Defective Adhesion, Migration and Homing Are Associated with Altered Rho GTPase Activity in Cells from Fanconi Anemia Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 350-350
Author(s):  
Xiaoling Zhang ◽  
Xun Shang ◽  
Lei Wang ◽  
Fukun Guo ◽  
Yi Zheng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fanconi Anemia ◽  
Rho Gtpase ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Autologous Transplantation ◽  
Hematopoietic Cells ◽  
Small Gtpase ◽  
Primary Fibroblast ◽  
And Migration

Abstract Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and predisposition to malignancy. One of the potential therapeutic options for patients with FA is collection of autologous multipotent hematopoietic progenitors prior to the development of severe pancytopenia for autologous transplantation and gene therapy. However, poor engraftment of FA hematopoietic cells represents a major obstacle for effective transplantation. Our current study attempted to investigate the mechanism underlying defective engraftment of FA bone marrow (BM) cells. Using BM cells from patients carrying mutations in the FA complementation group A (FA-A), we demonstrate that SDF (Stromal cell-derived factor)-1alpha – and integrin-mediated migration and adhesion, respectively, is defective in FA primary BM cells compared to those from normal donors (more than 2-fold decrease in both migration and adhesion compared to normal BM cells). Complementation of the FA-A defect by retrovirus gene transfer of FANCA gene almost completely restores the ability of the BM cells to migrate towards the chemokine SDF-1alpha. Similar results are obtained with primary fibroblast cells derived from a FA-A patient, which show 3-fold and 35% decrease in adhesion and migration, respectively, compared to FANCA-corrected cells. Furthermore, when transplanted into immunodeficient Nod/scid recipient mice, the FA-A BM cells exhibited significantly impaired homing function whereas normal cells were efficiently homed in the bone marrow. A significant decrease in the activity of the Rho GTPase Cdc42 in FA-A cells is found associated with the patient cell defective functions. Taken together, these data suggest that the FA proteins play a role in the regulation of cell adhesion and migration in addition to maintaining genomic stability, influencing homing and engraftment, possibly via the small GTPase signaling pathway. These findings may have implications in development of strategies for restoring engraftment function of FA hematopoietic cells.

Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 700-704 ◽  
Author(s):  
Kimberly A. Gush ◽  
Kai-Ling Fu ◽  
Markus Grompe ◽  
Christopher E. Walsh
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mitomycin C ◽  
Fanconi Anemia ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Hematopoietic Stem ◽  
Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

TNFα Overproduction in FANCC-Deficient Cells Is TLR8 Dependent.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 494-494
Author(s):  
Scott Vanderwerf ◽  
Johanna Svahn ◽  
Praveen Anur ◽  
Ricardo Pasquini ◽  
Grover C. Bagby
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Clonal Evolution ◽  
Hematopoietic Cells ◽  
Mononuclear Phagocytes ◽  
C Cells ◽  
Mutant Cells

Abstract Abstract 494 The Fanconi anemia (FA) proteins play a role in regulating genome stability but it is not clear that loss of genoprotection in FA hematopoietic cells accounts for the molecular pathogenesis of bone marrow failure so characteristic of this disease. Other factors are known to influence survival and replication of FA stem cells. For example, not only are FA progenitors and stem cells hypersensitive to the apoptotic effects of TNFα, FA cells over-produce TNFα. Most importantly over-production of and hypersensitivity to TNFα in hematopoietic cells of Fancc-/- mice results in bone marrow hypoplasia 1;2 and long-term ex-vivo exposure of murine Fancc -/- hematopoietic cells to both growth factors and TNFα results in the evolution of cytogenetically marked preleukemic clones.3 Therefore, the hematopoietic phenotype of FA is likely multifactorial and may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive. Methods: We sought to clarify the molecular basis of aberrant TNFα-production. We conducted gene expression microarray experiments using RNA samples from low density marrow cells obtained from 11 normal volunteers and 22 Fanconi anemia patients with uncomplicated marrow hypoplasia without clonal cytogenetic defects. Because the FA complex is known to enhance ubiquitinylation of FANCD2, we reasoned that the ubiquitinylation state of proteins involved in the TNF pathways might also be influenced by core FA proteins. Therefore, we conducted in vitro ubiquitinylation assays using hexahistidine-tagged ubiquitin and an ATP-recycling system added to lysates of FANCC-deficient lymphoblasts (HSC536) and control cells (isogenic cells complemented with WT FANCC cDNA). Following the ubiquitinylation reaction, ubiquitinylated proteins were affinity purified, digested and analyzed by 2D capillary LC-MS/MS. Mass spectra were obtained and peptide precursor-MS/MS spectrum pairs were analyzed using SEQUEST and support vector machine learning.4 Peptides identified only in one or the other cell line were considered. Results: Initially we anticipated focusing on the set of proteins uniquely ubiquitinated in normal cells. However, the transcriptomal results indicated that genes encoding proteins in the ubiquitin pathway were over-represented in the list of genes that were over-expressed in FA samples. Consequently, we examined both differential ubiquitination lists and found that a major regulator of TNF-gene expression, TLR8, appeared in the ubiquitinylated fraction only in mutant cells. In co-immunoprecipitation studies we confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells, and using RNAi determined that high level TNFα synthesis in mutant cells depended upon TLR8 and its downstream signaling intermediates IRAK-1 and IKK-alpha/beta. FANCC deficient THP1 blue cells were created using lentiviral shRNA targeting FANCC. These cells exhibited the MMC hypersensitive phenotype and over-expressed both TNFα and an NF-kappaB reporter gene (secreted embryonic alkaline phosphatase) in response to TLR8 agonists but not to other TLR agonists. Primary splenic macrophages from Fancc-/- mice were also hypersensitive to the TLR8 agonist R848. TNFα production in FA-C cells was suppressed by inhibitors of TLR8, p38 MAPK, IRAK, and IKK. Engineered point mutants of FANCC were capable of complementing the mitomycin C hypersensitivity phenotype of FANCC mutant cells but did not suppress TNFα overproduction in FANCC mutant cells. In conclusion, TNF over-expression in FANCC-deficient cells reflects the loss of FANCC function as a suppressor of TLR8 activation. In addition, FANCC suppresses TLR8 dependent production of TNFα in normal mononuclear phagocytes at least in part by suppressing either TLR8 ubiquitinylation or by inhibiting its association with an ubiquitinylated protein. Finally, this function of FANCC is independent of its function in protecting the genome from cross-linking agent-induced damage. In light of the role of TNFα in bone marrow failure and clonal evolution in this disease, control of TNF-production by targeting the TLR8 pathway might provide an opportunity to enhance hematopoietic activity and forestall clonal evolution in patients with this disorder. 1. Sejas DP, et al, J Immunol 2007;178:5277-5287. 2. Zhang × et al, J.Cell Sci. 2007;120:1572-1583. 3. Li J, et al, J.Clin.Invest. 2007;117:3283-3295, 4. Anderson DC, et al, J Proteome.Res 2003;2:137-146. Disclosures: No relevant conflicts of interest to declare.

Cytokinesis Failure In Fanconi Anemia Pathway Deficient Hematopoietic Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 878-878
Author(s):  
Kalindi Parmar ◽  
Patrizia Vinciguerra ◽  
Susana Godinho ◽  
Abigail Hamilton ◽  
David Pellman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Hematopoietic Cells ◽  
Fibroblast Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Binucleated Cells ◽  
Hoechst Staining

Abstract Abstract 878 Fanconi Anemia (FA) is a human genomic instability disorder characterized by progressive bone marrow failure, congenital abnormalities and high predisposition to cancer. Bone marrow failure in FA children is attributed partly to the excessive apoptosis and subsequent failure of the hematopoietic stem cell compartment. Understanding the mechanisms of bone marrow failure may allow better diagnosis and treatment for FA and other aplastic anemia patients. There are fourteen known Fanconi Anemia genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N, O). The FA pathway, regulated by these FA gene products, mediates DNA repair and promotes normal cellular resistance to DNA crosslinking agents. Recent studies suggest that besides maintaining genomic stability, the FA pathway may also play a role in mitosis since FANCD2 and FANCI, the two key FA proteins, are localized to the extremities of ultra-fine DNA bridges (UFBs) linking sister chromatids during cell division (Chan et al, Nat Cell Biol, 11:753-760, 2009; Naim and Rosselli, Nat Cell Biol, 11:761-768, 2009). Whether FA proteins play a direct role in cell division is still unclear. To dissect the mechanisms of bone marrow failure in FA, we have investigated the requirement of FA pathway during mitosis. Initially, we investigated the number of DNA bridges occurring during mitosis in FA-deficient and proficient cells by immunofluorescence and Hoechst staining. FA-deficient patient cell lines (FANCG-deficient and FANCD1/BRCA2-deficient cells) as well as Hela cells with shRNA-mediated knockdown of the FA pathway, displayed an increase in UFBs compared to the FA proficient cells during mitosis. The UFBs were coated by BLM (the RecQ helicase mutated in Bloom syndrome) in early mitosis. In contrast, the FA protein, FANCM, was recruited to the bridges at a later stage. Since the DNA bridges occluding the cleavage furrow potentially induce cytokinesis failure, we assessed FA-deficient cells for multinucleation. The increased number of DNA bridges correlated with a higher rate of binucleated cells in FA deficient Hela cell lines and FA patient-derived fibroblast cells. Moreover, an increase in binucleated cells was also detectable in FA-deficient primary murine bone marrow hematopoietic stem cells (Fancd2-/- cells and Fancg-/- cells) compared to the wild-type cells undergoing proliferation and in FA patient-derived bone marrow stroma cells compared to the stroma cells from normal human bone marrow. Interestingly, the increase in binucleated cells in FA-deficient murine hematopoietic stem cells correlated with the increase in apoptotic cells. Binuclearity, scored by immunostaining for microtubules and Hoechst staining for DNA, was the result of cytokinesis failure as observed by live cell imaging. Therefore, we investigated whether the FA-deficient cells are sensitive to the cytokinesis inhibitors. FA-deficient murine bone marrow lineage negative cells (Fancd2-/- cells) or FA human fibroblast cells were exposed to VX-680 (an inhibitor of Aurora kinases regulating cytokinesis) in culture for 72 hrs and cell survival was assessed. VX-680 caused increased toxicity (reduced cell viability and increased apoptosis) on FA-deficient cells in comparison to the wild-type cells. Enhanced inhibition of clonogenic growth of murine FA-deficient bone marrow cells (Fancd2-/- cells) compared to the wild-type cells was also observed by exposure to VX-680. These data indicated that FA pathway-deficient hematopoietic cells are hypersensitive to cytokinesis inhibitors. Collectively, our results underscore the importance of the FA pathway in mitosis and suggest that the cytokinesis failure observed in FA deficient hematopoietic cells could contribute to bone marrow failure in Fanconi anemia patients. Disclosures: No relevant conflicts of interest to declare.

Gender-related differences in the oxidant state of cells in Fanconi anemia heterozygotes

2011 ◽  
Vol 392 (7) ◽  
Author(s):  
Sandra Petrovic ◽  
Andreja Leskovac ◽  
Jelena Kotur-Stevuljevic ◽  
Jelena Joksic ◽  
Marija Guc-Scekic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Significant Difference ◽  
Heterozygous Carriers ◽  
Male Carriers ◽  
Female Carriers

Abstract Fanconi anemia (FA) is a rare cancer-prone genetic disorder characterized by progressive bone marrow failure, chromosomal instability and redox abnormalities. There is much biochemical and genetic data, which strongly suggest that FA cells experience increased oxidative stress. The present study was designed to elucidate if differences in oxidant state exist between control, idiopathic bone marrow failure (idBMF) and FA cells, and to analyze oxidant state of cells in FA heterozygous carriers as well. The results of the present study confirm an in vivo prooxidant state of FA cells and clearly indicate that FA patients can be distinguished from idBMF patients based on the oxidant state of cells. Female carriers of FA mutation also exhibited hallmarks of an in vivo prooxidant state behaving in a similar manner as FA patients. On the other hand, the oxidant state of cells in FA male carriers and idBMF families failed to show any significant difference vs. controls. We demonstrate that the altered oxidant state influences susceptibility of cells to apoptosis in both FA patients and female carriers. The results highlight the need for further research of the possible role of mitochondrial inheritance in the pathogenesis of FA.

scholarly journals Genomic Instability in Fanconi Anemia Results from a Combination of Chromosome Mis-Segregation in Mitosis and Unresolved Interphase DNA Damage

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 357-357 ◽  
Author(s):  
Donna Cerabona ◽  
Zahi Abdul Sater ◽  
Rikki Enzor ◽  
Grzegorz Nalepa
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Genomic Instability ◽  
Fanconi Anemia ◽  
Chromosomal Instability ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Biological Significance

Abstract Fanconi anemia (FA) is a complex genetic disorder characterized by bone marrow failure, multiple congenital anomalies, and genomic instability resulting in predisposition to cancer. Disruption of the FA signaling network impairs multiple genome-housekeeping processes, including DNA damage recognition and repair in interphase, DNA replication as well as high-fidelity chromosome segregation during mitosis. Recent data published by several groups, including our work (J Clin Invest 2013; 123: 3839-3847), implicated FA signaling in the control of several cell division events essential for chromosomal stability, including the spindle assembly checkpoint (SAC), centrosome maintenance, resolution of ultrafine anaphase bridges and cytokinesis. Understanding the mechanistic origins of chromosomal instability leading to carcinogenesis and bone marrow failure has important scientific and clinical implications. However, the relative contribution of the interphase and mitotic events leading to genomic instability in Fanconi anemia has not been systematically evaluated. In this work, we dissected the origins and mechanistic significance of chromosomal instability in Fanconi anemia ex vivo and in vivo. We employed the cytochalasin micronucleus assay to quantify the patterns of spontaneous and chemotherapy-induced genomic lesions in FA-A patient-derived primary fibroblasts and Fancc-/- mouse embryonic fibroblasts (MEFs). In this assay, dividing cells are treated with cytochalasin to inhibit cytokinesis and generate binucleated daughter cells. The presence of micronuclei in the resulting cells is indicative of genomic instability caused by either interphase DNA damage or chromosome mis-segregation. Centromere-negative micronuclei (CNMs) represent chromosomal fragments due to unresolved ds-DNA damage. Centromere-positive micronuclei (CPMs) result from whole-chromosome mis-segregation during mitosis. The frequency of both CPMs and CNMs was significantly increased in FA-deficient human and murine cells compared to gene-corrected isogenic control cells. These results indicate that genomic instability in FA is caused by a combination of interphase DNA damage and disordered mitosis. We confirmed the biological significance of these findings by showing that FA patient cells are hypersensitive to low concentrations of taxol (a spindle checkpoint-activating chemotherapeutic) similarly to mitomycin C (a cross-linking agent). Finally, we found increased frequency of micronuclei in Fancc-/- murine red blood cells compared to age-matched wild-type mice, which indicates that spontaneous chromosome mis-segregation occurs in FA-deficient bone marrow in vivo. Our study supports the emerging model of the FA family of proteins as holistic guardians of the genome during interphase and mitosis (see figure based on F1000Prime Rep. 2014; 6: 23, modified). This model furthers our understanding of genomic instability in Fanconi anemia and FA-deficient cancers, and opens new inroads towards targeted therapeutic interventions in these diseases. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

A High Throughput Small Molecule Screening Method for Fanconi Anemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3191-3191
Author(s):  
Anur Praveen ◽  
Jeffrey W Tyner ◽  
Scott Vanderwerff ◽  
Winifred Keeble ◽  
Grover C. Bagby
Keyword(s):  
Bone Marrow ◽  
Small Molecules ◽  
High Throughput ◽  
Fanconi Anemia ◽  
High Throughput Screening ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Bone Marrow Failure ◽  
Screening Method ◽  
Hematopoietic Cells

Abstract Abstract 3191 Poster Board III-128 The Fanconi Anemia (FA) proteins play an important role in regulating genome stability, but there is little evidence that the loss of the genoprotection per se, in FA cells accounts for the molecular pathogenesis of the bone marrow failure characteristic of this disease. Indeed, there is evidence that at least some of these proteins are multifunctional and participate in canonical signaling pathways in hematopoietic cells. FANCC deficient cells, for example, are hypersensitive to the apoptotic effects of TNFαa. In addition, FA-C cells over-produce TNFαa at least in part because FANCC ordinarily suppress the activation potential of toll-like receptor 8 (TLR8) (abstract submitted to this meeting). There is clear evidence that over-production of TNFαa and hypersensitivity to TNFαa in hematopoietic cells of Fancc-/- mice results in bone marrow hypoplasia (Sejas et al, 2007 and Zhang et al 2007) and that long-term ex-vivo exposure of murine Fancc -/- hematopoietic cells to both growth factors and TNFαa results in the evolution of cytogenetically marked preleukemic clones (Li et al 2007). Because the hematopoietic phenotype of FA may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive, we reasoned that suppression of TNFαa production by FA cells might enhance hematopoiesis. So we sought to develop a strategy to permit high throughput screening of small molecules designed to suppress TNFαa production specifically in FANCC deficient cells. Methods THP1 Blue cells (THP1B) have a stably integrated NF-kappaB reporter gene, secreted embryonic alkaline phosphatase (SEAP) and express SEAP and TNFαa in response to TLR ligands including the TLR8 ligand (R848). Each of five samples of THP1B cells were transduced with one of five lentiviral vectors expressing FANCC targeted shRNA. One of these vectors suppressed FANCC expression (by immunoblotting and RT-RT-PCR), suppressed FANCD2 levels in MMC exposed THP1B cells, induced chromosomal instability in the MMC assay and markedly enhanced R848-induced TNFαa production when compared to THP1B cells transduced with a non-targeted shRNA lentiviral vector. In multiwell plates, THP1B-shFANCC cells were exposed to multiple doses one of 81 small molecules including steroid hormones and inhibitors of tyrosine or serine threonine kinases. TNFαa (ELISA) and SEAP (QUANTI-blue colorimetry) were quantified in the supernatant media 24 hours after exposure to R848. Results 15 agents suppressed SEAP production without cytotoxicity and all of these suppressed TNFαa production as well. The same agents suppressed TNFαa production in two patient-derived FANCC-deficient cell lines (HSC536 and PD149) both of which over-express TNFαa in the ground state. Four p38 inhibitors (100nM-10μM) were analyzed and at 500 nM all suppressed SEAP and TNFαa by 90% or more. The Src family kinase inhibitor, Dasatinib (500nM) was also effective. Using Fancc-deficient mice exposed to TLR activating agents, in vivo preclinical studies designed to test the effectiveness of Dasatinib and one p38 inhibitor are underway, as are mechanistically focused multiplex assays in which known target molecules of these agents are suppressed using RNAi. Conclusions We have developed a reliable screening tool based upon the TNFαa-overproduction phenotype of FANCC deficient cells. Using it, we have identified inhibitors of p38 MAPK and Src family kinases that suppress TNFαa-overproduction in patient derived FANCC-deficient cells. The identification of these agents provides not only an opportunity to discover novel biochemical roles played by FANCC in innate immunity but also a strong rationale for evaluating such agents in preclinical models for marrow failure in FA. Disclosures No relevant conflicts of interest to declare.

Impaired Bone Marrow Microenvironment in Fanconi Anemia Murine Model Is Responsible for the Defective Hematopoietic Stem/ Progenitor Cell Mobilization.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3629-3629
Author(s):  
Yan Li ◽  
Shi Chen ◽  
Yongzheng He ◽  
Xiaohong Li ◽  
Fengchun Yang
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Fanconi Anemia ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Bone Marrow Microenvironment ◽  
Hematopoietic Stem

Abstract Abstract 3629 Poster Board III-565 Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by progressive bone marrow failure (BMF) and acquisition of malignancies. The only cure for BMF is a human leukocyte antigen (HLA)-matched BM transplantation from a family member or autologous stem cells before BMF develops. Therefore, mobilization of hematopoietic stem/progenitor cells (HSPCs) from BM into peripheral blood (PB) for collection has been a prerequisite for the therapy. However, patients with FA show a markedly decreased HSPC mobilization in response to the traditional mobilizing drug G-CSF and the mechanism(s) underlying the defect remains unknown. Mesenchymal stem/progenitor cells (MSPCs) have been known to be the common progenitor of a variety of cellular components in the bone marrow microenvironment. MSPCs express/secrete cytokines, extracellular matrix proteins and cell adhesion molecules, which regulate the homing, migration, proliferation and survival of HSPCs in vitro and in vivo. Recently, we reported that Fancg-/- MSPCs have a defect in hematopoietic supportive activity both in vitro and in vivo (Li et al. Blood, 2009). In the current studies, we show that Fancg-/- MSPCs have significant reduction in HSPC recruitment as compared to WT MSPCs in a transwell assay. Furthermore, Fancg-/- MSPCs have an alteration in the production of multiple cytokines/chemokines. Application of a neutralizing antibody to the cytokine blocked WT MSPC mediated HSPC migration in vitro. Furthermore, administration of the specific cytokine significantly increased HSPC mobilization in the Fancg-/- mice in vivo. These results demonstrated that an impaired BM microenvironment, specifically MSPCs in Fancg-/- mice, is contributory to defective HSPC mobilization. This study provides evidence of alternative clinical therapeutics for the mobilization of HSPCs in FA patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Pediatric MDS and bone marrow failure-associated germline mutations in SAMD9 and SAMD9L impair multiple pathways in primary hematopoietic cells

Leukemia ◽  
2021 ◽  
Author(s):  
Melvin E. Thomas ◽  
Sherif Abdelhamed ◽  
Ryan Hiltenbrand ◽  
Jason R. Schwartz ◽  
Sadie Miki Sakurada ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Failure ◽  
Transcriptional Profiling ◽  
Hematopoietic Cells ◽  
Protein Translation ◽  
Germline Mutations ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Cellular Environment ◽  
Primary Mouse

AbstractPediatric myelodysplastic syndromes (MDS) are a heterogeneous disease group associated with impaired hematopoiesis, bone marrow hypocellularity, and frequently have deletions involving chromosome 7 (monosomy 7). We and others recently identified heterozygous germline mutations in SAMD9 and SAMD9L in children with monosomy 7 and MDS. We previously demonstrated an antiproliferative effect of these gene products in non-hematopoietic cells, which was exacerbated by their patient-associated mutations. Here, we used a lentiviral overexpression approach to assess the functional impact and underlying cellular processes of wild-type and mutant SAMD9 or SAMD9L in primary mouse or human hematopoietic stem and progenitor cells (HSPC). Using a combination of protein interactome analyses, transcriptional profiling, and functional validation, we show that SAMD9 and SAMD9L are multifunctional proteins that cause profound alterations in cell cycle, cell proliferation, and protein translation in HSPCs. Importantly, our molecular and functional studies also demonstrated that expression of these genes and their mutations leads to a cellular environment that promotes DNA damage repair defects and ultimately apoptosis in hematopoietic cells. This study provides novel functional insights into SAMD9 and SAMD9L and how their mutations can potentially alter hematopoietic function and lead to bone marrow hypocellularity, a hallmark of pediatric MDS.

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