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The under-attribution of women’s contributions to scientific scholarship is well known and well studied. One measure of this under-attribution is the citation gap between men and women: the under-citation of papers authored by women relative to expected rates coupled with an over-citation of papers authored by men relative to expected rates. Here we explore this citation gap in contemporary physics. We find a global bias wherein papers authored by women are significantly under-cited, and papers authored by men are significantly over-cited. Moreover, we find that citation behaviour varies along several dimensions, such that imbalances differ according to who is citing, where they are citing and what they are citing. Specifically, citation imbalance in favour of man-authored papers is highest for papers authored by men, papers published in general physics journals and papers for which citing authors probably have less domain or author familiarity. Our results suggest that although deciding which papers to cite is an individual choice, the cumulative effects of these choices needlessly harm a subset of scholars. We discuss several strategies for the mitigation of these effects, including conscious behavioural changes at the individual, journal and community levels.

Background: Infectious diseases constantly represent the source of sickness as well as mortality in human beings. Herbal applications in human life through using plants for antibacterial and anticancer activity have shown the potential medicinal outcome. Objectives: To evaluate the antibacterial and anticancer activities of the crude extract of Matricaria aurea. Materials and Methods: The antibacterial activity of the crude flowers of M. aurea extract was examined against reference and clinical bacterial strains by agar well diffusion method. Minimum inhibitory concentrations and minimum bactericidal concentrations were determined by micro broth dilution assays using MH broth. Herbal extract was employed over human breast adenocarcinoma cell line (MCF-7), hepatocellular carcinoma cell line (HepG-2) and colorectal adenocarcinoma cell line (HCT-116) to optimize cancer cells proliferation by SRB assay. Results: The data has shown that the extract from M. aurea had significant antimicrobial activity against the tested microorganisms. The plant extract showed higher antibacterial activity against the reference strain of Streptococcus pyogenes. The MIC and MBC varied between 0.38-12.5 mg/ml and 3.1-200 mg/ml respectively. Synergy study elucidated the significant bacteriostatic effect of M. aurea extract on S. aureus and S. saprophyticus. The data of SRB assay deliver the potential anticancer activity through cell death. Conclusion: This study delivers innovative information that M. aurea possessed excellent bio-activities against pathogenic microbes and cancer cells, which drive attention for further research to explore the active components responsible for biological efficacies.

The use of immunotherapy to treat patients with myelodysplastic syndromes (MDS) shows promise but is limited by our incomplete understanding of the immunologic milieu. In solid tumors, CD141Hiconventional dendritic cells (CD141HicDCs) are necessary for antitumor immunosurveillance and the response to immunotherapy. Here, we found that CD141HicDCs are reduced in MDS bone marrow and based on the premise established in solid tumors, we hypothesized that reduced numbers of CD141HicDCs are associated with inferior overall survival in MDS patients. We found that MDS patients with reduced numbers of CD141HicDCs, but not other DC populations, showed reduced overall survival. To examine the basis for reduction in CD141HicDCs, we found fewer numbers of progenitors committed to DC differentiation in the MDS bone marrow and these progenitors expressed lower levels of interferon regulatory factor-8 (IRF8), a master regulator of CD141HicDC differentiation. To rescue impaired CD141HicDC differentiation, we used pharmacologic inhibition of lysine-specific demethylase 1A (LSD1) to promote CD141HicDC differentiation by MDS progenitors. These data reveal a previously unrecognized element of the MDS immunologic milieu. Epigenetic regulation of CD141HicDC differentiation offers an intriguing opportunity for intervention and a potential adjunct to immunotherapy for patients with MDS.

Background: Patients with intermediate and higher-risk MDS are generally treated with the DNA hypomethylating agents (HMAs) azacytidine and decitabine. These agents improve survival and cytopenias in ~50% of those treated; but only responders derive clinical benefit, and no pre-treatment response prediction has proven efficacy. There is thus an urgent need to develop new approaches to complement or enhance the response to standard-of-care HMA therapies. Studies suggest that HMAs might act in part via activation of anti-tumor immune mechanisms. Based upon this hypothesis a series of early phase trials combining immune checkpoint inhibitors (ICI: both anti-PD1/PDL1 and CTLA4) with azacitidine in MDS/AML patients were undertaken but clinical and immunological efficacy have proven disappointing. In these studies, the lack of antigen specificity made systematic assessment of the anti-MDS immune response and milieuchallenging. Antigen specific assessments are particularly important in MDS patients treated with ICIs because distinct from solid tumors, myeloid stem cell cancers involve the populations of antigen presenting cells that modify the capacity for T-cell response, such as dendritic cells. Our group has shown that MDS/AML patients treated with decitabine show increased expression of NY-ESO-1, a well-characterized tumor antigen whose expression is normally suppressed through dense promoter hypermethylation. In our prior Phase I study, MDS patients received vaccination against NY-ESO-1 in combination with standard decitabine. This combination was not only safe, but it also induced NY-ESO-1 specific CD4 +and CD8 +T cell responses in most patients. We hypothesized that combining anti-NY-ESO-1 vaccination (an antigen specific approach) with decitabine and an ICI would allow us to understand the immune synapse in patients with MDS. Methods: We developed an investigator-initiated Phase 1 trial in transplant-ineligible patients with MDS/low blast count AML adding an anti-PD-1 ICI to our established combination of decitabine + NY-ESO-1 vaccination (#NCT 0335871). This open-label, non-randomized single center Phase 1 study used an HLA unrestricted NY-ESO-1 vaccine (CDX-1401 (1 mg) + poly-ICLC (1.8 mg)) in combination with standard dose decitabine (20mg/m2/d x 5 days) and nivolumab (3 mg/kg every 2 weeks), 4 cycles of combination therapy were planned on study; patients deriving clinical benefit could continue treatment at the discretion of the treating physician. The study was conducted in accordance with the Declaration of Helsinki and approved by the Roswell Park Cancer Institute (RPCI) Internal Review Board. Six patients were enrolled, treated, and completed > 4 cycles of decitabine; serial bone marrow and peripheral blood samples were collected for analysis. The combination was safe. Results: Two patients developed CR and the remainder had stable disease as the best response to therapy (Table 1). The majority of patients mounted NY-ESO-1 specific CD4 +T cell responses associated with up-regulation of anti-PD-1 immunotherapy gene signatures in the CD4 +memory T cell compartment. No responses in CD8 +T cells were seen. In seeking explanation for the disparate findings in CD4 +and CD8 +T-cell compartments, we examined conventional dendritic cell populations associated with activation of CD8 +T cells and identified that our patients had markedly reduced numbers of conventional dendritic cells marked by high expression of CD141 (cDC1). This population is critical for a successful response to immunotherapy. cDC1 from MDS patients also showed reduced expression of genes that are key for optimal T-cell activation and expansion. Conclusions: These results suggest that the use of immunotherapy to induce effective, cytotoxic anti-tumor T cell responses depends upon the myeloid immunologic milieuin MDS patients. The critical importance of cDC1 function has recently been described and appreciated in the context of solid tumor immunotherapy. The observation of both numerical and functional defects of this cell population in patients with myeloid neoplasia provides a possible explanation for the disappointing efficacy of immunotherapies in this disease state. Approaches to augment the number and function of specific cDC1 populations in myeloid disease might overcome this defect and thereby enhance the efficacy of immunotherapy for patients with MDS.

We aimed to determine the effect of SGI-110 on methylation and expression of the cancer testis antigens (CTAs) NY-ESO-1 and MAGE-A in epithelial ovarian cancer (EOC) cells in vitroand in vivoand to establish the impact of SGI-110 on expression of major histocompatibility (MHC) class I and Intracellular Adhesion Molecule 1 (ICAM-1) on EOC cells, and on recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. We also tested the impact of combined SGI-110 and NY-ESO-1-specific CD8+ T-cells on tumor growth and/or murine survival in a xenograft setting. EOC cells were treated with SGI-110 in vitroat various concentrations and as tumor xenografts with 3 distinct dose schedules. Effects on global methylation (using LINE-1), NY-ESO-1and MAGE-Amethylation, mRNA, and protein expression were determined and compared to controls. SGI-110 treated EOC cells were evaluated for expression of immune-modulatory genes using flow cytometry, and were co-cultured with NY-ESO-1 specific T-cell clones to determine immune recognition. In vivoadministration of SGI-110 and CD8+ T-cells was performed to determine anti-tumor effects on EOC xenografts. SGI-110 treatment induced hypomethylation and CTA gene expression in a dose dependent manner both in vitroand in vivo, at levels generally superior to azacitidine or decitabine. SGI-110 enhanced the expression of MHC I and ICAM-1, and enhanced recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. Sequential SGI-110 and antigen-specific CD8+ cell treatment restricted EOC tumor growth and enhanced survival in a xenograft setting. SGI-110 is an effective hypomethylating agent and immune modulator and, thus, an attractive candidate for combination with CTA-directed vaccines in EOC.

Vachhani: Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Przespolewski:Jazz Pharmaceuticals: Other: PI on clinical trial. Thota:Incyte, Inc.: Speakers Bureau. Wang:Amgen: Other: Advisory role; Agios: Other: Advisory role; Pfizer: Other: Advisory role, Speakers Bureau; Stemline: Other: Advisory role, Speakers Bureau; Daiichi: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role; Jazz: Other: Advisory role; Abbvie: Other: Advisory role; Kite: Other: Advisory role. Griffiths:Boston Scientific: Consultancy; Genentech, Inc.: Research Funding; Abbvie, Inc.: Consultancy; Boston Scientific: Consultancy; Novartis Inc.: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; New Link Genetics: Consultancy; Genentech, Inc.: Research Funding; Onconova Therapeutics: Other: PI on a clinical trial; Appelis Pharmaceuticals: Other: PI on a clinical trial; Abbvie, Inc.: Consultancy, PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Persimmune: Consultancy; Partner Therapeutics: Consultancy; Persimmune: Consultancy; Novartis Inc.: Consultancy; Celgene, Inc: Consultancy, Research Funding; Celgene, Inc: Consultancy, Research Funding; Appelis Pharmaceuticals: Other: PI on a clinical trial; Partner Therapeutics: Consultancy; New Link Genetics: Consultancy.

Background:Patients with myelodysplastic syndromes (MDS) present across a clinical spectrum from mild disease to profound bone marrow failure and transformation to acute myeloid leukemia (AML). Those with lower risk disease are generally managed with watchful waiting and best supportive care (growth factors, blood and platelet transfusions and iron chelation; BSC), while those with higher risk disease are treated with repeated cycles of low dose “hypomethylating” chemotherapy (such as azacitidine or decitabine; HMA). A majority of patients with MDS, even those with lower risk disease, are likely to die of complications related to their diagnosis, mostly infections and bleeding but also leukemic transformation. As part of our standard approach to infection prevention, current clinical guidelines suggest annual vaccination against influenza. Patients with these disorders and their family members are advised to receive inactivated protein based vaccines rather than live vaccination approaches to limit infection risk. Most will receive high dose trivalent vaccination due to age. Despite these recommendations, limited data exist on the ability of patients with MDS across the spectrum of risk groups to respond to standard seasonal influenza vaccination. In light of the growing literature suggesting that patients with MDS have an altered immune environment, we hypothesized that they would show inferior response to standard vaccination. We sought to determine the response to influenza vaccination in patients with MDS receiving standard therapeutic management. Methods:A non-randomized study is currently ongoing at the Roswell Park Comprehensive Cancer Center for patients with MDS. Age-relevant family members are enrolled as a comparator population for vaccine response. Cohorts were stratified into 3 groups: healthy volunteers (Cohort 1), MDS patients receiving BSC (Cohort 2) and MDS patients actively receiving HMA (Cohort 3; Table 1). All participants are administered the yearly preparation of Sanofi Pasteur's Fluzone High-Dose Vaccine (containing trivalent inactivated strains: Influenza virus A (H1N1 and H3N2) and Influenza virus B). Baseline blood samples were collected prior to vaccination (day 0), and between days 25-90 and 115-185 post-vaccination. Serological responses to vaccination were determined by viral-neutralizing activity analyzed viamicroneutralization assay. Neutralizing antibody titers for the first year of the study were measured against seasonal influenza vaccine strains based upon the 2017-2018 vaccine product. Samples from the 2018-19 flu season are being analyzed. Results:To date 56 individuals have been recruited to the study over 2 years. Neutralizing antibody titers following vaccination are available for 20 individuals vaccinated in the 2017-18 flu season. Humoral immune responses to vaccination against different strains of Influenza virus A (H1N1 and H3N2) and Influenza virus B were observed across all cohorts (Figure 1). Response was deemed adequate if the titer for any vaccine component increased by >4 fold comparing the baseline to the day 25-90 time point. Cohort 1: 4/4 responded (100%); cohort 2: 4/4 responded (100%); cohort 3: 11/12 responded (92%). To better understand the effect of standard treatment for MDS on influenza vaccine response we are currently profiling immune cells pre and post vaccination using multi-parameter flow cytometry. Additional analyses are planned based on the number of HMA cycles received (<6, or ≥6) and cycle timing relative to vaccination. Conclusion:Patients with MDS respond to vaccination with Fluzone High Dose. Responses in patients with MDS were not statistically different from those seen in an age-relevant population of healthy family members. Additional individuals are being enrolled in order to assess whether standard HMA therapy impacts the response to influenza vaccination. These data suggest that MDS patients receiving BSC respond adequately to viral vaccination. Our preliminary data also show that patients receiving HMA therapy respond adequately to influenza vaccination. These data support the value of influenza vaccination in all patients with MDS and highlight the potential for anti-MDS immunotherapeutic vaccination strategies.

Sait: Celgene: Consultancy. Griffiths:Boston Scientific: Consultancy; Boston Scientific: Consultancy; Genentech, Inc.: Research Funding; Persimmune: Consultancy; Persimmune: Consultancy; Abbvie, Inc.: Consultancy; Genentech, Inc.: Research Funding; Novartis Inc.: Consultancy; Celgene, Inc: Consultancy, Research Funding; New Link Genetics: Consultancy; New Link Genetics: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Novartis Inc.: Consultancy; Partner Therapeutics: Consultancy; Partner Therapeutics: Consultancy; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Celgene, Inc: Consultancy, Research Funding; Abbvie, Inc.: Consultancy, PI on a clinical trial.

Background:Immunotherapeutic approaches for myelodysplastic syndrome (MDS) show promise, but progress is limited by our incomplete understanding of the immunologic milieu. In a recent Phase I trial, we found that MDS patients with higher numbers of CD141Hiconventional dendritic cells (cDCs) were more likely to respond to NY-ESO-1 vaccination. In solid tumor models, the CD141HicDC is critical for initiating anti-tumor immune responses but its impact in myeloid malignancies is unknown. In studies of primary human specimens and mouse models, we tested the hypothesis that MDS patients exhibit decreased quantity and quality of CD141HicDCs due to impaired myeloid differentiation. Methods:Bone marrow (BM) cells were collected from MDS patients (pre-treatment) and age matched healthy donors (HD; defined as absence of hematologic malignancy). We quantified DC populations, stem, progenitor cells and interferon regulatory factor-8 (IRF-8) expression using flow cytometry and RT-qPCR. Histone modifications were assessed by chromatin immunoprecipitation. To assess DC differentiation of progenitors, human CD34+ and mouse c-kit+ cells were expanded and differentiated in vitro. Results:We found fewer CD141HicDCs (p<0.0001), CD1c+cDCs (p<0.005) and plasmacytoid DCs (CD123+pDCs; p<0.005) overall in BM samples from MDS patients (n=71) compared to HD (n=17). We stratified MDS patients based on the relative number of DCs and found that only those patients with highest number of CD141HicDCs had superior survival (p < 0.05). No differences in survival were seen in patients stratified by the CD1c+cDCs (p = 0.96) and CD123+pDCs (p = 0.32) populations. We hypothesized that decreased numbers of CD141HicDCs and adverse survival in MDS patients resulted from impaired differentiation of DC progenitors. We showed that MDS patients (n=19) have fewer monocyte-DC progenitors (MDP) and common DC progenitors (CDP) compared to HD (n=11; p<0.01). We then hypothesized that MDS progenitors express lower levels of IRF8, a master regulator of CD141HicDC differentiation. IRF8 expression was significantly lower in CDPs from MDS patients compared to HD (p<0.05). Furthermore, MDS patients with lower levels of IRF8 (n=8) in their MDPs showed a trend towards production of fewer CDPs and significantly fewer CD141HicDCs compared to those with higher levels of IRF8 (n=10; p<0.005). These results suggest that approaches to increase IRF8 expression could enhance CD141HicDC differentiation. We hypothesized that inhibition of lysine-specific histone demethylase 1A (LSD1), which increases IRF8expression in myeloid leukemia cells, would induce CD141HicDC differentiation. Pharmacologic inhibition of LSD1 increased IRF8 expression (both mRNA and protein) in KG-1 cells, a model of human CD34+cells, and in HD and MDS CD34+progenitors (p<0.05). LSD1 inhibition in KG-1 cells resulted in increased H3K27 acetylation (3861-fold change) and H3K4 dimethylation (922.4-fold change) compared to PBS (p<0.05) at a region -70 kb to the IRF8transcriptional start site, a putative regulatory element that demonstrated the highest level of LSD1 binding. These data indicate that LSD1 inhibition alters histone modifications at the IRF8locus, resulting in increased expression of IRF8. Pharmacologic inhibition of LSD1 in HD CD34+cells increased the number of mature CD141HicDCs in 92% of specimens (n = 12; 3.4 fold-change) compared to PBS. Similarly, LSD1 inhibition in MDS CD34+cells increased the number of CD141HicDCs (n = 12) in 75% of patient specimens (16.8 fold-change) compared to PBS. IRF8 function is conserved between mice and humans. To test whether the effect of LSD1 inhibition on cDC differentiation was dependent on IRF8, we compared the effect of LSD1 inhibition on BM c-kit+cells from Irf8knock-out mice (Irf8-KO) and littermate controls (WT). LSD1 inhibition in WT c-kit+cells resulted in increased numbers of CD141HicDCs in vitro(p<0.05). By contrast, LSD1 inhibition of Irf8-KO c-kit+cells did not result in differentiation of CD141HicDCs. These data suggest that LSD1 inhibition drives CD141HicDCs differentiation through IRF8. Conclusion:These data reveal a previously unrecognized determinant of the immune microenvironment in MDS. The opportunity for epigenetic regulation of CD141HicDC differentiation in MDS offers an opportunity for intervention and a potential adjunct to immunotherapy for patients.

Immunotherapeutic approaches to treat patients with myeloid cancers show considerable promise. Delineation of the immunologic milieuin these patients is necessary to develop rationally-designed strategies. We have previously reported a phase I trial in which intermediate/high-risk(I/HR) MDS patients were vaccinated against NY-ESO-1 tumor antigen (anti-DEC-205-NY-ESO-1 fusion protein with poly-ICLC adjuvant; CDX-1401) in combination with decitabine (20 mg/m2/day x 5 days x 4 cycles). Vaccination of MDS patients induced NY-ESO-1-specific immune responses but there was significant intra-patient variability in the quantity and quality of these responses. CDX-1401 targets multiple populations of antigen presenting cells (APCs) including CD141Hiconventional dendritic cells (cDCs). Compared to other APCs, CD141HicDCs express higher levels of DEC-205, an antigen uptake receptor, and TLR3, the poly-ICLC receptor. These cells regulate anti-tumor immune responses in solid tumor models and are necessary for the efficacy of checkpoint inhibitors. Thus, we hypothesized that the observed variations in immune response to vaccine resulted from quantitative and/or qualitative differences in CD141HicDCs in MDS. Overall, in MDS patients compared with healthy donors, there was a 9-fold decrease in the frequency of CD141HicDCs (0.001 ± 0.002% versus0.009 ± 0.006% respectively; p < 0.001). In contrast with this finding, frequencies of the related CD1c+ cDC population were not different (0.18 ± 0.11% versus0.27 ± 0.61% respectively; p = 0.12). Strikingly, MDS patients with the highest frequency of CD141HicDCs demonstrated a greater functional response to vaccination (both humoral and T-cell mediated). Expression of DEC-205 in cDCs from MDS patients was not lower than in those from healthy donors, suggesting that response to vaccination was not mediated by differential expression of the vaccine target. To further analyze gene expression in cDCs, we performed RNA-seq analysis of DCs isolated from healthy donors (n = 3) and a vaccine responsive MDS patient from the study. Principal component analysis showed that gene expression profiles were similar in CD141Hiand CD1c+cDCs from MDS and normal patients, and further these profiles were distinct from plasmacytoid DCs and CD34+progenitors. This suggests that for MDS patients with detectable CD141Hipopulations, cDC function is intact. We expanded our observations to a larger cohort of untreated HR MDS patients and confirmed a selective decrease in the baseline number of bone marrow CD141HicDCs (n = 17; 0.002% ± 0.006%) compared to healthy donors (n = 5; 0.14 ± 0.01%; p < 0.001). We further characterized cDC progenitors in a subset of these patients using flow cytometry (n = 8). MDS patients demonstrated a population of bipotent cDC progenitors (early pre-DC) with an expected ratio of differentiation to committed CD1c+cDC progenitors and terminally differentiated CD1c+cDCs (p < 0.01). In contrast, the expected relationships between early pre-DCs and CD141HicDC committed progenitors or terminally differentiated CD141HicDCs was lost (p= 0.33 and 0.79 respectively). These data suggest a defect in the differentiation program for CD141HicDCs in MDS patients. We then hypothesized that decitabine treatment might promote a more normal cDC differentiation pattern. Using an in vitromodel of cDC differentiation, we found that decitabine treatment increased differentiation of MDS CD34+progenitors to CD1c+cDCs by an average of 1.72 ± 0.96 fold (n = 3). However, we were unable to detect a similar effect on CD141HicDC differentiation. Since in vitromodels do not reliably recapitulate the human environment, we analyzed serial bone marrow samples isolated from patients treated on study with decitabine and vaccination (n = 4). In these assays, 2/4 and 3/4 patients showed increased numbers of both CD141Hiand CD1c+cDC at the end of study compared to pre-treatment. Together, these data suggest that CD141HicDCs are selectively reduced in patients with HR MDS and that decitabine treatment promotes cDC differentiation in vitroand in MDS patients. Optimal immunotherapeutic approaches for MDS patients may therefore benefit from strategies that address defects in APC differentiation and function.

Karpf: Astex Pharmaceuticals: Research Funding. Wang:Incyte: Speakers Bureau; Immunogen: Research Funding. Griffiths:Astex Pharmaceuticals: Consultancy, Research Funding; Celgene Inc: Consultancy, Honoraria; Alexion Pharmaceuticals: Consultancy, Honoraria.

Background: The identification of suitable target antigens for immunotherapy in myeloid cancer has been a challenge. NY-ESO-1 is a recognized target in solid tumors, but in myeloid cancers expression is silenced by dense promoter hypermethylation. We have demonstrated standard dose decitabine induced expression of NY-ESO-1in the circulating blasts of AML patients. The level of expression was sufficient to trigger a cytotoxic response in NY-ESO-1 specific CD8+ T cells. We have previously reported our preliminary data demonstrating that vaccination against NY-ESO-1 in combination with decitabine is safe and resulted in NY-ESO-1 specific cellular immune responses in the first six treatment-na•ve myelodysplatic syndrome (MDS) patients on study. We now update our results following complete study accrual.

Wang: Immunogen: Research Funding. Griffiths:Celgene: Honoraria; Alexion Pharmaceuticals: Honoraria; Astex: Research Funding.

Griffiths: Alexion Pharmaceuticals: Honoraria; Astex: Research Funding; Celgene: Honoraria. Off Label Use: Decitabine is in routine clinical use in the United States for the management of unfit/elderly patients with AML. Patients received decitabine as standard of care off label in the hospital per Bloom et al PNAS 2010.

Background: Identification of suitable target antigens for immunotherapy has been a challenge in patients with myeloid malignancies. NY-ESO-1 has been identified as an immunotherapeutic target in solid tumors. Its use in myeloid cancer is limited due to silencing by dense promoter hypermethylation. We and others have demonstrated that decitabine can induce expression of NY-ESO-1in leukemia cell lines. We hypothesized that vaccination against NY-ESO-1 in combination with decitabine would be safe and result in NY-ESO-1expression sufficient to induce NY-ESO-1 specific humoral and cellular immune responses in treatment-na•ve myelodysplastic syndrome (MDS) patients.

Background: The development of immunotherapeutic strategies for the treatment of leukemia has shown considerable promise but targeting suitable myeloid leukemia antigens remains a significant challenge. Cancer testis antigens (CTA) have been identified as promising targets for immunotherapy in solid tumors, but dense promoter methylation silences their expression in acute myeloid leukemia, limiting their potential as targets. Patients with acute myeloid leukemia are frequently treated with hypomethylating agents (HMAs) and previous studies have established that exposure of leukemia cell lines to HMAs induces expression of CTAs. In this study, we tested the hypothesis that patients receiving HMAs exhibit increased expression of CTAs.

The DNA methyltransferase inhibitors (DNMTi) 5-azacitidine (Aza) and decitabine (Dac) are a standard of care for patients with myelodysplatic syndrome (MDS) and acute myeloid leukemia (AML). Many hypotheses exist for the mechanism of these agents, including re-expression of epigenetically silenced tumor suppressor genes and direct cytotoxicity. We and others have shown hypermethylation of WNT/β-catenin inhibitory genes in primary MDS and AML samples. Activation of WNT/β-catenin signaling has furthermore been shown to play a role in the development and relapse of AML. Inhibition of WNT/β-catenin signaling is proposed to be clinically beneficial in AML. Based on published and preliminary data demonstrating that AML cell lines exposed to Aza and Dac re-express WNT/b-catenin inhibitors and down-regulate WNT/β-catenin signaling, we hypothesized that patients with AML receiving Dac would demonstrate suppression of WNT/β-catenin signaling in circulating blasts.We obtained serial peripheral blood samples from 5 patients with AML during their first cycle of Dac therapy (20 mg/m2 per day for 10 days). These patients were ineligible for high-dose induction chemotherapy based on age, performance status or comorbid illness and were of intermediate or poor risk karyotype. All patients had circulating blasts at presentation, which remained stable or declined over time. Blast populations were identified using flow cytometric selection based upon the characteristics of the diagnostic sample. Global methylation was assessed (using LINE-1 as a surrogate) by bisulfite pyrosequencing of mononuclear cell DNA extracted from the peripheral blood. To determine whether reduced methylation was associated with decreases in WNT/β-catenin signaling, we performed imaging flow cytometry (ImageStream, Amnis Corporation) on viable blasts harvested pre-treatment and on days 5-9 of the first Dac cycle (at the methylation nadir). ImageStream was used to assess nuclear localization of β-catenin, a biochemical hallmark of active WNT signaling, in CD34+ blasts. Nuclear β-catenin was quantified using a similarity score: a log-transformed Pearson's correlation coefficient between the digitized images of immunostained β-catenin and a nuclear stain (DAPI). A resolution metric (Fishers discriminant ratio, Rd) was calculated to determine shifts in the population distributions of this similarity score between CD34+ blasts at diagnosis versus after Dac therapy; a negative Rd value indicates decreased similarity scores between β-catenin and DAPI in treated versus diagnostic samples and thus decreased nuclear β-catenin. To determine whether changes in nuclear β-catenin were associated with changes in gene transcription, we performed whole genome analysis of RNA extracted from diagnostic and follow up samples (n = 5 paired samples, Illumina Human-HT12v4 Expression BeadChip).As expected, Dac treatment significantly reduced global DNA methylation (LINE-1 methylation) from 77.0 ± 1.6% pre-treatment to a nadir of 60.9 ± 6.8% (p< 0.01, n = 6). During the methylation nadir (days 5 to 9), the cellular distribution of β-catenin in CD34+ blasts shifted from the nucleus to the cytoplasm as indicated by an average Rd value of -0.22 (n = 5). This Rd value is similar to that observed after treating U937 cells with 0.5 µM Dac. The total amount of β-catenin in CD34+ blasts from treated patients did not change compared to their pre-treatment levels (0.98 ± 0.40 fold, n = 5) indicating that the shift in the cellular distribution of β-catenin from the nucleus towards the cytoplasm was not accompanied by decreased protein level. Comparison of samples from day 5-9 of Dac treatment to pre-treatment samples revealed 128 genes with a ≥ 1.2-fold change (75 up, 53 down, p< 0.05) in mRNA levels. We did not observe changes in expression of WNT regulatory genes, suggesting that alternative mechanisms may explain the reduction in nuclear β-catenin following Dac therapy. Of the 10 genes up-regulated more than 1.5-fold, 4 (FOS, EGR1, CLEC12A, RNASE2) are associated with myeloid differentiation. Of these, FOS and EGR1 are negatively regulated by WNT/β-catenin signaling.Given that activation of WNT/β-catenin signaling can suppress hematopoietic differentiation, these data suggest a model in which Dac exposure reduces nuclear β-catenin, leading to increased expression of pro-differentiation genes.Wetzler: Teva: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Griffiths:Astex Pharmaceuticals: Research Funding; Celgene, Inc.: Honoraria; Alexion Pharmaceuticals: Honoraria.

The DNA methyltransferase inhibitors (DNMTi) 5-azacitidine (Aza) and decitabine (Dac) are a standard of care for patients with myelodysplatic syndrome (MDS) and acute myeloid leukemia (AML). Many hypotheses exist for the mechanism of these agents, including re-expression of epigenetically silenced tumor suppressor genes and direct cytotoxicity. We and others have shown hypermethylation of WNT/β-catenin inhibitory genes in primary MDS and AML samples. Activation of WNT/β-catenin signaling has furthermore been shown to play a role in the development and relapse of AML. Inhibition of WNT/β-catenin signaling is proposed to be clinically beneficial in AML. Based on published and preliminary data demonstrating that AML cell lines exposed to Aza and Dac re-express WNT/b-catenin inhibitors and down-regulate WNT/β-catenin signaling, we hypothesized that patients with AML receiving Dac would demonstrate suppression of WNT/β-catenin signaling in circulating blasts.

Abstract 1290

SGI-110 (Astex Pharmaceuticals, Inc.) is a dinucleotide hypomethylating agent whose active metabolite is decitabine (DAC). This drug demonstrates superior pharmacokinetics relative to the parent drug as a result of resistance to modification by cytidine deaminase, and is being investigated in myeloid malignancy in the phase I/II setting. We and others have demonstrated that WNT inhibitory genes including SFRP2 are epigenetically silenced in AML and that exposure to DNA methyltransferase inhibitors such as 5-Azacitidine (AZA) and DAC can re-express these genes and down-regulate β-catenin signaling in AML cell lines. We hypothesized that treatment with SGI-110 would have a similar effect upon the epigenetically silenced WNT inhibitor SFRP2 and further would down-regulate β-catenin signaling in AML cells in vitro.The AML cell lines HL60 and U937 were cultured in vitro using standard techniques and treated with phosphate buffered saline, 0.1, 1 or 5 μM SGI-110, 2μM AZA or 0.5μM DAC. Results presented are pooled data from a minimum of three biological replicates. Samples were harvested on day 5 and viable cells, DNA, RNA and protein obtained. β-catenin levels and cellular localization were quantified using imaging flow cytometry (ImageStream), DNA was extracted and bisulfite converted for analysis of gene specific and global DNA methylation by pyrosequencing (LINE-1, SFRP2), RNA was converted to cDNA for analysis by RT-PCR, and protein was obtained to confirm ImageStream results by Western blot. Nuclear translocation of β-catenin, indicative of its signaling activity, was assessed in individual cells by ImageStream using a similarity score: a log-transformed Pearson's correlation coefficient between the digitized images of immunostained β-catenin and a nuclear stain (DAPI). Shifts in the population (n=5,000) distributions of this similarity score were assessed by a resolution metric (Fishers discriminant ratio, Rd).Treatment of AML cell lines with 5μM SGI-110 was toxic, and in line with previous experiments in AML cell lines, above the IC90. Treatment at the lowest dose of SGI-110 had minimal effects upon viability, methylation, and mRNA and protein expression in both cell lines tested. Treatment with SGI-110 at the 1μM dose resulted in reductions in LINE-1 methylation in HL60 cells by 21% (from 82% to 61%), compared to 8% with AZA (to 74%) and 20% with DAC (to 62%). In U937 cells, LINE-1 methylation decreased by 40% (from 67% to 27%) after SGI-110 treatment compared to a 25% reduction with AZA (to 42%) and a 30% reduction with DAC (to 36%). SFRP2 methylation in HL60 and U937 decreased from 86 and 88% at baseline to 66 and 60% with SGI-110 at the 1μM dose, compared to 68% with AZA and to 61% with DAC. Expression of SFRP2 mRNA was observed following treatment with 1μM SGI-110 and with DAC, but was limited following AZA treatment. ImageStream analysis of total cellular β-catenin in HL-60 and U937 cells demonstrated 2.4-fold and 1.2-fold reductions in total β-catenin following 1μM SGI-110 treatment. These results were similar to those seen with DAC (1.8-fold and 1.3-fold in HL-60 and U937 cells respectively). AZA treatment appeared to have a greater effect on total β-catenin in U937 cells (1.3-fold reduction) than in HL-60 cells (0.84-fold reduction). Western blot confirmed reductions in β-catenin protein. We also observed decreased nuclear translocation of β-catenin after treatment of HL-60 and U937 cells with 1 μM SGI-110 (Rd = −0.58 and −0.21 respectively; the negative sign indicates a change in cellular distribution from the nucleus to the cytoplasm). Changes were comparable to those observed with DAC (Rd = −0.75 and −0.26 in HL-60 and U937 cells respectively). AZA treatment of U937 cells resulted in a shift in cellular distribution (Rd = −0.20) similar to that for DAC and SGI-110 but had no effect on β-catenin distribution in HL-60 cells (Rd= 0.00).SGI-110 is a novel DNMT inhibitor which demonstrates robust effects on LINE-1 methylation, SFRP2 mRNA expression, and β-catenin level and localization consistent with epigenetically mediated re-expression of the WNT inhibitor SFRP2. Both upregulated β-catenin signaling and SFRP2 methylation have been demonstrated to correlate with inferior survival in patients with myeloid malignancies. Re-expression of epigenetically silenced WNT inhibitory genes such as SFRP2 may abrogate β-catenin signaling in AML cells.Karpf: Astex Pharmaceuticals: Research Funding. Griffiths:Celgene: Honoraria; Astex Pharmaceuticals: Research Funding.