by Q-RT-PCR. Transfection of OCI-AML3 cells with a siRNAs cocktail targeting 4 of the CD34+ NF-κB activation genes dramatically repressed NF-κB activation as well as expression and promoter NF-κB association of JMJD3 gene, a known NF-κB transcriptional target. JMJD3 encodes a Jmjc-domain K27me3 demethylase, which positively regulates H3K4me3. We further characterized expression levels of 17 known histone demethylases known in human in 35 patients with MDS and identified JMJD3 as the only histone demethylase overexpressed in MDS CD34+ cells. siRNA targeting JMJD3 reduced expression and promoter H3K4me3 levels of several CHIP-Seq detected MDS- CD34+-NF-κB activation genes. Finally expression profile of JMJD3 and the panel CD34+-NF-κB activation genes in the 54 patients with MDS indicated that expression levels were consistently overexpressed in patients with higher-risk (high and int-2) disease compared to patients with lower (low and int-1) risk disease. In view of the known antiapoptotic and proliferative role of the NF-κB pathway, this data indicates that expression of upstream and downstream modulators of NF-κB signaling, regulated at the chromatin level by JMJD3, have a role in MDS progression and could serve as therapeutic targets. Through this novel in vivo CHIP-Seq analysis, we demonstrated that a positive regulatory loop exists in MDS CD34+ cells. This loop contains JMJD3 promoted gene activation through positive regulation of H3K4me3, which leads to NF-κB signaling activation, and then further promotion of JMJD3 expression and activation of the whole signaling cascade. Our study also demonstrates that in vivo CHIP-Seq can be used to discover disease specific targets. Disclosures: No relevant conflicts of interest to declare.

Description: The pathogenesis of MDS is multifactorial including both cell intrinsic alterations, such as mutations, and cell extrinsic stimuli such as immune deregulation. The prognosis of patients that lose response to hypomethylating agents (HMAs) is very poor and the mechanisms are not understood. Leukemia cells develop multiple resistance mechanisms to escape host immune response. Programmed death-1 (PD-1) is a negative costimulatory receptor on activated T lymphocytes. Expression of PD-1 ligands on tumor cells can induce immunosuppressive T-cells. Recent studies have indicated that demethylation of PD-1 leads to exhausted CD8+ T cells after chronic virus infection. We hypothesized that dysregulated PD-1 immune checkpoint signaling may be involved in pathogenesis of MDS and resistance to HMAs. We first evaluated the mRNA expression levels of PD-L1, PD-L2, PD-1 and CTLA4 by Q-PCR in bone marrow CD34+ cells from 124 patients which include 69 with MDS, 46 with CMML and 9 with AML. Seventy two (58%) patients were previously untreated. We observed aberrant up-regulated mRNA expression of PD-L1 in 39 patients (34% with fold 0-843.3), PD-L2 in 17 patients (14% with fold 0-22.5), PD-1 in 18 patients (15% with fold 0-76.7) and CTLA4 in 10 patients (8% with fold 0-25.1). No significant differences in gene expression were observed when comparing patients that had and had not received prior therapy. Statistically significant relationships were identified between elevated PD-1 expression and increased age (p=0.008), while elevated PD-L2 expression correlated significantly with female gender (p=0.005). Both elevated PD-L1 and CTLA4 expression correlated with MDS subtype, (p=0.034) and (p=0.012), respectively. Additionally, elevated CTLA4 expression correlated with higher white blood cell count (p=0.021), lack of prior therapy (p=0.02) and lower MDS IPSS score (p=0.027). We then performed an analysis of the impact on survival of the 4 gene expression in patients that had not received prior therapy. Patients with lower expression of PD-L1 had a non-significant trend towards better survival (31.5 months versus 16.2, p=0.24). Forty six of 124 patients analyzed received HMAs, and within these 46 patients lower PD-L1 expression correlated with a significantly improved overall response rate (67% vs 25%, p=0.038). Aberrant up-regulation of these 4 genes was also observed in peripheral blood mononuclear cell (PBMNC) from 61 MDS, CMML and AML patients. The relative expression of PD-L1 was significantly higher in MDS (p=0.018) and CMML (p=0.0128) compared to AML. Of interest, mRNA expression of these 4 genes was significantly higher in PBMNC than in CD34+ cells except PD-L1. By immunohistochemical (IHC) analysis, a strong correlation was observed between protein and mRNA expression. By IHC, we observed that leukemia blasts were positive for PD-L1 whereas stroma/non-blast cellular compartment was positive for PD-1 in bone marrow biopsies from MDS, CMML and AML patients. We then analyzed effect of HMAs on these four gene expression in cohort of 61 patients treated with different trials of epigenetic therapy. Treatment resulted in up regulated expression of PD-L1 in 57% of the patients (with maximum induction fold of 4.8), PD-L2 in 57% (maximum fold of 15.77), PD-1 in 58% (maximum fold of 50.26) and CTLA4 in 66% (maximum fold of 29.9). Of importance, patients resistant to therapy had increased gene expression compared to patients that achieved response (fold 5.3 vs 0.4 for PD-L1, 6.2 vs 0.4 for PD-L2, 3.0 vs 0.4 for PD-1 and 5.4 vs 0.7 for CTLA4). Comparing patients without and with expression induction, the median survival was 11.7 and 6.6 months (p=0.122) for PD-L1, 12.5 and 4.7 months (p=0.029) for PD-L2, indicating a better prognosis in patients without PD-L1 and PD-L2 induction treated with HMAs. To model this observation, we treated KG-1 and THP1 cells with different concentrations of decitabine (0 to 10 uM) and observed a dose dependent up-regulation of PD-L1 and PD-L2 in THP1 cells, and CTLA4, PD-1, PD-L1 in KG1 cells. Exposure to decitabine resulted in demethylation of PD-1 in these cell lines, and the demethylation effect was also observed in HMAs treated MDS and AML patients. This study suggests immune checkpoint PD-1/PD-1 ligands signaling may be involved in MDS pathogenesis and resistance mechanisms to HMAs. Blockade of this pathway can be a potential therapy in MDS and AML. Fig.1 PD-L1 expression in MDS CD34+ cells by IHC. Fig.1. PD-L1 expression in MDS CD34+ cells by IHC. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points VCAM-1/VLA-4 triggers reciprocal NF-κB activation in leukemia and stromal cells and mediates cross-talk between leukemia and stromal cells. VCAM-1/VLA-4 and NF-κB signaling plays a pivotal role in the development of leukemia chemoresistance.

Description: Background Risk of VTE is high in cancer patients, especially, in patients with APC. Treatment with chemotherapy further increases the risk. Purpose of the study was to evaluate the safety and efficacy of primary thromboprophylaxis with dalteparin in reducing the incidence of VTE in APC patients planned to start chemotherapy; and to determine the baseline risk factors/biomarkers predictive of VTE. Methods Patients with metastatic or locally advanced pancreatic cancer planned to start chemotherapy were randomized 1:1 to dalteparin vs control arms, stratified for the presence of metastasis and central venous catheter (CVC). The treatment arm received dalteparin 5000 U SQ daily for 16 weeks during chemotherapy and the control arm received chemotherapy alone. Bilateral compression ultrasound of the lower extremities was performed at baseline, and during study (weeks-8 and-16). In addition, blood was collected to identify biomarkers such as, plasma D-dimer levels, platelet activation markers (P-selectin), thrombin-antithrombin complex (TAT), prothrombin fragments 1 and 2 (F1+2), and cytokine levels. Univariate and multivariate logistic regression analysis of clinical and laboratory parameters were done to identify risk factors associated with the development of VTE. Results Of 87 patients enrolled, 75 were randomized to dalteparin (38 patients) or control (37 patients) arms; 8 did not meet the eligibility criteria (including 6 found positive for incidental VTE on screening ultrasound), and 4 withdrew consents before randomization. There were 41 males and 34 females; with median age 52 (range, 36-77 years). Over half of the patients (55% dalteparin arm and 54% control arm) completed 16 weeks on study. All 75 patients were evaluable for response in an intent-to-treat analysis. During the study, the incidence of VTE was 22% [8/37 patients; 2 pulmonary emboli (PE) and 6 deep vein thrombosis (DVT)] on the control arm as compared to 5% (2/38 patients; both DVT) on the dalteparin arm (p = 0.02). In the multivariate analysis, baseline plasma levels of D-dimer, ECOG performance status, presence of CVC, and prophylaxis with dalteparin were independent factors predictive of risk for VTE, as shown below. There was no statistically significant difference in overall survival between the two arms; however, there were higher proportion of patients with elevated baseline D-dimer levels in the dalteparin arm than the control arm (≥ 5000 ng/mL 16% vs 3%). Elevated baseline D-dimer level (≥ 5000 ng/mL) was also predictive of the presence of silent or asymptomatic VTE at screening for study entry (p=0.001), suggesting its potential value in identifying patients with silent VTE. Treatment with dalteparin was well tolerated; the main adverse events included minimal bruising (5/34, 15%), or pain (2/34, 6%) at the injection sites. There were no clinically significant bleeding episodes in the dalteparin arm. Conclusions The results of this study showed that the incidence of VTE is very high in patients with APC. Primary thromboprophylaxis with dalteparin was well tolerated and was associated with 75% reduction in the incidence of VTE in ambulatory patients with locally advanced or metastatic cancer while receiving chemotherapy. Baseline risk factors such as elevated D-dimer levels may help identify high risk patients for primary thromboprophylaxis as well as patients with the presence of asymptomatic VTE. Disclosures: Vadhan-Raj: Eisai: Research Funding. Off Label Use: Fragmin (Dalteparin): Prophylaxis of VTE in ambulatory cancer patients while receiving chemotherapy.

Description: Abstract 3845 Introduction: Matrix metalloproteinases (MMPs) belong to a unique family of zinc dependent endopeptidases, they are strictly regulated by their endogenous inhibitors called tissue inhibitor of MMPs (TIMPs). MMPs have been associated with tumorigenesis. In addition to their role in extracellular matrix turnover and cancer cell migration, MMPs regulate signaling pathways that control cell growth, inflammation, or angiogenesis and may even work in nonproteolytic manner. MMPs play a key role during invasion and metastasizing of cancer cells and they have been shown to be associated to invasive phenotype and poor prognosis in several solid tumors. Little is known about their role in MDS. A total of 23 different human MMPs and 4 TIMPs have been identified. To better understand the role of MMPs in MDS, we performed an expression profile screen study by QPCR to detect the expression level of all MMP and TIMP family members, except MMP23, in CD34+ cells from a cohort (N=10) of newly diagnosed, untreated patients with MDS, and compared the expression level with CD34+ cells from 5 normal donors. Of these 26 genes, we identified MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 as aberrantly up-regulated genes in MDS and expanded the study to a larger cohort of patients to correlate with clinical features and clinical outcomes. Methods: CD34+ cells from 98 newly diagnosed patients with MDS and 5 normal donors were evaluated for MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 expression profiling. CD34+ cells were sorted from patients and normal donor bone marrow. Total cellular RNA was isolated using Trizol, cDNA was obtained by using TaqMan reverse transcription reagent (Applied Biosystems). For real-time PCR, all assays were purchased from Applied Biosystems and analyzed with an Applied Biosystems Prism 7500 Sequencing detection system. GAPDH was used as internal control. Immunohistochemistry was used to detect MMP9 protein level in cytospin from CD34+ cells. MMP9 abtibody was obtained from R&D systems, MMP9 ELISA kit (R&D systems) was used to detect the MMP9 protein level in bone marrow plasma. Results: For the 98 MDS patients studied, 78% were older than 60 years; by IPSS score, 17 (17.3%) low risk, 35 (35.7%) INT-1, 24 (24.5%) INT-2, 10 (10.2%) high risk, 12 (12.2%) not available. By Cytogenetics, diploid 44 (45%), 20q- 7(7%), −5/5q- 7 (7%), −7/7q- 7 (7%), −5/5q- and −7/7q- 6 (6%), 8+ 6 (6%), IM 6 (6%), MISC 14 (14%). By QPCR and comparing MDS CD34+ cells with normal CD34+ cells, aberrant up-regulation (fold〉2) of MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 was detected in 40%, 93.8%, 94%, 84%, 15.6%, 45%, 21% and 27% of patients respectively. Up-regulation of MMP8, MMP9 and MMP25 were significant with a mean fold value 350.7 (0–3363.7), 1112.4 (0–15641) and 143.8 (0–1017.9) respectively. We performed MMP9 immunohistochemistry of MDS CD34+ cytospins from 16 pts with higher and lower MMP9 RNA relative expression level and found consistent protein expression level in cytoplasm. No elevated MMP9 protein levels were detected in bone marrow plasma. We then performed an analysis of associations with clinical variables and observed that relative expression value of MMP9 was associated with lower bone marrow blast (p=0.001) and longer survival (p=0.02) (figure 1). We did not found association between survival and the other 7 up-regulated genes. Conclusions: Bone marrow CD34+ cells from patients with MDS have abnormal up-regulation of MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3. MMP9 up-regulation is associated with longer survival. Our results suggest that MMP-9 could be a useful prognostic indicator for MDS and that this family of proteins needs further study in MDS. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3810 The pathogenesis of MDS is multifactorial including both cell intrinsic alterations, such as mutations, and cell extrinsic stimuli such as inflammatory signals and immune deregulation. It has been postulated that hypomethylating agents exert their effects in part via regulation of expression of cell surface antigens. The prognosis of patients that lose response to HMAs is very poor and the mechanisms of action of resistance to HMAs are not understood. Tumor cells develop multiple resistance mechanisms to escape host immune response and permit tumor cell growth which including T-cell dysfunction. Programmed death-1 (PD-1) is a negative costimulatory receptor on activated T lymphocytes which counters the activation signal provided by T cell receptor ligation. Tumor cells can express PD-1 ligands, PD-L1 and PD-L2, which can induce immunosuppressive T-cells. Recent studies have indicated that demethylation of the locus that encodes PD-1 leads to exhausted CD8+ T cells after chronic virus infection. We hypothesized that PD-1 signaling may be involved in resistance to HMAs. We first evaluated 8 myeloid leukemia cell lines, HL-60, NB4, THP1, U937, ML1, OCI-AML3, HEL and KG1, for expression of PD-1, PD-L1, PD-L2 and CTLA4 (another negative regulator of T-cell activation) by Q-PCR, except OCI-AML3, PD-L1 was detectable in all of these cell lines, whereas CTLA4 and PD-1 can only be detected in KG1. Subsequently, we analyzed the expression of PD-L1 by Q-PCR in CD34+ cells sorted from bone marrow of 129 newly diagnosed MDS patients. Compared with normal CD34+ controls, 38 (29%) of the patients had aberrant up-regulation of PD-L1 RNA expression (folds 2–814). We also analyzed 10 AML CD34+ samples from bone marrow and 3 of them were up-regulated. We then confirmed at the protein level using immunohistochemistry for the expression of PD1 and PD-L1 in 14 AML and 4 MDS bone marrow specimens. We observed that AML blasts were positive for PD-L1 whereas stroma/non-blast cellular compartment were positive for PD-1. We then analyzed the expression level of PD-1, PD-L1, PD-L2 and CTLA4 by Q-PCR in peripheral blood mononuclear cells of a cohort of patients with AML or MDS treated in different clinical trials of epigenetic therapy. These included: 18 patients from a phase 2 trial of vorinostat in combination with azacitidine (which included 11 patients that achieved complete remission (CR) and 7 resistant); 11 patients from a phase 2 study of low dose decitabine (all these patients were resistant to the therapy); 26 patients from phase I/II study of the combination of 5-azacitidine with lenalidomide (which include 5 CR patients and 21 resistant). Prior to therapy, PD-1, PD-L1, PD-L2 and CTLA4 expression was up-regulated in 25 (45%), 23 (42%), 13 (24%) and 5 (9%) of the patients respectively compared to normal peripheral blood mononuclear cells. After a course of therapy 24 (44%), 27 (49%), 20 (36%) and 31 (56%) of the patients acquired induced up-regulated expression of these genes. Of importance, in the group of patients treated with vorinostat in combination with azacitidine, patients resistant to therapy had more signigicant increase of gene expression compared to patients that achieved a CR (Figure 1). To model this observation, we treated KG-1 and THP1 cells with different concentrations of decitabine (0 to 10 uM) and observed a dose dependent up-regulation of PD-L1 and PD-L2 in THP1 cells, and CTLA4, PD-1, PD-L1 in KG1 cells at doses over 0.5 uM. Exposure to decitabine resulted in demethylation of PD-1 in these cell lines. In this study, we demonstrate aberrant expression of CTLA4, PD-1 and PD-1 ligands in MDS and AML patients and that HMAs can up-regulate the expression of these genes, suggesting that PD-1 signaling may be involved in resistance mechanisms to HMAs. Blockade of this pathway can be a potential therapy in MDS and AML. Fig 1. PD-L1 expression in the group of patients treated with vorinostat in combination with azacitidine. Fig 1. PD-L1 expression in the group of patients treated with vorinostat in combination with azacitidine. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2827 Introduction The myelodysplastic syndromes (MDS) are a group of leukemia characterized by bone marrow failure and increased risk of transformation to acute myelogenous leukemia (AML). Focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase (PTK), plays a key role in the integration of protein-mediated signal transduction. It is a critical mediator of the integrin signaling cascade, which modulates cell proliferation, apoptosis, adhesion, spreading and migration. FAK is upregulated in a wide variety of human cancers. It has been reported that expression of FAK in acute myeloid leukemia (AML) is associated with enhanced blast migration, increased cellularity and poor prognosis. Furthermore, FAK silencing inhibited leukemogenesis in BCR-ABL-transformed hematopoietic cells. FAK has been proposed a potential target in cancer therapy. In this study, we studied the expression patterns of FAK in 98 patients with MDS and performed preclinical studies of FAK inhibition in leukemia cell lines. Methods CD34+ cells from 98 newly diagnosed patients with MDS and 5 normal donor bone marrow specimens were sorted with CD34 isolation kit from Miltenyi Biotec. Total cellular RNA was isolated using Trizol, cDNA was obtained by using TaqMan reverse transcription reagent (Applied Biosystems). For real-time PCR, FAK assay were purchased from Applied Biosystems and analyzed with an Applied Biosystems Prism 7500 Sequencing detection system. GAPDH was used as internal control. Immunohistochemistry was used to detect FAK protein level in cytospin from MDS CD34+ cells. FAK antibody was obtained from abcam. FAK inhibitor was purchased from Santa Cruz Biotechnology. Results For the 98 MDS patients studied, 78% were older than 60 years; by IPSS score, 17 (17.3%) low risk, 35 (35.7%) INT-1, 24 (24.5%) INT-2, 10 (10.2%) high risk, 12 (12.2%) not available. By cytogenetics, diploid 44 (45%), 20q- 7 (7%), -5/5q- 7 (7%), -7/7q- 7 (7%), -5/5q- and -7/7q- 6 (6%), 8+ 6 (6%), IM 6 (6%), MISC 14 (14%). By real-time PCR, we observed FAK overexpression in 28% of MDS CD34+ cells (fold 2.2–26). We then analyzed FAK protein expression in 10 MDS CD34+ cell cytospin with either higher or lower RNA expression by QPCR using immunohistochemistry. The protein expression patterns were 100% consistent with RNA expression level. This result suggests that FAK expression is upregulated in MDS CD34+ cells. We then performed analysis of clinical associations between FAK expression and clinical characteristics. No association was observed in particular between FAK expression and survival. Because of the potential role of FAK as a therapeutic target, we then studied the effects of FAK inhibition in cell lines. We studied FAK expression level in leukemia cell lines of AML origin and found high protein expression level of FAK in AML leukemia cell line OCI-AML3 and KG1. We treated these cells with FAK inhibitor 14 and detected a dose dependent anti-proliferative effect on both cell lines. Using Annexin V - FITC analysis by flow cytometry, we found the FAK inhibition could induce apoptosis in both cell lines at concentrations of 10uM both at 24 hours and 48 hours. Conclusions This study shows that FAK is aberrantly expressed in MDS CD34+ cells, together with the anti-proliferative and apoptosis induction effect of FAK inhibitor, our study demonstrates that FAK may be a potential therapeutic target in MDS and AML patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 556 We have documented that deregulation of the Toll-like Receptor 2 (TLR2) centered innate immunity signals contribute to the pathogenesis of myelodysplastic syndromes (MDS). Of relevance, oncogenically active mutations of MYD88, a signal adaptor protein for TLR signal, have recently been identified as recurrent genetic lesions in both B-cell lymphoma and in chronic lymphocytic leukemia (CLL) (Vu N et al. Nature 2010 and Puente et al. Nature 2011). This information further supports the importance of innate immunity deregulation in leukemogenesis. To further characterize this pathway in MDS, we analyzed potential genetic alteration and expression level of MYD88 in patients of MDS. In a cohort of 40 MDS whole bone marrow mononuclear cell DNA, we first performed pyrosequencing analysis focusing on a list of previously reported MYD88 mutations (V217, W218, S219, I220, S222, M232, S243, L265, and T294). We did not detect mutation of any these hotspots on MYD88 in MDS. We then expanded the sequencing efforts to the entire coding region of MYD88 using an approach that combines PCR amplification and massive parallel sequencing. Still, no mutation of MYD88 was detected using this technique. We then examined the expression of MYD88 in CD34+ hematopoietic stem cells from 65 patients with MDS. In comparison to healthy donors, 26% of MDS patients (N=17) presented a more than 2 fold increase of MYD88 RNA, and 15% (N=10) had a 30%–90% increase. In average, MYD88 RNA level was 1.7 fold increased compared to control. Of potential clinical relevance, patients with higher MYD88 RNA expression in bone marrow CD34+ cells (above median value) (N=33) had a propensity of shorter period (24.4 mo) of overall survival (OS) in comparison to patients with lower levels of MYD88 expression (N=32) (32 mo)(P=0.05). We also found that patients with higher levels of MYD88 expression (split at 0.8 fold to controls) tended to have higher WBC (P=0.02). We have previously shown that blockade of the TLR2 mediated innate immunity signaling in MDS CD34+ cells could positively regulate erythroid lineage differentiation. To evaluate the potential of MYD88 blockade, we applied a 26 AA MYD88 inhibitory peptide that blocks its homodimerization (Invivogen, San Diego, CA) on primary CD34+ cells isolated from patients with lower-risk MDS (N=5). Methylcellulose medium supported colony formation assays indicated that the presence of MYD88 inhibitor led to an average of 60% increase for the numbers of erythroid colonies and a 30% increase for the numbers of total colonies. At the same time, we did not observe these effects of MYD88 blockade on the CD34+ cells isolated from the patients of higher-risk MDS (N=3). IL-8 is one of the key downstream transcriptional targets of the TLR-MYD88-NFkB innate immunity signaling that was documented to be elevated in bone marrow plasma of MDS. ELISA assays also indicated that blockade MYD88 in cultured MDS CD34+ cells led to a decrease of IL-8 concentration in medium. Taken together, these results indicated that MYD88 is overexpressed in hematopoietic stem cells of MDS and that blockade of MYD88 mediated innate immunity signaling may have therapeutic potential in treating patients with MDS. Disclosures: Kantarjian: Genzyme: Research Funding.