KIT Blockade Is Sufficient to Sustain Donor Hematopoietic Stem Cell Engraftment in Fanconi Anemia Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1206-1206
Author(s):  
Shanmuganathan Chandrakasan ◽  
Rajeswari Jayavaradhan ◽  
Ernst John ◽  
Archana Shrestha ◽  
Phillip Dexheimer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Conditioning Regimen ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Increased Risk

Abstract Background: Fanconi anemia (FA) is the most common cause of inherited bone marrow failure (BMF). Currently, the only curative option for the BMF in FA is an allogenic hematopoietic stem cell transplant (HSCT). However, due to the underlying DNA repair defect, FA patients poorly tolerate alkylating chemotherapy or irradiation based conditioning, which is necessary for donor engraftment. However, this results in significant short and long term morbidity/mortality and augments the inherent increased risk of malignancies in FA patients. To overcome the adverse effects associated with alkylating conditioning agents, alternate experimental approaches exploiting the inherent hematopoietic stem cell (HSC) defect in FA are of utmost clinical necessity. Objective: To develop a safe KIT blocking antibody (KIT-Ab) based HSCT conditioning regimen for FA that does not involve chemotherapy or irradiation. Method: High purity KIT-Ab was made from the ACK2 hybridoma and its specificity to KIT binding was validated using mast cell assay. Baseline peripheral blood cells and the bone marrow hematopoietic stem and progenitor cell (HSPC) compartment (Lin-Kit+Sca+ and Lin-Kit+Sca+CD150+CD48- cells) of FANCA-/- and FANCD2-/- murine models were analyzed. Mechanistic studies using sorted FA bone marrow HSPC were performed ex vivo. This was followed by definitive primary and secondary transplants experiments following injection of KIT-Ab. Results: Several features of FA hematopoietic stem/progenitor cells (HSPC) suggested their susceptibility to KIT-Ab blockade-mediated killing: (a) Expression of KIT was significantly lower in FANCA-/- HSPC, while expression of its ligand was higher in bone marrow stroma; (b) Moreover, genes associated with apoptosis/senescence, stress and inflammatory signaling that were upregulated in WT-HSPC following KIT-Ab blockade, were upregulated in FANCA-/- HSPC at baseline; (c) Furthermore, FANCA-/- HSPC demonstrated increased susceptibility to KIT-Ab mediated apoptosis and had a reduced proliferative capacity. In-vivo studies following ACK2 injection showed a marked reduction of colony-forming units (CFU-C) from both FANCA-/- and FANCD2-/- mice one week following injection, when compared to WT mice (48% and 76% decrease in CFU-C, respectively). Based on these findings, we evaluated the role of ACK2 as a sole HSCT conditioning regimen in FANCA-/- and FANCD2-/- mice. Indeed, definitive HSCT in both FANCA-/- and FANCD2-/- mice using KIT-Ab based conditioning resulted in donor HSC engraftment with multi-lineage chimerism, which progressively increased to 22-24% by 4-months, and was sustained in secondary transplants. Overall, we show that KIT-blockade alone is an adequate non-genotoxic HSPC-targeted conditioning in FA mice, and its clinical translation could circumvent the extensive transplant-related morbidity/mortality in this disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals How I treat MDS and AML in Fanconi anemia

Blood ◽  
2016 ◽  
Vol 127 (24) ◽  
pp. 2971-2979 ◽  
Author(s):  
Régis Peffault de Latour ◽  
Jean Soulier
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Clonal Evolution ◽  
Conditioning Regimen ◽  
Hematopoietic Stem ◽  
Long Term Follow Up

Abstract Fanconi anemia (FA) is the most frequent inherited cause of bone marrow failure (BMF). Most FA patients experience hematopoietic stem cell attrition and cytopenia during childhood, which along with intrinsic chromosomal instability, favor clonal evolution and the frequent emergence in their teens or young adulthood of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). To early identify and further predict bone marrow (BM) clonal progression and enable timely treatment, the follow-up of FA patients includes regular BM morphological and cytogenetic examinations. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment of FA patients with MDS or AML. Although questions remain concerning HSCT itself (including the need for pretransplant chemotherapy, the best conditioning regimen, and the optimal long-term follow-up of such patients especially regarding secondary malignancies), clonal evolution in the absence of significant BM dysplasia and blast cells can be difficult to address in FA patients, for whom the concept of preemptive HSCT is discussed. Illustrated by 3 representative clinical vignettes showing specific features of MDS and AML in FA patients, this paper summarizes our practical approach from diagnosis through treatment in this particular situation.

scholarly journals Oxymetholone Therapy of Fanconi Anemia Induces Hematopoietic Stem Cell Cycling By Suppressing Osteopontin Transcription

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2949-2949
Author(s):  
Qingshuo Zhang ◽  
Eric Benedetti ◽  
Matthew Deater ◽  
Kathryn Schubert ◽  
Angela Major ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematological Parameters ◽  
Hematopoietic Stem ◽  
Stem Cell Proliferation

Abstract Androgens are widely used for treating Fanconi anemia and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2-/- mice were used to assess the therapeutic efficacy of oxymetholone and its mechanism of action. 18-month old Fancd2-/- mice recapitulated key human Fanconi anemia phenotypes including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic oxymetholone treatment significantly improved these hematological parameters by stimulating the proliferation of hematopoietic stem and progenitor cells. RNAseq analysis implicated down-regulation of osteopontin as an important mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic oxymetholone therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure. Disclosures No relevant conflicts of interest to declare.

scholarly journals ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR ADULT PATIENTS WITH FANCONI ANEMIA.

2016 ◽  
Vol 8 ◽  
pp. 2016054 ◽  
Author(s):  
Hosein Kamranzadeh fumani ◽  
Mohammad Zokaasadi ◽  
Amir Kasaeian ◽  
Kamran Alimoghaddam ◽  
Asadollah Mousavi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Term Survival ◽  
Hematopoietic Stem

Background & objectives: Fanconi anemia (FA) is a rare genetic disorder caused by an impaired DNA repair mechanism which leads to an increased tendency toward malignancies and progressive bone marrow failure. The only curative management available for hematologic abnormalities in FA patients is hematopoietic stem cell transplantation (HSCT). This study aimed to evaluate the role of HSCT in FA patients.Methods: Twenty FA patients with ages of 16 or more who underwent HSCT between 2002 and 2015 enrolled in this study. All transplants were allogeneic and the stem cell source was peripheral blood and all patients had a full HLA-matched donor.Results: Eleven patients were female and 9 male (55% and 45%). Mean age was 24.05 years. Mortality rate was 50% (n=10) and the main cause of death was GVHD. Survival analysis showed an overall 5-year survival of 53.63% and 13 year survival of 45.96 % among patients.Conclusion: HSCT is the only curative management for bone marrow failure in FA patients and despite high rate of mortality and morbidity it seems to be an appropriate treatment with an acceptable long term survival rate for adolescent and adult group.

Genetics in Inherited Bone Marrow Failure Disorders

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-1-SCI-1
Author(s):  
Sioban Keel
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Accurate Diagnosis ◽  
Cell Transplant ◽  
Medical Monitoring ◽  
Hematopoietic Stem ◽  
New Genes ◽  
Increased Risk

The classical Inherited Bone Marrow Failure Syndromes (IBMFS) such as Fanconi anemia, Dyskeratosis Congenita, Shwachman-Diamond syndrome, and Diamond-Blackfan anemia are a heterogeneous group of disorders, all of which are characterized by impaired hematopoiesis, varying degrees of peripheral cytopenias and marrow hypoplasia and dysplasia. Many of these are associated with an increased risk of clonal dominance and evolution to myelodyplastic syndrome (MDS) and acute myeloid leukemia (AML). For the purposes of this talk, the familial MDS and acute leukemia predisposition syndromes are also included in the broad term IBMFS. The genes responsible for a subset of IBMFS have been identified and will be reviewed. However, the causative mutations in many patients presenting with seemingly inherited marrow failure remain unknown. Gene discovery in IBMFS has been difficult in large part due to the phenotypic heterogeneity of these syndromes. Some patients with IBMFS display a distinct clinical phenotype with associated syndromic abnormalities, others are variable and overlap with one another or with acquired MDS or idiopathic acquired aplastic anemia, and additional cases are more obscure and have evaded classification altogether. Accurate diagnosis of IBMFS inform patient care as it allows appropriate screening of siblings to avoid choosing an affected donor if marrow transplant is indicated and the selection of an appropriate transplant conditioning regiment to avoid undue toxicity. Additionally, accurate diagnosis allows appropriate medical monitoring and early intervention to successfully treat disease-specific non-hematologic medical complications. The application of next generation sequencing approaches for comprehensive genetic screening of IBMFS, including these cryptic or atypical presentations will be reviewed. In addition to providing accurate diagnoses in a subset of patients, genetic characterization in small family kindreds or even in single individuals presents unique opportunities to discover new genes and pathways contributing to dysfunctional hematopoiesis and clonal progression. The frequency of inherited mutations in known IBMFS genes among seemingly idiopathic acquired aplastic anemia patients or pediatric and younger adults with MDS referred for hematopoietic stem cell transplant will be reviewed. Future genetic studies are needed to characterize the secondary genetic events that lead to disease progression in IBMFS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rpl22 Deficiency Predisposes Hematopoietic Stem and Progenitor Cells to Leukemogenesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 899-899 ◽  
Author(s):  
Bryan Harris ◽  
Jaqueline Perrigoue ◽  
Rachel M. Kessel ◽  
Shawn Fahl ◽  
Stephen Matthew Sykes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stem Cell ◽  
Ribosomal Protein ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ectopic Expression ◽  
Hematopoietic Stem ◽  
Increased Risk

Abstract Mutations and deletions in ribosomal proteins are associated with a group of diseases termed ribosomopathies. Collectively, these diseases are characterized by ineffective hematopoiesis, bone marrow failure, and an increased risk of developing myelodysplastic syndrome (MDS) and subsequently acute myeloid leukemia (AML). This observation highlights the role of dysregulation of this class of proteins in the development and progression of myeloid neoplasms. Analysis of gene expression in CD34+ hematopoietic stem cells (HSC) from 183 MDS patients demonstrated that ribosomal protein L22 (Rpl22) was the most significantly reduced ribosomal protein gene in MDS. Interestingly, we observed that AML patients with lower expression of Rpl22 had a significant reduction in their survival (TCGA cohort, N=200, Log Rank P value <0.05). To assess the mechanism of reduced expression, we developed a FISH probe complementary to the RPL22 locus and assessed for deletion of this locus in an independent set of 104 MDS/AML bone marrow samples. Strikingly, we found that RPL22 deletion was enriched in high-risk MDS and secondary AML cases. We, therefore, sought to investigate whether reduced Rpl22 expression played a causal in leukemogenesis. Using Rpl22-/- mice, we found that Rpl22-deficiency resulted in a constellation of phenotypes resembling MDS. Indeed, Rpl22-deficiency causes a macrocytic reduction in red blood cells, dysplasia in the bone marrow, and an expansion of the early hematopoietic stem and progenitor compartment (HSPC). Since MDS has been described as a disease originating from the stem cell compartment, we next sought to determine if the hematopoietic defects were cell autonomous and resident in Rpl22-/- HSC. Competitive transplantation revealed that Rpl22-/- HSC exhibited pre-leukemic characteristics including effective engraftment, but a failure to give rise to downstream mature blood cell lineages. Importantly, there was a strong myeloid bias in those downstream progeny derived form Rpl22-/- HSC. Because human MDS frequently progresses to AML, we examined the potential for Rpl22-deficient HSC to be transformed upon ectopic expression of the MLL-AF9 oncogenic fusion. Indeed, Rpl22-deficient HSPC exhibited an increased predisposition to transformation both in vitro and in vivo, in MLL-AF9 knockin mice. To determine how Rpl22-deficiency increased the transformation potential of HSC, we performed whole transcriptome analysis on Rpl22-/- HSC. Interestingly, four expression signatures were observed that were consistent with the altered behavior exhibited by Rpl22-/- HSC. Rpl22-deficient HSC exhibited increased expression of: 1) genes associated with stem cell function, consistent with the basal expansion and effective engraftment of Rpl22-/- HSC upon adoptive transfer; 2) markers of the myeloid lineage, providing a potential explanation for the myeloid bias exhibited by Rpl22-/- HSC; 3) cell cycle regulators, consistent with the increased proliferation exhibited by Rpl22-/- HSC; and 4) components of the mitochondrial respiratory chain, a metabolic program on which leukemic stem cell function depends. Together, these data suggest that Rpl22 controls a program of gene expression that regulates the predisposition of HSPC to myeloid transformation. Disclosures No relevant conflicts of interest to declare.

scholarly journals TGF-β Inhibition Rescues Hematopoietic Stem Cell Defects and Bone Marrow Failure in Fanconi Anemia

2016 ◽  
Vol 18 (5) ◽  
pp. 668-681 ◽  
Author(s):  
Haojian Zhang ◽  
David E. Kozono ◽  
Kevin W. O’Connor ◽  
Sofia Vidal-Cardenas ◽  
Alix Rousseau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem

Is Not Age, But Comorbidities. Allogeneic Hematopoietic Stem Cell Transplantation In Patients Older Than 50 Years In Argentina

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5467-5467
Author(s):  
Mariano Berro ◽  
Juan Garcia ◽  
Ana Basquiera ◽  
Maria Marta Rivas ◽  
Maria Cecilia Foncuberta ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Chronic Gvhd ◽  
Conditioning Regimen ◽  
Disease Free Survival ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Transplant Outcome ◽  
Significant Difference

Abstract Materials and Methods We retrospectively reviewed 137 medical records of patients older than 50 years receiving an allogeneic hematopoietic stem cell transplant (HSCT) in 9 centers from Argentina. We evaluated the following characteristics: sex, age, diagnosis, stage, comorbidities (according to the HCT-CI score), type of donor, histocompatibility, source, conditioning and immunosupression. We analyzed the incidence and severity of acute Graft-vs-Host disease (aGVHD) with Chi Square, Overall Survival (OS) and Disease Free Survival (DFS) with Kaplan Meier and Relapse, Non Relapse Mortality y chronic GVHD (cGVHD) with CI. For multivariate analysis (MA) we included variables that in univariate had a p<0.2, used Cox regression model for time dependant outcomes and logistic regression for dichotomic variables, considering significant a p<0.05. Results Patients characteristics are listed in table 1. Between January 1997 and July 2013, 137 transplants were performed in adults older than 50 years, with a median follow up 1.3 years. Acute GVHD incidence was 41% (19% were grade II and 7.3% III-IV). The only variable associated with aGVH clinically significant (G II-IV) was AML that was protective (14% vs 35%, p<0.01; significant in MA, HR 0.29; 95% CI 0.12-0.72). Chronic GVHD incidence was 25%, extensive in 9.4% and the only risk factor for this outcome was MPN (1-3 years 40%-NA vs 12-20%, p=<0.01). Global OS 1 and 3 years was 44 and 20%, DFS was 33 and 20%, Relapse was 35 and 41% and NRM was 36 and 43% respectively. Co-morbid patients showed a significant increase in NRM (HCT.CI 0 vs 1 vs ≥2, 1-3 years 17-24%, 40-46% and 45-67%, p=0.01; significant in MA, for HCT.CI 0 vs ≥1, HR 2.4, 95% CI 1.12-5.25), as well as male patients (1-3 years 36-47% vs 23-27%, p=0.03), MPN (1-3 years 43-65% vs 29.34%, p=0.01) and Cyclosporine based immunosuppressant regimen (CSA) vs tacrolimus (1-3 years 47-53% vs 25-36%, p=0.01). AML patients experienced a higher relapse rate (1-3 years 50-50% vs 28-32%, p<0.01) as well as Fludarabine-Busulfan conditioning (1-3 years 45-48% vs 31-32%, p=0.02). Finally patients without comorbidities (HCT.CI 0 vs ≥1) had higher OS (1-3 years 54-30% vs 36-16%, p=0.03) and DFS (1-3 years 43-31% vs 30-15%, p=0.05) as well as tacrolimus vs CSA base regimen that had higher OS (1-3 years 49-25% vs 31-13%, p=0.01) and DFS (1-3 year 41-26% vs 20-11%, p<0.01; significant in MA, HR 0.56, 95% CI 0.33-0.98). Age (older than 60 vs younger), type of donor, use of myeloablative conditioning regimen and source did not showed any significant difference in the outcome analyzed. Conclusion HSCT is a valid therapeutic option for older patients. In this retrospective analysis of patients older than 50 years, we found that the main risk factors that impact in transplant outcome are patients comorbidities and not age, whereas transplant related toxicities increase with the number of comorbidities and therefore decrease OS and DFS. Beyond the fact that certain disease experienced more aGHVD (AML) or cGVHD and higher NRL (MPN) the other factor significantly related in transplant outcome was the use of tacrolimus vs CSA. Disclosures: No relevant conflicts of interest to declare.

The Zebrafish Provides Mechanistic Insights into the Role of Poly(A)-Specific Ribonuclease (PARN) in Hematopoietic Stem Cell (HSC) Homeostasis and Bone Marrow Failure

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 668-668
Author(s):  
Adam P Deveau ◽  
Andrew J Coombs ◽  
Santhosh Dhanraj ◽  
Gretchen Wagner ◽  
Yigal Dror ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Phosphohistone H3 ◽  
Biallelic Mutations ◽  
Insight Into

Abstract Development of tissues during embryogenesis and their homeostasis after formation are highly regulated by expression of coding and non-coding RNAs. Deadenylation is a core mechanism that regulates RNA function and fate by controlling turnover, abundance and maturation of RNA. Factors that promote or inhibit deadenylation control hematopoietic stem cell (HSC) homeostasis, and inhibition of deadenylation limits differentiation of the HSCs. Importantly, RNA biogenesis has emerged as a mechanism underlying several inherited bone marrow failure syndromes (IBMFSs), such as Diamond Blackfan anemia, dyskeratosis congenita (DC) and Shwachman-Diamond syndrome. Poly(A)-specific ribonuclease (PARN) is a major deadenylation factor and demonstrates high specificity for single-stranded poly (A) tails of various RNA species. We recently identified biallelic mutations in PARN as a cause of hematopoietic failure and profound hypomyelination, similar to the severe form of DC, Hoyeraal-Hreidersson syndrome. We developed a zebrafish model to characterize the hematopoietic phenotype of a patient identified to have severe inherited bone marrow failure resulting from a combined deletion of PARN on one allele and missense mutation in the other. Zebrafish posses a single parn ortholog. Zebrafish parn protein shares homology and high sequence identity (~64%) to its human counterpart. Embryos were injected with either translation start-site or splice-site-blocking morpholino at the one-cell stage. Both morpholino injections resulted in anemic embryos at 48 hours post fertilization (hpf), as evidenced by reduced o-dianisidine staining and gata1 expression by whole-mount in situ hybrization and GFP+ red cell numbers by fluorescence-activated cell sorting (FACS). Morphant embryos also demonstrated reduced expression of myeloid cell markers including l-plastin, myeloperoxidase, and macrophage expressed gene 1 and were leukopenic as evidenced by reduced number of GFP+ myeloid cells. FACS analysis revealed that fluorescently labeled HSCs were increased in parn morphants. Early hematopoietic markers, lmo2 and fli1, expressed in hemogenic and vascular tissue respectively, were also overexpressed in parn morphants. Furthermore, there was reduced global cell proliferation in morphant embryos as determined by phosphohistone H3 antibody staining. These findings suggest that the absence of parn results in a developmental arrest at the HSC stage with an inability to differentiate into leukocyte or erythroid lineages. Similarly, human cell culture data from PARN-deficient HSC/progenitor cells demonstrated markedly reduced colony forming capacity. By modeling parn deficiency in the zebrafish, we validate for the first time an IBMFS that results from biallelic mutations in a major deadenylating protein. Moreover, our zebrafish studies provide insight into the role of parn in maintaining HSC homeostasis/differentiation as the origin of the pancytopenia observed in this patient. Permanent knockouts in the zebrafish using CRISPR/Cas9 technology are underway, which will enable tracking the hematopoietic phenotype into adulthood. These studies have set the stage for critical translational research in a rare form of bone marrow failure as well as new insight into HSC regulation. Disclosures No relevant conflicts of interest to declare.

scholarly journals IFNy Stimulation Induces Hematopoietic Stem Cell Homing and Niche Relocalization

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3726-3726
Author(s):  
Marcus A Florez ◽  
Katie A Matatall ◽  
Laura Ortinau ◽  
Roman Jaksik ◽  
Marek Kimmel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Terminal Differentiation ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
The Impact

Interferon gamma (IFNy) is a pro-inflammatory cytokine that is upregulated during chronic infections and chronic diseases, such as aplastic anemia, and has been associated with pancytopenia and diminished hematopoiesis. Studies have shown that IFNy negatively regulates hematopoietic stem cell (HSC) homeostasis by decreasing self-renewal and promoting terminal differentiation. The tight regulation of HSC homeostasis is dependent upon the bone marrow (BM) microenvironment, or BM niche. The BM niche is composed of a network of cell types that provide elaborate cell-cell interactions, cellular metabolites, transcriptional regulators, and local and distant humoral and neural signals that allow for hematopoietic homeostasis. In particular, CXCL12-abundant reticular (CAR) cells are vital to HSC maintenance, as depletion of CXCL12, or its receptor, leads to HSC depletion. However, the mechanism which IFNy activates HSCs and influences its interaction with the BM niche is unknown. We hypothesize that IFNy promotes HSC terminal differentiation and loss of quiescence by altering HSC interactions with the BM niche. To assess changes in HSC interactions with the BM niche upon IFNy stimulation, we performed intravital imaging using CXCL12 GFP reporter mice before and after administration of recombinant IFNy. We found that HSCs stimulated with IFNy were significantly distanced from CAR cells compared to pre-treated controls. There was no change in distance with IFNy-receptor deficient HSCs, suggesting that movement away from the CAR cells was due to a cell autonomous IFNy-dependent mechanism. We performed gene expression analysis and transwell migration assays on HSCs from IFNy treated mice, and determined that there was no change in CXCL12 receptor (CXCR4) expression upon IFNy treatment, and IFNy did not alter migration towards CXCL12. These results suggest that HSC re-localization upon IFNy is independent of CXCL12 signaling. To explore the mechanism by which IFNy induces re-localization of HSCs, we first performed microarray analysis on HSCs from IFNy stimulated mice to assess what surface proteins were changed upon IFNy treatment. While there was no change in common HSC receptors thought to influence HSC homeostasis (cKit, Cdh2, Mpl, Itgb1, Itbg2, Itga4, and Itga1), we observed an increase in expression of bone marrow stromal antigen 2 (BST2). To explore the impact of BST2 on HSC homeostasis, quantification and proliferation analysis was performed on HSCs from Bst2-/- mice. Interestingly, Bst2-/- HSCs were significantly less proliferative and more abundant compared to controls. These studies suggest that BST2 may play a role in maintaining HSC homeostasis. The functional role of BST2 in cellular movement and adhesion has been studied in cancer. Increased BST2 expression has been associated with promoting the migration, adhesion and metastasis of various cancer cells. Since migration and adhesion is important for HSC homing, we assessed the effects of IFNy on HSC homing. Hematopoietic progenitors from IFNy-treated mice homed to the bone marrow with greater efficiency than PBS-treated controls, whereas progenitors from IFNy-receptor-deficientmice showed a decrease in homing. Additionally, WBM from IFNyR-/- had reduced engraftment than wildtype, consistent with a role for IFNy signaling in promoting HSC homing. The impact of BST2 on homing is currently being explored. In summary, we show that IFNy induces re-localization of HSCs away from quiescence-promoting CAR cells within the bone marrow niche via a mechanism that is independent of CXCL12 signaling. We further show that IFNy promotes HSC homing. The increased expression of BST2 on IFNy-stimulated HSCs appears to impact HSC proliferation and abundance in the bone marrow. Thus, BST2 may play a role in HSC activation and exit from quiescence. Expanding our understanding of the mechanism that drives HSC activation and terminal differentiation has important implications for patients who develop pancytopenia or bone marrow failure due to chronic inflammation. Disclosures No relevant conflicts of interest to declare.

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