Distinct Role of STAT3 and STAT5 Factors in MDS and AML

Introduction: Myelodysplastic Syndrome (MDS) refers to a heterogeneous group of clonal hematologic disorders characterized by hematopoietic dysfunctions and impaired differentiation in the hematopoietic stem and progenitor cells compartment. MDS is commonly referred to as a pre-leukemic stage due to the increased risk of progression to Acute Myeloid Leukemia (AML). AML is an aggressive hematologic malignancy characterized by the accumulation of immature myeloblasts in the bone marrow and the peripheral blood. Signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) are important regulators of several cellular processes including cell proliferation and survival. Abnormal STAT3 and STAT5 signaling has been implicated in various solid and hematologic malignancies, including MDS and AML, which renders them appealing targets for the development of novel therapeutic strategies.

Materials: STAT3, STAT5A or STAT5B knock-downs have been generated in two AML (MOLM-13 and Kasumi-1) and two MDS (MDS92 and MDS-L, gift of Prof. Kaoru Tohyama) cell lines, using shRNAs through lenti-viral delivery. The 4 biological replicates with the highest levels of knock-downs for STAT5A, STAT5B or STAT3 have been selected for each cell line and mRNA sequencing has been performed. To define the genome-wide binding of STAT5A, STAT5B and STAT3 in the two conditions, CUT&Tag has been performed in wild type MOLM-13, Kasumi-1 and MDS-L cells. Additionally, ATAC sequencing has been carried out in the same cell lines to investigate the chromatin conformation changes between the two conditions. Furthermore, STAT3, STAT5A and STAT5B knock-downs have been conducted using shRNAs through lenti-viral delivery in CD34+ enriched cells, isolated from bone marrow mononuclear cells of 14 high-risk MDS patients and 14 secondary (MDS-related) AML patients. The samples have been processed for mRNA-Sequencing to model the leukemic transformation in primary human cells.

Results: To elucidate the role of STAT3 and STAT5 in MDS to AML transformation, changes in STAT3 and STAT5 target gene networks have been identified. mRNA sequencing data from STAT3, STAT5A or STAT5B knock-downs in MDS and AML cell lines revealed a distinct role of the three factors in each state. The differential binding of each factor, explored through CUT&Tag, and the gene expression profiles in MDS and AML reciprocally reveal a different function of each factor, including pathways involved in cell cycle and apoptosis. Also, the direct and indirect target genes of STAT3, STAT5A and STAT5B in each condition, as well as a possible interplay between STAT3 and STAT5 activation in AML has been uncovered. The exploration of the chromatin accessibility landscapes between the MDS and AML conditions through ATAC sequencing has provided an insight into the chromatin conformation states, unveiling potential leukemia promoting networks, as well as the involvement of STAT3 and STAT5 pathways in the malignant transformation. Furthermore, a potential cross talk of STAT3 and STAT5 factors with the IKAROS family members, has been linked to AML transformation, as significant differential regulation has been detected between MDS and AML conditions after knock-down of STAT5A and STAT5B. The results are currently validated in primary hematopoietic cells of MDS and AML patients after STAT3, STAT5A or STAT5B knock-downs.

Conclusions: Our results show a different role between the two STAT5 factors (STAT5A and STAT5B) in MDS and AML conditions and delineate the role of STAT5A, STAT5B and STAT3 factors and their target gene networks in leukemic transformation, thus providing novel targets for therapeutic management of MSD and AML.

Funding: This project is funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813091 (ARCH)