Niche Heterogeneity Impacts Evolution of Myeloproliferative Neoplasms Driven By the Same Oncogenic Pathway

Different MPNs have distinct rates of malignant transformation (PMF>PV>ET). Although PV and ET can arise from hematopoietic stem/progenitor cells (HSPCs) with similarly activated JAK-STAT oncogenic pathway, the transformation rate into secondary myelofibrosis and leukemia is higher for PV than for ET. However, the underlying reasons are not fully clear. Whereas in some cases secondary mutations might cause transformation, it remains unclear whether distinct bone marrow (BM) microenvironments can influence the progression of MPNs or any preleukemic disorder. Previous studies have suggested that normal BM niches close to bone (endosteal) promote HSPC quiescence, whereas non-endosteal vessels permit transmigration of activated HSPCs. We hypothesized that PV and ET HSPCs might expand in different BM niches, which could thereby influence disease progression.

To address this question, we performed combined two-photon and confocal real-time intravital microscopy in the skull BM of WT mice transplanted with HSPCs from WT mice or MPN mice carrying the same driver mutation (JAK2^V617F) but showing phenotypically distinct diseases (ET or PV). ET HSPCs resembled WT HSPCs in their homing and engraftment near bone (22±15 and 26±13 μm, respectively) 3 days after i.v. injection into lethally-irradiated WT mice. In contrast, PV HSPCs located significantly further (31±21 μm) from bone, which was independently confirmed in a distinct PV model. The different homing of ET and PV HSPCs was similarly observed in non-irradiated WT recipients and was therefore independent of myeloablative conditioning. Following engraftment, ET and PV HSPCs preferentially expanded in endosteal and non-endosteal locations, respectively, over 7 months' follow up. Importantly, the asymmetric expansion of HSPCs in ET and PV was confirmed in human BM trephines. Human CD34+ HSPCs were significantly closer to bone in ET than in PV patients (86±2 vs. 109±6 μm). Together, these results suggest that mutated HSPCs preferentially occupy distinct BM niches in ET and PV.

Murine ET and PV HSPCs also differed in their dynamic interactions with the microenvironment. ET HSPCs migrated significantly faster (1 μm/min) than PV HSPCs (0.8 μm/min) and covered longer tracks after 1 h (57±2 and 35±1 μm, respectively). Moreover, ET HSPCs (but not PV HSPCs) migrated faster when closer to bone, suggesting an exploratory strategy of ET HSPCs to find endosteal niches. Separation of endosteal and non-endosteal BM fractions revealed increased abundance of integrin β3+ HSPCs in the endosteal BM of ET mice carrying JAK2^V617F or CALR^del52/del52 mutations, but not in different PV models (despite generally sharing oncogenic JAK-STAT activation). Furthermore, competitive gravity adhesion assay and interference reflection microscopy showed that ET HSPCs are more adhesive to ECM substrates of integrin β3, suggesting that integrin β3 might trigger endosteal lodging of ET HSPCs.

Asymmetric HSPC expansion caused differential microenvironmental remodeling possibly explaining differences in the pathophysiology and secondary outcomes of ET and PV. Non-endosteal sinusoids were dilated in different PV (but not ET) models, whereas CD31^hiSca1^hi arterioles and aberrant bone-forming integrin β1⁺ blood vessels increased only in ET mice. Similarly, sinusoidal vessels showed increased coverage by the integrin β1 ligand laminin α4 only in ET mice. Consequently, increased bone (μCT), osteoblasts and osteoclasts were found in ET but not PV mice. Increased expression of vascular-derived bone-forming factors (such as Bmp1 and Dll4) downstream of endothelial laminin α4/integrin β1 signaling might trigger osteosclerosis in ET mice.

Finally, we tested whether HSPC location might directly impact ET progression. In a separate study we found that β₃-adrenergic receptor (AR) signaling regulates Cxcl12-dependent BM HSPC localization (ASH abstract ID 116015). Therefore, we transplanted WT or ET donor BM cells into WT and β₃-AR KO mice. Measurement of peripheral blood counts over 28 weeks showed that the microenvironment lacking β₃-AR significantly worsened thrombocytosis and leukocytosis in ET, which correlated with redistribution of HSPCs and their progeny away from bone towards central BM.

Altogether, these results suggest that differential interactions with the microenvironment might impact disease progression in MPNs and possibly in other preleukemic disorders.