Mobilization Of Hematopoietic Progenitors Research Articles

Bone Marrow Sinusoidal Endothelial Cells Are a Site of Fgf23 Upregulation in Murine ß-Thalassemia

Fibroblast growth factor-23 (FGF23) is a bone-derived hormonal regulator of mineral metabolism with emerging functions in the bone marrow. FGF23 has been reported to negatively regulate erythropoiesis [PMID:24509850] and promote hematopoietic progenitor mobilization from the bone marrow [PMID:33512467]. Elevated FGF23 levels in the circulation have been described in iron-deficient subjects and also in subjects with the iron-loading anemia β-thalassemia. Recently, we reported bone marrow sinusoidal endothelial cells (BM-SEC) as a novel site of Fgf23 upregulation in mice with chronic iron-deficiency anemia and acute, phlebotomy-induced anemia [PMID:37417950]. We and others have detected Fgf23 mRNA elevation in total bone marrow of Hbb th3/+ mice, a model of non-transfusion dependent β-thalassemia that exhibits systemic iron loading. In the present study, we sought to clarify sites of Fgf23 promoter activity in Hbb th3/+ bone marrow using a reporter allele in which the enhanced green fluorescent protein (eGFP) coding sequence is knocked into the endogenous Fgf23 locus ( Fgf23 eGFP). We generated Hbb +/+ and Hbb th3/+ female littermates that carried either one ( Fgf23 +/eGFP) or zero ( Fgf23 +/+) copies of the Fgf23 reporter allele, which were raised on a standard rodent diet (200 mg/kg iron, 1.0% calcium, 0.7% phosphorus). Fgf23 eGFP/eGFP homozygotes were not generated due to known early growth retardation and mortality in Fgf23 null mice. After blood and tissue collection at 8 weeks of age, phenotypes of the 4 Hbb-Fgf23 genotype combinations were compared by two-way ANOVA. Compared to mice with intact Hbb and Fgf23 alleles ( Hbb +/+ Fgf23 +/+), Hbb th3/+ Fgf23 +/+ mice showed markedly elevated plasma levels of “total” FGF23 (sum of intact, active hormone and C-terminal cleaved fragments). Plasma total FGF23 levels in Hbb th3/+ Fgf23 +/+ and Hbb th3/+ Fgf23 +/eGFP mice were similar, indicating utility of the latter genotype for interrogating sites of increased Fgf23 expression in murine b-thalassemia. We therefore used anti-GFP immunohistochemistry in formalin-fixed bone marrow sections to assess Fgf23 eGFP reporter allele expression in the context of tissue architecture. Hbb th3/+ Fgf23 +/eGFP mice showed GFP expression in BM-SEC throughout the bone marrow, which was more intense than in non-anemic Hbb +/+ Fgf23 +/eGFP mice; controls lacking the Fgf23 eGFP allele confirmed anti-GFP antibody specificity. Hbb th3/+ mice also showed Fgf23 eGFP reporter expression in the thymic vasculature but not vasculature of the liver, spleen, or kidney. In contrast to their marked elevation in plasma total FGF23, Hbb th3/+ Fgf23 +/+ mice showed plasma levels of the intact, biologically-active FGF23 hormone that overlapped with levels in Hbb +/+ Fgf23 +/+ controls. Interestingly, compared with Hbb th3/+ Fgf23 +/+ mice, Hbb th3/+ Fgf23 +/eGFP mice showed 45% lower plasma intact FGF23, suggesting an effect of Fgf23 gene dosage. Given prior reported effects of FGF23 on hematopoiesis, we assessed if Fgf23 gene dosage modulated the severity of ineffective erythropoiesis in Hbb th3/+ mice. Compared to Hbb +/+ Fgf23 +/+ controls, Hbb th3/+ Fgf23 +/+ mice showed reductions in RBC, hemoglobin, and mean corpuscular hemoglobin, as well as increases in RDW and in spleen weight (reflecting extramedullary hematopoiesis). However, the severity of these parameters was not modified by heterozygous Fgf23 disruption ( Hbb th3/+ Fgf23 +/eGFP).While FGF23 has been reported to induce left ventricular hypertrophy [PMID:21985788], Hbb th3/+ Fgf23 +/+ and Hbb th3/+ Fgf23 +/eGFP mice showed similarly increased heart-to-body weight ratios compared to Hbb +/+ Fgf23 +/+ controls. In non-thalassemic mice, heterozygous Fgf23 disruption ( Hbb +/+ Fgf23 +/eGFP) caused a trend ( P=0.10) towards lower plasma intact FGF23 while not impacting hematological parameters or spleen weight. In summary, by showing that Fgf23 gene dosage modulates plasma intact FGF23 levels in Hbb th3/+ mice without altering severity of anemia or splenomegaly, our data suggest that moderate reductions in circulating levels of the intact FGF23 hormone do not alter the severity of ineffective erythropoiesis in Hbb th3/+mice at 8 weeks of age. Our finding that β-thalassemic mice exhibit Fgf23 upregulation in BM-SEC lays the groundwork for future investigation of the functional effects of locally-produced FGF23 in the thalassemic hematopoietic stem cell niche.

POSTER: MM-610 Impact of Induction Therapy on the Mobilization and Engraftment of Peripheral Blood Hematopoietic Progenitors for Autologous Transplantation in Patients With Multiple Myeloma. Experience of the University Hospital of Salamanca (Spain)

POSTER: MM-610 Impact of Induction Therapy on the Mobilization and Engraftment of Peripheral Blood Hematopoietic Progenitors for Autologous Transplantation in Patients With Multiple Myeloma. Experience of the University Hospital of Salamanca (Spain)

MM-610 Impact of Induction Therapy on the Mobilization and Engraftment of Peripheral Blood Hematopoietic Progenitors for Autologous Transplantation in Patients With Multiple Myeloma. Experience of the University Hospital of Salamanca (Spain)

MM-610 Impact of Induction Therapy on the Mobilization and Engraftment of Peripheral Blood Hematopoietic Progenitors for Autologous Transplantation in Patients With Multiple Myeloma. Experience of the University Hospital of Salamanca (Spain)

Mitigation of Multi-Organ Radiation Injury with ACE2 Agonist Diminazene Aceturate.

The renin-angiotensin system (RAS) is known to regulate the pathogenesis of radiation-induced injury as inhibitors of the RAS enzyme angiotensin converting enzyme (ACE) have established function as mitigators of multi-organ radiation injury. To further elucidate the role of RAS signaling during both the acute and delayed syndromes of radiation exposure, we have evaluated whether pharmacologic modulation of alternate RAS enzyme angiotensin converting enzyme 2 (ACE2) reduces the pathogenesis of multi-organ radiation-induced injuries. Here, we demonstrate pharmacologic ACE2 activation with the small molecule ACE2 agonist diminazene aceturate (DIZE) improves survival in rat models of both hematologic acute radiation syndrome (H-ARS) and multi-organ delayed effects of acute radiation exposure (DEARE). In the H-ARS model, DIZE treatment increased 30-day survival by 30% compared to vehicle control rats after a LD50/30 total-body irradiation (TBI) dose of 7.75 Gy. In the mitigation of DEARE, ACE2 agonism with DIZE increased median survival by 30 days, reduced breathing rate, and reduced blood urea nitrogen (BUN) levels compared to control rats after partial-body irradiation (PBI) of 13.5 Gy. DIZE treatment was observed to have systemic effects which may explain the multi-organ benefits observed including mobilization of hematopoietic progenitors to the circulation and a reduction in plasma TGF-beta levels. These data suggest the ACE2 enzyme plays a critical role in the RAS-mediated pathogenesis of radiation injury and may be a potential therapeutic target for the development of medical countermeasures for acute radiation exposure.

Mechanisms of improved erythroid progenitor growth with removal of chronic stress after trauma

BackgroundErythropoietic dysfunction after trauma and critical illness is associated with anemia, persistent inflammation, increased hematopoietic progenitor cell mobilization from the bone marrow, and reduced erythroid progenitor growth. Yet the duration and reversibility of these postinjury bone marrow changes remain unknown. This study sought to determine whether removal of chronic postinjury stress could induce improvements in erythroid progenitor growth. MethodsSprague-Dawley rats (n = 8–11/group) were assigned to the following: naïve, lung contusion and hemorrhagic shock, lung contusion and hemorrhagic shock plus daily chronic stress for 7 days followed by 7 days of routine handling to allow recovery (lung contusion and hemorrhagic shock + chronic stress 7), or lung contusion and hemorrhagic shock plus chronic stress for 14 days (lung contusion and hemorrhagic shock + chronic stress 14). Circulating CD117+CD71+ erythroid progenitors were detected by flow cytometry. Rodents were killed on day 14, and bone marrow erythroid progenitor growth and erythroid transcription factors were assessed. Differences were assessed by analysis of variance (P < .05). ResultsCompared to lung contusion and hemorrhagic shock + chronic stress 14, lung contusion and hemorrhagic shock + chronic stress 7 rodents had improved hemoglobin (8% ± 10% increase vs 6% ± 10% decrease) with fewer mobilized erythroid progenitors (898 × vs 1,524 cells), lower granulocyte-colony stimulating factor levels (3.1 ± 1.1 × pg/mL vs 5.9 ± 1.8 pg/mL), and improved erythroid progenitor growth. Cessation of stress had no impact on erythroid transcription factors GATA-1, GATA-2, LMO2, or KLF1. ConclusionImprovements in erythroid progenitor growth and reduced hematopoietic progenitor cell mobilization were seen 7 days after cessation of chronic stress and were associated with an improvement in hemoglobin. Early bone marrow erythropoietic functional recovery may result from resolution of hematopoietic progenitor mobilization rather than upregulation of pro-erythroid transcription factors. This study suggests that postinjury anemia is reversible and has the potential to improve with the cessation of stress.

Role of GATA2 in the Maintenance of the Bone Marrow Microenvironment

(Background) The bone marrow (BM) microenvironment comprises multiple stem cell niches derived from BM mesenchymal stem cells (BM-MSCs). Previous in vitro analyses have suggested that transcription factor GATA2 plays an important role in adipocyte differentiation and hematopoietic supporting activity of BM-MSCs (Kamata et al. Haematologica 2014). However, the role of GATA2 in BM-MSCs in vivo remains uncharacterized.(Method) Mice harboring a Gata2 exon 5 flanked by loxP sites (Gata2 -floxed mice, designated Gata2flox) were a kind gift from Dr. Camper (Charles et al. Molecular Endocrinology 2006). Gata2 was deletedin under the MSC-related promoters (Nestin , Prx1 , and Lepr ) (Matsuzaki et al. Cell Stem Cell 2014) based on the Cre-loxP system (Gata2f/f/ Nes -ER-Cre, Gata2f/f/ Prx1 -Cre, and Gata2f/f/ Lepr -Cre mice) (The Jackson Laboratory). For transplantation analysis, 2x105 BM cells from SJL (CD45.1+; The Jackson Laboratory) mice were intravenously injected into lethally irradiated CD45.2+Gata2flox/flox/ER-Cre or CD45.2+Gata2flox/flox mice (a total of 10 Gy). The recipient mice were injected intraperitoneally with 1 mg of tamoxifen (Sigma, USA) on days 29-31 and 36-38 after transplantation and subsequently analyzed on days 50-51. For the analysis, we focused on the CD45.1 donor cell fraction. To isolate MSCs, the compact bones were crushed with a pestle and treated with collagenase (Wako, Japan), after which Ter119-CD45- MSC fraction was separated using EasySep Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (StemCell Technologies, Canada). BM hematopoietic progenitors were separated and evaluated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For transcription profiling, a GeneChip Mouse Gene 2.0 ST Array (Affymetrix, USA) was used and the data was subsequently analyzed with the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/).(Results) After confirming Gata2 deletion under each BM-MSC-related promoter (Nestin , Prx1 , and Lepr ), we evaluated the hematological profile, total BM cell counts, BM progenitor frequency (LSK, CMP, GMP, MEP, and CLP), colony assay with sorted LSK fraction, BM histology, and colony-forming cell assay of BM-MSCs. Whereas we found a significant decrease in the colony number of Gata2flox/flox/ Prx1 -Cre mice, the phenotypic alterations in BM-MSC-specific conditional Gata2 knockout mice are somewhat weak, implying that Gata2 deletion with a single marker might be insufficient. Thus, we next transplanted normal BM cells into irradiated Gata2flox/flox/ER-Cre mice, after which Gata2 was knocked out by tamoxifen administration. In this case, Gata2 would be deleted in all cell types constituting the BM microenvironment but not the hematopoietic cells. Based on the analyses of the CD45.1+ donor cell fraction, we found a significant decrease in the percentage of the CMP fraction, whereas that of CLP was marginally increased (p = 0.081) by Gata2 knockout, suggesting that GATA2 expression in BM-MSCs might function in the differentiation of LSKs to CMPs.To elucidate the underlying molecular mechanisms, we performed expression profiling with the MSCs obtained from Gata2flox/flox/ER-Cre mice. Prior to the profiling analysis, RT-PCR analysis confirmed that the separated MSCs abundantly expressed MSC-related genes, including Nestin , Prx1 , Lepr , Cd29 , and Sca-1 . Based on the average of 2 independent datasets, we demonstrated that GATA2 knockout activated 110 and repressed 141 genes (> 2-fold). The repressed gene ensemble included adhesion molecules (Itga11 , Itgb3 ), extracellular matrix (Prg3 ), and genes involved in hematopoietic progenitor mobilization (Mmp9 , Elane , Ctsg ). Activated genes included Fabp5 , involved in fatty acid metabolism, suggesting that GATA2 knockout might accelerate adipocyte differentiation in vivo . Gene Ontology analysis revealed significant enrichment of genes related to cell adhesion (P = 4.8E−05) and chemotaxis (P = 2.7E−08), suggesting that the expressions for extracellular signals in BM-MSCs might be regulated by GATA2.(Conclusion) Besides its critical role in hematopoietic stem cells, GATA2 could also affect the function of BM-MSCs in vivo . Our data might offer a better understanding of the pathophysiology of bone marrow failure syndrome. DisclosuresNo relevant conflicts of interest to declare.

O-Fucose and Fringe-Modified Recombinant Notch Peptides As Decoy Molecules to Promote Hematopoietic Progenitor Mobilization

Nearly all autologous hematopoietic cell therapy (HCT) procedures are performed using mobilized hematopoietic progenitor cells (HPCs). Successful outcome is dependent on infusing an adequate number of functionally active HPCs. Mobilization strategy of AMD3100, in combination with G-CSF, increases total CD34+ cells mobilized, and often is used for HPC mobilization in myeloma and non-Hodgkin lymphoma patients. Unfortunately, this approach does not always lead to adequate HPC collection in patients who previously have been exposed to extensive cytotoxic therapy. Furthermore, suboptimal number of HPCs mobilized in some patients may result in slower hematopoietic reconstitution and increased risk of complications during the transplant. Hence developing new mobilization strategy is highly desired for patients who are considered poor-mobilizers. Notch is a signaling molecule important for stem cell self-renewal and fate determination in many tissues including the hematopoietic system. An important feature of Notch is its adhesive nature which was first described by cell aggregation assays in Drosophila studies. We previously reported that hematopoietic stem cell and progenitors (HSPCs) with faulty Notch-ligand interaction display increased cell cycling, decreased adhesion to marrow osteoblastic lineage cells, and enhanced egress from the marrow.Since Notch2 is the major Notch isotype expressed on HSPCs, we showed that prior treatment in mice with Notch2 blocking antibody but not Notch1 blocking antibody sensitizes HSPC to the mobilizing stimuli of G-CSF and AMD3100 with a 3~4 - fold increase in mobilization. We showed that transient Notch2 blockade leads to decreased CXCR4 expression by HSPCs with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. Notch2 blockade also increases cell cycling of HPCs during mobilization temporarily without affecting hematopoietic stem cell (HSC) homeostasis and self-renewal. Moreover, transient Notch2 blockade leads to greater HSPC homing to the marrow that is accompanied with a faster hematopoietic recovery and a competitive repopulating advantage of HSPCs.Herein, to search for alternative agents to Notch blocking antibody, we evaluate the ability of recombinant Notch peptides as decoy molecules to promote disengagement of Notch2-ligand interaction between HSPCs and niche supporting cells. By using OP9 cell-based binding assay and surface plasmon resonance (SPR) analyses, we showed that recombinant Notch111-13, Notch19-13, Notch18-13, Notch211-13, Notch29-13, and Notch28-13 peptide comprising EGF-like repeats 11-13, 8-13, and 9-13, of Notch1 or Notch2, respectively, all bind to Notch ligand Dll4, Dll1 and Jag2. Because EGF-like repeat 12 (EGF 12) has been shown located within the essential ligand binding domain, and the O -fucose attached to the threonine residue of Notch1 EGF12 acts as a surrogate amino acid to make specific and functional contact to residues of Notch ligand, we generated peptides in CHO cells with O -fucose attached to the threonine residue of EGF12 and further extended by Fringe. We found that binding affinities of the glycosylated peptides further increased compared to those of non-glycosylated peptides. Compared to Notch2 peptides, O -fucose and Fringe-modified Notch111-13 peptide shows the highest biding affinity to Notch ligand Dll4 with dissociation constant (Kd) reaching 34 nM. Similar to Notch2 blocking antibody, the O -fucose and Fringe-modified Notch111-13 peptide effectively outcompetes Notch2-ligand binding and thus blocks the adhesion of marrow HSPCs to the primary osteoblasts or OP9 cells expressing Notch ligands. Further, O -fucose and Fringe-modified Notch111-13 peptide can effectively induce HSPCs emigration from a 3D osteoblastic “spheroid niche”, an in vitro model of HSC niche. Compared to Notch2 blocking antibody, O -fucose and Fringe-modified Notch111-13 peptide caused a further decrease of HSPC cells remained in the “spheroid niche” and more HSPC emigration out of the “niche”.In summary, our findings suggest that recombinant Notch decoy peptides may be considered as alternatives to Notch2 blockade to be combined with the mobilizing regimen of AMD3100 and G-CSF for more effective HSPC mobilization. DisclosuresNo relevant conflicts of interest to declare.

TLR7/8 agonist treatment induces an increase in bone marrow resident dendritic cells and hematopoietic progenitor expansion and mobilization

There is accumulating evidence suggesting that toll-like receptor (TLR) signals play an important role in the regulation of hematopoietic stem/progenitor cells (HSPCs). TLR7/8 stimulation induces the myeloid differentiation of normal HSPCs and acute myeloid leukemia cells. However, the in vivo effect of TLR7/8 agonists on hematopoiesis is largely unknown. Here, we show that, similar to TLR4 and TLR2, treatment with the TLR7/8 agonist R848 induces an expansion of phenotypic hematopoietic stem cells (HSCs) with reduced repopulating potential and HSPC mobilization. In contrast to chronic TLR4 stimulation, treatment with R848 for 5 days did not induce a significant increase in myeloid-biased HSCs. Treatment with R848 results in a significant increase in classic dendritic cells (DCs) in the bone marrow, but a decrease in common dendritic cell progenitors and pre-DCs. Phenotypic analysis of DCs revealed that R848 treatment is associated with altered expression of certain chemokines, activation markers, and migratory receptors. Together, these data indicate that systemic administration of a TLR7/8 agonist has unique effects on hematopoiesis, including the expansion of DCs in the bone marrow, that might have clinical relevance to augment responses to certain immunotherapies, such as cancer vaccines and immune checkpoint blockade.

FGF-23 from erythroblasts promotes hematopoietic progenitor mobilization

Fibroblast growth factor 23 (FGF-23) hormone is produced by bone-embedded osteocytes and regulates phosphate homeostasis in kidneys. We found that administration of granulocyte colony-stimulating factor (G-CSF) to mice induced a rapid, substantial increase in FGF-23 messenger RNA in bone marrow (BM) cells. This increase originated mainly from CD45−Ter119+CD71+ erythroblasts. FGF-23 protein in BM extracellular fluid was markedly increased during G-CSF–induced hematopoietic progenitor cell (HPC) mobilization, but remained stable in the blood, with no change in the phosphate level. Consistent with the BM hypoxia induced by G-CSF, low oxygen concentration induced FGF-23 release from human erythroblast HUDEP-2 cells in vitro. The efficient mobilization induced by G-CSF decreased drastically in both FGF-23−/− and chimeric mice with FGF-23 deficiency, only in hematopoietic cells, but increased in osteocyte-specific FGF-23−/− mice. This finding suggests that erythroblast-derived, but not bone-derived, FGF-23 is needed to release HPCs from BM into the circulation. Mechanistically, FGF-23 did not influence CXCL-12 binding to CXCR-4 on progenitors but interfered with their transwell migration toward CXCL-12, which was canceled by FGF receptor inhibitors. These results suggest that BM erythroblasts facilitate G-CSF–induced HPC mobilization via FGF-23 production as an intrinsic suppressor of chemoattraction.

Modulation of the HGF/c-Met Axis Impacts Prolonged Hematopoietic Progenitor Mobilization Following Trauma and Chronic Stress.

Trauma and hemorrhagic shock trigger mobilization of hematopoietic progenitor cells (HPC) from bone marrow to peripheral blood. Hepatocyte growth factor (HGF), tyrosine-protein kinase Met (c-Met), matrix metallopeptidase 9 (MMP-9), and corticosterone regulate this mobilization process. We hypothesized that beta-blockade with propranolol and sympathetic outflow inhibition with clonidine following trauma and chronic stress would decrease hematopoietic progenitor cell mobilization. Sprague-Dawley rats were randomized to undergo three models of injury and stress: lung contusion, LC plus hemorrhagic shock (LCHS), or LCHS plus chronic restraint stress for 2 h daily (LCHS/CS). Propranolol and clonidine were administered by daily intraperitoneal injection until sacrifice on day seven. Bone marrow HGF, c-Met, and MMP-9 were measured by real-time PCR. Plasma corticosterone was measured by ELISA. Percentage HPC in peripheral blood was measured by flow cytometry. Propranolol and clonidine significantly decreased bone marrow MMP-9 expression, plasma corticosterone levels, and HPC mobilization, and significantly increased hemoglobin levels. HPC mobilization was greatest following LCHS/CS (5.4 ± 1.8) and was significantly decreased by propranolol (2.2 ± 0.9, P < 0.001) and clonidine (1.7 ± 0.5, P < 0.001). Hemoglobin (g/dL) was lowest following LCHS/CS (12.3 ± 1.2) and was significantly increased by propranolol (13.7 ± 0.4, P = 0.022) and clonidine (14.1 ± 1.1, P < 0.001). Severe injury was associated with increased bone marrow HGF, c-Met, and MMP-9, circulating corticosterone, HPC mobilization, and persistent anemia. Attenuating the neuroendocrine response to injury and stress with propranolol and clonidine reduced MMP-9 expression, corticosterone levels, HPC mobilization, and the degree of anemia.

TMIC-35. IDH1 MUTATION IN GLIOMA REPROGRAMS EARLY MYELOID DIFFERENTIATION IN THE BONE MARROW (BM) TO PRODUCE NON-IMUNESUPPRESSIVE NEUTROPHILS

Abstract Gliomas are the most common primary brain tumors; patients exhibit a poor prognosis. Mutations in isocitrate dehydrogenase (mIDH) are present in most patients with lower grade glioma (LGG), and are correlated with better prognosis and survival. We postulated that mIDH1 induces epigenetic reprogramming leading to alteration in immune cells’ function. To examine the role of mIDH1 in the tumor immune microenvironment (TME), we generated LGG glioma models using the sleeping beauty system (Koschmann et al., 2016, Nunez et al., 2019). We show that mIDH1 gliomas exhibit increased levels of CD11b+ Gr1+ myeloid derived suppressor cells in the tumor, BM, circulation and spleen of mice. We found that mIDH1 modifies the cytokines’ repertoire in the glioma microenvironment altering the phenotype and function of the tumor infiltrating CD45+/CD11b+/Gr-1+ myeloid cells, rendering them non-immunosuppressive. Production of these cells results from activation of the granulocytic differentiation program in the BM. This novel mechanism is mediated by tumor-derived granulocyte-colony stimulating factor (G-CSF) which elicits expansion and differentiation of hematopoietic stem cells, skewing hematopoiesis towards the immature myeloid lineage. Moreover, mIDH1 glioma derived G-CSF causes mobilization of hematopoietic stem cells (HSCs) and myeloid progenitors (MPs) from BM to spleen. Blocking G-CSF in mIDH1 bearing mice significantly restored HSCs, and MPs frequencies in the spleen to levels encountered in wtIDH1 glioma. Interestingly, blocking G-CSF restored the inhibitory function of the granulocytic CD11b+ Gr-1+ in mIDH1, and shortened the median survival (MS) of mIDH1 bearing mice to the same MS encountered in wtIDH1 glioma. Our results provide insights into novel epigenetic alterations triggered by mIDH1 which regulate myeloid cells’ heterogeneity and immunosuppression; a feature that can be harnessed to develop novel immunotherapeutic strategies.

Regulation of hematopoiesis by the chemokine system

Although chemokines are best known for their role in directing cell migration, accumulating evidence indicate their involvement in many other processes. This review focus on the role of chemokines in hematopoiesis with an emphasis on myelopoiesis. Indeed, many chemokine family members are an important component of the cytokine network present in the bone marrow that controls proliferation, retention, and mobilization of hematopoietic progenitors.

A demonstration of analytical similarity comparing a proposed biosimilar pegfilgrastim and reference pegfilgrastim

BackgroundRecombinant human granulocyte-colony stimulating factor (G-CSF, filgrastim) is used primarily to reduce incidence and duration of severe neutropenia and its associated complications in cancer patients that have received a chemotherapy regimen. The pegylated form of filgrastim, “pegfilgrastim”, is a long-acting form that requires only a once-per-cycle administration for the management of chemotherapy-induced neutropenia. Apobiologix, a division of ApoPharma USA, Inc., and Intas Pharmaceuticals Limited have co-developed a proposed pegfilgrastim biosimilar to US-licensed pegfilgrastim. MethodsThe analytical similarity of Apobiologix pegfilgrastim and US-licensed pegfilgrastim with respect to their physicochemical profile was established using a wide range of rigorous orthogonal analytical techniques. Biological function was compared using receptor binding analyses, in vitro proliferation assays, and in vivo hematopoietic progenitor mobilization. ResultsApobiologix pegfilgrastim and the US-licensed pegfilgrastim reference product were found to be highly similar analytically with respect to molecular mass, primary, secondary and tertiary protein structures, purity, charge, and hydrophobicity. No differences in receptor binding affinity were observed, and all samples demonstrated similar in vitro and in vivo bioactivity. ConclusionThese studies provide robust evidence supporting the structural and functional similarity between Apobiologix pegfilgrastim and the US-licensed reference pegfilgrastim, and hence their biosimilarity.

Mobilization of allogeneic peripheral blood stem cell donors with intravenous plerixafor mobilizes a unique graft

AbstractA single subcutaneous (SC) injection of plerixafor results in rapid mobilization of hematopoietic progenitors, but fails to mobilize 33% of normal allogeneic sibling donors in 1 apheresis. We hypothesized that changing the route of administration of plerixafor from SC to IV may overcome the low stem cell yields and allow collection in 1 day. A phase 1 trial followed by a phase 2 efficacy trial was conducted in allogeneic sibling donors. The optimal dose of IV plerixafor was determined to be 0.32 mg/kg. The primary outcome of reducing the failure to collect ≥2 × 106 CD34+/kg recipient weight in 1 apheresis collection to ≤10% was not reached. The failure rate was 34%. Studies evaluating the stem cell phenotype and gene expression revealed a novel plasmacytoid dendritic cell precursor preferentially mobilized by plerixafor with high interferon-α producing ability. The observed cytomegalovirus (CMV) viremia rate for patients at risk was low (15%), as were the rates of acute grade 2-4 graft-versus-host disease (GVHD) (21%). Day 100 treatment related mortality was low (3%). In conclusion, plerixafor results in rapid stem cell mobilization regardless of route of administration and resulted in novel cellular composition of the graft and favorable recipient outcomes. These trials were registered at clinicaltrials.gov as #NCT00241358 and #NCT00914849.

Mobilization of human immature hematopoietic progenitors through combinatory use of bortezomib and immunomodulatory drugs.

Combination use of the proteasome inhibitor bortezomib and the immunomodulatory drugs lenalidomide or thalidomide has provided superior outcomes in multiple myeloma over their single use; however, these combinations can produce significant toxicities. Unexpectedly, we found a small but significant increase in the population of immature granulocytes and erythrocytes/megakaryocytes in peripheral blood in 16 of 22 patients (73%) treated with dexamethasone in combination with bortezomib and immunomodulatory drugs (triplet), but not in any of 25 patients treated with either bortezomib or immunomodulatory drugs with dexamethasone (doublet). These immature cells gradually increased to a peak level (mean 2.6% per white blood cells) with triplet therapy, and disappeared immediately after therapy cessation. The numbers of circulating CD34+ cells and colony-forming cells derived from peripheral blood mononuclear cells increased after triplet therapy compared with those in patients treated by either bortezomib or immunomodulatory drugs plus dexamethasone. Furthermore, triplet regimen downregulated the expression of CXCR4, a chemokine receptor essential for bone marrow retention, on CD34+ cells, suggesting an unexpected effect on normal hematopoietic stem/progenitor cells through the reduced interaction with the bone marrow microenvironment. Our observations suggest that combination use should be carefully evaluated to exert synergistic anti-myeloma effects while avoiding unexpected adverse events.

The functional interplay between systemic cancer and the hematopoietic stem cell niche.

Hematopoietic cells are increasingly recognized as playing key roles in tumor growth and metastatic progression. Although many studies have focused on the functional interaction of hematopoietic cells with tumor cells, few have examined the regulation of hematopoiesis by the hematopoietic stem cell (HSC) niche in the setting of cancer. Hematopoiesis occurs primarily in the bone marrow, and processes including expansion, mobilization, and differentiation of hematopoietic progenitors are tightly regulated by the specialized stem cell niche. Loss of niche components or the ability of stem cells to localize to the stem cell niche relieves HSCs of the restrictions imposed under normal homeostasis. In this review, we discuss how tumor-derived factors and therapeutic interventions disrupt structural and regulatory properties of the stem cell niche, resulting in niche invasion by hematopoietic malignancies, extramedullary hematopoiesis, myeloid skewing by peripheral tissue microenvironments, and lymphopenia. The key regulatory roles played by the bone marrow niche in hematopoiesis has implications for therapy-related toxicity and the successful development of immune-based therapies for cancer.

Effect of administration timing of postchemotherapy granulocyte colony-stimulating factor on host-immune cell recovery and CD8+ T-cell response

Granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor, is a standard supportive therapy given during cancer treatment. It induces acceleration in neutrophil recovery through stimulation of mobilization of hematopoietic progenitors. Given that the latter is also induced by chemotherapy itself, the timing of administration of G-CSF postchemotherapy might impact the resultant overall effects. The present study aimed to determine the optimal timing of G-CSF postchemotherapy to exert its optimal effects on the immune cell recovery and its impact on antigen-specific CD8+ T-cell response. B6 mice were treated once with cyclophosphamide (4 mg/mouse; CTX) and then daily with G-CSF (5 g/mouse) from Days 1–5, 2–5 or 5–9 post-CTX treatment. The total numbers of various immune cell types were analyzed on Days 7, 9 and 12 post-CTX treatment. To evaluate effects on CD8+ T-cell response, a pmel-1 transgenic mouse model was used in combination with prime boost peptide vaccination therapy. The total number of white blood cells (WBC), neutrophils, monocytes, lymphocytes, granulocytes and dendritic cells (DC) were significantly increased after G-CSF treatment in particular when G-CSF was administered from Days 2–5 post-CTX treatment. Application of this timing of G-CSF and CTX treatment after adoptive transfer of T-cells followed by prime-boost vaccination with antigenic peptide did not block the expansion of the donor pmel-1 CD8+ T-cells. In conclusion, adjusting the timing of treatment with G-CSF postchemotherapy can optimize its promoting effects on recovery of myeloid cells without altering the associated antigen-specific immunity.

Mobilization of Human Immature Hematopoietic Progenitors after Combinatory Use of Bortezomib and Immunomodulatory Drugs

Introduction: Combination of proteasome inhibitor (PI) bortezomib and immunomodulatory drugs (IMiDs), lenalidomide or thalidomide, have revealed superior outcomes in multiple myeloma (MM) over their single use. PI and IMiDs possess their direct anti-tumor activity as well as exert the indirect pleiotropic biological effects by modulating immune response, cytokine production, cell adhesion molecule, anti-apoptotic factors, cell cycle regulators. We recently found that circulating immature granulocytes and erythrocytes/megakaryocytes frequently appeared in peripheral blood (PB) in patients treated with bortezomib and IMiDs. In this study, we tested whether this effect is derived from PI, IMiDs or combination, and tried to understand its underlying mechanism to further understand their combinatory effects to normal hematopoiesis.Patients & Methods: 47 patients aged 37 - 81 years with newly diagnosed or relapsed/refractory MM were enrolled into this study. PB or bone marrow (BM) samples were collected from 7 patients treated with bortezomib/lenalidomide/dexamethasone (VRD), 15 with bortezomib/thalidomide/dexamethasone (VTD), 6 with bortezomib/dexamethasone (BD), 19 with lenalidomide/dexamethasone (RD) regimen. Clonogenic progenitor assay (CFU-C) was performed using PB samples. Change in expression level of adhesion molecules on mobilized immature cells was analyzed with flowcytometric (FCM) analysis. We counted 250 differential WBC in May-Giemsa-stained PB smears under the microscope every other day after start of chemotherapy.Result: In PB analysis, a significant fraction of immature myeloid or erythro-megakaryocyte cells was morphologically documented in 16 out of 22 (73%) patients treated with VTD/VRD, but never in the patients treated with BD or RD (n=22). During the time course, circulating immature hematopoietic cells gradually increased after the commencement of VRD or VTD therapy, and reached to a peak on a median of 12 days (range, 4 - 24 days), which was documented after 2nd or 3rd administration of bortezomib in the most patients. In these patients, a mean peak percentage of immature cells per WBC was 2.6% (0 - 6.5%) during VRD/VTD therapy. We also performed FCM analyses to confirm mobilization of immature hematopoietic progenitors in peripherally during VRD/VTD therapy. Number of mobilized CD34+ cells was statistically higher in the VRD/VTD group than that in BD/RD group (0.070% vs. 0.017%, p=0.041). We extended an analysis of change in expression of adhesion molecules on mobilized immature cells in PB mononuclear cells, since down-regulation of these molecules resulted in release of stem/progenitor cells from marrow and mobilization into the circulation. We found a significant decrease in mean fluorescence intensity (MFI) of CXCR-4 expressed on PB CD34+ cells in VRD/VTD group compared with that in BD/RD groups (11.1 vs 15.2, p=0.033). Number of CFU-cells (CFU-C) increased after 2nd or 3rd administration of bortezomib during VRD/VTD therapy compared to that before therapy (Figure), which was coincided with the appearance of immature cells in PB. A number of CFU-C derived from 100,000 PB mononuclear cells was increased up to 45 in VRD/VTD group compared to 11 in BD/RD group (p=0.021).Discussion: In this study, we unexpectedly found a transient mobilization of the immature hematopoietic progenitors in the majority of patients who received concurrently PI and IMiDs, but not in the patients treated with either alone. IMiDs promote degradation of IKZF1, which regulate GATA-1 and PU.1, critical transcription factors for erythroid and granuloid lineage, resulting in a suppression of erythropoiesis and granulocyte maturation arrest with accumulation of immature granulocyte progenitors in the BM. In contrast, PI downregulate expressions such as CXCL-12/CXCR-4 and VLA-4/VCAM-1 by which hematopoietic progenitors anchor to BM microenvironments. Therefore, we hypothesize that accumulated immature progenitors in marrow by treatment with IMiDs, can be quickly released into peripherally by concurrent administration of bortezomib. It is still unknown whether the mechanism of mobilization effect is linked to their anti-myeloma effect. Thus, our observations provided the unexpected biological effects of these drugs. Combinatory use of new drugs should be carefully evaluated to maximize their benefit or prevent possible adverse events. [Display omitted] DisclosuresAkashi:Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

Abstract 4725: Hematopoietic stem cell niche activation and progenitor mobilization mediate cancer-associated immunosuppression and metastasis

Abstract Metastatic tumors have been shown to establish microenvironments in distant tissues that are permissive to disseminated tumor cells. Hematopoietic cells are essential components of this microenvironment, yet the events that initiate the hematopoietic response remain unclear. Further, the functional contribution of hematopoietic stem and progenitor cells (HSPCs) to metastatic progression has not been addressed. Here, we utilized two highly metastatic C57bl/6 murine cell lines capable of spontaneous metastasis following orthotopic injection to study how the hematopoietic response is initiated and can function to promote metastatic progression in immune competent tumor-bearing mice. In both the E0771 breast carcinoma model and M3-9-M rhabdomyosarcoma model we found that a primary tumor drove expansion of HSPCs within the bone marrow prior to establishment of metastatic lesions. To detect changes within the bone marrow microenvironment that accounted for the observed HSPC expansion in tumor-bearing mice, we performed qPCR analysis of whole femur lysate from control or pre-metastatic tumor-bearing mice. We found the bone marrow microenvironment of tumor-bearing mice expressed a transcriptional program of hematopoietic niche activation that promoted expansion as well as mobilization of hematopoietic progenitors, including downregulation of the HSPC homing cytokine CXCR4. Consistent with this, we detected elevated HSPCs in the circulation of newly diagnosed cancer patients as well as pre-metastatic tumor-bearing mice. By using a pharmacological antagonist of CXCR4 to mobilize HSPCs as seen in the tumor bearing setting, we demonstrated that circulating HSPCs were functionally capable of significantly enhancing metastasis in mice. To determine the mechanisms by which circulating HSPCs can promote metastatic progression, we tracked the developmental fate of flow cytometry-sorted HSPCs injected into the circulation of control or tumor-bearing pre-metastatic mice. We found that HSPCs localized within pre-metastatic tissues preferentially differentiated into the myeloid lineage. Further, depletion of these myeloid cells abrogated the metastasis-promoting effects of HSPC mobilization. Together, these data establish bone marrow niche activation as one of the earliest steps of the metastatic process and identify circulating HSPCs as a potential indicator of metastatic niche initiation. Citation Format: Amber J. Giles, Meera Murgai, Yorleny Vicioso, Steven Highfill, Miki Kasai, Linda Vahdat, Leonard Wexler, Crystal Mackall, David Lyden, Rosandra Kaplan. Hematopoietic stem cell niche activation and progenitor mobilization mediate cancer-associated immunosuppression and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4725. doi:10.1158/1538-7445.AM2015-4725

Pre-Clinical Studies of a Novel Recombinant Human Granulocyte Colony-Stimulating Factor

The human granulocyte colony-stimulating factor (G-CSF), a glycoprotein consisting of 174 amino acids, enhances the survival, proliferation, and differentiation of neutrophil precursors. G-CSF also induces the mobilization of hematopoietic stem cells and progenitors, which further stimulates recovery from neutropenia. Further studies indicates that Glu19 in the A helix of G-CSF molecule electrostatically inter-reacts with Arg288 of G-CSF-R. A recent study on the crystal structure of G-CSF, complexed with the cytokine homologous region of G-CSF-R, reveals that residues in the amino-terminus of G-CSF may act as additional contact sites with G-CSF-R.In this investigation, we designed and purified a novel recombinant human granulocyte colony-stimulating factor analog (rhG-CSFa), which has three extra amino acid residues (arginine, glycine, and serine) added at the amino-terminus. The structural modification resulted in more positive charge in the amino-terminus of G-CSFa, and was expected to further enhance the binding between the cytokine and its receptor. We optimized the purification procedure, achieving further improvements in refolding rate and overall yield. In preclinical studies, we evaluated the pharmacodynamics, pharmacokinetics, acute, subacute, and chronic toxicity of rhG-CSFa. Pharmacodynamics study in non-human primates demonstrated that intravenous rhG-CSFa induces typical peripheral neutrophil responses (with two peaks from treatment day 8 to day 20) and dramatic increases in the recovery rate in the animal model. More importantly, rhG-CSFa induced a higher peak of neutrophil recovery on day 7 than wild G-CSF did in monkeys, a result indicating that rhG-CSFa would provide greater benefit for patients receiving myeloid-suppressive therapies. In addition, rhG-CSFa maintained higher neutrophil counts than wild-type G-CSF did after cessation of G-CSF administration in monkeys; its effects persisted over 20 days post-treatment, which should be more helpful to patients for combating deleterious infections and preparing for the next round of treatment in clinic. In pharmacokinetics studies, the novel rhG-CSFa displayed a lower plasma clearance rate, 0.28 ml/min/kg (vs 0.5-0.7 ml/min/kg for wild-type G-CSF). This result suggests that rhG-CSFa may dissociate from its receptor at a slower rate than does wild-type G-CSF, which is possibly due to a tighter binding to its cognate receptor, and would result in a much extended plasma functional half-life. In further preclinical safety evaluation, no obvious toxicity or immunogenicity was observed, neither was any adverse drug reaction. In conclusion, we have generated a novel rhG-CSFa protein, with much higher yield, enhanced circulation half-life, improved thermal stability and greater bioactivity (without changing the overall conformation), compared with wild type G-CSF. Our preclinical studies strongly suggest that rhG-CSFa can be further explored for clinical trials for eventual applications in the clinic. DisclosuresRen:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment. Shi:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.