Supplemental Figure 5. Expression of DC associated cell surface proteins in CD141Hi cells post-decitabine.

Supplemental Figure 3. Effect of combination of NY-ESO-1 vaccine and decitabine treatment on the frequencies of regulatory T-lymphocytes (TRegs).

Supplemental Figure 2. Effect of combination of NY-ESO-1 vaccine and decitabine treatment on NY-ESO-1 expression.

Supplemental Figure 1. Lineages of CD11b+ myeloid cells isolated from MDS patients.

Supplemental Figure 4. Identification of conventional DC subsets in MDS patients.

AbstractPurpose: Treatment options are limited for patients with high-risk myelodysplastic syndrome (MDS). The azanucleosides, azacitidine and decitabine, are first-line therapy for MDS that induce promoter demethylation and gene expression of the highly immunogenic tumor antigen NY-ESO-1. We demonstrated that patients with acute myeloid leukemia (AML) receiving decitabine exhibit induction of NY-ESO-1 expression in circulating blasts. We hypothesized that vaccinating against NY-ESO-1 in patients with MDS receiving decitabine would capitalize upon induced NY-ESO-1 expression in malignant myeloid cells to provoke an NY-ESO-1–specific MDS-directed cytotoxic T-cell immune response.Experimental Design: In a phase I study, 9 patients with MDS received an HLA-unrestricted NY-ESO-1 vaccine (CDX-1401 + poly-ICLC) in a nonoverlapping schedule every four weeks with standard-dose decitabine.Results: Analysis of samples serially obtained from the 7 patients who reached the end of the study demonstrated induction of NY-ESO-1 expression in 7 of 7 patients and NY-ESO-1–specific CD4+ and CD8+ T-lymphocyte responses in 6 of 7 and 4 of 7 of the vaccinated patients, respectively. Myeloid cells expressing NY-ESO-1, isolated from a patient at different time points during decitabine therapy, were capable of activating a cytotoxic response from autologous NY-ESO-1–specific T lymphocytes. Vaccine responses were associated with a detectable population of CD141Hi conventional dendritic cells, which are critical for the uptake of NY-ESO-1 vaccine and have a recognized role in antitumor immune responses.Conclusions: These data indicate that vaccination against induced NY-ESO-1 expression can produce an antigen-specific immune response in a relatively nonimmunogenic myeloid cancer and highlight the potential for induced antigen-directed immunotherapy in a group of patients with limited options. Clin Cancer Res; 24(5); 1019–29. ©2017 AACR.See related commentary by Fuchs, p. 991

Current criteria for identifying cancer patients suitable for immunotherapy with immune checkpoint blockers (ICBs) are subjective and prone to misinterpretation, as they mainly rely on the visual assessment of CD274 (best known as PD-L1) expression levels by immunohistochemistry (IHC). To address this issue, we developed a RNA sequencing (RNAseq)-based approach that specifically measures the abundance of immune transcripts in formalin-fixed paraffin embedded (FFPE) specimens. Besides exhibiting superior sensitivity as compared to whole transcriptome RNAseq, our assay requires little starting material, implying that it is compatible with RNA degradation normally caused by formalin. Here, we demonstrate that a targeted RNAseq panel reliably profiles mRNA expression levels in FFPE samples from a cohort of ovarian carcinoma patients. The expression profile of immune transcripts as measured by targeted RNAseq in FFPE versus freshly frozen (FF) samples from the same tumor was highly concordant, in spite of the RNA quality issues associated with formalin fixation. Moreover, the results of targeted RNAseq on FFPE specimens exhibited a robust correlation with mRNA expression levels as measured on the same samples by quantitative RT-PCR, as well as with protein abundance as determined by IHC. These findings demonstrate that RNAseq profiling on archival FFPE tissues can be used reliably in studies assessing the efficacy of cancer immunotherapy.

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-1 in 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.

With recent advances in cellular biology, we now appreciate that modifications to DNA and histones can have a profound impact on transcription and function, even in the absence of changes to DNA sequence. These modifications, now commonly referred to as “epigenetic” alterations, have changed how we understand cell behavior, reprogramming and differentiation and have provided significant insight into the mechanisms underlying carcinogenesis. Epigenetic alterations, to this point, are largely identified by changes in DNA methylation and hydroxymethylation as well as methylation, acetylation, and phosphorylation of histone tails. These modifications enable significant flexibility in gene expression, rather than just turning genes “ON” or “OFF.” Herein we describe the epigenetic landscape in the regulation of gene expression with a particular focus on interrogating DNA methylation in myeloid malignancy.

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 vitro and in vivo and 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 vitro at various concentrations and as tumor xenografts with 3 distinct dose schedules. Effects on global methylation (using LINE-1), NY-ESO-1 and MAGE-A methylation, 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 vivo administration 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 vitro and 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.

Recent evidence suggests T cells are important for the survival of mice with myelodysplastic syndrome (MDS) (Gough, 2012). Furthermore, MDS and acute myeloid leukemia (AML) patients who received azacitidine and sodium valproate demonstrated an induction and, in some patients, an expansion of antigen-specific CD8 T cells. Importantly, of 4 patients who had a complete response, 3 patients developed antigen-specific CD8 T cells (Goodyear, 2010) suggesting that antigen-specific CD8 T cells may contribute to the antitumor response to azanucleosides.

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-1 in leukemia cell lines. We hypothesized that vaccination against NY-ESO-1 in combination with decitabine would be safe and result in NY-ESO-1 expression sufficient to induce NY-ESO-1 specific humoral and cellular immune responses in treatment-na•ve myelodysplastic syndrome (MDS) patients.

Cancer testis antigens (CTAs) are promising cancer associated antigens in solid tumors, but in acute myeloid leukemia, dense promoter methylation silences their expression. Leukemia cell lines exposed to HMAs induce expression of CTAs. We hypothesized that AML patients treated with standard of care decitabine (20mg/m2 per day for 10 days) would demonstrate induced expression of CTAs. Peripheral blood blasts serially isolated from AML patients treated with decitabine were evaluated for CTA gene expression and demethylation. Induction of NY-ESO-1 and MAGEA3/A6, were observed following decitabine. Re-expression of NY-ESO-1 and MAGEA3/A6 was associated with both promoter specific and global (LINE-1) hypomethylation. NY-ESO-1 and MAGEA3/A6 mRNA levels were increased irrespective of clinical response, suggesting that these antigens might be applicable even in patients who are not responsive to HMA therapy. Circulating blasts harvested after decitabine demonstrate induced NY-ESO-1 expression sufficient to activate NY-ESO-1 specific CD8+ T-cells. Induction of CTA expression sufficient for recognition by T-cells occurs in AML patients receiving decitabine. Vaccination against NY-ESO-1 in this patient population is feasible.

Recent evidence suggests that T cells are important for survival in an MDS mouse model (Gough SM, 2012). A study of azacitidine, a hypomethylating agent (HMA) with sodium valproate in MDS or AML showed induction of tumor antigen-specific CD8+ T cells correlating with CR (Goodyear O, 2010). These data suggest HMAs may enhance tumor recognition by CD8+ T cells. Dendritic cells (DCs), which arise from potentially malignant myeloid progenitors, are required for CD8 activation. CD58 (LFA3) on DCs interacts with CD8+ T cells to facilitate cell-cell interaction; CD58 also exerts costimulatory function (Bullens DM, 2001). In cancer, CD58 and members of the antigen presentation machinery, such as transport associated with antigen processing 1 (TAP-1) are downregulated by hypermethylation (Manning J, 2008). We hypothesized that leukemic blasts from patients treated with Dac would have altered expression of these genes and that leukemic cell lines exposed in vitro would enhance expression of CD58 and TAP-1.

The mechanism of clinical action for the FDA approved hypomethylating drugs azacitidine and decitabine remains unresolved and in this context the potential immunomodulatory effect of these agents on leukemic cells is an area of active investigation. Induced expression of methylated Cancer Testis Antigen (CTA) genes has been demonstrated in leukemic cell lines following exposure to hypomethylating drugs in vitro. SGI-110 is a novel hypomethylating dinucleotide with prolonged in vivo exposure and clinical activity in patients with MDS and AML. We demonstrate that this agent, like decitabine, produces robust re-expression of the CTAs NY-ESO-1 and MAGE-A, both in vitro and in leukemia-bearing AML xenografts. Upregulation of these genes in vitro was sufficient to induce cytotoxicity by HLA-compatible CD8+ T-cells specific for NY-ESO-1, a well-recognized and immunogenic CTA. Additionally, exposure to SGI-110 enhances MHC class I and co-stimulatory molecule expression, potentially contributing to recognition of CTAs. SGI-110, like the parent compound decitabine, induces expression of CTAs and might modulate immune recognition of myeloid malignancy.

Background: Mechanisms of endogenous PKC signal termination remain to be fully characterized. Results: Activated endogenous PKCα undergoes dynamin-dependent and -independent endocytic uptake and traffics through early and late endosomes for processing by lysosomes. Conclusion: Lysosomal degradation represents a novel mechanism of desensitizing PKC-mediated signaling. Significance: Multiple degradation mechanisms ensure strict control of the duration of PKC signaling in cells. Protein kinase C (PKC) isozymes undergo down-regulation upon sustained stimulation. Previous studies have pointed to the existence of both proteasome-dependent and -independent pathways of PKCα processing. Here we demonstrate that these down-regulation pathways are engaged in different subcellular compartments; proteasomal degradation occurs mainly at the plasma membrane, whereas non-proteasomal processing occurs in the perinuclear region. Using cholesterol depletion, pharmacological inhibitors, RNA interference, and dominant-negative mutants, we define the mechanisms involved in perinuclear accumulation of PKCα and identify the non-proteasomal mechanism mediating its degradation. We show that intracellular accumulation of PKCα involves at least two clathrin-independent, cholesterol/lipid raft-mediated pathways that do not require ubiquitination of the protein; one is dynamin-dependent and likely involves caveolae, whereas the other is dynamin- and small GTPase-independent. Internalized PKCα traffics through endosomes and is delivered to the lysosome for degradation. Supportive evidence includes (a) detection of the enzyme in EEA1-positive early endosomes, Rab7-positive late endosomes/multivesicular bodies, and LAMP1-positive lysosomes and (b) inhibition of its down-regulation by lysosome-disrupting agents and leupeptin. Only limited dephosphorylation of PKCα occurs during trafficking, with fully mature enzyme being the main target for lysosomal degradation. These studies define a novel and widespread mechanism of desensitization of PKCα signaling that involves endocytic trafficking and lysosome-mediated degradation of the mature, fully phosphorylated protein.

Abstract Background 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. Methods 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). Results 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. Conclusions 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. Disclosures: 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.

Since its discovery in 1997 ([Li and Sun, 1997][1]; [Li et al., 1997][2]; [Steck et al., 1997][3]), the phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5) P 3] phosphatase and tensin homolog (PTEN) has been established as one of the most frequently mutated tumor suppressor genes in human