Peripheral Blood Chimerism Research Articles

211At-Labeled Anti-CD45 Antibody as a Nonmyeloablative Conditioning for Canine DLA-Haploidentical Stem Cell Transplantation.

The α-emitter 211At deposits a high amount of energy within a few cell diameters, resulting in irreparable DNA double-strand breaks while minimizing off-target toxicity. We investigated the use of the 211At-labeled anti-CD45 monoclonal antibody (mAb) 211At-CD45-B10 as a nonmyeloablative conditioning regimen for dog-leukocyte-antigen-haploidentical hematopoietic cell transplantation. Methods: Seventeen healthy dogs were injected with either a 0.50 (n = 14) or 0.75 (n = 3) mg/kg dose of anti-CD45 mAb labeled with 211At (8.436-23.199 MBq [0.228-0.627 mCi/kg]) on day -3. Peripheral blood stem cells from dog-leukocyte-antigen-haploidentical donors were given on day 0. Peripheral blood chimerism was calculated by polymerase chain reaction assays, and blood clearance of the radioimmunoconjugate was studied using enzyme-linked immunosorbent assay and radioactivity measurements of serial blood samples. Results: All dogs achieved donor chimerism by day 28 (range, 27%-100%). The hematopoietic engraftment rate was 100%, though engraftment durability was variable. No difference in absorbed dose to blood was seen for the 2 mAb dosing levels studied. Neutropenia (0-29 cells/μL), lymphocytopenia (36-130 cells/μL), and thrombocytopenia (1.5-9 × 103/μL) with prompt recovery were observed. The main adverse nonhematologic event related to 211At-CD45-B10 was mild reversible transaminitis. Graft-versus-host disease was not seen. Twelve of the 17 dogs survived over 30 d, with donor chimerism ranging from 3% to 99%. Conclusion: The results suggest that nonmyeloablative conditioning with 211At-CD45-B10 could be used in haploidentical hematopoietic cell transplantation though with variable engraftment.

Hematopoietic Function Restoration by Transplanting Bone Marrow Niches In Vivo Engineered Using Carbonate Apatite Honeycomb Bioreactors

Hematopoietic stem cell (HSC) transplantation is used to treat blood and immunodeficient diseases. HSC expansion techniques must be developed to prevent complications and ensure reliable therapeutic efficacy. Hence, several studies have attempted in vitro expansion of HSCs using scaffolds but failed to mimic the diverse and complex nature of HSC environments. Herein, an artificial HSC microenvironment, bone marrow (BM) niches is created, through in vivo engineering using carbonate apatite honeycomb scaffolds and the potential of these scaffolds in restoring lost hematopoietic function and immunity is investigated. BM niches are generated in every honeycomb channel, wherein HSCs are gradually aggregated. Compared to the actual BM, the scaffolds exhibit a 9.9‐ and 78‐fold increase in the number of stored CD45− CD34+ side scatterlow cells that are mainly considered HSCs at 8 and 12 weeks, respectively. The transplantation of the honeycomb scaffold containing HSCs and BM niches into immunocompromised mice increases peripheral blood chimerism and restores hematopoietic function and the number of immunocytes (monocytes and lymphocytes) to normal levels. This study contributes to the development of efficient HSC transplantation techniques. Additionally, in vivo‐engineered integrated tissues using honeycomb scaffolds can be used to elucidate the interplay between the BM niches and resident cells.

The predictive value of T-cell chimerism for disease relapse after allogeneic hematopoietic stem cell transplantation.

Chimerism is closely correlated with disease relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, chimerism rate is dynamic changes, and the sensitivity of different chimerism requires further research. To investigate the predictive value of distinct chimerism for relapse, we measured bone marrow (BM), peripheral blood (PB), and T-cell (isolated from BM) chimerism in 178 patients after allo-HSCT. Receiver operating characteristic (ROC) curve showed that T-cell chimerism was more suitable to predict relapse after allo-HSCT compared with PB and BM chimerism. The cutoff value of T-cell chimerism for predicting relapse was 99.45%. Leukemia and myelodysplastic syndrome (MDS) relapse patients' T-cell chimerism was a gradual decline from 2 months to 9 months after allo-HSCT. Higher risk of relapse and death within 1 year after allo-HSCT. The T-cell chimerism rates in remission and relapse patients were 99.43% and 94.28% at 3 months after allo-HSCT (P = 0.009), 99.31% and 95.27% at 6 months after allo-HSCT (P = 0.013), and 99.26% and 91.32% at 9 months after allo-HSCT (P = 0.024), respectively. There was a significant difference (P = 0.036) for T-cell chimerism between early relapse (relapse within 9 months after allo-HSCT) and late relapse (relapse after 9 months after allo-HSCT) at 2 months after allo-HSCT. Every 1% increase in T-cell chimerism, the hazard ratio for disease relapse was 0.967 (95% CI: 0.948-0.987, P<0.001). We recommend constant monitoring T-cell chimerism at 2, 3, 6, and 9 months after allo-HSCT to predict relapse.

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids. We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

Allogeneic bone marrow transplantation for patients with treatment-refractory Crohn's Disease

Background & aimsDurable remissions of Crohn's Disease (CD) have followed myeloablative conditioning therapy and allogeneic marrow transplantation. For patients with treatment-refractory disease, we used reduced-intensity conditioning to minimize toxicity, marrow from donors with low Polygenic Risk Scores for CD as cell sources, and protracted immune suppression to lower the risk of graft-versus-host disease (GVHD). Our aim was to achieve durable CD remissions while minimizing transplant-related complications. MethodsDNA from patients and their HLA-matched unrelated donors was genotyped and Polygenic Risk Scores calculated. Donor marrow was infused following non-myeloablative conditioning. Patient symptoms and endoscopic findings were documented at intervals after transplant. ResultsWe screened 807 patients, 143 of whom met eligibility criteria; 2 patients received allografts. Patient 1 had multiple complications and died at day 332 from respiratory failure. Patient 2 had resolution of CD symptoms until day 178 when CD recurred, associated with persistent host chimerism in both peripheral blood and intestinal mucosa. Withdrawal of immune suppression was followed by dominant donor immune chimerism in peripheral blood and resolution of CD findings. Over time, mucosal T-cells became donor-dominant. At 5 years after allografting, Patient 2 remained off all medications but had mild symptoms related to a jejunal stricture that required stricturoplasty at 6 years. At 8 years, she remains stable off medications. ConclusionsThe kinetics of immunologic chimerism after allogeneic marrow transplantation for CD patients depends on the intensity of the conditioning regimen and the magnitude of immune suppression. One patient achieved durable improvement of her previously refractory CD only after establishing donor immunologic chimerism in intestinal mucosa. Her course provides proof-of-principal for allografting as a potential treatment for refractory CD, but an immunoablative conditioning regimen should be considered for future studies.(ClinicalTrials.gov, NCT01570348)

Evaluation of the effects of ischemia-reperfusion injury in rat isogenic and allogeneic muscle and skin transplant models.

Ischemia-reperfusion injury (IRI) is a phenomenon that affects transplant survival. The aim of our study was to examine the effects of IRI in isogenic and allogeneic muscle and skin transplantation models exposed to prolonged warm ischemia. Forty-eight Lewis rats and 16 Brown-Norway rats were used to create four groups: Isogenic Inguinal Flap Transplantation (IST), Isogenic Gastrocnemius Muscle Flap Transplantation (IMT), Allogeneic Inguinal Flap Transplantation (AST), and Allogeneic Gastrocnemius Muscle Flap Transplantation (AMT). Malonyldialdehyde (MDA) and superoxide dismutase (SOD) levels were measured on postoperative days 1, 7, 21, 35, 63, 100, and 120 in all groups. Donor-specific chimerism (DSC) in peripheral blood was evaluated in the allogeneic groups on postoperative days 7, 21, 35, 63, 100, and 120. The microRNA-21 and microRNA-205 levels were evaluated on postoperative days 1, 7, and 120 in all groups. At the end of the study, a histopathological examination was performed. A statistically significant difference was found between the groups in terms of MDA and SOD levels. DSC was detected in the AMT group. A significant increase in microRNA-205 was observed, especially in the AMT group. There was no significant difference in the number of functional muscle units between the muscle transplantation groups. The presence of DSC in the AMT group and the lack of a significant difference in the number of functional muscle units in the IMT and AMT groups are noteworthy findings.

Arid1b Loss Impairs Hematopoietic Stem Cell Function in Normal Hematopoiesis

Switch Sugar Non- Fermenting (SWI/SNF) nucleosome remodeling complexes are multi subunits complexes that utilize the energy from ATP to remodel chromatin, affecting cellular processes on chromatin including that of gene transcription. SWI/SNF complexes play important roles in normal development and differentiation as well as in oncogenesis. ARID1 proteins, ARID1A and ARID1B, are unique and mutually exclusive subunits of the canonical SWI/SNF complex BRG-1/BRM Associated Factor (BAF) complex. Arid1a is the most frequently mutated SWI/SNF subunit in cancers and plays a vital role in hematopoiesis. Arid1b is also frequently mutated in cancers, and its loss promotes Acute Myeloid Leukemia leukemogenesis. However, its role in normal hematopoiesis is not known. In this study we used hematopoietic specific Cre strains, VavCre and Mx1Cre, to delete Arid1b in a mouse genetic model to study its role in normal hematopoiesis. Analysis of steady state hematopoiesis in the VavCre model showed no significant differences in complete blood count (CBC) between Arid1b fl/flVavCre+ and VavCre+ mice. Further, bone marrow (BM) cellularity was not significantly different between control and experimental mice. Flow cytometry analysis of the percentage of mature myeloid, B cell, or T cell populations in the peripheral blood (PB), BM, or spleen (SPL) did not show significant differences between Arid1b fl/flVavCre+ and VavCre+ control mice. Further, we did not observe any significant differences in percentages of hematopoietic stem and progenitor cells (HSPCs), including HSCs, common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte/erythrocyte progenitors (MEPs), and common lymphoid progenitors (CLPs) in the BM of Arid1b fl/flVavCre+ mice versus VavCre+ controls. Analysis of Mx1Cre model showed similar results. Taken together, these results suggest that Arid1b is dispensable for steady state hematopoiesis. To assess the effect of loss Arid1b in a regenerative setting, we conducted non-competitive and competitive transplantation experiments in VavCre and Mx1Cre models. We transplanted BM cells from Arid1b fl/flCre+ or Cre+ mice (CD45.2) into lethally irradiated recipient CD45.1 mice with (CD45.1 at 1:1 ratio) or without competitor cells for competitive or non-competitive transplantations respectively. In Mx1Cre model, deletion of Arid1b was induced 4 weeks after transplantation. We monitored donor chimerism in PB over time as well as in the BM and SPL at end point. In both VavCre and Mx1Cre non-competitive transplants, we did not observe significant differences in CBC or donor chimerism in mature cells between Arid1b fl/flCre+ and control Cre+ mice in the PB over time. Further, no significant differences were observed between knockout and control mice in donor chimerism in mature cell populations in the BM or SPL, nor HSPC populations in the BM at end point (24-28 weeks). Deletion of Arid1b in the BM was confirmed. These results indicate that Arid1b loss does not affect regenerative potential of HSPCs in a non-competitive setting. In our competitive transplants, we observed significant decreases in PB donor chimerism for both myeloid and B cell populations over time in Arid1b fl/flVavCre+ compared to VavCre+ controls. Further, we observed significant decreases in donor chimerism of mature myeloid cells and B cells in the BM and SPL, as well as HSCs, CMPs, GMPs, and MEPs in the BM of Arid1b fl/flVavCre+ mice compared to VavCre+ controls at endpoint. However, there seemed to be incomplete penetrance as the observed phenotype varied in cohorts of different donor mice and was not significant in all cohorts. A similar phenotype was observed in the Mx1Cre model with the exception of a trend, albeit insignificant, towards a decrease in donor derived mature B cell populations in Arid1b fl/flMx1Cre+ compared to Mx1Cre+ mice in PB, SPL, and BM and no significant difference in HSCs in the BM at end point (20 weeks). Deletion of Arid1b in the BM was confirmed in both models. Overall, our results suggest that Arid1b deficient HSCs can regenerate the hematopoietic system but have defects in competitive transplantations with incomplete penetrance. Further work is needed to address the observed incomplete penetrance phenotype in competitive transplantations, and to understand the mechanism behind the requirement of Arid1b in normal hematopoiesis.

Randomized Trial of Prophylactic Donor Lymphocyte Infusion with Positively Selected CD4+ T Cells after HLA-Identical Allogeneic Stem Cell Transplantation

Introduction Allogeneic stem cell transplantation (alloSCT) can cure patients with hematological malignancies by T-cell mediated alloreactivity against patient hematopoiesis, the graft-versus-leukemia (GvL) effect. GvL can coincide with graft-versus-host disease (GvHD) when donor T cells target non-hematopoietic tissues. Increase of donor chimerism in bone marrow (BM) or peripheral blood (PB) after alloSCT indicates GvL response. After T-cell depleted alloSCT, donor lymphocyte infusion (DLI) can restore GvL. Dose and timing of DLI influence the degree of alloreactivity. Low dose of unmodified DLI as low as 0.3x10 6 T cells/kg 3 months after alloSCT appeared not to cause GvHD. (Zouwen et al. Ann Hematol, 2023) We hypothesized that infusion of a higher dose of purified CD4+ donor T cells would not cause GvHD, while inducing GvL by selective targeting of the MHC class II positive hematopoietic cells. Method A randomized controlled phase 2 trial was conducted in patients with hematological malignancies after HLA-identical sibling alloSCT with alemtuzumab added to the graft. Patients without GvHD or relapse were randomized at 2.5 months for prophylactic DLI with 1.0x10 6/kg CD4+ T cells (CD4+ DLI) 3 months after alloSCT or conventional care without DLI (CC). Both groups were eligible for prophylactic unmodified DLI at 6 months. Cumulative incidence (CI) of GvHD requiring immunosuppressive treatment 3 months after randomization was the primary endpoint. Changes in the trajectory of chimerism in BM and subsets in PB (CD4+ T cells, CD8+ T cells, NK cells, B cells and monocytes) were compared by means of mixed effects beta regression as secondary endpoints. Results 66 patients were included (table 1). CI of GvHD did not differ between groups (CD4+DLI: 6.1% vs CC: 9.1%, Gray's test p&amp;gt;0.9). Relapse was comparable (CD4+DLI: 15% vs CC: 12%), although heterogeneity of diseases prevented a formal comparison. In the CD4+ DLI group, BM chimerism was full donor (FD, &amp;gt;95% donor) at baseline in 23/33 patients, while 10/33 had mixed chimerism (MC, ≤95%). In the CC group, 23/33 were FD and 10/33 MC. Some patients with MC at baseline converted to FD in both groups (CD4+ DLI: 1/10 vs CC: 1/10). Changes in BM chimerism did not differ between CD4+ DLI and CC (p = 0.9). Chimerism of B cells, NK cells and monocytes in PB resembled bone marrow chimerism with most patients being FD at baseline. Changes in chimerism did not differ between CD4+ DLI and CC (p = 1.0, 0.5 and 0.9 for B cells, NK cells and monocytes respectively). In the CD4+ DLI group, 25/30 evaluable patients were MC in the CD4+ T-cell subset in PB at baseline. 3 month after randomization 7/25 had converted to FD, 17/25 remained MC and 1/25 was unevaluable. Of the 17 patient who remained MC, CD4+ donor chimerism increased (absolute increase &amp;gt;5%) in 13, decreased (&amp;gt;5%) in 2 and was stable in 2. In the CC group, 26/32 were MC in CD4+ T cells at baseline, of which 6 converted to FD, 18 remained MC and 2 were unevaluable. Of the 18 patients remaining MC, CD4+ chimerism increased in 5, decreased in 9 and remained stable in 4. The CD4+ donor chimerism increased significantly after CD4+ DLI compared to CC (p = 0.01) (Figure 1). In the CD4+ DLI group, 23/29 evaluable patients were MC in the CD8 subset at baseline. 7/23 converted to FD, 15/23 remained MC and 1 was unevaluable. Of these 15 MC patients at 3 months, 9 had increasing, 5 stable and 1 decreasing donor chimerism after 3 months. In CC, 22/31 were MC at baseline. 4/22 converted to FD at 3 months and 17/22 remained in MC. Of the 17 MC patients, 6 had increasing, 3 decreasing and 8 stable chimerism. The CD8+ donor chimerism increased significantly in the CD4+ DLI group compared to CC (p = 0.03). Conclusion CD4+ selected DLI at a dose of 1x10 6 CD4+/kg 3 months after HLA identical T-cell depleted alloSCT did not cause GvHD. Chimerism in BM leukocytes, B cells, NK cells or monocytes did not indicate GvL mediated by CD4+ DLI, possibly because most patients were already FD at baseline. CD4+ and CD8+ T-cell donor chimerism increased significantly after CD4+ DLI, suggesting a GVL-effect - earlier demonstrated in vitro for 4 selected patients. (Van Balen et al. Front Immunol, 2018) However, most patients did not convert to FD in the T-cell compartment, suggesting the GvL-effect of CD4+ DLI was limited. We considered this GvL-effect insufficient to prevent relapse and stress the importance of CD8+ donor T cells as main drivers of alloreactivity after DLI following HLA-identical alloSCT.

Dynamic Changes in Pre- and 3 Months Post-Transplant NGS MRD Status May Predict the Allogeneic Stem Cell Transplant Outcome of Patients with AML

Introduction: To deal with the high propensity for relapse and substantial genetic heterogeneity of AML, monitoring measurable residual disease (MRD) has become an effective approach to evaluate the response to chemotherapy and to predict relapse. However, the clinical implications of NGS MRD change after allogeneic hematopoietic cell transplantation (alloHCT) are still under investigation. The aims of this retrospective study are to verify whether alloHCT improved the outcome of AML patients with NGS MRD positive status in pre-transplant and to investigate how pre- and post-transplant NGS MRD status changes affect the outcome of patients with AML. Methods: This study analyzed patients of age more than 18 years who underwent alloHCT between 2018 and 2022 at single center. NGS mutation test was obtained at diagnosis, and every 3 months after stem cell transplant for 2 years with synchronized peripheral blood chimerism tests. Requirement for inclusion in this retrospective cohort study was availability of NGS mutation results at pre-transplant and at 3 months, as well as donor chimerism results after alloHCT. Patients with no mutations by NGS at diagnosis and at pre-transplant were excluded. Patients were classified by the NGS MRD status in pre-transplant and at 3 months post-transplant: Positive MRD to Positive MRD (Group A), Positive MRD to Negative MRD (Group B), Negative MRD to Positive MRD (Group C), and Negative MRD to Negative MRD (Group D). Patient characteristics are in Table 1. The Kaplan-Meier method and log-rank tests were used to estimate the survival and to compare the differences in survival. Statistical analyses were performed with GraphPad Prism version 9.1.0 (Prism Inc), Microsoft Excel (Microsoft Corporation). Results: Median follow-up was 21 months (range, 3-66). One hundred sixty-five patients met inclusion criteria. Among these, 104 (63.0%) were MRD positive, and 61 (37.0%) were MRD negative by NGS at pre-alloHCT. Fifty-one of the MRD positive patients (49%) became MRD negative by NGS at 3 months after alloHCT. The conversion rate to MRD negative from positive by NGS was not different by conditioning regimen (myeloablative (MA) 54.3% versus reduced intensity (RIC) 49.2%, p=0.67), or by AML risk group (intermediate 48.9% versus adverse 44.7%, p=0.84). Four years cumulative incidence of relapse (CIR) of each group was statistically different between Group A and the others (Group A 47.3%, Group B 17.7%, Group C 25.9%, and Group D 10.9%, p&amp;lt;0.001), but not different between Group B, C and D (p&amp;gt;0.5). Median OS was 17 months for Group A, 27 months for Group C, but the other groups did not reach. Four-year OS of each group was statistically different between Group A, Group B, and Group D (Group A 21.1% (95% CI: 2.0 - 53.8), Group B 55.2% (95% CI: 38.7 - 68.9), Group C 50.0% (95% CI: 20.8 - 73.6), and Group D 79.3% (95% CI: 63.7 - 94.9), respectively, p&amp;lt; 0.05). Among 12 patients who were MRD positive by NGS in post-alloHCT, but not in pre-alloHCT (Group C), 3 had clinical relapse, at 4 months, 6 months, and 26 months, respectively. Five patients had NGS mutations different from the initial diagnosis. They did not show clinical relapse and maintained 100% peripheral blood lymphoid (CD3) donor chimerism throughout the observation. Conclusion: This study showed alloHCT could convert 49% of pre-transplant MRD positive patients to MRD negative by NGS. However, there were no differences in conversion rate by intensity of conditioning regimen or by AML risk group. Combining pre- and 3 months post-transplant MRD status by NGS could discriminate prognostic subgroups in post-transplant MRD positive and negative group each and may be useful in designing post-alloHCT therapies.

Two Hit Theory for the Pathogenesis of Type 3 Congenital Amegakaryocytic Thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is an autosomal recessive disorder characterized by thrombocytopenia with an absence of megakaryocytes. It can lead to aplastic anemia or myelodysplastic syndrome (MDS). CAMT is linked to a mutation in the c-MPL gene on chromosome 1, which encodes the thrombopoietin receptor. In 2005, CAMT was categorized into three types based on the severity of c-MPL gene loss. Type 1 is the most severe, leading to early-onset pancytopenia and bone marrow abnormalities. Type 2 shows a temporary increase in platelets during infancy, followed by bone marrow failure at 3-6 years due to partial gene function. Type 3 has normal c-MPL function but produces ineffective megakaryocytes. We report a case of a patient with CAMT-like symptoms, without a c-MPL gene mutation, suggesting the presence of an alternative genetic defect. Methods Multi-institutional collaboration and review of medical records in addition to PubMed search for thrombocytopenia, bleeding complications, macrocytosis, trisomy 6, GATA2 variant, inherited bone marrow failure. Case Description A 12-year-old male with severe thrombocytopenia initially observed at 2 years of age. Bleeding complications included scalp hematoma, excessive bleeding during adenoidectomy, widespread petechiae, purpura, epistaxis, and gum bleeding. During this time platelet count ranged between 30-80,000 x10 3/uL. At 10 years of age, bleeding worsened with platelet counts of 6-20,000 x10 3/uL. Mean platelet volume (MPV) remained within the normal range. Simultaneously there was a decline in hemoglobin and the emergence of macrocytosis. Mother had moderate thrombocytopenia during pregnancy, older sibling with similar thrombocytopenia but asymptomatic. Clinical presentation made immune thrombocytopenia, autoimmune disorders, and immune dysregulation-induced platelet destruction unlikely. Inherited thrombocytopenia panel testing for 42 germline variants was negative. Bone marrow biopsy showed 60% cellularity and was remarkable for a decrease in megakaryocytes. Cytogenetic analysis revealed trisomy 6 in 7/20 metaphases. Molecular hematopathology identified a GATA2 variant in 50% of the alleles. Inherited BMF panel revealed heterozygosity for multiple variants of unknown significance (VUS), including c.5096A&amp;gt;G (p.Asp1699Gly) in BRCA2 gene, c.3898A&amp;gt;G (p.Ile300Val) in LYST gene, 1408A&amp;gt;G (p.Thr470Ala) in PALB2 gene, and c.7240G&amp;gt;A (p.Val2414Met) in tVPS13B gene. Another BMF panel revealed two additional heterozygous VUS - NBEAL2 and ADAMTS13. Testing for Fanconi anemia and dyskeratosis congenita were negative. Whole exome sequencing revealed heterozygosity for a 1.57 Mb duplication on chromosome 3q29. Additional molecular pathology panel testing for 468 genes, germline genetic testing for lysosomal disorders and mucopolysaccharidoses, were also negative. Repeat bone marrow testing demonstrated a progressive decrease in cellularity to 5-30% with trilineage hypoplasia. With extensive yet inconclusive diagnostic workup, worsening clinical presentation, and progressive hypocellularity of the bone marrow, he underwent an ABO incompatible stem cell transplantation (9:10, A HR) from a mismatched unrelated donor. The conditioning regimen was myeloablative with busulfan, cyclophosphamide, anti-thymocyte globulin, and post-transplant cyclophosphamide. Currently, he is 150 days post-transplant with peripheral blood chimerism demonstrating 97.7% donor on day 98. Platelet counts have normalized, and he has not required a transfusion for nearly 100 days. Discussion This case report highlights the concept of a “double hit” hypothesis. While homozygous mutations of PALB2 and BRCA2 are individually associated with Fanconi anemia and BMF syndromes, no hematological pathology has been documented in their heterozygous forms, raising the question of whether the co-expression of these variants in a heterozygous state could result in a similar disease phenotype as their homozygous counterparts. This finding prompts further exploration into the potential impact of combined variants on disease manifestation.

The DPP4 Dependent Transcriptome Identifies Pathways to Improve Mouse Hematopoietic Cell Function

Hematopoietic cell transplantation (HCT) is a curative treatment for hematologic disorders wherein healthy donor hematopoietic stem (HSCs) and progenitor cells (HPCs) collected from bone marrow (BM), mobilized peripheral blood (PB) or umbilical cord blood (CB) reconstitute an ablated hematopoietic system. There is a strong need to enhance functional competency of HSCs/HPCs for HCT to prevent complications like graft versus host disease (GVHD), relapse, and graft failure. Dipeptidyl peptidase 4 (DPP4) is a protein that cleaves N-terminal dipeptides from hematopoietic factors, impacting their function. Inhibition of DPP4 improves HSC/HPC competency in mouse models of HCT and clinically speeds neutrophil recovery after CB transplantation and reduces incidences of GVHD after PB transplantation. Thus, DPP4 inhibition is a viable component of a multi-pronged strategy to improve engraftment and prevent GVHD. While DPP4 inhibition improved early neutrophil recovery in clinical trials, the effects were modest, required high dose treatment, and effects on platelet recovery were unclear. Examining mechanisms by which DPP4 inhibition drives HSC/HPC functional competency may yield insight into targets that can enhance DPP4 driven improvements to HCT. We harvested BM from mice treated with the DPP4 inhibitor Diprotin A (DPA) and sorted immunophenotypically defined HSCs, multipotent progenitors (MPPs), common myeloid progenitors (CMPs), and granulocyte-macrophage progenitors (GMPs) for transcriptomic analysis. Linear modeling to examine differential gene expression controlling for cell type revealed that DPP4 inhibition gave strong enrichment of gene programs associated with mitochondrial function. Using a mitochondrial stress test and the Seahorse metabolic flux analyzer, we found that lineage depleted mouse BM cells treated ex vivo with DPA had lower basal oxygen consumption rates and spare respiratory capacity. These seemingly paradoxical data are consistent with reports that highly functional HSCs have higher mitochondrial mass but exhibit less mitochondrial activity. Thus, DPP4 inhibition may preserve high levels of functional competency by reducing cellular dependence on oxidative phosphorylation. To examine DPP4 inhibition effects on individual subpopulations of HSCs/HPCs, we modeled each cell type separately for differential gene expression. We found that gene programs associated with neutrophil development were significantly enriched in HSCs and MPPs from mice treated with DPA, including genes Elane, Lcn2, Prtn3 and Ctsg/h, concomitant with downregulation of platelet development programs in HSCs and GMPs from mice treated with DPA, including genes Gp6/9, P2ry12 and Clec1b. This suggests that DPP4 drives improved engraftment in part by priming HSCs/HPCs for neutrophil recovery but does not enhance platelet development. We then sought to target a platelet activating pathway in combination with DPP4 inhibition to improve HCT. Recipient mice were treated on days -2, -1, and 0 with 75 mg/kg DPA and day 0 with 50 µg/kg and days 1, 3, 5, and 7 with 25 µg/kg recombinant human Thrombopoietin (TPO), a platelet activating agonist. On day -1 mice were lethally irradiated and transplanted via tail vein with 2.5x10 5 whole BM donor cells on day 0. Complete blood counts 2 weeks following transplantation showed that mice treated with DPA alone exhibited worse platelet recovery, in line with our transcriptomic data. However, mice treated with the combination of DPA and TPO exhibited significantly better platelet recovery than vehicle controls and DPA alone. Further, neutrophil recovery trended towards improved recovery in the combination treatment compared to the single agent treatments. Flow cytometry of the PB revealed no differences in BM homing or in donor PB chimerism one month following HCT in the combination treatment compared to single agents (though all treatments showed improved homing over vehicle controls), suggesting the primary effects were on early recovery and not on initial or long-term engraftment. These data elucidate the DPP4 dependent transcriptome in the hematopoietic compartment, providing molecular insights into genes that drive enhanced functional competency of HSCs/HPCs. We also demonstrate that the DPP4 dependent transcriptome can reveal gene programs that can be targeted in combination to provide additive improvement on transplantation recovery.

Haploidentical Hematopoietic Cell Transplant with Post-Transplant Cyclophosphamide Expedites Donor-Derived Immune Reconstitution in a Pediatric Severe Aplastic Anemia Cohort

Severe aplastic anemia (SAA) is a life-threatening, idiopathic process involving the immune-mediated destruction of hematopoietic stem cells. Treatment involves immunosuppressive therapy (IST) and/or allogeneic hematopoietic cell transplantation (HCT). The latter has recently been optimized with use of a post-transplant cyclophosphamide (PTCy) based graft-versus-host disease (GvHD) platform, permitting successful HCT with only half-HLA-matched haploidentical donors. In pediatrics, this platform results in &amp;gt;90% overall survival with low rates of GvHD and graft failure. However, reports on donor chimerism and immune reconstitution are limited. We hypothesized that a PTCy platform would expedite CD3 donor engraftment and CD4 T cell recovery. In a phase III clinical trial, we treated three cohorts of pediatric (age &amp;lt;/=25 years) patients with SAA requiring alloHCT: MUD, MSD, and HAPLO. The MUD cohort included n=15 recipients of 7-8/8 HLA-matched unrelated donor grafts (n=5 7/8, n=10 8/8) with median age 14 years (range 2-17 years), following cyclophosphamide (Cy) 50 mg/kg, fludarabine (Flu) 200 mg/m 2, rabbit anti-thymocyte globulin (rATG) 9 mg/kg total dose rabbit and low dose total body irradiation (TBI) at 200 cGy. The MSD cohort included n=9 recipients of 8/8 HLA-matched sibling donor grafts with median age 15 years (range 12-24 years), following Cy 200 mg/kg and 9 mg/kg rATG. The HAPLO cohort included n=4 recipients of 4/8 HLA-matched related donor grafts with median age 8.5 years (range 2-21 years) following Cy 29 mg/kg, Flu 150 mg/m 2, 9 mg/kg rATG, and low dose TBI at 200 cGy. GvHD prophylaxis consisted of 30 days of mycophenolate mofetil, and 100 days of a calcineurin inhibitor (followed by an 8-10 week taper), with the HAPLO cohort additionally receiving post-transplant Cy (PTCy) 50 mg/kg/day on days +3 and +4. Peripheral blood lymphocyte counts and peripheral blood and bone marrow donor chimerism were evaluated on days +100, +180 and +365. Recipient characteristics were largely equivalent between cohorts, with 43% receiving IST prior to HCT. Clonality was overrepresented in the HAPLO cohort (2 of 4, 1 with monosomy 7 myelodysplastic syndrome and 1 with a large PNH clone), with only 2 of 15 in the MUD and none in the MSD cohorts. Active controlled infections at the time of HCT were additionally overrepresented in the HAPLO cohort, with disseminated pseudomonas in 1. The MUD cohort had 1 with Rhizopus invasive sinusitis (none in the MSD cohort). Achievement of neutrophil engraftment was statistically significantly delayed in the HAPLO cohort at a median of 17 days compared to 12 for MSD and 14 for MUD, p=0.004), however there were no episodes of primary or secondary graft failure. With only a single late death from intracranial hemorrhage in the MUD cohort, OS was equivalent at 2 years (100% in MSD and HAPLO cohorts, 93.3% in MUD). GvHD rates were low with only 1 (MUD) experiencing grade II acute GvHD, and 1 (MUD) experiencing mild chronic GvHD. Immune reconstitution varied by cohort with statistically significantly greater CD3 peripheral blood donor chimerism (p&amp;lt;0.001) as well as CD4 (p=0.04) and CD8 (p=0.03) T cell counts over time for the HAPLO recipients (CD3 chimerism in Fig 1, CD4 count in Fig 2; 2-way ANOVA fixed effects model). Considering a CD4 &amp;gt;200 cell/uL as clinically meaningful, we assessed the proportion of each cohort achieving this milestone at each timepoint. While each cohort had an increase in proportion achieving CD4&amp;gt;200 over time, the HAPLO cohort reached 50% with CD4 &amp;gt;200 at day +100, compared to 11% MUD and 0% MSD (p=0.09). At day +180, the proportion with CD4 &amp;gt;200 were 36% MUD, 25% MSD and 75% HAPLO (1 yr: 90% MUD, 100% MSD and HAPLO). Donor availability remains a challenge to successful transplant for patients with SAA, and haploidentical matches overcome donor limited accessibility. PTCy as GvHD prophylaxis is associated with low incidences of graft failure and severe GvHD. While PTCy might be anticipated to inhibit donor T cells and immune reconstitution, our data, albeit limited by small cohorts, demonstrates superior and more efficient CD3 donor engraftment and CD4 T cell count recovery following HAPLO HCT with PTCy compared to non-PTCy based MUD and MSD cohorts.

Cohesin Subunit RAD21 Regulates the Differentiation and Self-Renewal of Hematopoietic Stem and Progenitor Cells.

Recent studies suggest that chromosomal cohesin complex proteins are important in regulating hematopoiesis and may contribute to myeloid malignancies. To investigate the effects of perturbing the cohesin subunit protein RAD21 on normal hematopoiesis, we used conditional knockout (cKO) mouse models. While cohesin is vital for hematopoietic stem cell (HSC) function, Rad21 haploinsufficiency (Rad21Δ/+) led to distinct hematopoietic phenotypes. Our findings revealed that Rad21Δ/+ cells exhibited decreased hematopoietic reconstitution in competitive bone marrow transplantation assays. This reduction in peripheral blood chimerism was specifically observed in the lymphoid compartment, while the chimerism in the myeloid compartment remained unaffected. Rad21 haploinsufficiency also resulted in changes in the hematopoietic stem and progenitor cells (HSPC) and myeloid progenitor compartments, with a significant accumulation of granulocyte-macrophage progenitors in the bone marrow. We observed differential gene expression in Rad21Δ/+ LSK (Lin- Sca1-Kit+) cells, including genes required for HSPC function and differentiation, such as Setdb1, Hmga2, Ncor1, and Myb. In addition, we observed a notable decrease in the expression of genes related to the interferon response and a significant reduction in the expression of genes involved in the IL2-STAT5 signaling pathways. Our studies suggest that RAD21 protein and level of its post-translational modifications in the bone marrow cells may play a potential role in hematopoiesis. Overall, Rad21 haploinsufficiency impairs hematopoietic differentiation and increases HSC self-renewal.

Increased heterozygosity in low-pass sequencing data allows identification of blood chimeras in cattle.

In about 90% of multiple pregnancies in cattle, shared blood circulation between fetuses leads to genetic chimerism in peripheral blood and can reduce reproductive performance in heterosexual co-twins. However, the early detection of heterosexual chimeras requires specialized tests. Here, we used low-pass sequencing data with a median coverage of 0.64× generated from blood samples of 322 F1 crosses between beef and dairy cattle and identified 20 putative blood chimeras through increased levels of genome-wide heterozygosity. In contrast, for 77 samples with routine SNP microarray data generated from hair bulbs of the same F1s, we found no evidence of chimerism, simultaneously observing high levels of genotype discordance with sequencing data. Fifteen out of 18 reported twins showed signs of blood chimerism, in line with previous reports, whereas the presence of five alleged singletons with strong signs of chimerism suggests that the in-utero death rate of co-twins is at the upper limit of former estimates. Together, our results show that low-pass sequencing data allow reliable screening for blood chimeras. They further affirm that blood is not recommended as a source of DNA for the detection of germline variants.

HIV-1 remission and possible cure in a woman after haplo-cord blood transplant

Previously, two men were cured of HIV-1 through CCR5Δ32 homozygous (CCR5Δ32/Δ32) allogeneic adult stem cell transplant. We report the first remission and possible HIV-1 cure in a mixed-race woman who received a CCR5Δ32/Δ32 haplo-cord transplant (cord blood cells combined with haploidentical stem cells from an adult) to treat acute myeloid leukemia (AML). Peripheral blood chimerism was 100% CCR5Δ32/Δ32 cord blood by week 14 post-transplant and persisted through 4.8 years of follow-up. Immune reconstitution was associated with (1) loss of detectable replication-competent HIV-1 reservoirs, (2) loss of HIV-1-specific immune responses, (3) invitro resistance to X4 and R5 laboratory variants, including pre-transplant autologous latent reservoir isolates, and (4) 18months of HIV-1 control with aviremia, off antiretroviral therapy, starting at 37months post-transplant. CCR5Δ32/Δ32 haplo-cord transplant achieved remission and a possible HIV-1 cure for a person of diverse ancestry, living with HIV-1, who required a stem cell transplant for acute leukemia.

Non-Clinical Cell Therapy Development Using the NCG Mouse Model as a Test System.

The NCG triple immunodeficient mice on a NOD/Nju background lack functional/mature T, B, and NK cells, and have reduced macrophage and dendritic cell function. This study characterized the NCG mouse model for toxicity, engraftment and tumorigenicity assessments of cell therapies, using CD34+ hHSPC adult mobilized cells with two myeloablation regimens.Mice received sub-lethal irradiation or busulfan and were then injected intravenously with CD34+ hHSPCs (1.0 x 106 cells/mouse) or PBS (control), while positive control animals received 2 x 106 HL-60 cells/mouse. hCD34+ cell donors were treated with the mobilizing agent G-CSF prior to leukapheresis. Following injections, mouse blood samples were collected to assess engraftment rates by flow cytometry with body weights recorded periodically up to 20 weeks post-cell injection. No significant clinical signs or body weight changes were observed. At week 10 post-cell injection, the peripheral blood chimerism of hCD45+ cells was above 20%. While mCD45+ concentration was constant between week 10 and 17 in whole blood samples, hCD45+ concentration and chimerism slightly decreased at week 17. However, chimerism remained above 10%, with busulfan-treated mice presenting higher values. Chimerism was further assessed by quantifying human Alu sequences in blood and multiple organs using qPCR. Alu sequences were most abundant in the spleen and bone marrow, while lowest in the testes. In the positive control group, expected mortalities due to tumorigenesis were observed between days 27 and 40 post-cell injection. Overall, study results may be used to inform study design and potential toxicological endpoints relevant to non-clinical cell therapy development.

Combination of 5-Azacytidine and Pegifna Is Able to Overcome Resistance in JAK2-V617F Positive MPN with Loss of Dnmt3a

Pegylated interferon alpha (IF) can induce molecular remissions in subset of JAK2-V617F-positive MPN patients by targeting long-term hematopoietic stem cells (LT-HSCs). Patients with additional somatic mutations in genes involved in LT-HSC self-renewal have been reported to have poorer responses to IF. We found that loss of Dnmt3a increases competitiveness and self-renewal of JAK2-V617F-positive LT-HSCs and confers resistance to IF treatment in MPN mouse models and in HSCs and progenitor cells from MPN patients. Here we examined whether the resistance of double mutant JAK2-V617F;Dnmt3aΔ/Δ hematopoietic cells to IF can be overcome by addition of arsenic trioxide (At), or 5-azacytidine (Az). To test the effects of the combination treatments in vivo, we generated cohorts of mice expressing JAK2-V617F (VF) alone, or in combination with a homozygous deletion of the Dnmt3a gene (Dnmt3aΔ/Δ). We used bone marrow cells from VF or VF;Dnmt3aΔ/Δ mice that express a GFP reporter gene mixed with a 10-fold excess of bone marrow cells from a wildtype (WT) mouse to perform competitive transplantations into lethally irradiated WT recipient mice. After 7 weeks, recipient mice were randomized into 6 treatment groups including a vehicle control group. These mice were treated for 12 weeks with IF (25µg/kg; s.c. once per week), At (5mg/kg; i.p. every second day), or Az (2mg/kg; i.p. daily for two weeks followed by a break of 2 weeks), or combinations of IF+At and IF+Az (Figure 1). Peripheral blood parameters were normalized by At and Az treatment arms in both single-mutant and double-mutant mice (Figure 1A). Spleen and liver weight was decreased in all treatment groups and genotypes, except in VF;Dnmt3aΔ/Δ mice treated with IF alone, which showed a trend towards increased spleen and liver weights. Expression of GFP allowed us to follow the contribution of cells derived from the VF or VF;Dnmt3aΔ/Δ donor mice, respectively. In VF recipient mice, a pronounced decrease in GFP chimerism of peripheral blood lineages was observed in the groups treated with a combination of IF+At, or IF+Az (Figure 1A). LT-HSC from bone marrow and spleen showed reduction in GFP chimerism upon treatment with Az alone, compared to vehicle and further reduction below 10% was observed in combination of Az and IF (Figure 1B). Double-mutant VF;Dnmt3aΔ/Δ recipient mice remained resistant to the single agent regiments, but showed a significant reduction of GFP chimerism in peripheral blood and in LT-HSCs when treated with a combination of IF+Az and to a lesser degree also when treated with IF+At (Figure 1B). Thus, a combination of pegIFNa with 5-azacytidine is a promising approach to target MPN cells carrying mutations in JAK2 and Dnmt3a genes that could be also considered as a treatment option for therapy-resistant forms of MPN in patients carrying JAK2-V617F and loss-of-function DNMT3A mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recovery of Highly Functional Human Hematopoietic Stem and Progenitor Cells from 27-Year Old Cryopreserved Umbilical Cord Blood

Umbilical cord blood (CB) is a promising source of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) for hematopoietic cell transplantation (HCT). CB is cryopreserved following collection, allowing for the amassment of nearly 800,000 CB units in public banks and an estimated 4 million in private banks. Cryopreservation of CB is essential for successful CB transplantation, and it is critical to understand how long CB can be stored with efficient recovery of engraftable HSCs. Prior work demonstrated efficient recovery of engraftable HSCs from 10-, 15-, and 21-year old cryopreserved CB. Here, we demonstrate efficient recovery of highly engraftable HSCs from 27-year old cryopreserved CB units. 3 CB units cryopreserved for >27 years were thawed with clinical protocols, and total nucleated cells were recovered with 76-87% viability. 3-6 fresh CB units were used as a comparison for cryopreserved units. Immunophenotyping assays to determine numbers of HSCs/HPCs showed that cryopreserved CB units had on average 4.25x103 CD34+ cells per 1x106 nucleated low density CB cells (LDCB), similar to fresh CB with 4.55x103CD34+ per 1x106 LDCB. Within the CD34+ population, cryopreserved CB when compared to fresh CB had on average equal or greater numbers of stringently immunophenotypically defined HSCs (9.1x103 vs 6.2x103), multipotent progenitors (MPPs) (2.1x105 vs 8.6x104), and multilymphoid progenitors (MLPs) (1.9x104 vs 1.0x104) per 1x106 CD34+ cells. Conversely, there were fewer numbers of common myeloid progenitors (CMPs) (2.9x104 vs 1.1x105), megakaryocyte-erythroid progenitors (MEPs) (4.5x104 vs 1.0x105), and granulocyte macrophage progenitors (GMPs) (4.8x104 vs 1.4x105). Colony forming unit (CFU) assays demonstrated that cryopreserved CB units exhibited lower functional HPC numbers per 1x106 CD34+ enriched cells including CFU-GM (1.9x104 vs 8.3x104), BFU-E (1.2x104 vs 3.4x104), and CFU-GEMM (1.1x104 vs 5.8x104). In ex vivo analysis of HSC/HPC proliferative capacity, cryopreserved CB units showed similar or greater capacity for expansion in growth stimulating media compared to fresh CB, including HSCs (7.7-fold vs 6.2-fold increase), MPPs (3.5-fold vs 4-fold increase), MLPs (44-fold vs 1.7-fold increase), CFU-GM (29-fold vs 13.3-fold increase), and CFU-GEMM (28.6-fold vs 4-fold increase). For in vivo functional HSC analysis, 10,000 CD34+ enriched cells were transplanted to sublethally irradiated NSG mice. Cryopreserved CB units exhibited early and late human engraftment in peripheral blood (PB) at months 1, 2, 4, and 5. Bone marrow (BM) human chimerism at month 5 ranged from low (0.5%) to robust levels of engraftment (52.7%). Limiting dilution analysis revealed that 2 cryopreserved CB units had similar SCID repopulating cell (SRC) frequency, ranging from 314 to 1392 SRCs per 1x106 CD34+ cells while the third had a much lower 65 to 393 SRCs per 1x106 CD34+ cells. The third CB unit also exhibited lower average human chimerism in the BM (0.55%) than the other 2 units (15% and 16.2%). However, the SRC frequencies in all 3 CB units are similar to those in fresh CB units published by our group and others (range 51 to 929 SRCs per 1x106 CD34+ cells). Secondary transplants showed that all 3 CB units have secondary repopulation capacity demonstrated by human chimerism in PB. These data suggest functional HSCs with short- and long-term engrafting and sustained self-renewal capacity can be cryopreserved for at least 27 years. We show that different cryopreserved CB units contain variable functional HSC frequency. Similar results in ex vivo characterization suggest cryopreservation is not the cause of these functional differences, but rather there were functional differences in the CB units prior to cryopreservation. This underscores the need to have reliable measures of CB functionality prior to cryopreservation for more efficient selection of donor units and to test potential effects of cryopreservation on individual units. Transcriptomic studies are ongoing to determine molecular programs that may contribute to differences in SRC frequency. These data will provide insight into mechanistic explanations for differences in HSC function in different CB units. Authors’ note: This project was conceived by the authors prior to the death of Hal E. Broxmeyer. Dr. Broxmeyer initiated the study as a continuation of his life's work demonstrating the utility of CB for HCT. This work is dedicated to Hal.

Downregulation of UBA1 in Myelodysplastic Syndrome

Somatic loss-of-function mutations of major E1 ubiquitin activating enzyme gene UBA1 were identified in VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) and myelodysplastic syndromes (MDS) recently, suggesting that impairment of UBA1 activity contributes to pathogenesis of these diseases that have systemic inflammatory disorders. We sought to determine whether abnormal expression of UBA1 also occurs in MDS and contributes to development of disease. We analyzed RNA sequencing results obtained from the CD34+ bone marrow hematopoietic stem and progenitor cells (BM HSPC) in a cohort containing treatment naive MDS (N=55), chronic myelomonocytic leukemia (CMML, N=22), and heathy donors (N=12). Since UBA1 is an X-chromosome linked gene that escapes X-chromosomal silencing in females, all donors in this cohort were males. Results indicated that the level of UBA1 RNA was significantly downregulated in MDS but not in CMML. In the subtypes of MDS-low blasts (N=23, including MDS with ring sideroblasts, multilineage dysplasia, and single lineage dysplasia), forty-eight percent (N=11) had an over 1.5-fold downregulation of UBA1 RNA expression. Based on gene set enrichment assay, the UBA1 downregulation in MDS-low blasts was associated with altered autophagy and innate immune signaling such as complement, IL6, and TNF signals. Moreover, we also identified that the UBA1 downregulation in BM CD34+ cells of MDS-low blasts co-occurred with SF3B1 mutations detected by targeted next generation sequencing in BM mononuclear cells: Among the 11 MDS-low blasts patients with downregulated UBA1 expression, six patients (55%) carried mutant SF3B1, whereas none of the 12 patients with normal UBA1 expression had SF3B1 mutation. In another word, all SF3B1 mutant patients with MDS-low blasts (N=6) presented decreased UBA1 RNA. To characterize whether impairment of UBA1 affects hematopoietic functions of BM HSPC, we applied TAK-243, an UBA1 specific inhibitor, to BM CD34+ cells isolated from healthy donors (N=5, all males) and cultured in vitro. Colony formation unit (CFU) assays were performed to evaluate hematopoietic repopulating activity. Results indicated the CFU in CD34+ cells treated by TAK-243 was significantly reduced compared with cells treated by vehicle control (P<0.05). Moreover, when hematopoietic colonies formed by CD34+ cells were collected from methylcellulose plates and characterized by flow cytometry analysis, the frequency of CD71-/ CD235+ erythroid cells in TAK-243 treated plates was significantly lower than that in control plates (P<0.05). These results implicated that impairment of UBA1 activity perturbed hematopoietic functions of BM HSPC, particularly the erythropoietic function. We next evaluated in vivo hematopoietic impact of UBA1 impairment by applying TAK-243 to wildtype male B6 mice. Compared to vehicle control treated mice (N=4), mice treated by TAK-243 (N=4) exhibited significant decreases in hemoglobin and white cell counts (P<0.05) in peripheral blood (PB) and splenomegaly. In BM compartment, TAK-243 treated mice had decreased CD71+ and Ter119+ erythroid populations and increased Gr1+/ CD11b+ myeloid population (P<0.05). TAK-243 treatment also altered BM HSPCs, including reduced megakaryocyte-erythroid progenitor (MEP) and increased granulocyte-monocyte progenitor (GMP) in BM, as well as reduced CD150+/ CD48- stem cells in the BM Lin-/ Sca1+/ cKit+ (LSK) population. Moreover, BM LSK isolated from TAK-243 treated mice (N=4) formed significantly reduced number of colonies compared to vehicle control treated mice (N=4) in CFU assay. Consistently, in competitive transplantation assay, repopulating activity of BM cells of TAK-243 treated mice was significantly impaired, as measured by the donor-derived CD45.2 chimerism in PB of the CD45.1 recipients. Moreover, we performed bulk RNA-Seq in BM LSK of drug treated mice, which revealed that the ribosomal function and cell cycle regulation signals in TAK-243 treated mice were significantly affected. In summary, our study indicated that, in addition to somatic mutations, there was also a significant downregulation of UBA1 expression in BM HSPC of MDS. Compromised UBA1 function impairs hematopoietic function of BM HSPC, which could be an important mechanism for pathogenesis of MDS.

Metformin Treatment Abrogates the Competitive Advantage of Dnmt3a R878H HSPCs By Enhancing DNA Methylation Activity

Clonal hematopoiesis of indeterminate potential (CHIP) refers to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) carrying leukemogenic mutations in individuals without evidence of a blood disorder. CHIP carriers are at an increased risk of blood cancers and cardiovascular diseases. Targeting the clonal expansion of mutant HSPCs has the potential to mitigate CHIP-related illnesses. The most commonly mutated gene in CHIP is DNMT3A, which encodes an enzyme that catalyzes the methyl transfer from S-adenosylmethionine (SAM) to cytosines in DNA. The heterozygous DNMT3A hotspot mutation at R882 exerts a dominant negative effect on the wild-type (WT) allele, resulting in decreased DNA methylation activity. Prior studies have shown that murine HSPCs carrying mutant Dnmt3a R878H (equivalent to R882 in humans) have an advantage over WT cells in competition assays. Here, we aimed to first identify differences in the functional properties between Dnmt3a-mutated and WT HSPCs. Given the crosstalk between mitochondrial metabolism and DNA methylation, we investigated potential differences in mitochondrial activity and found that the level of mitochondrial respiration was higher in Dnmt3aR878H/+ HSPCs than in WT cells. To determine if this translated to differential dependencies, we silenced the expression of critical subunits in the each of five electron transport chain (ETC) complexes. Knockdown of NDUFV1 in Complex I was the most effective in reducing the competitive advantage of Dnmt3aR878H/+ cells in vitro. To explore translational relevance of this finding, we tested the impact of metformin, a pharmacologic inhibitor of Complex I, on Dnmt3a-mutated HSPCs. Treatment with metformin at clinically relevant concentrations suppressed the competitive advantage of mutant cells in vitro. Importantly, this effect was rescued by expression of NDI1, a metformin-resistant yeast analog of Complex I. To extend these findings, we conducted a competitive repopulation assay between CD45.2+ Dnmt3aR878H/+ and CD45.1+ Dnmt3a+/+ HSPCs. Metformin treatment abrogated the in vivo competitive advantage of mutant cells over an 8-month period based on peripheral blood chimerism. To decipher the mechanism of action, we performed transcriptome and metabolome profiling of mutant HSPCs treated with or without metformin for 1 month. GSEA analysis of the transcriptome data revealed a downregulation of genes associated with stemness in metformin-treated cells. Intriguingly, we found that metformin increased the expression of genes involved in one-carbon metabolism, which generates SAM. Consistent with these findings, metabolomics analysis showed that the levels of several key metabolites in one-carbon metabolism, including SAM, were higher in metformin-treated cells compared with controls. Genetic and pharmacologic inhibition of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism, as well as the addition of exogeneous S-adenosylhomocysteine (SAH), an inhibitor of SAM-dependent methyltransferases, effectively rescued the effect of metformin on mutant cells in vitro. Based on the above findings, we hypothesized that metformin treatment might enhance the residual DNA methylation activity found in Dnmt3aR878H/+ mutated cells by increasing SAM concentration. In support of this hypothesis, we found that exogeneous SAM reproduced the effect of metformin, and HSPCs with homozygous Dnmt3aR878H mutations were insensitive to metformin. Furthermore, we performed DNA methylome profiling of untreated and metformin-treated mutant and WT HSPCs using RRBS. This analysis revealed that the hypomethylated regions (HMRs) in untreated Dnmt3aR878H/+ HSPCs relative to WT controls were enriched for regions of repressed chromatin with H3K27 methylation. Importantly, metformin treatment increased the level of DNA methylation at these regions in mutated HSPCs. In summary, our findings demonstrate that Dnmt3aR878H/+ HSPCs depend on ETC activity and particularly complex I activity to maintain their competitive advantage over WT cells. We propose a model in which inhibition of complex I activity with metformin increases one-carbon metabolism and SAM level, thereby restoring DNA methylation at HMRs and the differentiation of mutant HSPCs. Our findings have important implications for the development of preventive interventions against DNMT3A-mutated CHIP.