Investigating the Complex Interplay Between Leukemic Stem Cells, Their Myeloid Progeny and Cells from the Bone Marrow Microenvironment in Generating Myeloproliferative Diseases

Leukemic stem cells (LSCs) are operationally defined as the cell population within the tumor capable of transplanting leukemia to recipient mice. Considering the importance of the bone marrow (BM) microenvironment in maintaining normal hematopoietic functions, and the recent evidence suggesting that dysfunctional microenvironment can drive myeloproliferative disorder (MPD), we questioned

• how microenvironmental deregulations contribute to the aberrant functions of primary LSCs (pLSCs) in MPD pathogenesis; and

• whether transplanted LSCs (tLSCs) could recreate these microenvironmental deregulations.

To address these questions, we used our junB-deficient mouse model of MPD (junB^flox/floxMore-Cre) in which deletion of junB occurs in both the hematopoietic system (starting from the hematopoietic stem cell (HSC) compartment) and the microenvironment; and where pLSCs have been identified as aberrantly behaving HSCs. To study the bone morphology changes in primary junB-deficient mice (mainly hematopoietic-derived bone-resorbing osteoclasts and bone-forming osteoblasts) and the impact of tLSC on bone morphology of transplanted wild type recipients, we performed dual-energy X-ray absorptiometry (DEXA) analysis. Primary mice were found to have decreased bone mineral density (BMD) in both cortical and trabecular bone, whereas no changes were observed in transplanted mice that were still at early stages of MPD development (approximately 3–6 months post transplantation). We are currently following these transplanted animals to determine whether disease progression leads to a decrease in BMD. Conversely, we also transplanted wild-type hematopoietic cells into junB-deficient mice to determine whether the junB-deficient BM microenvironment could by itself drive MPD development. Preliminary analysis revealed a limited expansion of wild-type myeloid cells, which did not progress into frank MPD over time but correlated with a partial correction of the BMD loss afflicting the junB-deficient recipient mice. To identify which junB-deficient hematopoietic cells are responsible for remodeling the BM microenvironment, we then used mice with junB deletion restricted to cells of the granulocytic/macrophage lineage (junB^flox/floxhMRP8-Cre-ires/GFP or junB GM-deficient mice). While young junB GM-deficient mice did not display any major changes in the myeloid lineage, around half of the old junB GM-deficient mice (~10–15 months) displayed MPD development. Although, we are still investigating the phenotype and transplantability of this particular MPD, we observed a strict correlation between its development and BMD loss, clearly demonstrating the involvement of junB-deficient myeloid cells in this remodeling process. Altogether, these results indicate that BMD loss is most likely a secondary consequence of MPD development, which results from the progressive over-production of junB-deficient myeloid cells. These dysfunctional junB-deficient myeloid cells impact on the BM microenvironment thereby creating a “leukemic niche”, which in turn can contribute to and/or support the expansion of the myeloid compartment. To directly study the effect of junB-deficient hematopoietic cells on cells of the osteoblastic lineage, we then used a flow cytometry-based approach to enumerate osteo-lineage cells (OSBs) in primary and transplanted junB-deficient mice. Upon MPD development, we observed in both cases increased numbers of functionally aberrant OSBs, which were able to induce overproliferation of wild-type HSCs in an ex vivo assay. We are currently analyzing how junB-deficient hematopoietic cells (mainly myeloid cells) can deregulate the function of wild type OSBs, and extending our analysis to determine the number and activity of osteoclasts in both junB-deficient mice (primary and transplanted) and junB GM-deficient mice. Altogether, our findings suggest that the primary function of junB-deficient LSC is to establish and maintain a constant production of dysfunctional myeloid cells, which then in turn alter the normal function of constituents of the BM microenvironment leading to both BM niche remodeling and MPD pathogenesis. Our future studies are aimed at understanding how the complex interplay between LSCs, their myeloid progeny and cells from the BM microenvironment is established at the molecular level.