The Transcriptional Impact of 5q Deletion in MDS at Single Cell Resolution
Authors: Diaz-Mazkiaran Aintzane , Serrano Guillermo , Berastegui Nerea , Garcia-Olloqui Paula , Huerga Sofia , Pierola Ana Alfonso , Ullate-Agote Asier , Ariceta Beñat , Ainciburu Marina , Vilas-Zornoza Amaia , San-Martin Patxi , Aguirre-Ruiz Paula , Lamo De Espinosa Jose Maria , Acha Pamela , Calvete Oriol , Jimenez Tamara , Molero Antonieta , Montoro Julia , Diez-Campelo Maria , Valcarcel David , Sole Francesc , Ochoa Idoia , Ezponda Teresa , Prosper Felipe , Hernaez Mikel 
Published: December 2023

Introduction: Myelodysplastic syndromes with 5q deletion (del(5q) MDS) are characterized by two commonly deleted regions (CDRs): 5q31 and 5q32-33. Although several genes within these CDRs have been associated with disease pathophysiology, the molecular basis underlying its origin is still unknown. Lenalidomide represents the first therapeutic approach for del(5q) MDS, but patients experience loss of responsiveness within 2-3 years, highlighting the need of characterizing the mechanisms impairing treatment response. While previous studies have identified dysregulated genes and pathways in the disease, they overlooked that the hematopoietic system of these patients is composed of a mixture of cells with and without the deletion (del(5q) and non-del(5q) cells), which hampers the ability to unravel the real transcriptional impact of the deletion.
Methods: We isolated by FACS and performed single-cell RNA sequencing (scRNAseq, 10XGenomics Chromium platform) of CD34+ cells from 4 untreated patients, 2 patients treated with lenalidomide (partial (PCR) and complete (CCR) cytogenetic responders), and 3 healthy elderly donors. To identify del(5q) and non-del(5q) cells, the copy number alteration (CNA) inference methods CopyKat and CaSpER were applied. To unveil the transcriptional alterations prompted by the deletion, differential expression (DE), gene regulatory network (GRN) and cell-to-cell communication (CCC) analyses were performed (Figure 1).
Results: CNA methods robustly identified del(5q) cells and non-del(5q) cells, which was consistent with the del(5q) percentage obtained by karyotyping. At diagnosis, del(5q) cells were detected in all hematopoietic progenitors, including the hematopoietic stem cells (HSC), suggesting that the deletion occurs in the earliest stages of hematopoietic differentiation. Although the distribution of del(5q) cells was highly heterogeneous among patients (Figure 2), a positive enrichment of del(5q) cells was detected in erythroid progenitors, underscoring the role of this genetic lesion in the anemia characterizing del(5q) MDS.
To delve into the transcriptional mechanisms differentiating del(5q) and non-del(5q) cells, we performed DE and GRN analyses. Only 7 genes were downregulated in del(5q) cells; however, most play a key role in MDS and other tumors, such as PRSS21, MAP3K7CL, and CCL5. GRN analyses identified regulons JARID2, IRF1 and KAT6B, known to be key for proper hematopoietic differentiation, showing less activity in del(5q) cells. Likewise, the regulons RERE and KDM2A, which downregulated genes involved in ribosomal processes, were more active in del(5q) cells, supporting the concept that cells harboring the deletion may have a more prominent role in promoting altered hematopoiesis. However, comparisons against healthy donor cells suggested that non-del(5q) cells could share similar, albeit attenuated, abnormal behavior to del(5q) cells. In line with these results, CCC analyses identified minimal differential interactions in del(5q) cells with respect to non-del(5q) cells, indicating that the deletion does not confer a differential communicative potential to the cells. Importantly, several interactions present in MDS cells were absent in healthy cells, such as the AGTRAP-RACK1, whose members have been postulated as potential therapeutic targets for promoting proliferation in other cancers.
After lenalidomide, del(5q) and non-del(5q) cells from PCR and CCR patients showed higher protein degradation and erythropoietin signaling, as well as higher activity of the PD-L1/PD-1 pathway, suggesting a potential immunosuppressive mechanism of these cells after treatment. Even if several transcriptional alterations detected at diagnosis were reverted after lenalidomide, ribosomal translation processes were still altered, even at time of CCR. These results evidence that, after lenalidomide, non-del(5q) progenitor cells revert some of the transcriptional alterations that present at diagnosis, while maintaining other lesions that could be relevant for abnormal hematopoiesis, and potentially, for the future relapse of the patients.
Conclusions: This study provides the first characterization of del(5q) and non-del(5q) cells at the single-cell resolution and reveals previously unknown transcriptional alterations that could contribute to disease pathogenesis or less responsiveness to lenalidomide.