Low Plasma Citrate Levels and Specific Transcriptional Signatures Associated with Quiescence of CD34+ Progenitors Predict Azacitidine Therapy Failure in MDS/AML Patients

Abstract

To better understand the molecular basis of resistance to azacitidine (AZA) therapy in myelodysplastic syndromes (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), we performed RNA sequencing on pre-treatment CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 25 MDS/AML-MRC patients of the discovery cohort (10 AZA responders (RD), six stable disease, nine progressive disease (PD) during AZA therapy) and from eight controls. Eleven MDS/AML-MRC samples were also available for analysis of selected metabolites, along with 17 additional samples from an independent validation cohort. Except for two patients, the others did not carry isocitrate dehydrogenase (IDH)1/2 mutations. Transcriptional landscapes of the patients’ HSPCs were comparable to those published previously, including decreased signatures of active cell cycling and DNA damage response in PD compared to RD and controls. In addition, PD-derived HSPCs revealed repressed markers of the tricarboxylic acid cycle, with IDH2 among the top 50 downregulated genes in PD compared to RD. Decreased citrate plasma levels, downregulated expression of the (ATP)-citrate lyase and other transcriptional/metabolic networks indicate metabolism-driven histone modifications in PD HSPCs. Observed histone deacetylation is consistent with transcription-nonpermissive chromatin configuration and quiescence of PD HSPCs. This study highlights the complexity of the molecular network underlying response/resistance to hypomethylating agents.

Noncoding RNAs and Their Response Predictive Value in Azacitidine-treated Patients With Myelodysplastic Syndrome and Acute Myeloid Leukemia With Myelodysplasia-related Changes

“…In the process of epigenetic modulation, DNMT1 (a maintenance DNA methyltransferase) was defined as one of the core PCGs related to the AZA response. Previously, it was demonstrated that DNMT1 serves as a pharmacologic target of HMAs (65), and down-regulation of DNMT1 expression in AZA nRESPs has been attributed to noncycling status and reduced replication of HSPCs in these patients (18). Another core PCG of epigenetic mechanisms involved in the response to AZA was the EZH2 gene, which was also specifically down-regulated in our cohort of AZA nRESPs.…”

“…Current research activities are usually focused on the identification of an ideal, single biomarker whose alterations (such as a mutational change or transcription deregulation) would be predictive of treatment response with high sensitivity and specificity. The patient capability to respond to AZA and the identification of prediction markers have been repeatedly addressed (11,12,18). However, given the genomic heterogeneity of the disease, no universal biomarker has been identified thus far.…”

“…Previously, the quiescent phenotype of HSCs was proposed as a key factor in the prediction of treatment outcome in MDS (64). In a preceding publication, it was identified that the BM of AZA RESPs contains actively cycling cells poised for erythromyeloid differentiation, whereas in nRESPs, BM HSPCs display a signature of a quiescent state (repression of genes related to active cell cycling, replication, and DNA damage response) (18). These metabolic pathways seem to be closely interconnected with epigenetic modifications; the noncycling status has been associated with decreased expression of the HIST1 cluster and therefore with the transcription-nonpermissive chromatin configuration maintaining cell quiescence (18).…”

“…In a preceding publication, it was identified that the BM of AZA RESPs contains actively cycling cells poised for erythromyeloid differentiation, whereas in nRESPs, BM HSPCs display a signature of a quiescent state (repression of genes related to active cell cycling, replication, and DNA damage response) (18). These metabolic pathways seem to be closely interconnected with epigenetic modifications; the noncycling status has been associated with decreased expression of the HIST1 cluster and therefore with the transcription-nonpermissive chromatin configuration maintaining cell quiescence (18). In this study, it was confirmed that AZA nRESPs display suppressed expression of epigenetic regulators and many chromatin related genes (especially those from the HIST1H and HIST2H families) in addition to genes involved in recombinational repair.…”

“…To provide biological insights into the contribution of noncoding RNAs (ncRNAs) to the AZA response or resistance and to propose novel appropriate molecular markers able to predict the response, whole transcriptome RNA sequencing (RNA-seq) was performed in CD34+ BM cells in patients with higherrisk MDS and AML with myelodysplasia-related changes (AML-MRC) before AZA therapy. A more detailed analysis of PCG data was not the major aim of this study, as they have already been addressed in a previous study (18). This study focused on the noncoding transcriptome and paid special attention to the examination of various classes of ncRNAs as predictive markers of the AZA response and compared their power to predict the response with the predictive power of PCGs.…”