Critical Role of Lama4 for Hematopoiesis Regeneration and Acute Myeloid Leukemia Progression

Impairement of normal hmatopoiesis and leukemia progression are two well-linked processes during leukemia development and controlled by the bone marrow (BM) niche. Extracellular matrix proteins including laminin are important BM niche components. However, their role in hematopoiesis regeneration and leukemia is unknown. Laminin α4 (Lama4), a major receptor-binding chain of several laminins, is altered in BM niches in mice with acute myeloid leukemia (AML). So far, the impact of Lama4 on leukemia progression remains unknown. We here report that Lama4 deletion in mice resulted in impaired hematopoiesis regeneration following irradiation-induced stress, which is accompanied with altered BM niche composition and inflammation. Importantly, in a transplantation-induced MLL-AF9 AML mouse model, we demonstrate accelerated AML progression and relapse in Lama4-/-mice. Upon AML exposure, Lama4-/- mesenchymal stem cells (MSCs) exhibited dramatic molecular alterations including upregulation of inflammatory cytokines that favor AML growth. Lama4-/- MSCs displayed increased anti-oxidant activities and promoted AML stem cell proliferation and chemoresistance to cytarabine, which was accompanied by increased mitochondrial transfer from the MSCs to AML cells and reduced reactive oxygen species in AML cells in vitro. Similarly, we detected lower levels of reactive oxygen species in AML cells from Lama4-/- mice post-cytarabine treatment. Notably, LAMA4 inhibition or knockdown in human MSCs promoted human AML cell proliferation and chemoprotection. Together, our study for the first time demonstrates a critical role of Lama4 in impeding AML progression and chemoresistance. Targeting Lama4 signaling pathways may offer potential new therapeutic options for AML.

Arhgap21 Deficiency Results in Increase of Osteoblastic Lineage Cells in the Murine Bone Marrow Microenvironment

ARHGAP21 is a member of the RhoGAP family of proteins involved in cell growth, differentiation, and adhesion. We have previously shown that the heterozygous Arhgap21 knockout mouse model (Arhgap21+/−) presents several alterations in the hematopoietic compartment, including increased frequency of hematopoietic stem and progenitor cells (HSPC) with impaired adhesion in vitro, increased mobilization to peripheral blood, and decreased engraftment after bone marrow transplantation. Although these HSPC functions strongly depend on their interactions with the components of the bone marrow (BM) niche, the role of ARHGAP21 in the marrow microenvironment has not yet been explored. In this study, we investigated the composition and function of the BM microenvironment in Arhgap21+/− mice. The BM of Arhgap21+/− mice presented a significant increase in the frequency of phenotypic osteoblastic lineage cells, with no differences in the frequencies of multipotent stromal cells or endothelial cells when compared to the BM of wild type mice. Arhgap21+/− BM cells had increased capacity of generating osteogenic colony-forming units (CFU-OB) in vitro and higher levels of osteocalcin were detected in the Arhgap21+/− BM supernatant. Increased expression of Col1a1, Ocn and decreased expression of Trap1 were observed after osteogenic differentiation of Arhgap21+/− BM cells. In addition, Arhgap21+/− mice recipients of normal BM cells showed decreased leucocyte numbers during transplantation recovery. Our data suggest participation of ARHGAP21 in the balanced composition of the BM microenvironment through the regulation of osteogenic differentiation.

Stromal inflammation is a targetable driver of hematopoietic aging

Hematopoietic aging is marked by a loss of regenerative capacity and skewed differentiation from hematopoietic stem cells (HSC) leading to impaired blood production. Signals from the bone marrow (BM) niche tailor blood production, but the contribution of the old niche to hematopoietic aging remains unclear. Here, we characterize the inflammatory milieu that drives both niche and hematopoietic remodeling. We find decreased numbers and functionality of osteoprogenitors (OPr) and expansion of pro-inflammatory perisinusoidal mesenchymal stromal cells (MSC) with deterioration of the sinusoidal vasculature, which together create a degraded and inflamed old BM niche. Niche inflammation, in turn, drives chronic activation of emergency myelopoiesis pathways in old HSCs and multipotent progenitors (MPP), which promotes myeloid differentiation at the expense of lymphoid and erythroid commitment and hinders hematopoietic regeneration. Remarkably, niche deterioration, HSC dysfunction and defective hematopoietic regeneration can all be ameliorated by blocking IL-1 signaling. Our results demonstrate that targeting IL-1 as a key mediator of niche inflammation is a tractable strategy to improve blood production during aging.

Aging of the Hematopoietic Stem Cell Niche: New Tools to Answer an Old Question

The hematopoietic stem cell (HSC) niche is a specialized microenvironment, where a complex and dynamic network of interactions across multiple cell types regulates HSC function. During the last years, it became progressively clearer that changes in the HSC niche are responsible for specific alterations of HSC behavior. The aging of the bone marrow (BM) microenvironment has been shown to critically contribute to the decline in HSC function over time. Interestingly, while upon aging some niche structures within the BM are degenerated and negatively affect HSC functionality, other niche cells and specific signals are preserved and essential to retaining HSC function and regenerative capacity. These new findings on the role of the aging BM niche critically depend on the implementation of new technical tools, developed thanks to transdisciplinary approaches, which bring together different scientific fields. For example, the development of specific mouse models in addition to coculture systems, new 3D-imaging tools, ossicles, and ex-vivo BM mimicking systems is highlighting the importance of new technologies to unravel the complexity of the BM niche on aging. Of note, an exponential impact in the understanding of this biological system has been recently brought by single-cell sequencing techniques, spatial transcriptomics, and implementation of artificial intelligence and deep learning approaches to data analysis and integration. This review focuses on how the aging of the BM niche affects HSCs and on the new tools to investigate the specific alterations occurring in the BM upon aging. All these new advances in the understanding of the BM niche and its regulatory function on HSCs have the potential to lead to novel therapeutical approaches to preserve HSC function upon aging and disease.

Mesenchymal Stromal Cells (MSCs) from Myelodysplastic Syndromes (MDS) Are Not Clonally Mutated I n Vivo

Introduction There is increasing evidence for an active role of the bone marrow (BM) microenvironment in the pathogenesis of Myelodysplastic Syndromes (MDS). Genetically engineered murine models have shown that isolated mutations in the BM niche can disrupt the non-mutated hematopoietic compartment and induce MDS-like phenotypes. However, it is still unclear whether primary MDS in humans may possibly be associated with acquired mutations non-hematopoietic BM stroma cells. Although chromosomal aberrations and mutations have been described in in ex vivo expanded MSC cultures from MDS and AML patients, little validation has been performed to address whether such molecular lesions were not clonal outgrowths resulting from the strenuous and massively expansive cell culture procedures. Materials and Methods We performed whole exome sequencing on paired ex vivo expanded MSCs and native BM samples of n=98 MDS and associated myeloid neoplasia cases treated at the Department of Hematology and Oncology of the Medical Faculty Mannheim, Heidelberg University, Germany (median age 73 years, range 44-86). As controls, we included a cohort of n=28 samples from healthy subjects (median age 75 years, range 36-84). MSCs were expanded adherently on plastic dishes by seeding 5x10e6 mononuclear cells in StemMACS MSC Expansion Medium XF (Miltenyi Biotec) for a median of 34 days, (95% confidence interval 22-50d). Whole exome sequencing was carried out using Nextera DNA Flex Tagmentation kit (Illumina) with IDT xGene Research probe v1 at a median coverage at 88x with BM MNC as germline control accounting for possible LOH in the BM sample. Validation experiments were performed by deep re-sequencing of single CFU-F colonies (n=4 patients), sequencing of serial cultures (n=7 patients) and re-sequencing of primary sorted native bone marrow MSCs from n=9 patients. Results In the exome sequencing analyses of ex vivo expanded MSCs we discovered multiple recurrent mutations in MSCs of MDS patients including but not limited to genes such as ZFX (n=8/98) and RANK (n=5/98). MSCs from MDS patients displayed an overall higher mutational burden and increased replicative stress as determined by gH2AX and RPA staining, which correlated with the mutational burden and shorter telomeres as compared to healthy controls. The analysis of mutational signatures revealed that MDS MSCs were distinct compared to healthy MSCs. Furthermore, we found that MDS MSCs displayed increased senescence assessed by flow bGAL staining and associated inflammatory gene expression determined by IL6 qPCR/ELISA for n=32 cases. To investigate whether acquired mutations in MSCs were driven by the ex vivo expansion we performed individualized amplicon based deep re-sequencing of serial culture passages and different BM aspirations for n=7 patients as well as single colony re-sequencing in n=4 patient cases. Furthermore, we re-sequenced primary sorted CD45-,CD235a-,CD31+/-,CD271+/- BM cells of n=9 cases. All of these validation experiments indicated that the discovered mutations were associated with expansion in culture and but not present in clonally relevant cell populations in the primary BM in vivo. Discussion Together with previously published data of the BM niche of myeloid neoplasms, our results add to the notion that MSCs in MDS are molecularly and functionally altered. Nevertheless, our current comprehensive sequencing analyses leave little doubt that if acquired mutations in the stroma of MDS patients play a role in MDS disease initiation at all, then at such a low clonal and possibly locally confined level, that they are not detectable with currently feasible sample acquisition and methodology. In our current study, we discovered no evidence for acquired mutations in BM derived MSCs in MDS. Disclosures Schmitt: Affimed GmbH: Research Funding. Flach: Gilead: Current Employment. Hofmann: BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Nowak: Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; Celgene: Honoraria; AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding; Affimed: Research Funding; Takeda: Honoraria.

Single-Cell Multi-Omic Analysis Uncovers Comprised Immune Function and Primary Resistance Mechanism in Acute Myeloid Leukemia

Background: Approximately 20% of AML patients do not respond to induction chemotherapy (primary resistance) and 40-60% of patients develop secondary resistance, eventually leading to relapse followed by refractory disease (RR-AML). Diversified molecular mechanisms have been proposed for drug resistance and RR phenotype. However, we still cannot predict when relapse will occur, nor which patients will become resistant to therapy. Single-cell multi-omic (ScMo) profiling may provide new insights into our understanding of hematopoietic stem cell (HSC) differentiation trajectories, tumor heterogeneity and clonal evolution. Here we applied ScMo to profile bone marrow (BM) from AML patients and healthy controls. Methods: AML samples were collected at diagnosis with institutional IRB approval. Cells were stained with a panel of 62 DNA barcoded antibodies and 10x Genomics Single Cell 3' Library Kit v3 was used to generate ScMo data. After normalization, clusters were identified using Uniform Manifold Approximation and Projection (UMAP) and annotated using MapCell (Koh and Hoon, 2019). We analyzed 23,933 cells from 4 adult AML BM samples, and 39,522 cells from 2 healthy adults and 3 sorted CD34+ normal BM samples. Gene set enrichment analysis (GSEA) and Enrichr program were used to examine underlying pathways among differentially expressed genes between healthy and AML samples. Results: We identified 16 cell types between the AML and normal samples (Fig 1a) amongst 45 clusters in the UMAP projection (Fig 1b). Comparative analysis of the T cell clusters in AML samples with healthy BM cells identified an "AML T-cell signature" with over-expression of genes such as granzymes, NK/T cell markers, chemokine and cytokine, proteinase and proteinase inhibitor (Fig 2a). Among them, IL32 is known to be involved in activation-induced cell death in T cells and has immunosuppressive role, while CD8+ GZMB+ and CD8+ GZMK+ cells are considered as dysfunctional or pre-dysfunctional T cells. Indeed, Enrichr analysis showed the top rank of phenotype term - "decreased cytotoxic T cell cytolysis". We next examined whether NK cells, are similarly dysfunctional in the AML ecosystem. The "AML NK cell signature" includes Fc Fragment family, IFN-stimulated genes (ISGs), the effector protein-encoding genes and other genes when compared to normal NK cells (Fig 2b). GSEA analysis revealed "PD-1 signalling" among the top 5 ranked pathways in AML-NK cells, though no increase in PD-1 protein nor PDCD1 gene were identified in these cells. Inhibitory receptor CD160 was expressed higher in AML samples along with exhaustion (dysfunction) associated genes TIGIT, PRF1 and GZMB (Fig 2c). Enrichr analysis uncovered enrichment of "abnormal NK cell physiology and "impaired natural killer cell mediated cytotoxicity". Similarly, the "AML monocyte signature" was significantly enriched with genes in "Tumor Infiltrating Macrophages in Cancer Progression and Immune Escape" and "Myeloid Derived Suppressor Cells in Cancer Immune Escape". We also analyzed HSPC component in one pair of cytogenetically matched, untreated complete remission (CR) /RR AML pair (Fig 2d). Notably, half of the 10 genes overexpressed in RR-AML, CXCR4, LGALS1, S100A8, S100A9, SRGN (Serglycin), regulate cell-matrix interaction and play pivotal roles in leukemic cells homing bone marrow niche. The first 4 of these genes have been demonstrated as prognostic indicators of poor survival and associated with chemo-resistance and anti-apoptotic function. Furthermore, single-cell trajectory analysis of this CR/RR pair illustrated a change in differentiation pattern of HSPCs in CR-AML to monocytes in RR-AML. We are currently analyzing more AML samples to validate these findings. Conclusions: Our ScMo analysis demonstrates that the immune cells are systematically reprogrammed and functionally comprised in the AML ecosystem. Upregulation of BM niche factors could be the underlying mechanism for RR-AML. Thus, reversing the inhibited immune system is an important strategy for AML therapy and targeting leukemic cell-BM niche interaction should be considered for cases with high expression of these molecules on AML HSPCs. Note: J.Z. and J.A.S. share co-first authorship. Figure 1 Figure 1. Disclosures Scolnick: Proteona Pte Ltd: Current holder of individual stocks in a privately-held company. Xu: Proteona Pte Ltd: Current Employment. Ooi: Jansen: Honoraria; Teva Pharmaceuticals: Honoraria; GSK: Honoraria; Abbvie: Honoraria; Amgen: Honoraria. Lovci: Proteona Pte Ltd: Current Employment. Chng: Aslan: Research Funding; Takeda: Honoraria; Johnson & Johnson: Honoraria, Research Funding; BMS/Celgene: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Antengene: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria.

Hematopoietic Stem Cell Regeneration through Paracrine Regulation of the Wnt5a-Prox1 Signaling Axis

The crosstalk between bone marrow (BM) microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration after injury. Here we show that deletion of the genes encoding the DNA repair-deficient syndrome Fanconi anemia (FA), Fanca and Fancc, in mice dampens HSC regeneration through both direct effects on HSCs and indirect effects on BM niche cells. Specifically, Fanca- or Fancc-deficiency compromises hematologic recovery and dampens HSC regeneration following irradiation. FA HSCs show persistent upregulation of the Wnt target Prox1, a homeobox transcription factor, in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improves long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in wild-type (WT) HSC mimics the defective repopulation phenotype of FA HSCs. By analyzing paracrine factors in Wnt signaling, we found that WT mice, but not FA mice, show significant induction by TBI of BM stromal Wnt5a protein, which is produced in LepR +CXCL12 + BM stromal cells. Wnt5a treatment of irradiated FA mice enhances hematopoietic recovery and HSC regeneration. Conversely, Wnt5a neutralization in co-cultured LepR + BM stromal cells inhibits HSC regeneration and hematopoietic recovery following TBI. Mechanistically, Wnt5a secreted by LepR +CXCL12 + BM stromal cells inhibits b-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSPCs. The detrimental effect of deregulated Wnt5a-Prox1 signaling on HSC regeneration and hematopoietic recovery is also observed in aged mice. Irradiation induces upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improves HSC regeneration and hematopoietic recovery after irradiation. Finally, treatment of aged mice with Wnt5a enhances hematopoietic repopulation. Collectively, these findings identify the novel paracrine Wnt5a-Prox1 signaling axis in regulating HSC regeneration under conditions of injury and aging. Disclosures No relevant conflicts of interest to declare.

Inhibition of Fibroblast Growth Factor-23 (FGF-23) Rescues Bone and Hematopoietic Stem Cell Niche Defects in Beta-Thalassemia, Uncovering the Missing Link between Hematopoiesis and Bone

The bone marrow (BM) niche regulation and interactions with hematopoietic stem cells (HSC) have been extensively studied in steady state conditions and malignancies, but are still underexplored in hematological inherited disorders. We provided the first demonstration of impaired HSC function caused by an altered BM niche in a non-malignant disease, beta-thalassemia (BT) (Aprile et al., Blood 2020). BT is a globally widespread congenital hemoglobin disorder, resulting in severe anemia, ineffective erythropoiesis and multi-organ secondary complications, including bone alterations. Correction of the genetic defect is achieved by transplantation of HSC from healthy donors or autologous HSC from patients upon gene therapy. Since the quality and the engraftment of HSC depend on the BM microenvironment, niche-HSC crosstalk plays a crucial role for transplantation outcome. During the analysis of different components of the niche, we found reduced bone density in BT th3 mice, along with a defective HSC supporting activity by the BM stromal niche. Interestingly, osteoporosis is a constant hallmark in BT patients. We investigated the mechanisms underlying bone and HSC niche defects focusing on the role of fibroblast growth factor-23 (FGF-23), a hormone mainly secreted by osteocytes, but also by erythroid cells, which negatively modulates bone metabolism. Since FGF-23 is stimulated by the anemia-related factor erythropoietin (EPO), we hypothesized that the high EPO levels in BT might contribute to increase FGF-23, potentially affecting bone and BM niche homeostasis. We found high levels of circulating FGF-23 in th3 mice (wt vs. th3: 290.5±27.3 vs. 1823±136.1 pg/ml, p<0.0001) and in BT patients (HD vs. THAL: 94.7±1.8 vs. 117.2±5.3 RU/ml, p<0.01), associated to its increased expression by bone and BM erythroid cells. In vivo neutralization of EPO axis was sufficient to normalize FGF-23 levels (th3 vs. th3+anti-EPO: 1591±162.2 vs. 496.1±33.3 pg/ml, p<0.001), thus demonstrating the causative role of EPO. EPO stimulation and signaling inhibition strategies highlighted the involvement of Erk1/2 and Stat5 pathways in activating Fgf-23 transcription in bone and BM erythroid cells, respectively. To provide proof of concept data on the contribution of FGF-23 to BT bone and stromal niche alterations, we inhibited FGF-23 signaling. In vivo administration of FGF-23 blocking peptide rescued the trabecular bone density in th3 mice (th3 vs. th3+FGF23inh: 138.2±4.9 vs. 166.9±5.2 mg/cm 3, p<0.01). Short-term inhibition treatment (38 hours) was sufficient to enhance bone mineralization by acting on Alkaline Phosphatase and on the expression of the main regulators of mineralization Dmp1 and Mepe by osteocyte, whereas long-term administration (12 days) restored also osteoblast number and bone deposition. Importantly, FGF-23 inhibition normalized the expression of key niche molecules, such as Jagged-1 and osteopontin, involved in the functional crosstalk between HSC and the stromal niche. Consistently, the treatment restored the frequency of th3 HSC by expanding the pool of quiescent cells (th3 vs. th3+FGF23inh: 0.026 vs. 0.045% on Lin neg BM cells, p<0.01). FGF-23 inhibition had also a positive anti-apoptotic effect on the expanded BM erythroid compartment (th3 vs. th3+FGF23inh: 61.6±1.3 vs. 51.1±2.1% of BM Ter119 + cells, p<0.001), promoting the maturation of early erythroid precursors (th3 vs. th3+FGF23inh: 8.5±1 vs. 16.4±1.1% of BM Pro-Erythroblasts, p<0.0001), as already shown in models of secondary anemias. Evidence in BT patients showed negative correlations between FGF-23 levels and markers of bone homeostasis (e.g. osteocalcin R 2=0.88, p<0.05) and positive correlations with makers of ineffective erythropoiesis (e.g. circulating reticulocytes R 2=0.83, p<0.05), thus positioning FGF-23 as the molecule at the crossroads of erythropoiesis and bone metabolism in BT. Our findings uncover an underexplored role of FGF-23 in bone and BM niche defects in a primary anemia, as a condition of chronic EPO stimulation, and propose FGF-23 as the missing link between hematopoiesis and bone regulation. The inhibition of FGF-23 signaling might provide a novel strategy to ameliorate bone compartment and restore HSC-BM niche interactions in BT by a 'two birds with one stone' approach, with a potential translational relevance in improving HSC transplantation and gene therapy. Disclosures Cappellini: Celgene: Consultancy, Research Funding; Vifor: Consultancy; La Jolla: Research Funding; Protagonist Therapeutics: Research Funding; IONIS Pharmaceuticals: Consultancy; CRISPR Therapeutics: Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Targeting PI3K-AKT-mTOR Signaling in Multiple Myeloma Mesenchymal Stem Cells Mediates Antiproliferative Effect on Myeloma Cells

Introduction: Multiple myeloma (MM) is a B-cell malignancy characterized by an abnormal proliferation and infiltration of malignant plasma cells in the bone marrow (BM). Mesenchymal stromal cells (MSCs) represent a crucial component of the BM niche and mediate essential signalling via cytokines and cell-cell interactions. The interplay of MM cells and BM-MSC is complex and relies on multiple signaling pathways leading to MM progression and therapeutic resistance. Objectives: MM remains an incurable disease so far. Distinctive for this disease is a long-lasting polarization of the BM niche influencing MM progression and prognosis. We, therefore, focussed on MSCs to identify enrichment for different hallmark gene sets and their aberrant signaling contributing to the pathogenesis of the disease, therapy response and to further identify novel therapeutic strategies. Methods: BM-MSCs were isolated from patients with MM at diagnosis (MM-D-MSC) and in remission (MM-R-MSC) as well as from donors with other malignant diseases (CTR-MSC). RNA sequencing and Western Blot were used for examination of enriched pathways. Various functional assays for proliferation, apoptosis and cell cycle were performed either using a mono-culture or co-culture protocol of MSC and the MM-cell lines MM.1S and SKMM2 treating the cells with the pan-PI3K-inhibitor GDC-0941. Results: MM-D-MSCs supported the growth of myeloma cell lines better (3 fold, p<0.01) than MM-R- and CTR-MSCs. Our results demonstrate that MM-D-MSCs have a distinct gene expression profile compared to CTR-MSC indicating potential nodes of crosstalk and therapeutic importance. Amongst others, the PI3K-AKT-mTOR hallmark gene set was significantly enriched in MM-D-MSCs as compared to CTR-MSCs (p<0.001). We confirmed these findings on a proteomic level. We found evidence for the upregulation of PI3Kα, AKT, pAKT and mTOR in MM-D-MSC comparing to the other MM-R- and CTR-MSCs (p<0.05). We treated these MSC and the MM-cell lines MM.1S and SKMM2 with the PI3-Kinase inhibitor GDC-0941. The treatment reduced the signaling PI3Kα, AKT and mTOR in both, MSC and MM-cells. As stated MM-D-MSC supported the growth of myeloma cells better than other MSC types. However, upon GDC-0941 treatment, the proliferation of MM-D-MSCs was significantly reduced compared to the other MSC-types. In addition, the inhibition of proliferation of myeloma cell lines MM1S and SKMM2 was much more pronounced when they were cocultured with MM-D-MSC (32 and 34 %, p=0.04) compared to the growth of myeloma cells in coculture with MSC types, either in remission or other malignancies. Conclusion: We here identified functionally distinct differences in MM-D-MSCs compared to MM-R-MSCs or CTR-MSCs. Our data further provides a deeper insight into the molecular signature of MM-MSCs, a predictive of patient prognosis and treatment outcome. Targeting MSCs as a crucial part of the MM-BM niche by using PI3K-inhibitors could contribute to novel therapeutic strategies to effectively block MM-MSC interaction improving overall patient survival. Disclosures Raab: Roche: Consultancy; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Khandanpour: BMS/Celgene: Honoraria; Sanofi: Honoraria, Research Funding; Pfizer: Honoraria; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria; GSK: Honoraria.

Inducible Sbds Deletion Impairs Bone Marrow Niche Capacity to Engraft Donor Bone Marrow After Transplantation

Bone marrow (BM) niche-derived signals are critical for facilitating engraftment after hematopoietic stem cell (HSC) transplantation (HSCT). HSCT is required for restoration of hematopoiesis in patients with inherited bone marrow failure syndromes (iBMFS). Shwachman-Diamond syndrome (SDS) is a rare iBMFS associated with mutations in SBDS. Previous studies have demonstrated that SBDS deficiency in osteolineage niche cells causes bone marrow dysfunction that promotes leukemia development. However, it is unknown whether BM niche defects caused by SBDS deficiency also impair efficient engraftment of healthy donor HSC following HSCT, a hypothesis that could explain morbidity seen after clinical HSCT for patients with SDS. Here, we report a mouse model with inducible Sbds deletion in hematopoietic and osteolineage cells. Primary and secondary BM transplantation (BMT) studies demonstrated that SBDS deficiency within BM niches caused poor donor hematopoietic recovery and specifically poor HSC engraftment after myeloablative BMT. We have additionally identified multiple molecular and cellular defects within niche populations that are driven by SBDS deficiency and that are accentuated or develop specifically following myeloablative conditioning. These abnormalities include altered frequencies of multiple niche cell subsets including mesenchymal lineage cells, macrophages and endothelial cells; disruption of growth factor signaling, chemokine pathway activation, and adhesion molecule expression; and p53 pathway activation, and signals involved in cell cycle arrest. Taken together, this study demonstrates that SBDS deficiency profoundly impacts recipient hematopoietic niche function in the setting of HSCT, suggesting that novel therapeutic strategies targeting host niches could improve clinical HSCT outcomes for patients with SDS.