Matthew J. Christopher

CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance

Haematopoietic stem cells (HSCs) primarily reside in the bone marrow where signals generated by stromal cells regulate their self-renewal, proliferation and trafficking. Endosteal osteoblasts and perivascular stromal cells including endothelial cells, CXCL12-abundant reticular cells, leptin-receptor-positive stromal cells, and nestin–green fluorescent protein (GFP)-positive mesenchymal progenitors have all been implicated in HSC maintenance. However, it is unclear whether specific haematopoietic progenitor cell (HPC) subsets reside in distinct niches defined by the surrounding stromal cells and the regulatory molecules they produce. CXCL12 (chemokine (C–X–C motif) ligand 12) regulates both HSCs and lymphoid progenitors and is expressed by all of these stromal cell populations. Here we selectively deleted Cxcl12 from candidate niche stromal cell populations and characterized the effect on HPCs. Deletion of Cxcl12 from mineralizing osteoblasts has no effect on HSCs or lymphoid progenitors. Deletion of Cxcl12 from osterix-expressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, results in constitutive HPC mobilization and a loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from endothelial cells results in a modest loss of long-term repopulating activity. Strikingly, deletion of Cxcl12 from nestin-negative mesenchymal progenitors using Prx1–cre (Prx1 also known as Prrx1) is associated with a marked loss of HSCs, long-term repopulating activity, HSC quiescence and common lymphoid progenitors. These data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow, and that expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

Expression of the G-CSF receptor in monocytic cells is sufficient to mediate hematopoietic progenitor mobilization by G-CSF in mice

Expression of the G-CSF receptor on bone marrow monocytes is sufficient to trigger HSC mobilization in response to G-CSF, in part via effects on osteoblast lineage cells.

Suppression of CXCL12 production by bone marrow osteoblasts is a common and critical pathway for cytokine-induced mobilization

Current evidence suggests that hematopoietic stem/progenitor cell (HSPC) mobilization by granulocyte colony-stimulating factor (G-CSF) is mediated by induction of bone marrow proteases, attenuation of adhesion molecule function, and disruption of CXCL12/CXCR4 signaling in the bone marrow. The relative importance and extent to which these pathways overlap or function independently are uncertain. Despite evidence of protease activation in the bone marrow, HSPC mobilization by G-CSF or the chemokine Grobeta was abrogated in CXCR4(-/-) bone marrow chimeras. In contrast, HSPC mobilization by a VLA-4 antagonist was intact. To determine whether other mobilizing cytokines disrupt CXCR4 signaling, we characterized CXCR4 and CXCL12 expression after HSPC mobilization with Flt3 ligand (Flt3L) and stem cell factor (SCF). Indeed, treatment with Flt3L or SCF resulted in a marked decrease in CXCL12 expression in the bone marrow and a loss of surface expression of CXCR4 on HSPCs. RNA in situ and sorting experiments suggested that the decreased CXCL12 expression is secondary to a loss of osteoblast lineage cells. Collectively, these data suggest that disruption of CXCR4 signaling and attenuation of VLA-4 function are independent mechanisms of mobilization by G-CSF. Loss of CXCL12 expression by osteoblast appears to be a common and key step in cytokine-induced mobilization.

Regulation of neutrophil homeostasis.

Purpose of reviewNeutrophils are an essential component of the innate immune response and a major contributor to inflammation. Consequently, neutrophil number in the blood is tightly regulated. Herein, we review recent studies that have greatly advanced our understanding of the mechanisms controlling neutrophil homeostasis. Recent findingsAccumulating evidence shows that stromal derived factor-1 (CXCL12) through interaction with its major receptor CXCR4 provides a key retention signal for neutrophils in the bone marrow. Granulocyte colony-stimulating factor induces neutrophil release from the bone marrow, in major part, by disrupting stromal derived factor-1/CXCR4 signaling. Granulocyte colony-stimulating factor expression is regulated by a novel feedback loop that senses neutrophil emigration into tissues. Specifically, engulfment of apoptotic neutrophils by tissue phagocytes initiates a cytokine cascade that includes interleukin-23, interleukin-17, and ultimately granulocyte colony-stimulating factor. SummaryGranulocyte colony-stimulating factor plays a central role in the dynamic regulation of neutrophil production and release from the bone marrow in response to environmental stresses. Recent studies have begun to elucidate both the pathways linking neutrophil clearance to granulocyte colony-stimulating factor expression and the mechanisms by which the factor induces neutrophil release from the bone marrow. These studies may lead to novel strategies to modulate neutrophil responses in host defense and inflammation.

Association Between Mutation Clearance After Induction Therapy and Outcomes in Acute Myeloid Leukemia

IMPORTANCE Tests that predict outcomes for patients with acute myeloid leukemia (AML) are imprecise, especially for those with intermediate risk AML. OBJECTIVES To determine whether genomic approaches can provide novel prognostic information for adult patients with de novo AML. DESIGN, SETTING, AND PARTICIPANTS Whole-genome or exome sequencing was performed on samples obtained at disease presentation from 71 patients with AML (mean age, 50.8 years) treated with standard induction chemotherapy at a single site starting in March 2002, with follow-up through January 2015. In addition, deep digital sequencing was performed on paired diagnosis and remission samples from 50 patients (including 32 with intermediate-risk AML), approximately 30 days after successful induction therapy. Twenty-five of the 50 were from the cohort of 71 patients, and 25 were new, additional cases. EXPOSURES Whole-genome or exome sequencing and targeted deep sequencing. Risk of identification based on genetic data. MAIN OUTCOMES AND MEASURES Mutation patterns (including clearance of leukemia-associated variants after chemotherapy) and their association with event-free survival and overall survival. RESULTS Analysis of comprehensive genomic data from the 71 patients did not improve outcome assessment over current standard-of-care metrics. In an analysis of 50 patients with both presentation and documented remission samples, 24 (48%) had persistent leukemia-associated mutations in at least 5% of bone marrow cells at remission. The 24 with persistent mutations had significantly reduced event-free and overall survival vs the 26 who cleared all mutations. Patients with intermediate cytogenetic risk profiles had similar findings. [table: see text]. CONCLUSIONS AND RELEVANCE The detection of persistent leukemia-associated mutations in at least 5% of bone marrow cells in day 30 remission samples was associated with a significantly increased risk of relapse, and reduced overall survival. These data suggest that this genomic approach may improve risk stratification for patients with AML.

Granulocyte Colony-Stimulating Factor Induces Osteoblast Apoptosis and Inhibits Osteoblast Differentiation

Long‐term treatment of mice or humans with granulocyte colony‐stimulating factor (G‐CSF) is associated with a clinically significant osteopenia characterized by increased osteoclast activity and number. In addition, recent reports have observed a decrease in number of mature osteoblasts during G‐CSF administration. However, neither the extent of G‐CSFs suppressive effect on the osteoblast compartment nor its mechanisms are well understood. Herein, we show that short‐term G‐CSF treatment in mice leads to decreased numbers of endosteal and trabecular osteoblasts. The effect is specific to mature osteoblasts, because bone‐lining cells, osteocytes, and periosteal osteoblasts are unaffected. G‐CSF treatment accelerates osteoblast turnover in the bone marrow by inducing osteoblast apoptosis. In addition, whereas G‐CSF treatment sharply increases osteoprogenitor number, differentiation of mature osteoblasts is impaired. Bone marrow transplantation studies show that G‐CSF acts through a hematopoietic intermediary to suppress osteoblasts. Finally, G‐CSF treatment, through suppression of mature osteoblasts, also leads to a marked decrease in osteoprotegerin expression in the bone marrow, whereas expression of RANKL remains relatively constant, suggesting a novel mechanism contributing to the increased osteoclastogenesis seen with long‐term G‐CSF treatment. In sum, these findings suggest that the hematopoietic system may play a novel role in regulating osteoblast differentiation and apoptosis during G‐CSF treatment.

G-CSF regulates hematopoietic stem cell activity, in part, through activation of Toll-like receptor signaling.

Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte colony-stimulating factor (G-CSF) is often induced with infection and has a key role in the stress granulopoiesis response. However, its effects on HSCs are less clear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect associated with induction of Toll-like receptor (TLR) expression and signaling. The G-CSF-mediated expansion of HSCs is reduced in mice lacking TLR2, TLR4 or the TLR signaling adaptor MyD88. Induction of HSC quiescence is abrogated in mice lacking MyD88 or in mice treated with antibiotics to suppress intestinal flora. Finally, loss of TLR4 or germ-free conditions mitigates the G-CSF-mediated HSC repopulating defect. These data suggest that low-level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling.

Primary acute myeloid leukemia cells with IDH1 or IDH2 mutations respond to a DOT1L inhibitor in vitro

Primary acute myeloid leukemia cells with IDH1 or IDH2 mutations respond to a DOT1L inhibitor in vitro

A genomic analysis of Philadelphia chromosome-negative AML arising in patients with CML

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome, an acquired clonal abnormality resulting from translocation of chromosomes 9 and 22, and the generation of the BCR–ABL fusion oncogene. The development of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of CML, as TKI therapy leads to inhibition of BCR–ABL activity, suppression of the BCR–ABL-containing clone and restoration of normal hematopoiesis in the vast majority of cases.

DNMT3A R882 -associated hypomethylation patterns are maintained in primary AML xenografts, but not in the DNMT3A R882C OCI-AML3 leukemia cell line

DNMT3A mutations act as dominant negative alleles in vitro and are associated with focal regions of DNA hypomethylation in primary acute myeloid leukemia (AML) samples and non-leukemic hematopoietic cells. In primary AML cells, this hypomethylation manifests both as methylation loss and attenuated CpG island hypermethylation relative to normal hematopoietic stem/ progenitor cells. Although DNMT3A mutations have a clear effect on DNA methylation in AML cells, the functional consequences of these changes are not yet clear. Future study of the downstream effects of mutant DNMT3A-associated hypomethylation will require model systems to investigate the genomic targets that are affected, and to understand whether these changes alter gene regulation in ways that promote leukemogenesis. Examples of model systems include genetically modified mice, patient-derived xenografts, and human cell lines containing DNMT3A mutations. The methylation phenotypes of mice lacking Dnmt3a, or expressing mutant Dnmt3a alleles, have been reported previously, but much less is known about whether alterations in methylation caused by DNMT3A alleles are retained in either patient-derived xenografts or human AML cell lines, and whether these models could therefore be used to accurately represent DNMT3A-dependent methylation changes in AML cells. To address this question, we performed whole-genome bisulfite sequencing (WGBS) using DNA from OCIAML3 cells, which is the only leukemia cell line currently known to have a native DNMT3A mutation. We also evaluated four xenografts derived from a primary AML sample containing the DNMT3A mutation. The OCI-AML3 line was obtained from the DSMZ cell collection and cultured via recommended conditions before DNA extraction at two independent passages for WGBS. The presence of the DNMT3A allele in these cells was verified via targeted sequencing prior to methylation analysis (Supplementary Figure S1), as were the recurrent NPM1 exon 12 insertion (NPMc) and the NRAS mutation. No functional mutations were identified in other recurrently mutated AML genes with roles in epigenetic modification, such as IDH1, IDH2, ASXL1, EZH2, or TET2. Two missense variants of unknown significance were present in TET1 (Supplementary Table S2), which is not frequently mutated in AML samples. Importantly, we saw no evidence for amplification of the wild-type DNMT3A allele in this cell line (data not shown). We also extracted two replicate DNA samples from comparator AML cell lines that are wild-type for DNMT3A, including Kasumi-1 and NB4, which have a t(8;21) translocation (creating the RUNX1RUNX1T1 fusion gene) and a t(15;17) translocation (resulting in a PML-RARA fusion), respectively. Patientderived AML xenografts were generated in two independent humanized NSG mice (NSG-SGM3) from a primary AML sample with the DNMT3A mutation (along with NPM1 and FLT3-ITD mutations; AML 721214, described as AML88 in ref. ; Supplementary Table S1) via tail vein injection of 1 million cells. Mice