Hypomethylating Treatment Research Articles

Targeting protein methylation in pancreatic cancer cells results in KRAS signaling imbalance and inhibition of autophagy

Pancreatic cancer cells with mutant KRAS require strong basal autophagy for viability and growth. Here, we observed that some processes that allow the maintenance of basal autophagy in pancreatic cancer cells are controlled by protein methylation. Thus, by maintaining the methylation status of proteins such as PP2A and MRAS, these cells can sustain their autophagic activity. Protein methylation disruption by a hypomethylating treatment (HMT), which depletes cellular S-adenosylmethionine levels while inducing S-adenosylhomocysteine accumulation, resulted in autophagy inhibition and endoplasmic reticulum stress-induced apoptosis in pancreatic cancer cells. We observed that by reducing the membrane localization of MRAS, hypomethylation conditions produced an imbalance in KRAS signaling, resulting in the partial inactivation of ERK and hyperactivation of the PI3K/AKT–mTORC1 pathway. Interestingly, HMT impeded CRAF activation by disrupting the ternary SHOC2 complex (SHOC2/MRAS/PP1), which functions as a CRAF-S259 holophosphatase. The demethylation events that resulted in PP2A inactivation also favored autophagy inhibition by preventing ULK1 activation while restoring the cytoplasmic retention of the MiT/TFE transcription factors. Since autophagy provides pancreatic cancer cells with metabolic plasticity to cope with various metabolic stress conditions, while at the same time promoting their pathogenesis and resistance to KRAS pathway inhibitors, this hypomethylating treatment could represent a therapeutic opportunity for pancreatic adenocarcinomas.

Identification of Predictive Factors for Overall Survival and Response during Hypomethylating Treatment in Very Elderly (≥75 Years) Acute Myeloid Leukemia Patients: A Multicenter Real-Life Experience.

Simple SummaryIntensive induction strategies are rarely used for older patients in community on-cology practice, with comorbidities being the major cause of contraindication. We conducted a multicentric retrospective study to evaluate activity and safety in a real-life setting of hypomethylating drugs (HMAs) in patients older than 75 years with AML. In multivariate analysis, age (≥80), Charlson comorbidity index (≥3), creatinine clearance and the type of best response (≥PR) during treatment maintained independent significance in predicting survival. Furthermore, our data show that HMAs have similar efficacy compared to pivotal trials and are well tolerated in a setting of very elderly patients with several co-comorbidities.Elderly patients represent the most challenging and hard-to-treat patient population due to dismal characteristics of the disease, such as secondary-acute myeloid leukemia (AML), enrichment of unfavorable molecular genes (TP53) and comorbidities. We conducted a multicentric retrospective study to evaluate activity and safety in a real-life setting of hypomethylating drugs (HMAs) in patients older than 75 years with AML. Between September 2010 and December 2021, 220 patients were treated, 164 (74.5%) received AZAcitidine and 56 DECitabine; most patients (57.8%), received more than four cycles of HMAs. The best response obtained was CR in 51 patients (23.2%), PR in 23 (10.5%) and SD in 45 (20.5%); overall transfusion independence was obtained in 47 patients (34%), after a median of 3.5 months. The median OS (mOs) was 8 months (95% CI 5.9–10.2), with 1- and 2-years OS of 39.4% (95% CI 32.7–46) and 17.4% (95% CI 11.7–23.1), respectively; similar mOS was observed according to HMA treatment (AZA 8.3 vs. DEC 7.8 months, p = 0.810). A subset of 57 long survivors (44 in AZA group and 13 in DEC group) received at least 12 cycles of HMAs, their mOS was 24.3 months. In multivariate analysis, age (≥80), Charlson comorbidity index (≥3), creatinine clearance and the type of best response (≥PR) during treatment maintained independent significance in predicting survival. Infectious complications, most frequently pneumonia (35) and septic shock (12), were lethal in 49 patients (22.2%). Our data show that HMAs have similar efficacy compared to pivotal trials and are well tolerated in a setting of very elderly patients with several co-comorbidities.

Dynamics of PD-1 expression are associated with treatment efficacy and prognosis in patients with intermediate/high-risk myelodysplastic syndromes under hypomethylating treatment

Hypomethylating agents (HMAs) are widely used in patients with higher-risk MDS not eligible for stem cell transplantation. However, the general response rate by HMAs is lesser than 50% in MDS patients, while the relapse rate is high. Emerging evidence indicates that demethylating effects committed by HMAs may facilitate the up-regulation of a range of immune checkpoints or cancer suppressor genes in patients with MDS, among which the programmed death protein 1 (PD-1) and its ligands are demonstrated to be prominent and may contribute to treatment failure and early relapse. Although results from preliminary studies with a limited number of enrolled patients indicate that combined administration of PD-1 inhibitor may yield extra therapeutic benefit in some MDS patients, identifications of this subgroup of patients and optimal timing for the anti-PD-1 intervention remain significant challenges. Dynamics of immune checkpoints and associated predictive values during HMA-treatment cycles remained poorly investigated. In this present study, expression levels of immune checkpoints PD-1 and its ligands PD-L1 and PD-L2 were retrospectively analyzed by quantitative PCR (Q-PCR) in a total of 135 myelodysplastic syndromes (MDS) cohort with higher-risk stratification. The prognostic value of dynamics of these immune checkpoints during HMA cycles was validated in two independent prospective cohorts in our center (NCT01599325 and NCT01751867). Our data revealed that PD-1 expression was significantly higher than that in younger MDS patients (age ≤ 60) and MDS with lower IPSS risk stratification (intermediate risk-1). A significantly up-regulated expression of PD-1 was seen during the first four HMA treatment cycles in MDS patients, while similar observation was not seen concerning the expression of PD-L1 or PD-L2. By utilizing binary logistic regression and receiver operating characteristic (ROC) models, we further identified that higher or equal to 75.9 PD-1 expressions after 2 cycles of HMA treatment is an independent negative prognostic factor in predicting acute myeloid leukemia (AML) transformation and survival. Collectively, our data provide rationales for monitoring the expression of PD-1 during HMA treatment cycles, a higher than 75.9 PD-1 expression may identify patients who will potentially benefit from the combined therapy of HMA and PD-1 inhibitors.

H3K9 dimethylation safeguards cancer cells against activation of the interferon pathway.

Activation of interferon genes constitutes an important anticancer pathway able to restrict proliferation of cancer cells. Here, we demonstrate that the H3K9me3 histone methyltransferase (HMT) suppressor of variegation 3–9 homolog 1 (SUV39H1) is required for the proliferation of acute myeloid leukemia (AML) and find that its loss leads to activation of the interferon pathway. Mechanistically, we show that this occurs via destabilization of a complex composed of SUV39H1 and the two H3K9me2 HMTs, G9A and GLP. Indeed, loss of H3K9me2 correlated with the activation of key interferon pathway genes, and interference with the activities of G9A/GLP largely phenocopied loss of SUV39H1. Last, we demonstrate that inhibition of G9A/GLP synergized with DNA demethylating agents and that SUV39H1 constitutes a potential biomarker for the response to hypomethylation treatment. Collectively, we uncovered a clinically relevant role for H3K9me2 in safeguarding cancer cells against activation of the interferon pathway.

Epigenetic Silencing of Tumor Suppressor lncRNA NKILA: Implication on NF-κB Signaling in Non-Hodgkin's Lymphoma.

The long non-coding RNA (lncRNA) NKILA, localized to 20q13.31, is a negative regulator of NF-κB signaling implicated in carcinogenesis. As a CpG island is embedded in the promoter region of NKILA, it is hypothesized as a tumor suppressor lncRNA silenced by promoter DNA methylation in non-Hodgkin’s lymphoma (NHL). By pyrosequencing-verified methylation-specific PCR, NKILA methylation was detected in 1/10 (10%) NHL cell lines, but not in normal peripheral blood buffy coats or tonsils. NKILA methylation correlated with the repression of NKILA in cell lines. Hypomethylation treatment with 5-Aza-2′-deoxycytidine resulted in promoter demethylation and the re-expression of NKILA. In 102 NHL primary samples, NKILA was methylated in 29 (51.79%) diffuse large B-cell lymphoma (DLBCL) and 4 (20%) peripheral T-cell lymphoma cases, but unmethylated in all 26 mantle cell lymphoma cases. Mechanistically, the knockdown of NKILA resulted in promoting IkBα phosphorylation, associated with nucleus translocation of total p65 and phosphorylated p65 in SU-DHL-1 cells, hence constitutive NF-κB activation. Functionally, the knockdown of NKILA in SU-DHL-1 cells led to decreased cell death and increased cellular proliferation. Collectively, NKILA was a tumor suppressor lncRNA frequently hypermethylated in DLBCL. Promoter DNA methylation-mediated NKILA silencing resulted in increased cellular proliferation and decreased cell death via the repression of NF-κB signaling in NHL.

Treatment of higher-risk myelodysplastic syndrome

Introduction. The myelodysplastic syndromes are a group of clonal haematopoietic stem cell disorders characterized by cytopenia, dysplasia, ineffective hematopiesis, recurrent genetic abnormalities, and increased risk of developing acute myeloid leukemia. In this paper, we present the review and recommendations for treatment of high risk myelodysplastic syndromes on behalf of the Serbian myelodysplastic syndromes group. Material and Methods. A literature review was conducted using the following bibliographic databases: Google Scholar, MEDLINE and Kobson. The recommendations treatment of high risk myelodysplastic syndromes are based on expert opinion based on review of literature and contemporary recommendations for treatment of high risk for myelodysplastic syndromes. Recommendations. Higher-risk myelodysplastic syndromes should be defined in patients risk group with > 3.5 IPSS-R score. Allo- HSCT is recommended in fit higher-risk patients with IPSS-R > 3.5 as well as in fit lower-risk patients with poor risk features according to EBMT/ELN International expert panel and myelodysplastic syndromes right group. Acute myeloid leukemia like or hypomethylation treatment before Allo-HSCT is indicated in patients with myelodysplastic syndromes with ? 10% of blasts. Azacitidine is recommended in intermediate-2 and high risk IPSS patients who are not eligible for transplantation with minimal number of six cycles to define response. Acute myeloid leukemia like treatment is recommended in fit higher-risk for patients with myelodysplastic syndromes with excess of blasts, good performance status, without substantial comorbidities, and with no poor/very poor cytogenetics/genetics. Conclusion. The treatment of fit higher-risk patients should be based on allo-SCT. In patients who are not candidates for transplant hypomethylation treatment is indicated as well as acute myeloid leukemia like treatment in selected patients.

Cancer testis antigens in myelodysplastic syndromes revisited: a targeted RNA-seq approach

ABSTRACT Cancer-Testis antigens (CTA) are named after the tissues where they are mainly expressed: in germinal and in cancer cells, a process that mimics many gametogenesis features. Mapping accurately the CTA gene expression signature in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) is a prerequisite for downstream immune target-discovery projects. In this study, we take advantage of the use of azacitidine to treat high-risk MDS and CMML to draw the CTAs landscape, before and after treatment, using an ad hoc targeted RNA sequencing (RNA-seq) design for this group of low transcript genes. In 19 patients, 196 CTAs were detected at baseline. Azacitidine did not change the number of CTAs expressed, but it significantly increased or decreased expression in nine and five CTAs, respectively. TFDP3 and DDX53, emerged as the main candidates for immunotherapeutic targeting, as they showed three main features: i) a significant derepression on day +28 of cycle one in those patients who achieved complete remission with hypomethylating treatment (FC = 6, p = .008; FC = 2.1, p = .008, respectively), ii) similar dynamics at the protein level to what was observed at the RNA layer, and iii) to elicit significant specific cytotoxic immune responses detected by TFDP3 and DDX53 HLA-A*0201 tetramers. Our study addresses the unmet landscape of CTAs expression in MDS and CMML and revealed a previously unrecognized TFDP3 and DDX53 reactivation, detectable in plasma and able to elicit a specific immune response after one cycle of azacitidine.

Decitabine-Induced Early Platelet Response, a Predictor of Favorable Outcome during Hypomethylating Treatment of MDS, Is Associated with In Vivo Megakaryocytic Differentiation

Introduction Despite broad use of hypomethylating agents (HMAs) in MDS/AML treatment, the number of established outcome predictors is still very limited (Stomper and Lübbert, Sem Hematol 2019). One of them, the early, isolated and sometimes massive increase of platelets, is recurrently observed in patients with MDS treated with HMAs. It is not observed with non-HMA cytidine analogs such as low-dose cytarabine. In HMA-treated MDS patients, an early platelet increase is a predictor of overall and leukemia-free survival (van den Bosch et al., Leuk Res 2004; van der Helm et al., Br J Haematol 2011). HMAs induce cellular differentiation in vitro, by gene reactivation in the malignant cells. However, evidence for HMA-induced in vivo differentiation is still very limited. We hypothesized that the megakaryocytic cell lineage in MDS is a target for HMA-induced cellular maturation in vivo. Methods We systematically analyzed the bone marrow morphology of 34 higher-risk MDS patients (median age: 71.5 years, range 51-79) before and after 1 cycle of treatment with the HMA decitabine (DAC). All patients had been treated at a single center within 3 prospective clinical trials (Wijermans et al., Ann Hematol 2005; Lübbert et al., J Clin Oncol 2011). One treatment cycle consisted of 45 mg/m2 DAC per day (15 mg/m2 intravenously over 4 hours every 8 hours) for 3 consecutive days, repeated 6 weeks later. The early platelet response was evaluated after 1 cycle of DAC treatment. Based on the criteria of the International Working Group, an absolute increase in platelet count of 30x109/l or more compared to the pre-treatment count was defined as a platelet response. The histological analysis of the bone marrow specimens was performed by an experienced hematopathologist blinded to the treatment timepoints. Up to 200 megakaryocytes (MK) per sample were quantified at a magnification of 400 x using chloroacetate esterase staining. Results Thirteen of 34 patients (38%) showed a platelet response already after 1 cycle of DAC treatment, 21 (62%) did not. The median pre-treatment platelet count did not differ in patients with or without an early platelet response (median of 34x109/l in both groups, range 7-169 and 8-265, respectively). After 1 cycle of DAC treatment, patients with a platelet response had a median platelet count of 117x109/l (range 78-281), patients without this response had a median platelet count of 32 x109/l (range 4-155). Overall survival (OS) was measured from the time of early platelet response assessment after completion of 1 treatment cycle, i.e. after 6 weeks. The presence of a platelet response after 1 DAC cycle was associated with a longer OS compared to the absence of this early platelet response: median of 26.6 versus 14.0 months (p=0.04). Both pre- and post-treatment bone marrow biopsies of patients with an early platelet response showed higher numbers of MK, as well as significant differences in MK morphology compared to biopsies from patients without an early platelet response. Regarding MK numbers, increased MK density in specimens of patients with an early platelet response was observed both before (mean MK number per high power field 6.2 vs. 2.6, p=0.02) and after the application of DAC (mean MK number 10.4 vs. 3.1, p=0.01). Regarding MK maturation stage, more pre-treatment juvenile MK (on average 32.4% vs. 20.5% of all MK, p=0.03) and MK with typical myeloproliferative stigmata (present in 5/13 vs. 2/21 biopsies) were observed in patients with an early platelet response, compared to patients without this response. Regarding the induction of megakaryocytic maturation during this early treatment phase, more post-treatment "naked", mature MK nuclei indicative of active platelet shedding (on average 9.5% vs. 3.8% of all MK, p=0.01), were noted in patients with an early platelet response than in patients without an early platelet response. Conclusions This is, to the best of our knowledge, the first systematic hematopathological analysis of changes in quantitative and morphological MK features in bone marrow specimens of MDS patients during HMA treatment. DAC, which has in vitro differentiation-inducing effects on megakaryoblastic cells, induced maturation also of dysplastic MK in vivo in higher-risk MDS patients with an early platelet response to this HMA. The predictive value of an early platelet increase, a very easy-to-determine parameter, during this type of treatment is confirmed. Disclosures No relevant conflicts of interest to declare.

Enhanced polo-like kinase 1 expression in myelodysplastic syndromes.

BackgroundCancer is characterized by uncontrolled cellular proliferation, and Polo-like kinase 1 (PLK1), a key regulator of the cell cycle, is overexpressed in many cancers, including acute leukemia and lymphoma. However, the dynamics of PLK1 transcription in myelodysplastic syndromes (MDS) are unknown. This study aimed to investigate the transcript dynamics of PLK1 and determine its role in the pathophysiology of MDS.MethodsPLK1 mRNA obtained from the bone marrow samples of 67 patients with MDS, 16 patients with secondary acute myeloid leukemia (sAML), and 10 healthy controls were analyzed using quantitative real-time PCR and compared according to various clinical parameters.ResultsThe median PLK1 expression levels differed slightly, but not significantly, between MDS and sAML patients [661.21 (range, 29.38–8,987.31) vs. 1,462.05 (32.22–5,734.09), respectively], but were significantly higher (P<0.001) than the levels in the healthy controls [19.0 (1.60–49.90)]. Further analyses of PLK1 levels according to the WHO classification of MDS, prognostic risk groups, karyotype risk groups, marrow blast percentage, and depth of cytopenia did not reveal any significant associations. In patients progressing to sAML, PLK1 expression levels differed significantly according to the presence or absence of resistance to hypomethylation treatment (2,470.58 vs. 415.98, P=0.03).ConclusionPLK1 is upregulated in MDS patients; however, its role in the pathophysiology of MDS is unclear. Gene upregulation in cases with pharmacotherapeutic resistance warrants further investigation.

The forkhead box C1 (FOXC1) transcription factor is downregulated in acute promyelocytic leukemia.

Forkhead box (FOX) genes encode transcription factors, which regulate embryogenesis and play an important role in hematopoietic differentiation and in mesenchymal niche maintenance. Overexpression of the family member FOXC1 has been reported in solid tumors and acute myeloid leukemia (AML).We studied FOXC1 expression and function in acute promyelocytic leukemia (APL) and normal hematopoietic progenitors. FOXC1 mRNA and protein levels were significantly lower in primary marrow samples from 27 APL patients, as compared to samples obtained from 27 patients with other AML subtypes, and 5 normal CD34+ hematopoietic cells. FOXC1 expression significantly increased in APL samples at the time of remission following consolidation treatment. In cell lines overexpressing PML-RARA, and in the NB4 t(15;17)-positive cell line, FOXC1 expression was lower than in other non-APL cell lines, and increased following treatment with all-trans retinoic acid (ATRA), due to functional binding of ATRA to the FOXC1 promoter region. Reduced FOXC1 expression was also associated to DNA hypermethylation of the +354 to +568 FOXC1 region, both in primary APL, and in NB4 cells. Treatment of NB4 cells with decitabine demethylated FOXC1 and upregulated its expression.Our findings indicate that FOXC1 is consistently repressed in APL due to hypermethylation and the presence of the PML-RARA rearrangement. A potential role of hypomethylating treatment in advanced APL remains to be established.

Hypomethylating treatment induces tumor specific CD8+ T-cell recognition to osteosarcoma

Objective To investigate feasible immunotherapy strategy using tumor specific cell against osteosarcoma, and to analyze the therapeutic effect of adoptive cellular infusion therapy on osteosarcoma. Methods Decitabine (DAC) was employed as a hypomethylating agent for the treatment in osteosarcoma cell lines HOS and U2OS. After treatment, the expression of cancer-testis antigen (CTA) was evaluated by PCR and Western Blot. In animal studies, human osteosarcoma cell line HOS, which was transfected by luciferase and HLA-A0201 in previous, was inoculated into immune deficient NOD-SCID mice to establish osteosarcoma xenografts. Ex-vivo expanded CTA specific homo CD8+ T-ells were labeled with DiR and injected into the mice via the tail vein. In vivo imaging system was utilized to detect the distribution of administrated CD8+ T-cells. In addition, the progression of tumor xenografts was monitored. Moreover, mouse K7M2 osteosarcoma cell line was used to establish animal models in immune competent BALB/c mice. Immune competent models were utilized to evaluate the effectiveness of hypomethylating treatment in regarding to spontaneous immune attack against tumors. Flow cytometry was used to analyze the proportion of intratumoral lymphocytes and the status of these effector antitumor immune cells, and to reveal the effect of hypomethylating treatment in facilitating lymphocyte infiltration and activation. Results The expression of all the evaluated cancer/testis antigens were elevated in HOS and U2OS osteosarcoma cell lines after hypomethylating treatment with DAC. The proliferation of in vitro cultured osteosarcoma cells can be significantly suppressed after at least 5 d treatment with DAC. Besides, DAC alone controlled osteosarcoma cell proliferation. In immune deficient mouse models, hypomethylating pre-treatment resulted in successful T-cell homing to tumor sites. Moreover, the combination treatment with DAC and CTA specific T-cell adoptive transfer significantly suppressed tumor proliferation. In immune competent mouse models, hypomethylating treatment with DAC improved autologous T-cell infiltration into the tumor, and strengthened the activity of intratumoral CD8+ T-cells, elevated the secretion of IFN-gamma, granzyme B and perforin by CD8+ T-cells. Conclusion Hypomethylating treatment is able to suppress osteosarcoma cell proliferation, improve the expression of CTA in osteosarcoma cells, and consequently provide optimal environment for CTA specific T-cell adoptive therapy. Key words: Osteosarcoma; Cell line, tumor; Animal experimentation

Dynamic prognostic value of the revised international prognostic scoring system following pretransplant hypomethylating treatment in myelodysplastic syndrome.

This study aimed to analyze the use of the revised International Prognostic Scoring System (IPSS-R) assessed after hypomethylating treatment (HMT) for patients with myelodysplastic syndrome (MDS) undergoing an allogeneic stem cell transplantation (SCT). Among 115 patients who received pre-SCT HMT, comparison analysis of the prognostic values between the IPSS-R at the time of HMT (IPSS-R@HMT) and at the time of SCT after HMT (IPSS-R@SCT) showed a significantly higher predictive power for overall survival (OS) of the latter. Alteration in IPSS-R risk occurred in 60%, while the patients with 'down-staged' IPSS-R@SCT showed better OS compared with those with 'unchanged' or 'up-staged' risk. On multivariate analysis in all 201 patients, IPSS-R@SCT, monosomal karyotype, treatment failure to pre-SCT treatment, and high hematopoietic cell transplantation-comorbidity index were independently associated with OS. Constructed using these factors, the MDS Transplantation Prognostic Scoring System (MTPSS) identified four risk groups with 4-year OS of 76.4% in low, 61.4% in intermediate-1 and 21.9% in intermediate-2 risk groups, whereas all in the high risk group died within 2 years after SCT (P<0.001). Our study emphasizes the need for further studies aiming to evaluate a transplantation prognostic model such as the MTPSS to make appropriate decisions for transplantation in MDS.

Better transplant outcome with pre-transplant marrow response after hypomethylating treatment in higher-risk MDS with excess blasts.

Hypomethylating treatment (HMT) has been suggested as a feasible bridge to hematopoietic stem cell transplantation (HSCT), but controversies exist around influences of HMT response on transplant outcomes. To assess the safety and influences of pre-transplant HMT focusing on debulking effects and transplant outcomes, we retrospectively analyzed consecutive HSCT-eligible patients who received HMT for higher-risk MDS with excess blasts. Of all 98 patients, 11 patients failed to proceed to HSCT and HMT-related mortality occurred in 8 patients. When excluding 9 patients who refused HSCT, 87% of scheduled HSCT (77 of 89) was performed after a median of 3 cycles (range, 1-8) of HMT. The 4-year overall survival after HMT (n = 98) and HSCT (n = 77) was 44.0% and 53.6%, respectively. Transplant outcomes were significantly different by the final response at HSCT; marrow response group (complete remission, marrow complete remission with or without hematologic improvement) showed significantly better 4-year disease-free survival compared to no marrow response group (n = 36, 87.3% vs. n = 41, 10.7%, P < 0.001). This difference between the groups was also evident in overall survival (90.9% vs. 8.6%, P < 0.001) and cumulative incidences of relapse (6.5% vs. 45.4%, P < 0.001) and treatment-related mortality (6.2% vs. 43.9%, P < 0.001). These observations indicate that pre-transplant HMT is a feasible bridging treatment in patients with excess blasts regarding high success rate of proceeding to transplantation and good survival rate. Marrow response at HSCT regardless of concomitant hematological improvement is an independent predictor of better survival, suggesting that immediate HSCT rather than continuing HMT should be performed once marrow response is achieved.

Relationship between somatic mutations and the response to the treatment of de novo patients with myelodysplastic syndromes: reports from the 57th American Society of Hematology annual meeting

Research progress of somatic mutations and the response to the treatment of de novo myelodysplastic syndromes (MDS) patients in the 57th American Society of Hematology (ASH) annual meetings was reviewed. The optimal methods and therapy time for patients with high-risk de novo MDS remained an area of ongoing investigation. The clinical prognostic scoring system does not include the molecular genetic abnormalities and DNA metlylation of histone/nuclear chromatin modifications, which may predict the effect of hypomethylation (HMA). Treatment of HMA may change the expression of genes related with prognosis, and the response rate to the HMA treatment was significantly increased for TET2-mutated patients with high-variant allele frequencies. The overall grade of recommendation for choosing HMA therapy over induction chemotherapy in high-risk MDS based on molecular genetic mutations was 2C, according to less-associated toxicity and increased responses primarily in TET2-mutated disease. Further prospective studies are needed to evaluate the long-term effects of HMA therapy, particularly in TET2-mutated patients. Key words: Myelodysplastic syndromes; Somatic mutations; American Society of Hematology annual meeting

Feasibililty of Azacitidine As Bridge to Allogeneic Stem Cell Transplantation in Patients with Higher-Risk MDS or Low-Blast Count AML: Results of the BMT-AZA Multicenter Prospective Study

Background Allogeneic SCT remains the only curative option in myelodysplastic syndromes (MDS). We prospectively evaluated in a multicenter phase 2 GITMO (Italian Bone Marrow Transplantation Group) trial, the feasibility of allogeneic stem cell transplantation (ASCT, primary protocol end-point) after induction treatment with azacitidine (Vidaza, Celgene). According to the protocol, patients had to perform at least 4 and up to 12 cycles of azacitidine as induction treatment before ASCT. Patients not eligible for ASCT could continue treatment until disease progression.Methods Between November 2010 and September 2014, 102 patients were enrolled by 20 Italian hematology centers. Nine patients never started treatment due to progressive disease (PD, n=2), refusal (n=3), or unknown causes (n=4). Of remaining 93 patients, 70 had with IPSS Int-2/High-risk MDS, 15 WHO-defined acute myeloid leukemia (AML) and 8 chronic myelomonocytic leukemia (CMML). There were 32 females and 61 males with median age 59 yrs (range 21-66.5 yrs). At treatment start, ECOG performance status was: 0 in 66 patients, 1 in 17 and 2 in 10 patients, while MDS HCT-index was: low (0) in 43 patients, intermediate (1-2) in 31 and high (>2) in 18 patients. All 93 patients started azacitidine s.c. at the standard dose of 75 mg/sqm/day, 7 days/month, at a median of 0.8 months (range 0-105 months) from initial MDS diagnosis.Results At response assessment after a median of 4.5 cycles (range 1-11), 25% of patients (n=19) achieved complete remission (CR), 3% marrow-CR (n=2), 14% partial remission (PR, n=11), 9% hematologic improvement (HI, n=7), 35% had stable disease (SD, n=27) and 14% PD (n=11). ASCT was performed in 48 patients (52%), after a median of 4.5 azacitidine cycles (range: 1-11). Data on ASCT are presently available for 45 patients, transplanted with bone marrow (n=10 pts) or peripheral blood stem cells (n=35 pts) from an HLA-identical donor (sibling in 11, and MUD in 34 cases). Forty-five patients did not receive ASCT due to: disease relapse or progression (n=16, 35%), adverse events (n=12, 27%), refusal (n=5, 11%), or other causes (n=12, 27%).At a median follow-up of 18.5 months (range 0.2-31.5), 43 patients are alive (25 received ASCT), and 50 patients died. Causes of death are reported in Table 1. Median overall survival (OS) for the whole patient cohort was 13.4 months (95% CI: 10.9-18.6, Figure 1). Disease status after azacitidine was predictive of significantly improved survival for patients in CR/PR/HI, compared to SD or PD (n=77 patients who completed at least 4 cycles, Figure 2). ASCT considered as time-dependent covariate was associated to a significantly longer survival (p=0.008, HR 0.4, 95% CI: 0.2-0.8). Stratification of MDS according to IPSS and karyotype was not associated to survival.Conclusions Our study shows that allogeneic transplantation following azacitidine is feasible in HR-MDS/CMML and AML, with about 50% of patients being able to undergo HSCT. These data favourably compare with previous studies in MDS on ASCT preceded by conventional chemotherapy. Disease status at the time of HSCT confirms its significant prognostic role for survival, also in the context of hypomethylating treatment, where stabilization of disease also represents a therapeutic target. Disease relapse or progression remain the major causes of death indicating the need for other strategies aimed at improving disease control in MDS. [Display omitted] [Display omitted] [Display omitted] DisclosuresVoso:Celgene: Honoraria, Research Funding. Off Label Use: Azacitidine used as induction treatment before allogeneic stem cell transplantation in MDS. Finelli:Janssen: Other: Speaker; Novartis: Other: Speaker; Celgene: Other: Speaker, Research Funding.

Research progress on choice of hypomethylating treatment in myelodysplastic syndromes

The clinical manifestation and outcome of myelodysplastic syndrome(MDS) are highly heterogeneous. It is important to choose personal treatment plan according to risk groups, age, performance status and compliance. The overall response rate of hypomethylaing treatment in MDS is 40%-60%. Hypomethylating agents should be considered for fit patients who have 5% or more bone marrow blasts or who are with adverse cytogenetic characteristics. Platelets count ≥100×109/L and white blood cell count <3.0×109/L independently predict better response. The treatment of patients with HLA-DR1501 phenotype remains controversial. TET2 mutation, DNMT3A mutation, ASXL1 wild type are independent predictors of better response. This review summaries the current research progress of hypomethylating treatment in MDS on prognostic group, clinical indicators and gene mutation. Key words: Myelodysplastic syndromes; Hypomethylating agents; Decitabine; Gene mutation

Epigenetic therapy of myelodysplastic syndromes and acute myeloid leukemia.

This review will discuss issues arising along with the expanding use of hypomethylating treatment (HMT) in the management of acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). HMT has been shown to induce responses in MDS and AML, and azacitidine (Vidaza, Celgene) has been shown to prolong survival in higher-risk MDS. Recent studies have supported the idea that disease stability may also be a treatment goal, whereas treatment interruption in responding patients is associated with rapid disease relapse and death. In AML, a modest but significant survival advantage has been shown for HMT by censoring patients at the time of subsequent AML therapy, but the major limitation remains the short duration of responses. Unfortunately, some of the strategies to overcome these limitations have failed, including the combination of HMT to histone-deacetylase inhibitors, which has not definitively shown to significantly prolong survival. Molecules interfering with other pathways impacting the survival and proliferation of blasts, used alone or in combination, including guadecitabine, selinexor, or inhibitors of IDH2 mutations, are more promising approaches. Hypomethylating drugs are the first successful treatment for elderly patients with higher-risk MDS and are effective for some AML subtypes. Translational studies will hopefully identify patients with a favorable profile of response to these drugs, and help to identify newer targets for combination treatments.

Decitabine Induced the Cell Differentiation, Decreased Malignant Potential, and Increased Susceptibility to Apoptosis Via a Death Receptor Pathway with Hypomethylation of MS4A3, PAR-4, APITD1, LOX in Myelodysplastic Syndrome Cell Line, MDS-L

Introduction: Myelodysplastic syndrome (MDS) is a group of clonal myeloid disorders characterized by ineffective hematopoiesis and cytopenias. Treatment strategies are based on the balance between patients’ predicted lifespan and the anticipated risk-benefit of intervention. Current guidelines recommend to classify patients into low-risk and high-risk groups. Hypomethylating agents azacitidine and decitabine are recommended for patients with high-risk MDS, who are not eligible for intensive therapy including stem cell transplantation. Azacitidine showed a survival benefit, however, treatment with this agent has not been standardized. To further optimize the use of hypomethylating agents, the target molecules of the agents should be identified to select patients, who would best benefit from the hypomethylating treatment. In the present study, the cellular drug sensitivity, the cell differentiation, the induction of apoptosis, and DNA methylation status after the treatment with decitabine were extensively investigated using cultured MDS cell line MDS-L that had been previously established.Methods: MDS-L cells, which had been established in the previous study (Br J Haematol, 87,235-242,1994) were used. This cell line is a blastic subline derived from the bone marrow blasts of a 50-year Japanese male patients with MDS. Trypan dye exclusion assay was used for determining growth inhibition effects. The cellular morphology was observed by staining the cells with May- Grunwald-Giemsa dye. Cell surface markers were detected by using flow cytometry. The transcript of Wilms’ tumor 1 was measured by real time RT-PCR. The induction of apoptosis was determined either by the sub-G1 population in the cell cycle analysis or the caspase cleavage. The enzymatic activity of DNA methyltransferase 1 (DNMT1) was determined by using a DNMT1 activity assay kit (Active Motif, Carlsbad, CA). DNA methylation array was performed using Infinium Human Methylation 450 BeadChip (Illumina, San Diego, CA).Results: The 50%-growth inhibitory concentration of decitabine was 64 nM in MDS-L cells. The cells were incubated with a non-toxic concentration (5 nM) of decitabine for 6 months (named as MDS-L-DAC cell line). MDS-L-DAC cells proliferated more slowly than untreated MDS-L cells (doubling time:51 h). Morphologically, MDS-L-DAC cells were maturated with a lobulated nuclei and cytoplasmic granulation. Differentiation induction was also indicated by an increased CD15 positivity (9.3→30.5%) and a decreased CD34 positivity (98.8→6.4%) in MDS-L-DAC cells. Wilms’ tumor 1 transcript level was also reduced (1,400,000→420,000 copies/μgRNA) in MDS-L-DAC cells, suggesting a decreased malignant potential by the effect of decitabine. Decitabine induced greater amount apoptosis (sub-G1 :12.3%) was with caspase 3 and caspase 8 cleavage in MDS-L-DAC cells, but not in MDS-L cells (sub-G1: 5.6%). The enzymatic activity of DNMT1 was successfully inhibited by decitabine in MDS-L-DAC cells (11.2→4.5OD/h/mg). The epigenome-wide association study using DNA methylation array compared the methylation status at CpG islands between MDS-L cells and MDS-L-DAC cells. Demethylation occurred in MS4A3 (Membrane-spanning 4-domains subfamily A member 3), PAR-4 (Prostate apoptosis response 4), CD40LG (CD40 ligand), APITD1 (Apoptosis-inducing, TAF 9-like domain 1), LOX (Lysyl Oxidase), and AMPH (Amphipysin) in MDS-L-DAC cells. Western blotting confirmed that the corresponding protein levels were increased.Discussion and Conclusions: MS4A3 is associated with myeloid differentiation. Caspase-8-mediated PAR-4 cleavage is required for tumor necrosis factor α-induced apoptosis. Low expression of APITD1 interferes with the ability for apoptosis through the p53 pathway. LOX is one of the commonly methylated genes in gastric cancer. Thus, the long-term incubation with decitabine at a low concentration demethylated these gene promoters and regained their protein expression. Decitabine induced the cell differentiation, decreased malignant potential, and increased susceptibility to apoptosis via a death receptor pathway, provably due to the functions of these proteins. DisclosuresNo relevant conflicts of interest to declare.

Association Between Down-Regulation of EZH2 and Abnormal Karyotype, Response to Hypomethylation Treatment, and Patient Survival in Myelodysplastic Syndromes

EZH2 encodes a key histone methylation regulatory molecule. Genetic mutations of EZH2 occur in ~10% patients with MDS and are associated with poor prognosis. However, the expression patterns of EZH2 are less well studied in MDS. To characterize these expression patterns, we assessed EZH2 mRNA expression in primary patient bone marrow CD34+ cells (n=78) (age: 33-91; IPSS: low 24%, Int-1 31%, Int-2 27%, High 14%; Karyotype: diploid 62%, 5q-/7q- 23%, others 15%). QRTPCR assays indicated that 47% (n=37) of patients had reduced EZH2 mRNA expression (less than 50% of controls), but this finding was not statistically significant (p= 0.159). Subtype analyses based on various karyotypes revealed that EZH2 is significantly underexpressed in patients bearing chromosome 7 or 7q deletions (mean=0.4 fold, p=0.006). Seventy-five percent of patients with 7 or 7q deletions have EZH2 expression less than 50% of that of controls. Chromosome 7 deletions were also associated with lower EZH2 expression than that seen in diploidy and other cytogenetic abnormalities (p=0.041). Wepreviously found that the overexpression of a group of innate immune genes contributes to MDS pathogenesis and is related to deregulation of histone methylation. Because EZH2 is a key regulator of histone methylation, we assessed the relationship between deregulation of these genes and EZH2 under-expression. To exclude the effects of cytogenetic defects and EZH2 mutations, we studied only the subset of patients with normal karyotypes and wildtype EZH2. We surveyed capture deep sequencing results of 32 of the diploid patients from the cohort that had sequencing data available. Three patients carry EZH2 mutations, including missense (nt148511133), nonsense (nt148524257), and splicing mutations (nt148524257). In the remaining 29 patients with normal karyotypes and wildtype EZH2, 14 (48%) had EZH2 under-expression. We then compared mRNA expression of 11 innate immune genes known to overexpress in MDS between the patients with EZH2 underexpression and others. We observed that mRNA levels of all 11 immune genes tested were higher in the EZH2 underexpression group and statistically significant (p<0.05) for the genes JMJD3, IL-8, IL-1B, TLR-2, and S100-A9.We then performed survival analysis for EZH2 expression in MDS. Surprisingly, multivariate analysis in the whole cohort indicated that EZH2 underexpression is associated with better overall survival (OS) (HR 0.23, 95% CI (0.07-0.72); p=0.013). We also performed analysis in the subset without chromosome 7 deletion and observed a similar association (HR 0.18 (0.06-0.55) p=0.012). To investigate whether this result was related to responses to therapy, we reviewed treatment records and found that 61% of patients in the cohort (n=53) received hypomethylating agents (HMA). In this HMA treatment subset, non-responders (n=27) tended to have lower EZH2 expression than responders (n=26) (mean EZH2 of 0.497 vs 0.944, p=0.12). However, we noticed that in the subset of HMA responders, EZH2 expression was significantly lower (p=0.02) in patients who achieved longer responses (more than 12 months, n=15) than in those who progressed or relapsed within 12 months following treatment. We are currently investigating whether this impact of EZH2 underexpression on HMA responses contributes to its effect on OS. Taken together, the results of this study indicate that underexpression of EZH2 in the bone marrow hematopoietic progenitor cell compartment may have unique effects on the molecular pathogenesis, prognosis, and treatment of MDS and may do so through a unique mechanism that differs from that of previously characterized EZH2 mutations. Further investigations are also required to determine the relationships between EZH2, HMA-based treatments, and patient survival. DisclosuresGarcia-Manero:Epizyme, Inc: Research Funding.

Why methylation is not a marker predictive of response to hypomethylating agents

The azanucleotides azacitidine and decitabine have been shown to induce hematologic response and prolong survival in higher-risk myelodysplastic syndromes. They are inhibitors of DNA methyltransferase-1 and induce DNA-hypomethylation. Induction of apoptosis is also clinically relevant, in particular during the first treatment cycles, when cytopenia is a frequent side-effect. Since the hypomethylating effect is reversible, and the malignant clone has been shown to persist in most responding patients, several cycles are necessary to achieve and maintain responses, while treatment interruption is associated with rapid relapse. Methylation studies have shown global and gene-specific hypermethylation in myelodysplastic syndromes, but there seems to be little relation between the degree of demethylation following hypomethylating treatment and hematologic response. The presence of concurrent genomic hypermethylation and hypomethylation may impair the predictive power of current detection techniques. This scenario has been complicated by the identification of epigenetic enzyme mutations, including TET2, IDH1/2, DNMT3A and EZH2, which are important for response to hypomethylating treatment. Changes in azanucleotide metabolism genes may also play a role. In the future, methylation analysis concentrating not only on promoters, but also on gene bodies and intergenic regions, may identify key genes in patients with the highest probability of response to azanucleotides and allow a patient-tailored approach.