Accelerated Progression in Lower-Risk Myelodysplastic Neoplasms: Attenuated Cell Cycle, DNA Damage Response, and Augmented Oncogenic Transcriptome Signatures

Patients with myelodysplastic neoplasms (MDS) face the risk of transformation to acute myeloid leukemia (AML). Although prognostic scoring systems exist for risk stratification and treatment decision making in MDS patients (Garcia-Manero G. Am J Hematol. 2023;98(8):1307-1325), disease management remains challenging due to the heterogeneity of clinical courses and long-term outcomes. The natural history of patients with lower-risk MDS (LR-MDS) is very heterogeneous and some LR-MDS patients experience rapid progression despite a generally favorable prognosis.

The aim of our study was to uncover the molecular mechanisms underlying accelerated progression in hematopoietic stem/progenitor cells of patients with LR-MDS regardless of driver mutations. We focused on the transcriptome of bone marrow (BM) CD34 ⁺ cells from diagnostic samples using RNA-seq and used various bioinformatic pipelines to examine differentially expressed protein-coding genes, and long non-coding RNAs (lncRNAs) as well as differential alternative splicing events.

RNA-seq dataset of CD34 ⁺ ribodepleted RNA samples from 53 LR-MDS patients without accelerated progression (stMDS) and 8 who progressed within 20 months (prMDS) showed 845 differentially expressed genes (ІlogFCІ > 1, FDR < 0.01) between these groups. CD34 ⁺ cells of prMDS exhibited a transcriptional pattern of quiescent-like cell state with overall decreased metabolism signatures and significantly reduced lineage differentiation compared to stMDS CD34 ⁺ cells. Gene set enrichment analysis (GSEA) showed that cell cycle- and cell cycle checkpoint- associated genes and activation of ATR in response to replication stress were all significantly under-represented in prMDS CD34 ⁺ BM (Fig. 1A). Cellular pathways from GO Biological Processes associated with cellular responses to stress and DNA damage responses (DDR) were downregulated in prMDS compared to stMDS. The only GO terms that were significantly upregulated in prMDS were processes related to cell-matrix adhesion or cell-cell adhesion. Indeed, key transcription factor controlling changes in cell-cell adhesion, ZEB1, was significantly overexpressed in prMDS BM CD34 ⁺ cells, whereas E-cadherin expression ( CDH1) was decreased; the expression levels of CDH1 and ZEB1 showed a significant moderate negative correlation (Fig. 1B).

Additionally, prMDS samples exhibited high levels of aberrant splicing and global lncRNA expression. Analysis of aberrant transcripts, deregulated lncRNAs and their targets suggested their contribution to the attenuation of DDR pathways in prMDS. Given that the downregulation of DDR gene expression was the major feature distinguishing the prMDS CD34 ⁺ cell transcriptome from that of stMDS in our cohort, we selected - from the transcriptional profile of the top 50 downregulated genes in GSEA - a prognostic DDR-associated gene signature consisting of 19 genes. Using regularized Cox regression analysis of the expression profile of 19 DDR-associated genes, the probability of a progression-free disease was calculated in our cohort. High DDR gene expressors had a significantly higher probability of progression-free disease (p < 0.001) compared to low DDR gene expressors (Fig. 2A).

Among the top 50 upregulated genes in the prMDS vs. stMDS samples, ranked by GSEA, overexpression of previously identified markers of leukemic progression and/or poor MDS/AML survival ( SPNS2, MN1, DOCK1 and others) was demonstrated. Interestingly, overexpression of NEK3, a gene not previously associated with MDS/AML progression, showed a predictive power for accelerated progression in LR-MDS with the best significance of curve separation among the genes tested (Fig. 2B). Furthermore, NEK3 expression was an independent prognostic factor (p<0.001) for progression-free disease in a multivariate analysis of important clinical and genetic factors. To conclude, our data suggest that DDR gene expression signature and NEK3 expression appear to be a significant predictor of LR-MDS progression and may be applicable at the time of diagnosis for the prognostic stratification of patients with LR-MDS, regardless of their mutational status.

Supported by AZV (NU21-03-00565) and (NU23-03-00401); MH CZ-DRO (UHKT, 00023736); Program EXCELES, ID Project No. LX22NPO5102, and UP Young Researcher Grant Competition, ID Project JG_2023_016.