Characterizing Molecular Pathways in Waldenström’s Macroglobulinemia and Myelodysplastic/Myeloproliferative Neoplasms with Neutrophilia Using Single-Cell RNA Sequencing Technology

Hematologic malignancies are characterized by remarkable cellular and molecular heterogeneity, underlying their variable clinical behavior and therapeutic response. Single-single-cell RNA sequencing (scRNA-seq) technologies have recently gained increasing success, enabling the study of biological heterogeneity by analyzing transcriptome expression at single cell resolution. This thesis investigates through single cell transcriptomics two hematologic contexts, Waldenström’s macroglobulinemia (WM) and myelodysplastic/myeloproliferative neoplasms with neutrophilia (MDS/MPN-N), with the overarching aim of characterizing molecular pathways driving lineage differentiation and microenvironmental interactions, and identifying novel therapeutic opportunities. The first part of the thesis focuses on WM, a lymphoplasmacytic lymphoma characterized by excess IgM secretion and recurrent MYD88 and CXCR4 mutations. Despite significant advances, the role of the immune and stromal microenvironment in disease progression and therapeutic resistance remains incompletely understood. This work dissected the molecular interplay between malignant cells and their niche, uncovering pathways sustaining tumor growth. Using patient-derived samples and co-culture models, we showed that WM cells remodel the immune microenvironment by promoting the expansion and activation of regulatory T cells (Tregs). Transcriptomic profiling revealed that Tregs from WM patients display enhanced activation of NF-κB, MAPK, and PI3K/AKT pathways. ScRNA-seq was used to investigate tumor–immune crosstalk and cellular heterogeneity, leading to the identification of the CD40/CD40-ligand (CD40L) axis as a key mediator of the B-cell/Treg crosstalk. Pharmacologic inhibition of CD40/CD40L signaling reduced Treg induction and suppressive function, and decreased WM cell proliferation, suggesting that blockade of this pathway could reprogram the immunosuppressive milieu and restrain tumor growth. Building on these insights, the second part of the thesis focuses on MDS/MPN-N, a rare myeloid disorder characterized by overlapping dysplastic and proliferative features, neutrophilia, and a high degree of clonal heterogeneity. To study this clonal disorder, we developed a conditional mouse model where the expression of mutated SETBP1, an oncogene frequently active in the context of MDS/MPN-N, is restricted to the hematopoietic bone marrow, and causes a myeloid leukemia very similar to its human counterpart. Given these premises and taking into account the absence of disease-specific therapies, I subsequently studied the effect of the epigenetic modulator azacitidine (aza) in a longitudinal cohort of three MDS/MPN-N patients who were treated with this compound as an off-label therapy. Aza improved hematologic parameters, restoring granulocytic and erythroid maturation, reducing transfusion dependence, and stabilizing leukocyte and platelet counts. Longitudinal molecular monitoring through scRNA-seq showed that aza reshapes stem and progenitor cell behavior rather than acting solely via cytotoxicity. To dissect the cellular basis of these effects, scRNA-seq was employed to analyze hematopoietic stem and progenitor populations before and after azacitidine treatment. This analysis revealed that aza promotes differentiation along specific hematopoietic lineages, reduces immature progenitors, and restores erythroid lineage commitment through suppression of PU.1 and reactivation of the GATA2-to-GATA1 transcriptional switch. Key regulators of stem cell fate, including MECOM and EYA1, were modulated, supporting a shift toward physiologic proliferation and maturation. Overall, this thesis demonstrates that WM and MDS/MPN-N are shaped by complex interactions between differentiation programs and the microenvironment. The findings illustrate how scRNA-seq approaches can uncover dynamic cellular responses to therapy and highlight potential targets in heterogeneous hematologic malignancies.