Leukemia-Induced Dysregulation of Bone Marrow Skeletal Stem Cells (SSC) Subpopulations and Their Hematopoietic Supportive Function

Specialized bone marrow microenvironment (BMM) niches are essential for hematopoietic stem cell (HSC) maintenance and differentiation, and include perivascular cells with mesenchymal stem cells (MSC) capacity. The role of BMM niches in regulating LSC growth remains unclear. We have shown that cytokines altered in CML BMM contribute a competitive growth advantage to CML LSC over normal HSC (Zhang et al. Cancer Cell. 2012; 21(4):577). However, the effect of leukemic hematopoiesis on BM MSC and their progeny, and the effect of such alterations on the regulation of LSC versus HSC growth, is not well studied. Recently rigorous single-cell analyses and lineage tracing has been used to delineate a BM skeletal stem cell (SSC) hierarchy (Chan et al. Cell. 2015, 160(1-2):285). Eight distinct subpopulations were identified within the CD45-Ter119-Tie2-AlphaV+ BM mesenchymal cells, of which the mSSC (Thy-6C3-CD105-CD200+) subpopulation generated all of the other subpopulations. Two multipotent progenitor populations, pre-BCSP (Thy-6C3-CD105-CD200-) and BCSP (Thy-6C3-CD105+) gave rise to bone, cartilage and hematopoietic supportive stroma. The 6C3 (Thy-6C3+CD105+) and HEC (Thy-6C3+CD105-) populations gave rise to stroma, the BLSP (Thy+6C3-CD105-) and Thy (Thy+6C3-CD105+CD200-); populations formed bone, and the PCP (Thy+6C3-CD105+CD200+) population formed cartilage. To examine how these mesenchymal populations related to those targeted in the Cre-transgenic mouse lines commonly used to study BMM, we performed lineage tracing by crossing Prx-1-Cre, Sp7-Cre, BGLAP-Cre, and UBC-Cre mice with Rosa26^CAG-tdTomato mice, in which tdTomato is expressed by cells targeted for Cre-mediated recombination. Interestingly, mSSC and 6C3 cells were targeted in all Cre lines, while the Thy population was only targeted in Prx1-Cre mice. We examined SSC population distribution and function following CML development using the SCL-tTA-BCR-ABL transgenic mouse model. The frequency and absolute number of bone-forming Thy (WT: 442.5±65.66; CML: 2501±705.5; p=0.012, n=14) and stroma-forming 6C3 (WT:1061±193.7; CML: 2271±358.5; p=0.009, n=14) cells were significantly increased in BM of CML mice compared to wild-type (WT) mice, while primitive mSSC, pre-BCSP and BCSP populations remained unchanged. Expression of major hematopoietic regulatory molecules, transcription factors and cell cycle regulatory genes in CML and WT BMM subsets was measured by qPCR analysis using the Fluidigm system. Integrin α4 and Tenascin levels were reduced, whereas PDGFRβ, BMP2 and Hif-1α levels were increased in CML compared to normal mSSC. Several WNT pathway members were significantly upregulated in CML compared to WT 6C3 stromal cells, whereas CXCL12, KitL, IL-3, and IGF-1 were reduced. Expression of cell cycle regulators, Hedgehog and Notch pathway members remained unchanged. In ongoing experiments to investigate whether these subsets have altered HSC supportive capacity towards normal and CML stem cells, we co-cultured FACS sorted normal or CML LTHSC with FACS sorted normal or CML mSSC, BCSP, 6C3, and Thy cells for 3 days followed by transplantation into lethally irradiated WT mice. Normal LTHSC engraftment at 4 weeks post-transplant was enhanced by coculture with normal mSSC but not with CML mSSC. In contrast, there was increased engraftment of normal LTHSC co-cultured with CML compared to normal BCSP, Thy or 6C3 cells. CML LTHSC engraftment at 4 weeks was not enhanced by either normal or CML mSSC, but was enhanced following co-culture with normal as well as CML Thy and 6C3 cells. These results indicate that SSC subpopulations from CML BM demonstrate altered capacity to support normal LTHSC compared to their normal counterparts, but that CML and WT SSC populations may have similar effects on CML LTHSC. This experiment is ongoing with results of longer-term engraftment pending. In conclusion, we have further characterized a phenotypically-defined skeletal stem cell hierarchy and identified significant alterations in distribution of SSC subpopulations within CML compared to WT BM, with significantly altered expression of important hematopoietic regulatory molecules and HSC supportive function. We hypothesize that leukemia-induced SSC alterations may confer a growth advantage to CML LSC over normal HSC, and that characterization of key regulatory mechanisms could facilitate therapeutic intervention.