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• An in vivo model of MDS displays time-dependent defects in HSPCs and in microenvironmental populations.• Normalization of the marrow microenvironment alters disease progression and transformation and improves hematopoietic function.In vitro evidence suggests that the bone marrow microenvironment (BMME) is altered in myelodysplastic syndromes (MDSs). Here, we study the BMME in MDS in vivo using a transgenic murine model of MDS with hematopoietic expression of the translocation product NUP98-HOXD13 (NHD13). This model exhibits a prolonged period of cytopenias prior to transformation to leukemia and is therefore ideal to interrogate the role of the BMME in MDS. In this model, hematopoietic stem and progenitor cells (HSPCs) were decreased in NHD13 mice by flow cytometric analysis. The reduction in the total phenotypic HSPC pool in NHD13 mice was confirmed functionally with transplantation assays. Marrow microenvironmental cellular components of the NHD13 BMME were found to be abnormal, including increases in endothelial cells and in dysfunctional mesenchymal and osteoblastic populations, whereas megakaryocytes were decreased. Both CC chemokine ligand 3 and vascular endothelial growth factor, previously shown to be increased in human MDS, were increased in NHD13 mice. To assess whether the BMME contributes to disease progression in NHD13 mice, we performed transplantation of NHD13 marrow into NHD13 mice or their wild-type (WT) littermates. WT recipients as compared with NHD13 recipients of NHD13 marrow had a lower rate of the combined outcome of progression to leukemia and death. Moreover, hematopoietic function was superior in a WT BMME as compared with an NHD13 BMME. Our data therefore demonstrate a contributory role of the BMME to disease progression in MDS and support a therapeutic strategy whereby manipulation of the MDS microenvironment may improve hematopoietic function and overall survival. (Blood. 2016;127(5):616-625)

Duchenne muscular dystrophy in boys progresses rapidly to severe impairment of muscle function and death in the second or third decade of life. Current supportive therapy with corticosteroids results in a modest increase in strength as a consequence of a general reduction in inflammation, albeit with potential untoward long-term side effects and ultimate failure of the agent to maintain strength. Here, we demonstrate that alternative approaches that rescue defective autophagy in mdx mice, a model of Duchenne muscular dystrophy, with the use of rapamycin-loaded nanoparticles induce a reproducible increase in both skeletal muscle strength and cardiac contractile performance that is not achievable with conventional oral rapamycin, even in pharmacological doses. This increase in physical performance occurs in both young and adult mice, and, surprisingly, even in aged wild-type mice, which sets the stage for consideration of systemic therapies to facilitate improved cell function by autophagic disposal of toxic byproducts of cell death and regeneration.

Inv(3q26) and t(3:3)(q21;q26) are specific to poor-prognosis myeloid malignancies, and result in marked overexpression of EVI1, a zinc-finger transcription factor and myeloid-specific oncoprotein. Despite extensive study, the mechanism by which EVI1 contributes to myeloid malignancy remains unclear. Here we describe a new mouse model that mimics the transcriptional effects of 3q26 rearrangement. We show that EVI1 overexpression causes global distortion of hematopoiesis, with suppression of erythropoiesis and lymphopoiesis, and marked premalignant expansion of myelopoiesis that eventually results in leukemic transformation. We show that myeloid skewing is dependent on DNA binding by EVI1, which upregulates Spi1, encoding master myeloid regulator PU.1. We show that EVI1 binds to the −14 kb upstream regulatory element (−14kbURE) at Spi1; knockdown of Spi1 dampens the myeloid skewing. Furthermore, deletion of the −14kbURE at Spi1 abrogates the effects of EVI1 on hematopoietic stem cells. These findings support a novel mechanism of leukemogenesis through EVI1 overexpression.

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem and progenitor cells and represent the most common cause of acquired marrow failure. Hallmarked by ineffective hematopoiesis, dysplastic marrow, and risk of transformation to acute leukemia, MDS remains a poorly treated disease. Although identification of hematopoietic aberrations in human MDS has contributed significantly to our understanding of MDS pathogenesis, evidence now identify the bone marrow microenvironment (BMME) as another key contributor to disease initiation and progression. With improved understanding of the BMME, we are beginning to refine the role of the hematopoietic niche in MDS. Despite genetic diversity in MDS, interaction between MDS and the BMME appears to be a common disease feature, and therefore represents an appealing therapeutic target. Further understanding of the interdependent relationship between MDS and its niche is needed to delineate the mechanisms underlying hematopoietic failure and how the microenvironment can be clinically targeted. This review will provide an overview of data from human MDS and murine models supporting a role for BMME dysfunction at several steps of disease pathogenesis. While no models or human studies so far have combined all these findings, we will review current data identifying BMME involvement in each step of MDS pathogenesis, organized to reflect the chronology of BMME contribution as the normal hematopoietic system becomes myelodysplastic and MDS progresses to marrow failure and transformation. Although microenvironmental heterogeneity and dysfunction certainly add complexity to this syndrome, data are already demonstrating that targeting microenvironmental signals may represent novel therapeutic strategies for MDS treatment.

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The efficient clearance of dead and dying cells, efferocytosis, is critical to maintain tissue homeostasis. In the bone marrow microenvironment (BMME), this role is primarily fulfilled by professional bone marrow macrophages, but recent work has shown that mesenchymal stromal cells (MSCs) act as a non-professional phagocyte within the BMME. However, little is known about the mechanism and impact of efferocytosis on MSCs and on their function. To investigate, we performed flow cytometric analysis of neutrophil uptake by ST2 cells, a murine bone marrow-derived stromal cell line, and in murine primary bone marrow-derived stromal cells. Transcriptional analysis showed that MSCs possess the necessary receptors and internal processing machinery to conduct efferocytosis, with Axl and Tyro3 serving as the main receptors, while MerTK was not expressed. Moreover, the expression of these receptors was modulated by efferocytic behavior, regardless of apoptotic target. MSCs derived from human bone marrow also demonstrated efferocytic behavior, showing that MSC efferocytosis is conserved. In all MSCs, efferocytosis impaired osteoblastic differentiation. Transcriptional analysis and functional assays identified downregulation in MSC mitochondrial function upon efferocytosis. Experimentally, efferocytosis induced mitochondrial fission in MSCs. Pharmacologic inhibition of mitochondrial fission in MSCs not only decreased efferocytic activity but also rescued osteoblastic differentiation, demonstrating that efferocytosis-mediated mitochondrial remodeling plays a critical role in regulating MSC differentiation. This work describes a novel function of MSCs as non-professional phagocytes within the BMME and demonstrates that efferocytosis by MSCs plays a key role in directing mitochondrial remodeling and MSC differentiation. Efferocytosis by MSCs may therefore be a novel mechanism of dysfunction and senescence. Since our data in human MSCs show that MSC efferocytosis is conserved, the consequences of MSC efferocytosis may impact the behavior of these cells in the human skeleton, including bone marrow remodeling and bone loss in the setting of aging, cancer and other diseases.

While data suggest that, in the myelodysplastic syndromes (MDS), the bone marrow microenvironment (BMME) is abnormal, the lack of examination of the BMME in a robust in vivo model has limited progress in the understanding of reciprocal MDS-BMME interactions. If microenvironmental defects contribute to disease progression, targeting the BM niche may offer an alternative approach for therapeutic benefit. We sought to define the MDS BMME in a well-established transgenic murine model that recapitulates hallmark features of human MDS. In this model, hematopoietic tissue-specific expression of the NUP98-HOXD13 (NHD13) fusion gene is driven by Vav regulatory elements, resulting in peripheral cytopenias by 16 weeks of age and mortality from transformation to leukemia at a median time of 11 months of age. Mice were analyzed at 15-36 weeks of age, when the MDS phenotype is prominent in the absence of leukemia. Flow cytometric quantification of BM stroma in 23-week old NHD13 mice showed a 6.5-fold increase in frequency of CD51+/Sca1- osteoblastic cells (OBC) compared to WT (p<0.05). CD51+/Sca1+ multipotent stromal cells (MSC) and CD31+/Sca1+ endothelial cells were also significantly increased in NHD13 compared to WT mice. This was not due to loss of hematopoietic cells in the marrow of NHD13 mice. While an expansion of functional MSCs and osteoblastic cells could result in skeletal changes, micro CT imaging of the femora and tibiae of 20-week old NHD13 mice revealed no differences in skeletal parameters compared to WT mice. These data suggest that the expanded osteolineage cells in NHD13 mice are not functional bone-forming cells. While stromal populations were not altered in bone-associated cells of 23-week old NHD13 compared to WT mice, 36-week old NHD13 mice also showed increased bone-associated OBCs, MSCs, and endothelial cells. Therefore, there are significant time-dependent shifts in critical stromal populations in this in vivo model of MDS, which may contribute to an abnormal BMME. To determine if the MDS BMME contributes to hematopoietic failure, NHD13 BM (CD45.2) was transplanted with WT competitor BM (CD45.1) in a 1:1 ratio into lethally irradiated NHD13 or WT (CD45.2) recipients, thus exposing the same MDS hematopoietic cells to either MDS or WT microenvironments. Using this transplantation paradigm, we previously reported improvement of hematopoiesis when NHD13 BM is exposed to a WT BMME. Surprisingly, CD45.1+ WT competitor-derived cells exhibited myeloid skewing when transplanted into NHD13 recipients compared to WT recipients, suggesting that interaction of WT BM with an MDS BMME can induce myeloid skewing, a feature of the NHD13 model. NHD13 BM was next transplanted non-competitively into lethally irradiated NHD13 and WT mice. At 10 weeks post-transplant, WT recipients had a 2.5-fold increase in peripheral leukocytes (p<0.05), significant improvement of anemia, and significant mitigation of BM long term-HSC loss compared to NHD13 recipients, suggesting that WT BMME components can rescue hematopoietic function in MDS. Together, our studies strongly suggest that a murine model recapitulates MDS microenvironmental abnormalities, and that exposure of MDS hematopoietic cells to a non-malignant microenvironment is sufficient to improve hematopoietic function. Thus, improvement of the BM microenvironment represents a novel therapeutic strategy to ameliorate hematopoietic function in MDS. Disclosures Becker: Millenium: Research Funding. Calvi:Fate Therapeutics: Patents & Royalties.