Abstract:
Chronic myeloid leukemia (CML) is a myeloproliferative disorder originating from BCR::ABL1-transformed leukemic stem cells (LSCs). The advent of tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1 has revolutionized the treatment of CML and led to deep molecular remission in most patients. However, despite the benefits, TKI-insensitive LSCs remain in most patients for prolonged periods, standing in the way of a complete cure. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still the only established curative therapy for patients with TKI failure. But this is frequently impractical for elderly patients who make up the majority of CML cases.
The bone marrow microenvironment (BMME) plays a pivotal role in the pathogenesis of leukemia by regulating and protecting LSCs, and disease relapse is tightly linked to BMME. The interaction of leukemic cells with the BMME is bidirectional. It has been speculated that leukemic cells hijack and destroy normal hematopoietic stem cell supportive niches, and transform the BM into leukemia growth-supportive niches. This skewed BMME is associated with drug resistance and relapse, yet the underlying mechanisms and interactions remain incompletely understood. Therefore, characterizing the phenotypic, functional, and spatial properties of each cell within the BMME could facilitate a “two-pronged” treatment targeting both the leukemic cells and their interactions with the BMME to create an unfavorable environment for leukemia progression.
During my thesis, we established a spatiotemporal single-cell atlas of the BMME in a murine model of CML. We analyzed femoral sections from wild-type and CML mice at 7, 14 and 21 days post-induction. Using a 54-marker CODEX panel, we profiled 2,033,725 cells in 55 tissue regions and identified 41 cell types through unsupervised clustering and supervised annotation. During leukemic progression, we observed an expansion of myeloid and progenitor cell populations, increased PD-L1+ leukemic cells, the upregulation of PD-1 on CD4+ and CD8+ T cells, and a profound loss of B cells, plasma cells and bone cells. Spatial mapping revealed leukemia-specific cellular neighborhoods enriched in PD-1+CD8+ T cells, suggesting localized immune cell exhaustion.
We next investigated how leukemia reshapes vascular niches. In advanced CML, we observed a massive angiogenic expansion in the BM; however, these neovessels were immature, characterized by exuberant branching, marked loss of pericyte support, and a displacement of hematopoietic stem/progenitors and other committed progenitors away from the vasculature. Consistently, 3D BM imaging revealed large contiguous territories of disorganized vessels, indicating global disruption of perivascular niche integrity.
We further analyzed megakaryocyte biology and immune–stromal cross-talk. Early-stage CML showed increased contacts between plasmacytoid dendritic cells and megakaryocytes, whereas advanced CML featured heightened megakaryocyte emperipolesis of non-leukemic granulocytes. Megakaryocytes were morphologically irregular in CML mice and BM trephine biopsies from CML patients. In contrast, in mice with acute myeloid leukemia, vasculature and megakaryocytes were reduced, while remaining megakaryocytes retained normal morphology. Laser-capture microdissected megakaryocytes from newly diagnosed CML patients had reduced expression of cytoskeleton genes, which was reversed in advanced cases treated with tyrosine kinase inhibitors. 3D imaging revealed depleted megakaryocytes in the diaphysis, underscoring region-specific pathology.
To extend these spatial profiling approaches toward clinically relevant human material, an additional part of this thesis focused on the establishment of multiplex imaging workflows for formalin-fixed paraffin-embedded (FFPE) bone marrow. While the murine studies were based on fresh-frozen tissue, translation to human disease requires robust analysis of archival FFPE specimens, which remain the standard format for clinical bone marrow biopsies. In this context, a 65-plex CODEX antibody panel for human FFPE bone marrow was developed and optimized. Using a clinically annotated cohort of 109 patients with myelodysplastic syndromes (MDS) who underwent allogeneic hematopoietic cell transplantation, six tissue microarrays were generated from archived bone marrow biopsies, with three cores per patient to account for spatial heterogeneity. This part of the project established the technical and conceptual basis for high-dimensional spatial profiling of human MDS bone marrow and for investigating whether tissue architecture and microenvironmental organization may provide information relevant to transplant outcome.
Taken together, this thesis demonstrates that myeloid malignancies are associated with profound remodeling of the BMME. In CML, leukemic progression was linked to vascular immaturity, spatially organized immune suppression, and megakaryocyte dysplasia, all of which may contribute to leukemic persistence and resistance. In parallel, the translational extension to human FFPE BM established a framework for applying high-dimensional spatial proteomics to clinically annotated patient material. The core CML findings summarized here were previously reported in the preprint and the subsequently published Blood article arising from the same study, or which I am the sole first author, whereas the human FFPE MDS component represents an additional translational extension included in this dissertation.
Chronic myeloid leukemia