INTRODUCTION: Previous studies have shown that osteocytes, the matrix-embedded cells in bone, control bone modeling and remodeling through direct contact with adjacent cells and via secreted factors that can reach cells in the bone marrow microenvironment (BMM). Osteocytes express several receptors including G protein-coupled receptors (GPCRs) and mice lacking the stimulatory subunit of G-proteins (Gsα) in osteocytes have abnormal myelopoiesis, skeletal abnormalities and reduced adipose tissue. This study aimed at evaluating the effects of osteocyte-secreted factors on myeloid cell proliferation and differentiation in vitro. To investigate cross-talk between osteocytes and the BMM, we established osteocytic cell lines lacking Gsα expression to study the molecular mechanisms by which osteocytes control myeloid cell proliferation and differentiation. METHODS: CRISPR/Cas9 was used to knockout Gsα in the osteocytic cell line Ocy454. Conditioned media (CM) from differentiated Ocy-GsαCtrl and Ocy-GsαKO cells were used to treat myeloid cells and bone marrow mononuclear cells (BMNCs) isolated from long bones of 6-8-week-old C57/BL6 mice. BMNCs were cultured with Macrophage Colony Stimulating Factor (M-CSF), Receptor Activator of Nuclear Factor Kappa β Ligand (RANKL) to induce osteoclast differentiation. Proliferation, TRAP staining, TRAP activity, resorption pit assay, F-actin ring formation and mRNA expression were used to evaluate cell proliferation, differentiation and function of the induced osteoclasts. Proteomics analysis of CM was performed to identify osteocyte-secreted factors capable of controlling myelopoiesis and osteoclastogenesis. RESULTS: Myeloid cells treated with CM from Ocy-GsαKO showed a significant increase in cell proliferation compared to Ocy-GsαCtrl CM and non-treated control. BMNCs treated with CM from Ocy-GsαCtrl and Ocy-GsαKO showed a significant increase in cell proliferation as compared to non-treated control. Osteoclast differentiation was significantly suppressed by CM from Ocy-GsαCtrl and further suppressed by CM from Ocy-GsαKO compared to non-treated control. Osteoclasts exposed to CM from Ocy-GsαKO showed a significant defect in activity and function as compared to cells exposed to CM from Ocy-GsαCtrl and non-treated cells. Osteoclast apoptosis was significantly enhanced by Ocy-GsαCtrl and Ocy-GsαKO CM compared to non-treated control. Among osteocyte secreted factors, we identified neuropilin-1 (NRP-1) as a Gsα-dependent osteocytic factor capable of suppressing osteoclastogenesis. CM from Ocy-GsαKO in which M-CSF was reduced by shRNA demonstrated decrease in BMNCs proliferation, demonstrating that osteocytes are also important sources of this cytokine. CONCLUSIONS: Osteocytes produce several Gsα-dependent and -independent secreted factors capable of supporting myelopoiesis, promoting macrophage proliferation and suppressing osteoclast formation. We identified osteocyte-derived NRP-1 as a novel factor capable of decreasing osteoclastogenesis. In addition, we found that M-CSF secreted by osteocytes is responsible in part for BMNC proliferation. Future studies should focus on determining the role of osteocyte-mediated NRP-1 and other secreted factor(s) in control of myelopoiesis and osteoclastogenesis. / 2020-06-26T00:00:00Z

Atherosclerosis propagates when innate immune cells, myeloid-derived macrophages, undergo unregulated uptake of cholesterol-rich modified low-density lipoproteins (LDL). Excess storage and retention of this cholesterol leads to development of lipid-laden macrophage foam cells, that accumulate within the intima of arteries as developing plaque. Formation of atherosclerotic lesions reduces blood flow and can further lead to more serious complications such as myocardial infarction, stroke, and cardiovascular disease. AMP-activated protein kinase (AMPK), a master regulator of cellular energetics, has been shown to participate in many anti-atherogenic pathways within myeloid cells such as (but not limited to) the inhibition of cholesterol synthesis and stimulation of reverse cholesterol transport. However, a recent report described a pro-atherogenic role for myeloid AMPK, showing it is expression required for myeloid cell recruitment and longevity within the atherosclerotic microenvironment. Despite this, multiple reports all corroborate describing a protective role for systemic pharmacological AMPK activation. We sought to determine the consequence of modified LDL variants in myeloid AMPK signaling and to further clarify the role of myeloid AMPK signaling within atherosclerosis. In cultured macrophages primed with modified LDL variants underwent AMPK activation, which was also associated with increased markers of autophagy. In an in vivo model of intermediate atherosclerosis, we observed that neither myeloid AMPK expression nor systemic AMPK-activating therapy influenced lesion myeloid content, necrosis, or autophagic markers. Furthermore, despite a suggestive trend, both myeloid AMPK and AMPK-therapy did not significantly influence lesion size in male or female mice. Interestingly, we found that in animals lacking AMPK signaling to only one substrate, HMGCR (the rate limiting enzyme in cholesterol synthesis), knock-in mice developed accelerated atherosclerosis when compared to their wild-type littermate. Furthermore, we determined that AMPK signaling to HMGCR in the hematopoietic compartment alone is enough to protect against atherogenesis. Taken together, these studies show the benefit of interrogating specific AMPK-regulated pathways in the context of atherosclerosis, and sheds light on the benefit of utilization of single point mutation knock-in models opposed to global or cell type-specific knockout models for investigations into AMPK within atherosclerosis.

In patients with acute myeloid leukemia (AML), cytarabine-based chemotherapy usually achieves remission, but this is commonly followed by relapse and chemo-resistance. In this study, we aim to establish next-generation sequencing (NGS)-based microRNA expression profiling and pathway analysis to identify pathways regulated differentially between chemo-sensitive and -resistant AML as potential therapeutic targets. MicroRNA expression profiles differ significantly between chemo-sensitive and chemo-resistant AML cells and reflect differences in the activity of intracellular signaling cascades. Alterations in signaling pathway activities contribute to treatment resistance and thus represent potential drug targets. Our microRNA-led approach indicates a role for activin receptor type 2A in ARA-C resistance of AML cells and suggests activin receptor signaling to be a candidate pathway for targeted therapy.

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The Gadd45 family of proteins is known to play a central role as cellular stress sensors that modulate the response of mammalian cells to different stressors, including oncogenic stress. Gadd45a expression is regulated during myeloid cell differentiation, and is also induced in response to acute stimulation with cytokines, myeloablation and inflammation. The proto-oncogene C-myc plays a pivotal role in growth control, differentiation and apoptosis in hematopoietic cells. Deregulated Myc in hematopoietic cells blocks the differentiation program and prevents normal homeostatic cellular apoptosis, which alters the balance of cell populations, often participating in leukemogenesis. The status of Gadd45a expression has been shown to impact on different cancers, including breast cancer and leukemia. How the stress response gene Gadd45a modulates oncogenic stress imparted by deregulated c-Myc in myeloid cells has not been investigated. We hypothesized that Gadd45a and its interacting partner proteins can modulate specific pro-survival or pro-apoptotic signaling pathways, altering the cellular response to oncogenic myc in myeloid cells. Gadd45a may play different roles in proliferating and differentiating cells, and myeloid cells in vivo are at all stages of myeloid development. Therefore, to understand how Gadd45a status impacts on proliferating and differentiating myeloid cells, we decided to study the effect of loss of Gadd45a in myc-expressing cells that are either proliferating or stimulated to undergo differentiation. Therefore, to address this issue we utilized bone marrow from wild-type (wt) and Gadd45a null mice, and retrovirally infected these cells to express constitutive Myc or empty vector control. Using these cells we have shown that bone marrow deficient in Gadd45a and expressing constitutive Myc, display decreased apoptosis under proliferating conditions, yet increased apoptosis in media containing the differentiation inducing cytokine GM-CSF. We show that in expansion media loss of Gadd45a in the presence of Myc elicits its function through the activation of p38, with evidence supporting a role for PU.1 and Mcl-1 expression, which are downstream of p-p38. In contrast, deregulated C-Myc and loss of Gadd45a does not signal through p-38 in GM-CSF, but surprisingly there is a decrease in cytokine receptor expression. This data demonstrates that Gadd45a may be required for optimal cytokine receptor expression in myeloid cells, which can impact on survival of the cells. Although in primary bone marrow Gadd45a status had no effect on differentiation of Myc expressing cells, the loss of Gadd45a in Hoxb8 generated cell lines shifted differentiation towards increased neutrophils. Determining the role of Gadd45a on the biological outcome of myeloid cells in response to deregulated c-Myc will provide vital information in understanding the function of Gadd45a in the development and progression of Myc expressing myeloid leukemia. / Molecular Biology and Genetics

AML is a cancer of the blood and bone marrow characterized by the presence of highly proliferative and abnormally differentiated myeloblasts. Previous work from the Bhatia lab utilized the orthotopic xenograft model in order to isolate a population of leukemic regenerating cells (LRC) that exists prior to relapse. Affymatrix analysis of LRCs revealed up-regulation of 248 genes that can act as unique targets to prevent relapse. In order to screen compounds against all 248 targets, it is important to develop an in vitro model that is able to appropriately recapture the functional and molecular markers of LRCs. Primary AML samples were treated with 5-doses of 0.15 μM, 1 μM AraC, or DMSO control and several outcomes were measured. In vitro AraC treatment was not able to recapitulate the progenitor frequency curve and CD34 expression curve observed in vivo. Additionally, we were not able to see a consistent increase in select LRC targets DRD2, GLUT2, FUT3, and FASL via flow cytometry. Despite an increase in the mRNA levels of LRC genes 24h after treatment with 0.15 μM AraC, long term analysis could not be completed due to poor RNA quality and low expression of LRC-targets. Primary AML cells were co-culture with mouse MS-5 stromal cell line order to study the effects of mesenchymal stromal cells on AML response to AraC. Co-culture with MS-5 cells had different effects on select primary AML cells. AML 14939 showed an increase in CD34 and LRC targets DRD2 and FUT3 following AraC treatment when co-cultured with MS-5 cells; while A374 showed no differences between DMSO and AraC treated groups. Overall, these findings suggest the LRC signature is not induced by treatment with AraC alone. Complex interactions between AML cells and their bone marrow niche during AraC treatment plays an important role in the development of LRCs prior to AML relapse. / Thesis / Master of Science (MSc) / AML is a cancer of blood cells characterized by the presence of rapidly dividing cancer cells termed myeloblasts. AML has a high rate of disease relapse. The Bhatia lab modelled AML relapse in a mouse and discovered an unique population of cells that exist prior to relapse termed LRCs. LRCs express distinctive genes that can act as targets for the development of new therapies to prevent relapse. In order to screen potential relapse preventing compounds, we set out to recapture AML relapse using cells in a dish. AML cells from patients were treated with chemotherapy reagent AraC and the number of cancer progenitors and the expression of specific LRC proteins were measured. AraC did not increase the level of 3 out of 4 LRC proteins studied. We determined the LRCs were not caused by AraC treatment, and the physiology of the bone marrow environment plays an important role in inducing relapse.

Yes / The HOX genes encode a family of transcription factors that have key roles in both development and malignancy. Disrupting the interaction between HOX proteins and their binding partner, PBX, has been shown to cause apoptotic cell death in a range of solid tumors. However, despite HOX proteins playing a particularly significant role in acute myeloid leukemia (AML), the relationship between HOX gene expression and patient survival has not been evaluated (with the exception of HOXA9), and the mechanism by which HOX/PBX inhibition induces cell death in this malignancy is not well understood. In this study, we show that the expression of HOXA5, HOXB2, HOXB4, HOXB9, and HOXC9, but not HOXA9, in primary AML samples is significantly related to survival. Furthermore, the previously described inhibitor of HOX/PBX dimerization, HXR9, is cytotoxic to both AML-derived cell lines and primary AML cells from patients. The mechanism of cell death is not dependent on apoptosis but instead involves a regulated form of necrosis referred to as necroptosis. HXR9-induced necroptosis is enhanced by inhibitors of protein kinase C (PKC) signaling, and HXR9 combined with the PKC inhibitor Ro31 causes a significantly greater reduction in tumor growth compared to either reagent alone. / Funded in part through a grant to RA from the Cultural Bureau of the Kingdom of Saudi Arabia.