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The unique interactions of leukemia cells with the bone marrow (BM) microenvironment (niche) are critical for disease progression and resistance to treatment. We have recently found that the vascular adhesion molecule E-selectin is a key niche component mediating acute myeloid leukemia (AML) chemoresistance, highlighting E-selectin as a promising therapeutic target. In this study, we found canonical E-selectin receptors CD44 and CD162 to be crucial for E-selectin adhesion, as mouse AML cells lacking both receptors failed to bind to E-selectin. We then developed an in vitro model to assess the chemo-sensitivity of mouse AML blasts adhering to various vascular adhesion molecules; this showed that E-selectin uniquely boosts AML cell survival to chemotherapy, but only when CD44/CD162 are present. Likewise when transplanted into recipient mice, CD44/CD162-/- AML cells were significantly more sensitive to chemotherapy compared to wildtype AML. Together these results suggest that CD44 and/or CD162 are key E-selectin receptors involved in AML chemoresistance. To validate these findings in human, we used CRISPR-Cas9 gene editing to selectively suppress CD44 or CD162 from the human AML cell line KG1a. Using our in vitro chemo-sensitivity assay, we showed that E-selectin could not promote AML survival in the absence of either CD44 or CD162, confirming our findings in mice. However interestingly, KG1a cells could still bind to E-selectin in the absence of CD44 or CD162, suggesting the involvement of several E-selectin receptors that can play different roles – either binding or signalling. To conclude, we described a novel form of niche-mediated chemoresistance that can be modelled in vitro, and identified CD44 and CD162 as key AML cell surface receptors involved. These findings highlight blockade of E-selectin or its receptors as a novel strategy to improve the treatment of AML.

Neurological heterotopic ossification (NHO) is the abnormal formation of bone in soft tissues as a consequence of spinal cord or traumatic brain injury. NHO causes pain, ankyloses, vascular and nerve compression and delays rehabilitation in this high-morbidity patient group. The pathological mechanisms leading to NHO remain unknown and consequently there are no therapeutic options to prevent or reduce NHO. Genetically modified mouse models of rare genetic forms of heterotopic ossification (HO) exist, but their relevance to NHO is questionable. Consequently, we developed the first model of spinal cord injury (SCI)-induced NHO in genetically unmodified mice. Formation of NHO, measured by micro-computed tomography, required the combination of both SCI and localized muscular inflammation. Our NHO model faithfully reproduced many clinical features of NHO in SCI patients and both human and mouse NHO tissues contained macrophages. Muscle-derived mesenchymal progenitors underwent osteoblast differentiation in vitro in response to serum from NHO mice without additional exogenous osteogenic stimuli. Substance P was identified as a candidate NHO systemic neuropeptide, as it was significantly elevated in the serum of NHO patients. However, antagonism of substance P receptor in our NHO model only modestly reduced the volume of NHO. In contrast, ablation of phagocytic macrophages with clodronate-loaded liposomes reduced the size of NHO by 90%, supporting the conclusion that NHO is highly dependent on inflammation and phagocytic macrophages in soft tissues. Overall, we have developed the first clinically relevant model of NHO and demonstrated that a combined insult of neurological injury and soft tissue inflammation drives NHO pathophysiology.

The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, suggesting that MYB may be a therapeutic target in these diseases. However, realization of this potential requires a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis, and an approach for developing an effective therapeutic. We previously showed that the interactionof c-MybwiththecoactivatorCBP/p300 is essential for its transforming activity. Here, by using cells from Booreana mice which carry a mutant allele of c-Myb, we show that this interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type mice, Booreana cells transduced with AML1-ETO9a or MLL-AF9 retroviruses fail to generate leukemia upon transplantation into irradiated recipients. Finally, we have begun to explore themolecularmechanisms underlying these observations by gene expression profiling. This identified several genes previously implicated in myeloid leukemogenesis and HSC function as being regulated in a c-Myb-p300-dependent manner. These data highlight the importance of the c-Myb-p300 interaction inmyeloid leukemogenesis and suggest disruption of this interaction as a potential therapeutic strategy for acute myeloid leukemia. Refereed/Peer-reviewed

Potent functional HSCs are enriched at the endosteal bone marrow (BM), which comprises ∼10% of total BM. To identify novel niche factors that regulate HSCs, we performed a gene expression microarray seeking genes overexpressed in the endosteal relative to central BM. Prostacyclin/prostaglandin I2 (PGI2) synthase (Ptgis) was one of the highest enriched genes in the endosteal versus central BM (27-fold). PTGIS is the sole enzyme for biosynthesis of PGI2. PGI2 has no reported roles in HSC regulation in the BM and was chosen for further investigation. We found PGI2 analogue iloprost treatment of sorted BM lineage- Kit+ Sca1- (LKS+) cells in vitro for 7 days potently reduced proliferation and differentiation compared to vehicle control. BM cells pulse treated ex vivo with iloprost for 1 hour also resulted in 14-fold increased multilineage reconstitution potential compared to vehicle controls in competitive transplant assay. Iloprost administration in vivo partially rescued BM HSC reconstitution potential in mice sub-lethally (6.5 Gy) irradiated or administered pro-inflammatory granulocyte colony stimulating factor (G-CSF) for 3 days. These data suggest that PGI2 protects HSC from stress. Mechanistically, we found iloprost pulse treatment in vitro was associated with increased pSTAT3 and cell cycle progression in HSCs. Additionally, we found ∼4-fold greater proportion of untreated LKS+ HSPC homing to the BM of recipients co-administered iloprost + G-CSF compared to vehicle + G-CSF at 4 hours post-transplant. We found that G-CSF administration also decreased Scf and Cxcl12 mRNA expression in BM, which were partially rescued with iloprost co-administration. These data suggest that PGI2 protects HSC during stress by acting directly on HSCs and indirectly to preserve BM niche functions. In summary, we identified PGI2 as a novel HSC niche factor abundant in the endosteal BM.

The central macrophage (MΦ) in erythroblastic islands (EI) is critical to erythropoiesis by providing iron and growth factors, and mediating enucleation of the maturing erythroblasts. As MΦ are key effectors of inflammation, we investigated the effect of lipopolysaccharides (LPS) on erythropoiesis in vivo. LPS (2.5mg/kg/d for 2 days) in C57BL/6 mice caused the whitening of the bone marrow (BM) with dramatically decreased erythroblast and reticulocyte numbers. Imaging flow cytometry was used to visualize structural changes and quantify numbers of EI. LPS treatment in vivo caused a marked loss of EI in the BM. This suppressive effect of LPS on BM erythropoiesis was TLR4-dependent as it was absent in TLR4 KO mice. By qRT-PCR and flow cytometry, TLR4 is not expressed by BM erythroblasts but by myeloid cells including EI MΦ. Together with the fact that addition of 100ng/mL LPS into BFU-E assays does not inhibit BFU-E colony growth, these suggest that the suppressive effect of LPS on erythroblasts is indirectly mediated. It is known that LPS mobilizes HSC in a G-CSF-dependent manner. As MΦ express the G-CSF receptor (GCSFR), we explored LPS effects in GCSFR KO mice. Surprisingly, although HSC did not mobilize in response to LPS in GCSFR KO mice, BM erythropoiesis was still suppressed with reduced numbers of erythroblasts. Unexpectedly however, the BM from LPS-treated GCSFR KO mice was still red with high numbers of erythrocytes. The explanation of this paradox is that LPS treatment causes BM vascular leakage in GCSFR KO mice with blood plasma volume in the BM 2.9-fold higher compared to LPS-treated WT mice (measured by i.v. injection of Evans Blue). In conclusion our data show that LPS suppresses medullar erythropoiesis indirectly in a G-CSF-independent manner but GCSFR-mediated signaling is necessary to maintain the integrity of the BM vasculature in response to LPS.

Despite the fact that many hypoxia-inducible genes are important in hematopoiesis, the spatial distribution of oxygen in the bone marrow (BM) has not previously been explored in vivo. Using the hypoxia bioprobe pimonidazole, we showed by confocal laser scanning microscopy that the endosteum at the bone-BM interface is hypoxic, with constitutive expression of hypoxia-inducible transcription factor-1α (HIF-1α) protein in steady-state mice. Interestingly, at the peak of hematopoietic stem and progenitor cell (HSPC) mobilization induced by either granulocyte colony-stimulating factor or cyclophosphamide, hypoxic areas expand through the central BM. Furthermore, we found that HSPC mobilization leads to increased levels of HIF-1α protein and increased expression of vascular endothelial growth factor A (VEGF-A) mRNA throughout the BM, with an accumulation of VEGF-A protein in BM endothelial sinuses. VEGF-A is a cytokine known to induce stem cell mobilization, vasodilatation, and vascular permeability in vivo. We therefore propose that the expansion in myeloid progenitors that occurs during mobilization depletes the BM hematopoietic microenvironment of O2, leading to local hypoxia, stabilization of HIF-1α transcription factor in BM cells, increased transcription of VEGF-A, and accumulation of VEGF-A protein on BM sinuses that increases vascular permeability.Disclosure of potential conflicts of interest is found at the end of this article.

Advances in tissue-engineering have resulted in a versatile tool-box to specifically design a tailored microenvironment for hematopoietic stem cells (HSCs) in order to study diseases that develop within this setting. However, most current in vivo models fail to recapitulate the biological processes seen in humans. Here we describe a highly reproducible method to engineer humanized bone constructs that are able to recapitulate the morphological features and biological functions of the HSC niches. Ectopic implantation of biodegradable composite scaffolds cultured for 4 weeks with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone organ including a large number of human mesenchymal cells which were shown to be metabolically active and capable of establishing a humanized microenvironment supportive of the homing and maintenance of human HSCs. A syngeneic mouse-to-mouse transplantation assay was used to prove the functionality of the tissue-engineered ossicles. We predict that the ability to tissue engineer a morphologically intact and functional large-volume bone organ with a humanized bone marrow compartment will help to further elucidate physiological or pathological interactions between human HSCs and their native niches.

Harvest of granulocyte colony-stimulating factor (G-CSF)-mobilized hematopoietic stem cells (HSCs) begins at day 5 of G-CSF administration, when most donors have achieved maximal mobilization. This is based on surrogate markers for HSC mobilization, such as CD34(+) cells and colony-forming activity in blood. However, CD34(+) cells or colony-forming units in culture (CFU-C) are heterogeneous cell populations with hugely divergent long-term repopulation potential on transplantation. HSC behavior is influenced by the vascular bed in the vicinity of which they reside. We hypothesized that G-CSF may mobilize sequentially cells proximal and more distal to bone marrow venous sinuses where HSCs enter the blood. We addressed this question with functional serial transplantation assays using blood and bone marrow after specific time points of G-CSF treatment in mice. We found that in mice, blood collected after only 48 hours of G-CSF administration was as enriched in serially reconstituting HSCs as blood collected at 5 days of G-CSF treatment. Similarly, mobilized Lin(-)CD34(+) cells were relatively enriched in more primitive Lin(-)CD34(+)CD38(-) cells at day 2 of G-CSF treatment compared with later points in half of human donors tested (n = 6). This suggests that in both humans and mice, hematopoietic progenitor and stem cells do not mobilize uniformly according to their maturation stage, with most potent HSCs mobilizing as early as day 2 of G-CSF.

Rb (retinoblastoma protein) inhibits E2F-1-induced cell death. We now show that the ability of Rb to inhibit E2F-1-induced cell death is dependent on a functional LXCXE-binding site in Rb, thereby suggesting that proteins that bind the LXCXE-binding site in Rb may regulate the anti-apoptotic activity of Rb. HDAC1, an LXCXE protein that plays a critical role in Rb-mediated transcription repression, abrogates the effect of Rb on E2F-1-induced cell death. In contrast, RF-Cp145, another LXCXE protein, cooperates with Rb to inhibit E2F-1-induced cell death. Both proteins exert their effect in an LXCXE-dependent manner. Rb regulates E2F-induced cell death by acting upstream of p73. Rb represses the p73 promoter. Our results further suggest a model in which Rb-E2F-1 complexes mediate the anti-apoptotic activity of Rb through active repression of target genes without recruiting HDAC1.

Since the publication of our paper “Regulation of p21WAF1/Cip1 Stability by Wisp39, an Hsp90 Binding Protein” by Jascur et al., (2005, Molecular Cell 17, 237–249), it has come to our attention that the WISp39 gene reported in our paper is identical to the DIR1 gene (Robson et al., 1999xA novel human stress response-related gene with a potential role in induced radioresistance. Robson, T., Joiner, M.C., Wilson, G.D., McCullough, W., Price, M.E., Logan, I., Jones, H., McKeown, S.R., and Hirst, D.G. Radiat. Res. 1999; 152: 451–461Crossref | PubMed | Scopus (36)See all ReferencesRobson et al., 1999).