ALBERT

Description: Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are unclear. 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. RNA profiling and flow cytometry studies of HSCs from G-CSF treated mice show that multiple toll- like receptors (TLRs) are upregulated in HSCs upon G-CSF treatment, and gene set enrichment analysis shows enhancement of TLR signaling in G-CSF-treated HSCs. G-CSF-induced expansion of phenotypic HSCs is reduced in mice lacking the TLR signaling adaptors MyD88 or Trif, and the induction of quiescence is abrogated in mice lacking these adaptors. Furthermore, loss of TLR4 mitigates the G-CSF-mediated HSC repopulating defect. Interestingly, baseline HSC function is also dependent on TLR signaling. We show that HSC long-term repopulating activity is enhanced in Tlr4-/- and MyD88-/- mice, but not Trif-/- mice. One potential source of TLR ligands affecting HSC function in the bone marrow is the gut microbiota. Indeed, we show that in mice treated with antibiotics to suppress intestinal flora, G-CSF induced HSC quiescence and hematopoietic progenitor mobilization are attenuated. Moreover, in germ free mice, HSC long-term repopulating activity is enhanced. Collectively 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. Our finding of enhanced TLR signaling upon G-CSF treatment, and the mitigation of G-CSF’s effects in mice deficient for TLR signaling or commensal organisms, suggest that TLR antagonists and/or agonists may ultimately be used clinically to enhance engraftment following bone marrow transplantation or applied toward the treatment of patients with bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

Description: Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene, which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately a block in granulocytic differentiation. To test this model, we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice, though without significant basal UPR activation, are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover, inaction of protein kinase RNA-like ER kinase (Perk), one of the major sensors of ER stress, either alone or in combination with G193X Elane, had no effect on basal granulopoiesis. However, inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN.

Description: Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Most cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. Herein, we report the preliminary description of a novel transgenic mouse line that expresses G192X Ela2, reproducing the G193X ELA2 mutation found in some patients with SCN. The G192X mutation was introduced into the murine Ela2 locus by homologous recombination in embryonic stem cells. Heterozygous or homozygous G192 Ela2 “knock-in” mice were healthy with no apparent developmental defect. While expression of Ela2 mRNA was normal, no mature NE protein was detected in the neutrophils of homozygous G192X Ela2 mice. However, in granulocytic precursors (mainly promyelocytes/myelocytes) a small amount of heavily glycosylated mutant NE protein was detected. Together, these observations suggest that G192X NE protein is retained in the endoplasmic reticulum (ER) and rapidly degraded. Consistent with ER stress and induction of the UPR, a significant increase in BiP/GRP78 and ATF6 mRNA expression in mutant granulocytic precursors were observed. Surprisingly, G192X Ela2 mice have normal basal granulopoiesis. The number of circulating neutrophils, granulocytic differentiation in the bone marrow, and number and cytokine responsiveness of myeloid progenitors were comparable to wild type mice. In summary, the G192X Ela2 mice appear to reproduce the NE protein misfolding and UPR activation observed in human SCN granulocytic precursors. However, expression of G192X Ela2 is not sufficient to disrupt basal granulopoiesis in mice. Studies of stress granulopoiesis are underway.

Description: Shwachman-Diamond Syndrome (SDS) is a rare multisystem disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, and metaphyseal chondrodysplasia. Recent studies show that mutations of SBDS, a gene of unknown function, are present in the majority of patients with SDS. In the present study, we show that most, but not all, patients classified based on rigorous clinical criteria as having SDS had compound heterozygous mutations of SBDS. Full-length SBDS protein was not detected in leukocytes of SDS patients with the most common SBDS mutations, consistent with a loss-of-function mechanism. In contrast, SBDS protein was expressed at normal levels in SDS patients without SBDS mutations. These data confirm the absence of SBDS mutations in this subgroup of patients and suggest that SDS is a genetically heterogeneous disorder. The presence (or absence) of SBDS mutations may define subgroups of patients with SDS who share distinct clinical features or natural history.

Description: Severe congenital neutropenia (SCN) is an inherited disorder of granulopoiesis that is associated with a markedly increased risk of developing acute myeloid leukemia (AML) or myelodysplasia (MDS). Somatic mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), are strongly associated with the development of AML/MDS in SCN. These mutations invariably produce a truncated G-CSFR that, though remaining ligand-dependent, transmits a hyperproliferative signal. Transgenic mice carrying a targeted (knock-in) mutation of Csf3r (termed d715) reproducing a mutation found in a patient with SCN have an exaggerated neutrophil response to G-CSF treatment but do not develop AML/MDS. Moreover, we recently showed expression of the d715 G-CSFR confers a strong clonal advantage at the hematopoietic stem cell level that is dependent upon exogenous G-CSF. Collectively, these data suggest that CSF3R truncation mutations are an initiation or early progression factor for leukemic transformation. However, there is, as yet, scant direct evidence supporting this hypothesis. Previous studies have established that activating mutations of receptor tyrosine kinases, such as internal tandem duplications of FLT3, are able to cooperate with PML-RARα to induce AML. Since the CSF3R mutations in SCN also are “activating”, we asked whether the d715 G-CSFR could cooperate with PML-RARα to induce AML in mice. PML-RARα transgenic mice were intercrossed with d715 G-CSFR mice (all inbred 〉 10 generations onto a C57BL/6 background) to generate the cohorts listed in Table 1. A separate cohort for each genotype was treated chronically with pegylated G-CSF (1 mg/kg every 4–5 days for 6 months) to simulate the high level of serum G-CSF present in patients with SCN. Complete blood counts were performed at 3 months intervals and documented a similar increase in neutrophil counts in all mice treated with G-CSF. The cumulative incidence of AML and median follow-up for each cohort is shown in Table 1. None of the mice without the PML-RARα transgene, regardless of G-CSF treatment, developed AML, confirming that the d715 G-CSFR is not sufficient to induce AML. In mice carrying the PML-RARα transgene but not treated with G-CSF, a nonsignificant trend to increased AML was observed in mice expressing the d715 G-CSFR (P=0.12). However, in mice carrying the PML-RARα transgene and treated with G-CSF, the presence of the d715 G-CSFR significantly increased the penetrance (P=0.009) and reduced the latency of AML. In all cases, the leukemia was characterized by leukocytosis, splenomegaly, and a high percentage of blasts in the bone marrow and spleen that co-express Gr1 and c-Kit. These data provide the first direct evidence that the CSF3R mutations present in patients with SCN are leukemogenic and provide further support for the proposition that patients who acquire CSF3R mutations be considered for early stem cell transplantation. The cumulative incidence of AML and median follow-up for each cohort PML-RAR CSF3R G-CSF Rx N Median follow-up Cumulative AML% No WT No 20 454 0.0% No d715 No 45 250 0.0% No WT Yes 20 488 0.0% No d715 Yes 47 311 0.0% Yes WT No 57 286 6.4% Yes d715 No 67 267 11.9% Yes WT Yes 54 322 20.4% Yes d715 Yes 50 311 44.0%

Description: Granulocyte colony-stimulating factor (G-CSF) is the principal cytokine regulating granulopoiesis. Truncation mutations of the G-CSF receptor (G-CSFR) are associated with the development of acute myeloid leukemia in patients with severe congenital neutropenia. Although increased proliferative signaling by a representative G-CSFR truncation mutation (termed d715) has been documented, the molecular basis for this hyperproliferative phenotype has not been fully characterized. Given the accumulating evidence implicating Src family kinases in the transduction of cytokine receptor signals, the role of these kinases in the regulation of G-CSF signaling was examined. We show that Hck and Lyn, Src family kinases expressed in myeloid cells, are negative regulators of granulopoiesis that act at distinct stages of granulocytic differentiation. Whereas Hck regulates the G-CSF-induced proliferation of granulocytic precursors, Lyn regulates the production of myeloid progenitors. Interestingly, d715 G-CSFR myeloid progenitors were resistant to the growth-stimulating effect of treatment with a Src kinase inhibitor. Together, these data establish Lyn and Hck as key negative regulators of granulopoiesis and raise the possibility that loss of Src family kinase activation by the d715 G-CSFR may contribute to its hyperproliferative phenotype.

Description: Abstract 387 A shared feature of many bone marrow failure syndromes is their propensity to develop myelodysplasia (MDS) or acute myeloid leukemia (AML). The molecular mechanisms that underlie this susceptibility are largely unknown. Severe congenital neutropenia (SCN) is an inherited disorder of granulopoiesis that is associated with a marked increased risk of developing MDS/AML. Somatic mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), that truncate the carboxy-terminal tail are associated with the development of MDS/AML in SCN. Transgenic mice carrying a ‘knock-in’ mutation of their Csf3r (termed d715 G-CSFR) reproducing a mutation found in a patient with SCN have normal basal granulopoiesis but an exaggerated neutrophil response to G-CSF treatment. We previously reported that the d715 G-CSFR is able to cooperate with the PML-RARƒÑ oncogene to induce AML in mice. Herein, we summarize data supporting the hypothesis that alterations in the bone marrow microenvironment induced by G-CSF contribute to oxidative DNA damage in hematopoietic stem/progenitors cells (HSPCs) and possibly leukemic transformation. We previously showed that G-CSF treatment is associated with a marked loss of osteoblasts in the bone marrow, thereby potentially disrupting the osteoblast stem cell niche (Semerad, Blood 2005). Of note, patients with SCN chronically treated with G-CSF are prone to develop osteopenia, suggesting that osteoblast suppression by G-CSF also may occur in humans. We first asked whether the d715 G-CSFR was able to mediate this response. Wild-type or d715 G-CSFR were treated with G-CSF for 1–7 days and osteoblast activity in the bone marrow measured by expression of CXCL12 and osteocalcin. Consistent with previous reports, a decrease in osteocalcin and CXCL12 was not apparent until after 3 days of G-CSF treatment and reached a maximum after 7 days. Surprisingly, the magnitude of osteoblast suppression was greater in d715 G-CSFR compared with wild-type mice. The fold-decrease in osteocalcin mRNA from baseline in wild-type mice was 147 ± 70.1 versus 1,513 ± 1091 in d715 G-CSFR mice (p 〈 0.001). Likewise, a greater fold-decrease in CXCL12 mRNA was observed. We next assessed oxidative stress in c-KIT+ Sca+ lineage− (KSL) progenitors after G-CSF treatment. In both wild-type and d715 G-CSFR KSL cells no increase in reactive oxygen species (ROS) was observed at baseline or 12 hours after a single dose of G-CSF. However, after 7 days of G-CSF, a significant increase (3.4 ± 0.1 fold; p = 0.009) in ROS was observed in d715 G-CSFR but not wild-type KSL cells. To determine whether oxidative stress contributed to DNA damage, histone H2AX phosphorylation (pH2AX) was measured by flow cytometry. No increase in pH2AX was observed after short-term (less than 24 hour) G-CSF treatment. However, a modest but significant (1.9 ± 0.1 fold; p = 0.0007) increase in pH2AX was observed in d715 G-CSFR but not wild-type KSL cells after 7 days of G-CSF. To determine whether increased oxidative stress was casually linked to DNA damage, we co-administered the antioxidant N-acetyl cysteine (NAC) during G-CSF treatment. As expected, induction of ROS in KSL cells was markedly suppressed by NAC administration. Importantly, the increase in pH2AX levels in d715 G-CSFR KSL cells induced by G-CSF was completely blocked by NAC administration. Finally, to determine whether alterations in the bone marrow microenvironment, specifically decreased CXCL12 expression, contributed to DNA damage, we treated mice with AMD3100, a specific antagonist of CXCR4 (the major receptor for CXCL12). Treatment of wild-type or d715 G-CSFR mice with a single dose of G-CSF (3 hour time point) or with AMD3100 alone did not induce H2AXp. However, co-administration of AMD3100 with a single dose of G-CSF induced modest but significant H2AXp in d715 G-CSFR KSL cells (5.74 ± 1.06 fold; P

Description: The number of circulating neutrophils is tightly regulated in order to effectively protect against microbial pathogens while minimizing damage to host tissue. Homeostatic control of neutrophils in the blood is achieved through a balance of neutrophil production, release from the bone marrow, and clearance from the circulation. Accumulating evidence suggests that signaling by the chemokine CXCL12, through its major receptor CXCR4, may play a key role in controlling neutrophil homeostasis. Indeed, gain-of-function mutations of CXCR4 are responsible for most cases of WHIM syndrome, a syndrome that features impaired neutrophil release from the bone marrow. Conversely, we previously reported that mice carrying a myeloid-specific deletion of CXCR4 (CXCR4f/−LysM+/Cre mice) display constitutive neutrophil release. Moreover, we provided data suggesting that neutrophil mobilization by G-CSF or Groβ are dependent on CXCR4 signaling, as neutrophil mobilization by these agents was absent in CXCR4f/−LysM+/Cre mice. These data firmly establish CXCR4 signaling as a key regulator of neutrophil release from the bone marrow under basal and stress conditions. Though controversial, there also is evidence that CXCR4 may play a role in neutrophil clearance from the blood by selectively trapping and removing aged neutrophils in the bone marrow. In this study, we examine the role of CXCR4 in neutrophil clearance using CXCR4f/−LysM+/Cre mice. Strain-matched wild type or CXCR4f/−LysM+/Cre mice were treated with a single injection of BrdU to label newly synthesized neutrophils. A similar percentage of myeloid cells in the bone marrow were labeled in wild type and CXCR4f/−LysM+/Cre mice, suggesting that the loss of CXCR4 does not affect granulocytic cell proliferation. Consistent with its role in regulating neutrophil release, the transit time for labeled neutrophils to appear in the circulation was significantly reduced in CXCR4f/−LysM+/Cre mice (45 hours) compared with wild type mice (72 hours). The half-life (t1/2 ) of neutrophils in the blood was calculated using the formula N=N0e−λt where N0 = the peak number of labeled cells, N = the number of cells at time t and λ = the decay constant. Surprisingly, no difference in the circulating neutrophil half-life was observed in CXCR4f/−LysM+/Cre mice compared to wild type mice (18.3 ± 13.6 hours vs.12.7 ± 9.5 hours respectively, P=0.43). We next performed adoptive transfer experiments to determine the site of neutrophil clearance. Specifically, an equivalent number of bone marrow neutrophils from wild type or CXCR4f/−LysM+/Cre mice were injected intravenously into recipient mice. Donor neutrophils were identified based on differential Ly5 gene expression. By 3 hours post-infusion, the majority of donor neutrophils were cleared from the blood. Compared to wild type neutrophils, CXCR4−/− neutrophils showed reduced homing to the bone marrow [number of donor neutrophils per femur: 6.7 ± 0.3 x 104 (wild type) compared to 2.6 ± 0.8 x 104 (CXCR4−/−); P

Description: There is evidence suggesting that N-cadherin expression on osteoblast lineage cells regulates hematopoietic stem cell (HSC) function and quiescence. To test this hypothesis, we conditionally deleted N-cadherin (Cdh2) in osteoblasts using Cdh2flox/flox Osx-Cre mice. N-cadherin expression was efficiently ablated in osteoblast lineage cells as assessed by mRNA expression and immunostaining of bone sections. Basal hematopoiesis is normal in these mice. In particular, HSC number, cell cycle status, long-term repopulating activity, and self-renewal capacity were normal. Moreover, engraftment of wild-type cells into N-cadherin–deleted recipients was normal. Finally, these mice responded normally to G-CSF, a stimulus that mobilizes HSCs by inducing alterations to the stromal micro-environment. In conclusion, N-cadherin expression in osteoblast lineage cells is dispensable for HSC maintenance in mice.

Description: Abstract 563 Granulocyte-colony stimulating factor (G-CSF) is the most common agent used to mobilize hematopoietic stem/progenitor cells (HSPCs) for stem cell transplantation. We previously showed that HSPC mobilization by G-CSF is associated with significant changes in the bone marrow microenvironment. Most notably, there is a marked loss of mature osteoblasts in the bone marrow following G-CSF treatment. These findings raise the possibility that the hematopoietic compartment plays a hitherto unrecognized role in maintaining bone homeostasis. Bone marrow transplantation experiments demonstrated that G-CSF does not directly suppress osteoblast function but acts through a transplantable hematopoietic intermediary. The identify of this hematopoietic intermediary and the signals generated by these cells that suppress osteoblasts are unknown. Earlier work from our lab suggested that mature neutrophils and B- and T-lymphocytes are dispensable for G-CSF-induced osteoblast loss. We therefore hypothesized that bone marrow monocytes play a role in regulating osteoblasts both during G-CSF treatment and at steady state. Consistent with this hypothesis, we previously reported that purified murine monocytes/macrophages stimulated the growth and terminal differentiation of osteoblasts in vitro. To further test this hypothesis, we generated a transgenic mouse in which G-CSFR expression is largely confined to monocytic cells. Specifically, we expressed the G-CSFR and GFP under control of the CD68 promoter. CD68 is a glycoprotein whose expression is restricted to the surface of cells of the monocytic lineage. Two founder lines were identified and backcrossed to G-CSFR-/- mice, generating mice (termed CD68:G-CSFR) in which the G-CSFR expression is predicted to be restricted to monocyte lineage cells. Consistent with this prediction, we detected G-CSFR expression on monocytes but not granulocytes or lymphocytes from CD68:G-CSFR mice. At baseline, CD68:G-CSFR mice are neutropenic but had normal levels of circulating monocytes, B and T cells, and colony-forming cells (CFU-C). Wild-type, G-CSFR-/-, and CD68:G-CSFR mice were treated with G-CSF for 7 days and HSPC mobilization and osteoblast activity in the bone marrow were assessed. As expected, G-CSFR-/- mice had no discernible response to G-CSFR. In contrast, the CD68:G-CSFR mice had a strong mobilization response to G-CSF, with an increase in circulating CFU-C (CFU-C per ml ± SEM: 6,400 ± 1,008) that was comparable to wild-type mice (5,500 ± 2,000). Osteoblast activity was assessed by quantitative RT-PCR for osteocalcin and CXCL12 on bone marrow aspirates. Consistent with previous reports, in wild-type mice, G-CSF treatment resulted in a 38-fold decrease in osteocalcin expression [relative osteocalcin mRNA to beta-actin ± SEM: 18.1 ± 9.08 (baseline) and 0.47 ± 0.21 (G-CSF)] and a 4.9-fold decrease in CXCL12 expression [5.84 ± 3.12 (baseline); 1.19 ± 0.45 (G-CSF)]. A similar 81-fold decrease in osteocalcin (0.22 ± 0.09) and 7.5-fold decrease in CXCL12 (0.77 ± 0.18) expression were observed in CD68:G-CSFR mice after G-CSF treatment. Together, these data demonstrate that G-CSFR expression in monocytes is sufficient to generate the signals required for the suppression of osteoblast activity and HSPC mobilization in mice. Disclosures: No relevant conflicts of interest to declare.