Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

Ionizing radiation and chemotherapy deplete hematopoietic stem cells and damage the vascular niche wherein hematopoietic stem cells reside. Hematopoietic stem cell regeneration requires signaling from an intact bone marrow (BM) vascular niche, but the mechanisms that control BM vascular niche regeneration are poorly understood. We report that BM vascular endothelial cells secrete semaphorin 3 A (SEMA3A) in response to myeloablation and SEMA3A induces p53 – mediated apoptosis in BM endothelial cells via signaling through its receptor, Neuropilin 1 (NRP1), and activation of cyclin dependent kinase 5. Endothelial cell – specific deletion of Nrp1 or Sema3a or administration of anti-NRP1 antibody suppresses BM endothelial cell apoptosis, accelerates BM vascular regeneration and concordantly drives hematopoietic reconstitution in irradiated mice. In response to NRP1 inhibition, BM endothelial cells increase expression and secretion of the Wnt signal amplifying protein, R spondin 2. Systemic administration of anti - R spondin 2 blocks HSC regeneration and hematopoietic reconstitution which otherwise occurrs in response to NRP1 inhibition. SEMA3A – NRP1 signaling promotes BM vascular regression following myelosuppression and therapeutic blockade of SEMA3A – NRP1 signaling in BM endothelial cells accelerates vascular and hematopoietic regeneration in vivo.

Magnetism-Induced Cell-Targeting Transplantation Improves Early Hematopoietic Reconstitution in a Murine Bone Marrow Transplantation Model

Background Early hematopoietic reconstitution is essential for improving survival and reducing complications after hematopoietic stem/progenitor cell (HSC/HPC) transplantation (HSCT/HPCT). Increasing HSC/HPC homing to the bone marrow is a potential approach for promoting hematopoietic reconstitution. Methods We proposed the transplantation of HSCs/HPCs with a magnetism-induced cell-targeting transplantation (MagIC-TT) strategy. HSCs/HPCs were magnetized with CD45 microbeads. The biological characteristics (morphology, proliferation, viability, and ferroptosis) and target migration ability of these cells were studied in vitro. The hematopoietic reconstitution experiments were constructed in vivo in autologous and allogeneic bone marrow transplantation models with grouping showed as Table 1. The therapeutic effects were assessed by survival, donor chimerism, routine blood examination and histological analysis. We also performed transcriptomic sequencing for further mechanistic studies. Results The biological characteristics was found no significant difference between the MagIC-TT and non-MagIC-TT groups, while migration ability was greatly improved with MagIC-TT (Data not showed). The survival rate was higher in the MagIC-TT group and significantly different in the allogeneic model (P<0.05). Hematopoietic reconstitution of donor chimerism, WBCs, HGB, and PLTs was faster in the MagIC-TT groups (within 22 days) (Figure 1). Confocal observation showed that more donor cells (eGFP +) were retained in the injected femur of the MagIC-TT group than in the femur of the non-MagIC-TT group 7 days after transplantation (P<0.05) (Figure 2). The severity of aGVHD (Assessed by survival, body weight, back arching, fur losting, diarrhea etc.) was reduced in the MagIC-TT groups in the allogeneic model (P<0.05) (Data not showed). Transcriptome sequencing revealed differentially expressed genes (DEGs) involved in localization/locomotion and pathways, cytokine-cytokine receptor interactions and chemokine signaling pathways between the two groups (Figure 3). Conclusion The MagIC-TT strategy improves HSC/HPC homing, resulting in faster hematopoietic reconstitution in a murine bone marrow transplantation model. Key words Magnetism; Hematopoietic Stem Cell Transplantation (HSCT); Cell Homing; Hematopoietic Reconstitution; Ferroptosis Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

High-Density Clonal Analysis Reveals Highly Active Contribution of Multipotent Hematopoietic Stem Cells during Early Phases of Hematopoietic Recovery after Transplantation

Recovery after conditioning and transplantation of hematopoietic stem and progenitor cells (HSPC) is thought to be biphasic, with short-term engrafting progenitors driving the recovery for 6-9 months and multipotent hematopoietic stem cells (HSCs) providing long-term repopulation. Recent clonal tracking data from autologous human gene therapy trials seems to support this model (Biasco et al. 2016, Cell Stem Cell; Six et al. 2020, Blood). These recent reports investigating the contribution of HSCs in patients are based on the longitudinal tracking of thousands of gene-marked cells using retroviral integration site analysis (ISA). While this technology is very reliable to follow gene therapy patients and monitor the potential outgrowth of dominant or malignant clones, low sensitivity and high error rates require significant data exclusion and sophisticated statistical tests to ensure data reliability (Adair et al. 2020, Molecular Therapy MCD). Lack of sensitivity can be overcome by increasing the frequency (high density) of sampling. However, limited material from patients remains a bottleneck for improved data quality and, consequently, correct interpretation of such complex datasets. To overcome the limitations of ISA and determine the onset of HSC contribution we performed high-density sampling for ISA in nonhuman primates (NHPs) transplanted with gene-modified HSCs. In the first month of hematopoietic recovery weekly blood samples were taken to enhance data density and increase the reliablity to detect clones with low abundance. Animals were followed up to 5 years to confirm that identified HSC clones persist long-term. Finally, clonal tracking data from the NHPs was used to inform a simulation of hematopoietic reconstitution, determine the temporal involvement of HSCs, and refine the phases of hematopoietic recovery after myeloablation and HSC transplantation. In contrast to the current biphasic model, contribution of multipotent HSCs clones was detected in the very first blood samples taken 2 to 3 weeks post-transplant during neutrophil recovery. HSC clones found in these early time points persisted long-term throughout the entire follow-up and were detected in bone marrow CD34 + cells 4 years later. Most surprisingly, multipotent HSCs became the dominant source for mature blood cells in the peripheral blood as early as 50 days post-transplant. To understand the observed kinetics of HSC contribution and change in clonal diversity in our dataset, we simulated the clonal outgrowth and differentiation of multipotent clones. Simulations predicted that hematopoietic recovery is primarily HSC driven and HSC contribution follows a stochastic pattern. Finally, to confirm that HSCs proliferation and differentiation is a stochastic process, in vitro experiments in colony-forming cell (CFC) assays were carried out. As predicted, the decision of individual HSCs to either grow into a larger pool or differentiate and get lost followed the same kinetics as observed in vivo. Here, we show evidence that long-term persisting multipotent HSCs actively contribute during early hematopoietic reconstitution after myeloablation and HSC transplantation. Enhanced sampling showed that multipotent HSCs produce neutrophils during recovery and become the predominant source of mature blood cells as early as 50 days post-transplant. Most importantly, observed changes in the clonal diversity during early recovery suggest a stochastic engraftment of HSCs rather than a bi-phasic reconstitution initially driven by short-term progenitors. These findings should have important implications for the design of ex vivo and in vivo HSC gene therapy and genome editing approaches. Figure 1 Figure 1. Disclosures Radtke: 47 Inc.: Consultancy; Ensoma Inc.: Consultancy. Kiem: Homology Medicines: Consultancy; VOR Biopharma: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company.

A FLI1-KLF6 Axis Regulates Aging in Human Hematopoietic Stem and Progenitor Cells and Normalization of KLF6 Levels in Aged Cells Leads to Their Rejuvenation

Aging causes a gradual decline in hematopoietic stem cell (HSC) function, which increases the risk for hematological malignancies. While much has been done in murine models, human HSC aging impairment is less understood. We recently showed that Krüppel-like transcription factor 6 (KLF6) is among the top downregulated genes during human HSC aging, which correlates with H3K27ac loss at several upstream putative enhancers. Moreover, loss of KLF6 in human CD34 + cells resulted in impaired in vitro differentiation, increased colony-forming potential and a transcriptional profile similar to that of aged CD34 + CD38 - cells. We hypothesized that age-acquired deregulation of KLF6 may be a key player in age-related HSC dysfunction and sought to fully characterize this. Thus, we isolated CD34 + cells from young (<32 y.o) and aged (>65 y.o.) healthy donors and performed CRISPR-Cas9 genome editing and transcriptional activation of KLF6, respectively, followed by epigenetic and transcriptional reprogramming, in vivo hematopoietic reconstitution, and analysis of DNA damage, apoptosis, and reactive oxygen species (ROS) levels. KLF6 knock-out (KO) and non-targeting control (NTC) cells from young healthy donors were engrafted into immunodeficient NSGS mice. Hematopoietic reconstitution analysis showed that KLF6 KO cells led to increased myeloid and reduced lymphoid reconstitution in peripheral blood (PB; p<1.62 -7) and an increase in immunophenotypically defined HSC and CD34 + CD38 - progenitor fractions in the bone marrow (BM; p=0.02, and p=0.04, respectively). H3K27ac analysis of KLF6 KO cells revealed a loss of 3,390 ChIP-seq peaks (FDR < 0.05) and 285 peaks gained. Functional annotation using ChIP-Enrich showed that H3K27ac loss associates with myeloid homeostasis, erythroid differentiation and oxidative stress (FDR < 0.05). Three putative enhancer (E) regions upstream of the KLF6 locus showed loss of H3K27ac with aging. Depletion of the E1 but not E2 or E3 regions phenocopied in vitro and in vivo findings of KLF6 KO. Transcription factor (TF) ChIP-seq data analysis revealed FLI1, ERG, and RUNX1 binding overlapping the E1 region. Knockdown of FLI1 but not ERG or RUNX1 led to an increase in KLF6. Notably, FLI1 mRNA levels, but not ERG or RUNX1, are increased during normal aging. We next performed in vitro KLF6 activation in aged CD34 + (KLF6a) cells using a dCas9-VP64 system to test if we could rejuvenate these cells. KLF6a cells exhibited a decrease in their in vitro myeloid differentiation potential, compared to aged NTC CD34 + cells (p<0.0041), and behaved instead similar to young controls. ChIP-seq analysis of KLF6a showed marked decrease of H3K4me1 (n=3,273 peaks) with relatively few regions with increased H3K4me1 (n=602) (FDR < 0.05). In contrast, we observed an increase in H3K27ac (n=3,361 peaks) with only 71 peaks lost compared to aged NTC (FDR < 0.05). Regions that gained H3K27ac in KLF6a were associated with platelet activation, cell junction and adhesion. In vivo analysis of KLF6a cells injected into NSGS mice revealed a significant reduction in the PB myeloid fraction compared to NTC (p<1.2-8), with a concomitant expansion in the lymphoid compartment (p<4.4 -11). BM composition analysis at week 16 showed a decrease in the HSC fraction in KLF6a cells (p=0.0029) as well as a reduction in CD34 +CD38 -, CD34 +CD38 + and MEPs (p=0.036, p<0.0001 and p=0.041, respectively). We next examined the impact of KLF6 modulation on DNA damage and observed that young human KLF6 KO cells had a significant increase in gH2AX and 53BP1 (p<0.0001, for both) whereas KLF6a in aged CD34 + cells exhibited reduced gH2AX and 53BP1 foci in comparison to aged NTC (p<0.0001, for both). In addition, apoptotic levels in KLF6 KO cells were higher than in NTC cells (p=0.006) whereas aged KLF6a cells showed a reduction in the incidence of apoptotic cells compared to NTC (p=0.019). Finally, ROS analysis in young KLF6 KO showed increased levels of total and mitochondrial ROS compared to NTC (p=0.0008 and p<0.0001, respectively) whereas both ROS fractions were reduced in KLF6a cells (p=0.0002 and p<0.0001, respectively). In summary, these results show that the FLI1-KLF6 axis plays a key role in regulating HSPC aging and that KLF6 is required for normal HSPC function and differentiation. In addition, normalization of KLF6 levels in aged HSPCs resulted in reprogramming and rejuvenation HSPCs, confirming the central role of this TF in aging HSPC biology. Disclosures No relevant conflicts of interest to declare.

The CADM1 tumor suppressor gene is a major candidate gene in MDS with deletion of the long arm of chromosome 11

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoietic stem-cell disorders characterized by ineffective hematopoiesis leading to peripheral cytopenias and in a substantial proportion of cases to acute myeloid leukemia. The deletion of the long arm of chromosome 11, del(11q), is a rare but recurrent clonal event in MDS. Here, we detail the largest series of 113 cases of MDS and myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) harboring a del(11q) analyzed at clinical, cytological, cytogenetic and molecular levels. Female predominance, a survival prognosis similar to other MDS, a low monocyte count and dysmegakaryopoiesis were the specific clinical and cytological features of del(11q) MDS. In most cases, del(11q) was isolated, primary and interstitial encompassing the 11q22-23 region containing ATM, KMT2A and CBL genes. The common deleted region at 11q23.2 is centered on an intergenic region between CADM1 (also known as TSLC1, Tumour Suppressor in Lung Cancer 1) and NXPE2. CADM1 was expressed in all myeloid cells analyzed in contrast to NXPE2. At the functional level, the deletion of Cadm1 in murine Lineage-Sca1+Kit+ cells modifies the lymphoid to myeloid ratio in bone marrow although not altering their multi-lineage hematopoietic reconstitution potential after syngenic transplantation. Together with the frequent simultaneous deletions of KMT2A, ATM and CBL and mutations of ASXL1, SF3B1 and CBL, we show that CADM1 may be important in the physiopathology of the del(11q) MDS, extending its role as tumor-suppressor gene from solid tumors to hematopoietic malignancies.

Hematopoietic stem and progenitor cells integrate Bacteroides-derived innate immune signals to promote gut tissue repair

Bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) are often activated by bacterial insults to replenish the host hemato-immune system, but how they integrate the associated tissue damage signals to initiate distal tissue repair is largely unknown. Here, we showed that acute gut inflammation expands HSPCs in the BM through GM-CSFR activation, and directs them to inflamed mesenteric lymph nodes for further differentiation into myeloid cells specialized in gut tissue repair. We also identified that this process is exclusively mediated by Bacteroides, a commensal gram-negative bacteria, that activates innate immune signaling. In contrast, chronic gut inflammation reduces HSC potential for hematopoietic reconstitution and immune response against infection. Similarly, microbial signals contribute to aging-associated HSPC expansion. These findings establish a cross-organ communication that promotes tissue regeneration, but if sustained, impairs tissue homeostasis that may be relevant to aging and chronic disorders.

Hematopoietic recovery after transplantation is primarily derived from the stochastic contribution of hematopoietic stem cells

Reconstitution after hematopoietic stem cell (HSC) transplantation is assumed to occur in two distinct phases: initial recovery mediated by short-term progenitors and long-term repopulation by multipotent HSCs which do not contribute to hematopoietic reconstitution during the first 6-9 months. We have previously reported the transplantation and exclusive engraftment of the HSC-enriched CD34+CD45RA-CD90+ phenotype in a nonhuman primate model. Here, we closely followed the clonal diversity and kinetics in these animals. Enhanced sampling and high density clonal tracking within the first 3 month revealed that multipotent HSCs actively contributed to the early phases of neutrophil recovery and became the dominant source for blood cells as early as 50 days after transplant. Longitudinal changes in clonal diversity supported a stochastic engraftment of HSCs with the majority of HSCs clones vanishing early during neutrophil recovery and a smaller fraction of HSC clones expanding into bigger pools to support long-term hematopoiesis. In contrast to the bi-phasic model, we propose that hematopoietic recovery after myeloablation and transplantation is primarily derived from HSCs in a stochastic manner rather than in two phases by independent cell populations.

A Retrospective Observation of Treatment Outcomes Using Decitabine-Combined Standard Conditioning Regimens Before Transplantation in Patients With Relapsed or Refractory Acute Myeloid Leukemia

Hypomethylating agents, decitabine (DAC) and azacitidine, can act as prophylactic and pre-emptive approaches after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and a non-intensive bridging approach before allo-HSCT. However, they are rarely used as a part of conditioning regimens in patients with relapsed or refractory acute myeloid leukemia (AML). This retrospectively study included a total of 65 patients (median, 37; range, 13–63) with relapsed or refractory AML who were treated by allo-HSCT after myeloablative conditioning regimens without or with DAC (high-dose DAC schedule, 75 mg/m2 on day −9 and 50 mg/m2 on day −8; low-dose DAC schedule, 25 mg/m2/day on day −10 to −8). DAC exerted no impact on hematopoietic reconstitution. However, patients who were treated with the high-dose DAC schedule had significantly higher incidence of overall survival (OS, 50.0%) and leukemia-free survival (LFS, 35.0%), and lower incidence of relapse (41.1%) and grade II–IV acute graft versus host disease (aGVHD, 10.0%) at 3 years, when compared with those treated with standard conditioning regimens or with the low-dose DAC schedule. In conclusion, high-dose DAC combined with standard conditioning regimens before allo-HSCT is feasible and efficient and might improve outcomes of patients with relapsed or refractory AML, which provides a potential approach to treat these patients.