TLE4 Is a Critical Mediator of Osteoblast and Runx2-Dependent Bone Development

Healthy bone homeostasis hinges upon a delicate balance and regulation of multiple processes that contribute to bone development and metabolism. While examining hematopoietic regulation by Tle4, we have uncovered a previously unappreciated role of Tle4 on bone calcification using a novel Tle4 null mouse model. Given the significance of osteoblasts in both hematopoiesis and bone development, this study investigated how loss of Tle4 affects osteoblast function. We used dynamic bone formation parameters and microCT to characterize the adverse effects of Tle4 loss on bone development. We further demonstrated loss of Tle4 impacts expression of several key osteoblastogenic genes, including Runx2, Oc, and Ap, pointing toward a potential novel mechanism for Tle4-dependent regulation of mammalian bone development in collaboration with the RUNX family members.

Immuno-Modulation of Hematopoietic Stem and Progenitor Cells in Inflammation

Lifelong blood production is maintained by bone marrow (BM)-residing hematopoietic stem cells (HSCs) that are defined by two special properties: multipotency and self-renewal. Since dysregulation of either may lead to a differentiation block or extensive proliferation causing dysplasia or neoplasia, the genomic integrity and cellular function of HSCs must be tightly controlled and preserved by cell-intrinsic programs and cell-extrinsic environmental factors of the BM. The BM had been long regarded an immune-privileged organ shielded from immune insults and inflammation, and was thereby assumed to provide HSCs and immune cells with a protective environment to ensure blood and immune homeostasis. Recently, accumulating evidence suggests that hemato-immune challenges such as autoimmunity, inflammation or infection elicit a broad spectrum of immunological reactions in the BM, and in turn, influence the function of HSCs and BM environmental cells. Moreover, in analogy with the emerging concept of “trained immunity”, certain infection-associated stimuli are able to train HSCs and progenitors to produce mature immune cells with enhanced responsiveness to subsequent challenges, and in some cases, form an inflammatory or infectious memory in HSCs themselves. In this review, we will introduce recent findings on HSC and hematopoietic regulation upon exposure to various hemato-immune stimuli and discuss how these challenges can elicit either beneficial or detrimental outcomes on HSCs and the hemato-immune system, as well as their relevance to aging and hematologic malignancies.

Progress in experimental research on SPRED protein family

The Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology-1 (EVH-1) domain (SPRED) family of proteins was discovered in 2001. These Sprouty-related tyrosine kinase-binding proteins negatively regulate a variety of growth factor-induced Ras/ERK signaling pathways. In recent years, SPRED proteins have been found to regulate vital activities such as cell development, movement, and proliferation, and to participate in pathophysiological processes such as tumor metastasis, hematopoietic regulation, and allergic reactions. The findings of these studies have important implications regarding the involvement of SPRED proteins in disease. Early studies of SPRED proteins focused mainly on various tumors, cardiovascular diseases, and organ development. However, in recent years, great progress has been made in elucidating the role of SPRED proteins in neuropsychiatric, inflammatory, endocrine, and ophthalmic diseases. This article provides a review of the experimental studies performed in recent years on the SPRED proteins and their role in the pathogenesis of certain diseases.

WASH is required for the differentiation commitment of hematopoietic stem cells in a c-Myc–dependent manner

Hematopoiesis is fully dependent on hematopoietic stem cells (HSCs) that possess the capacity to self-renew and differentiate into all blood cell lineages. WASH, Wiskott–Aldrich syndrome protein (WASP) and SCAR homologue (WASH) is involved in endosomal sorting as an actin-nucleating protein. Here, we show that conditional WASH deletion in the hematopoietic system causes defective blood production of the host, leading to severe cytopenia and rapid anemia. WASH deficiency causes the accumulation of long-term (LT)-HSCs in bone marrow and perturbs their differentiation potential to mature blood lineages. Importantly, WASH is located in the nucleus of LT-HSCs and associates with the nucleosome remodeling factor (NURF) complex. WASH assists the NURF complex to the promoter of c-Myc gene through its VCA domain-dependent nuclear actin nucleation. WASH deletion suppresses the transcriptional activation of c-Myc gene and impairs the differentiation of LT-HSCs. WASH acts as an upstream regulator to modulate c-Myc transcription for hematopoietic regulation.

Characterization Of RING Finger E3-Ubiquitin Ligase Cereblon In Hematopoietic Regulation

Characterization of RING finger E3-Ubiquitin Ligase Cereblon in Hematopoietic Regulation Cereblon is a RING-domain E3 ubiquitin ligase (UbL) that is the direct protein target for thalidomide and lenalidomide. Inhibition of this molecule mediates antiproliferative activity in myeloma cells and in activated B-cell-like subtype diffuse large B-cell lymphoma. In addition to its anticancer properties, lenalidomide potentiates T-cell effector function through the engagement of the CD28-mediated co-stimulatory pathway by a CRBL-dependent mechanism that is poorly characterized. Human and animal studies show CRBL to be ubiquitously expressed in the hematopoietic compartment and in neurons, where a nonsense mutation causes a mild autosomal recessive non-syndromic intellectual disability. Ubiquitin ligase activity underlies suppression of growth factor signaling and maintains homeostasis within the hematopoietic compartment. C-Cbl, cbl-b, GRAIL, and ITCH represent four partially overlapping E3-UbL regulators of cytokine signaling and hematopoietic regulation. To understand the role of CRBL in hematopoetic development and function, we studied a CRBL-deficient mouse strain. All mice used in this study were bred and maintained under specific pathogen-free conditions according to institutional guidelines. The strategy used to create germline crbn deletion (crbl-/-) was described previously, although these mice were not investigated for aberrations in hematopoietic development. First, crbl-/- mice were viable, fertile and normal in appearance without limb malformations. Consistent with a role for CRBL in the hematopoietic development, crbl-/- mice exhibited marked changes in their hematopoietic profiles, including lymphoid hyperplasia, a markedly expanded peripheral white blood cell count and neutrophil expansion. In the T-cell compartment, splenic lymphocytes numbers were increased with demargination of the T-cells into B-cell zones by immunohistochemical staining. Double-negative thymocytes ratio were altered with an accumulation of DN1 and DN3 cells and lower DN4. In contrast, differentiated cell lineages such as mature T-cells, including CD4 and CD8 single-positive (SP) thymocytes, SP peripheral T-cells and naïve and memory T-cells were similar in number to wild-type littermates. The mature T-cells, similar to Cbl-b KO mice showed superior proliferation and IL-2 production in the absence of CD28 co-ligation. Collectively, these histological changes are indicative of a role for crbl in negatively regulating cytokine and receptor signaling events that control cell proliferation or survival. Interestingly, this phenotype overlaps partially with c-cbl and cbl-b KO mice suggesting that they could be coordinately regulating similar pathways. In summary, our findings demonstrate a novel role for crbl, the molecular target of thalidomide and lenalidomide in hematopoiesis and suggest that this molecule may act by an unknown mechanism in concert with other E3 UbLs involved in cytokine receptor signaling. This study provides the first characterization of crbl genetic deficiency in a murine model and demonstrates a unique regulatory relationship between CRBL and other known E3 UbLs. Disclosures: McDaniel: Celgene: Employment.

A Gene-Targeting Approach for the Biological Significance of AML1/Runx1 Arginine-Methylation

Abstract 1309 Acute Myeloid Leukemia 1 (AML1; also called as Runx1: Runt-related transcription factor 1) belongs to the Class II group of leukemia-associated mutation-target genes, and it encodes the DNA-binding subunit of the hetero-dimeric transcription factor complex, Core-Binding Factor (CBF). CBF plays pivotal roles in initial hematopoietic development during embryogenesis and in cellular differentiation of thrombotic and lymphatic lineages throughout adult life. Recent researches revealed that cellular AML1 polypeptide is processed with post-translational modifications, including phosphorylation, acethylation, ubiquitination, and methylation. Biological significance of these modifications on the AML1's function as the hematopoietic regulator, however, largely remains to be elucidated. In this study, we focused on the arginine-methylation as an initial step towards the comprehensive understanding for the AML1-regulating mechanism through these modifications. Arginine residues just downstream to the Runt-domain, which is located at N-terminal region of the molecule and functions as the binding site to DNA and CBF beta: the hetero-dimerization partner, are recently reported to be methylated, resulting in the inhibition of the corepressor-binding thus enhancing its trans- activating activity. In order to elucidate biological significance of these methylations, we performed a series of genetic experiments: First, we generated the non-methylatable double arginine-to-lysine (RRKK) mutant of AML1 at these residues, which should keep AML1-corepressor-binding. When this mutant was subjected to the luciferase reporter-assay using a target-gene construct, it showed lower trans- activating activity in comparison to that for wild-type molecule, as expected. However, this loss-of-function mutation appeared to be dispensable at least for in vitro function for hematopoietic regulation in that this RRKK mutant did rescue hematopoietic differentiation of the AML1-deficient murine ES cells in culture when expressed from a knock-in allele as was the case for the wild-type cDNA of mouse AML1. To further evaluate the biological activity of this mutant in the context of an entire animal, we introduced this mutant cDNA into AML1/Runx1 locus of mouse ES cells by means of a targeted-insertion (knock-in) strategy. Germline mutant mouse lines were successfully established, following blastocyst-injection of these ES cell clones. Heterozygous mice were healthy and fertile, and genotyping for the live pups generated from heterozygotes-crossing revealed that this arginine-mutant allele segregated according to the Mendelian ratio. Homozygous AML1RRKK/RRKK mice were born alive and grew up adult, circumventing the mid-embryonic death due to hematopoietic block that was originally described for the AML1-deficient mice, thus the in vitro notion that these arginine-methylations were not essential for the early hematopoietic development described above was further underscored. There were no significant differences so far observed in peripheral blood cell counts among mice of the AML1RRKK/RRKK or AML1WT/WT genotypes, in comparison to their wild-type littermates. Preliminary studies revealed that AML1RRKK/RRKK mice showed imbalance of the peripheral T cell populations, implying that these methylations may have roles in these cellular lineages. We are currently focusing on further examination of these mutant mice, paying special attention to the cellular lineages where genetic manifestations were observed for AML1 haploinsufficient mice and/or conditional AML1-deficient mice. We hope that these efforts will unveil the biological significance of the AML1 methylation in hematopoietic regulation. Disclosures: No relevant conflicts of interest to declare.

SDF-1α regulation in breast cancer cells contacting bone marrow stroma is critical for normal hematopoiesis

Breast cancer cells (BCCs) show preference for the bone marrow (BM). An animal model showed 2 populations of BCCs in the BM with regard to their cycling states. An in vitro model of early BC entry into BM showed normal hematopoiesis. Here, we show a critical role for BCC-derived SDF-1α in hematopoietic regulation. The studies used a coculture of BM stroma and BCCs (cell lines and stage II BCCs). Northern blots and enzyme-linked immunosorbent assay (ELISA) showed gradual decreases in SDF-1α production in BCCs as they contact BM stroma, indicating partial microenvironmental effects caused by stroma on the BCCs. SDF-1 knock-down BCCs and increased exogenous SDF-1α prevented contact inhibition between BCCs and BM stroma. Contact inhibition was restored with low SDF-1α levels. Long-term culture-initiating assays with CD34+/CD38–/Lin– showed normal hematopoiesis provided that SDF-1α levels were reduced in BCCs. Gap junctions (connexin-43 [CX-43]) were formed between BCCs and BM stroma, with concomitant interaction between CD34+/CD38–/Lin– and BM stroma but not with the neighboring BCCs. In summary, SDF-1α levels are reduced in BCCs that contact BM stroma. The low levels of SDF-1α in BCCs regulate interactions between BM stroma and hematopoietic progenitors, consequently facilitating normal hematopoiesis.