Bone Marrow Transplantation Model Research Articles

After Bone Marrow Transplantation, the Cell-Intrinsic Th2 Pathway Promotes Recipient T Lymphocyte Survival and Regulates Graft-versus-Host Disease.

Recipient T cells can aggravate or regulate lethal and devastating graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). In this context, we have shown before that intestinal immune conditioning with helminths is associated with survival of recipient T cells and Th2 pathway-dependent regulation of GVHD. We investigated the mechanism of survival of recipient T cells and their contribution to GVHD pathogenesis in this helminth infection and BMT model after myeloablative preparation with total body irradiation in mice. Our results indicate that the helminth-induced Th2 pathway directly promotes the survival of recipient T cells after total body irradiation. Th2 cells also directly stimulate recipient T cells to produce TGF-β, which is required to regulate donor T cell-mediated immune attack of GVHD and can thereby contribute to recipient T cell survival after BMT. Moreover, we show that recipient T cells, conditioned to produce Th2 cytokines and TGF-β after helminth infection, are fundamentally necessary for GVHD regulation. Taken together, reprogrammed or immune-conditioned recipient T cells after helminth infection are crucial elements of Th2- and TGF-β-dependent regulation of GVHD after BMT, and their survival is dependent on cell-intrinsic Th2 signaling.

Abstract 5136: Surgical stress promotes long-lasting protumorigenic changes in bone marrow derived progenitor cells

Abstract Introduction: Surgery is a crucial intervention for cancer patients. However, the perioperative period is characterized by an increased risk for accelerated growth of metastatic disease. Our recent work shows that the protumorigenic systemic inflammatory changes and the formation of neutrophil extracellular traps (NETs) persist months after surgery in both mice and humans, and parallel micrometastatic tumor growth and recurrence. Here we hypothesized that surgery may induce long-term sustained epigenetic and transcriptomic changes in bone marrow derived granulocytes-monocytes progenitor cells (GMPs) leading to functional reprogramming of mature neutrophils and persistent systemic release of these protumorigenic NET-forming neutrophils. Methods: Eight-week-old mice were subjected to non-lethal surgical stress (laparotomy) for 30 mins. Seven days after, mice were inoculated with 1x106 MC38 (murine colorectal cancer cells) subcutaneously. For bone marrow (BM) transplant model, a group of B6 congenic (CD45.1) mice were subjected to laparotomy. BM derived cells were harvested 7 days after the procedure and orthotopically transplanted into B6 CD45.2 irradiated (1000 rads) mice. After the establishment of hematopoiesis (6 weeks), MC38 cancer cells were implanted into the recipient mice. Results: Mice that underwent laparotomy had significantly increased tumor volume at 3 weeks compared to control mice, that only underwent anesthesia (Laparotomy vs control **p<0.01). While the intratumoral flowcytometry analysis showed no difference in the frequency of infiltrating immune cells, quantitative (q) PCR analysis revealed significant plasticity in the tumor infiltrating neutrophils (TANs) observed by an increase in the expression of genes (MCP1, Arg1) associated with pro-inflammatory (N2) type neutrophils compared to control. A persistent increased level of NETs was also observed within the tumors and circulation of mice several weeks after laparotomy. ATAC-seq and Bulk-RNA-seq analysis further revealed major epigenetic and transcriptomic changes in TANs, circulating neutrophils and GMPs. To substantiate long-lasting surgical effects, tumor burden was assessed in the BM recipient mice and showed to be significantly increased in mice that had received BM cells from laparotomy mice versus BM cells from sham group. Conclusion: Surgery promotes long-term rewiring of the bone marrow derived cells resulting in increased tumor growth. Understanding the underlying molecular mechanism may help with therapeutic interventions to prevent protumorigenic surgical effects and improve patient outcomes. Citation Format: Hamza O. Yazdani, Tony Haykal, Ruiqi Yang, Celine Tohme, David A. Geller, Samer Tohme. Surgical stress promotes long-lasting protumorigenic changes in bone marrow derived progenitor cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5136.

Effect of lethal total body irradiation on bone marrow chimerism, acute graft-versus-host disease, and tumor engraftment in mouse models: impact of different radiation techniques using low- and high-energy X-rays.

Effects of X‑ray energy levels used for myeloablative lethal total body irradiation (TBI) delivery prior to bone marrow transplantation (BMT) in preclinical mouse models were examined. In mouse models, single-fraction myeloablative TBI at alethal dose was delivered using two different X‑ray devices, either low (160 kV cabinet irradiator) or high energy (6MV linear accelerator), before semi-allogeneic hematopoietic stem-cell transplantation (HSCT) to ensure bone marrow (BM) chimerism, graft-versus-host disease (GVHD), and tumor engraftment. Recipient mice were clinically followed for 80days after bone marrow transplantation (BMT). Flow cytometry was performed to assess donor chimerism and tumor engraftment in recipient mice. Both X‑ray irradiation techniques delivered a10 Gy single fraction of TBI, presented alethal effect, and could allow near-complete early donor chimerism on day13. However, low-energy irradiation increased Tcells' alloreactivity compared to high-energy irradiation, leading to clinical consequences for GVHD and tumor engraftment outcomes. The alloreactive effect differences might be attributed to the distinction in inflammatory status of irradiated recipients at donor cell infusion (D0). Delaying donor cell administration (D1 after lethal TBI) attenuated Tcells' alloreactivity and clinical outcomes in GVHD mouse models. Different X‑ray irradiation modalities condition Tcell alloreactivity in experimental semi-allogeneic BMT. Low-energy X‑ray irradiator induces apost-TBI inflammatory burst and exacerbates alloreactive reactions. This technical and biological information should be considered in interpreting GVHD/ graft-versus-leukemia effect results in mice experimental models of BMT.

Single cell and lineage tracing studies reveal the impact of CD34+ cells on myocardial fibrosis during heart failure

BackgroundCD34+ cells have been used to treat the patients with heart failure, but the outcome is variable. It is of great significance to scrutinize the fate and the mechanism of CD34+ cell differentiation in vivo during heart failure and explore its intervention strategy.MethodsWe performed single-cell RNA sequencing (scRNA-seq) of the total non-cardiomyocytes and enriched Cd34-tdTomato+ lineage cells in the murine (male Cd34-CreERT2; Rosa26-tdTomato mice) pressure overload model (transverse aortic constriction, TAC), and total non-cardiomyocytes from human adult hearts. Then, in order to determine the origin of CD34+ cell that plays a role in myocardial fibrosis, bone marrow transplantation model was performed. Furthermore, to further clarify the role of CD34 + cells in myocardial remodeling in response to TAC injury, we generated Cd34-CreERT2; Rosa26-eGFP-DTA (Cre/DTA) mice.ResultsBy analyzing the transcriptomes of 59,505 single cells from the mouse heart and 22,537 single cells from the human heart, we illustrated the dynamics of cell landscape during the progression of heart hypertrophy, including CD34+ cells, fibroblasts, endothelial and immune cells. By combining genetic lineage tracing and bone marrow transplantation models, we demonstrated that non-bone-marrow-derived CD34+ cells give rise to fibroblasts and endothelial cells, while bone-marrow-derived CD34+ cell turned into immune cells only in response to pressure overload. Interestingly, partial depletion of CD34+ cells alleviated the severity of myocardial fibrosis with a significant improvement of cardiac function in Cd34-CreERT2; Rosa26-eGFP-DTA model. Similar changes of non-cardiomyocyte composition and cellular heterogeneity of heart failure were also observed in human patient with heart failure. Furthermore, immunostaining showed a double labeling of CD34 and fibroblast markers in human heart tissue. Mechanistically, our single-cell pseudotime analysis of scRNA-seq data and in vitro cell culture study revealed that Wnt-β-catenin and TGFβ1/Smad pathways are critical in regulating CD34+ cell differentiation toward fibroblasts.ConclusionsOur study provides a cellular landscape of CD34+ cell-derived cells in the hypertrophy heart of human and animal models, indicating that non-bone-marrow-derived CD34+ cells differentiating into fibroblasts largely account for cardiac fibrosis. These findings may provide novel insights for the pathogenesis of cardiac fibrosis and have further potential therapeutic implications for the heart failure.

Improved Antitumor Effect of NK Cells Activated by Neutrophils in a Bone Marrow Transplant Model.

The licensing process mediated by inhibitory receptors of the Ly49 C-type lectin superfamily that recognizes self-major histocompatibility complex (MHC) class I in mice is essential for the proper antitumor function of natural killer (NK) cells. Several models for NK cell licensing can be exploited for adoptive immunotherapy for cancer. However, the appropriate adoptive transfer setting to induce efficient graft versus tumor/leukemia effects remains elusive, especially after hematopoietic stem cell transplantation (HSCT). In our previous experiment, we showed that intraperitoneal neutrophil administration with their corresponding NK receptor ligand-activated NK cells using congenic mice without HSCT. In this experiment, we demonstrate enhanced antitumor effects of licensed NK cells induced by weekly intraperitoneal injections of irradiated neutrophil-enriched peripheral blood mononuclear cells (PBMNCs) in recipient mice bearing lymphoma. Bone marrow transplantation was performed using BALB/c mice (H-2d) as the recipient and B10 mice (H-2b) as the donor. The tumor was A20, a BALB/c-derived lymphoma cell line, which was injected subcutaneously into the recipient at the same time as the HSCT. Acute graft versus host disease was not exacerbated in this murine MHC class I mismatched HSCT setting. The intraperitoneal injection of PBMNCs activated a transient licensing of NK subsets expressed Ly49G2, its corresponding NK receptor ligand to H-2d, and reduced A20 tumor growth in the recipient after HSCT. Pathological examination revealed that increased donor-oriented NK1.1+NK cells migrated into the recipient tumors, depending on neutrophil counts in the administered PBMNCs. Collectively, our data reveal a pivotal role of neutrophils in promoting NK cell effector functions and adoptive immunotherapy for cancer.

Bone-marrow derived cells do not contribute to new beta-cells in the inflamed pancreas.

The contribution of bone-marrow derived cells (BMCs) to a newly formed beta-cell population in adults is controversial. Previous studies have only used models of bone marrow transplantation from sex-mismatched donors (or other models of genetic labeling) into recipient animals that had undergone irradiation. This approach suffers from the significant shortcoming of the off-target effects of irradiation. Partial pancreatic duct ligation (PDL) is a mouse model of acute pancreatitis with a modest increase in beta-cell number. However, the possibility that recruited BMCs in the inflamed pancreas may convert into beta-cells has not been examined. Here, we used an irradiation-free model to track the fate of the BMCs from the donor mice. A ROSA-mTmG red fluorescent mouse was surgically joined to an INS1Cre knock-in mouse by parabiosis to establish a mixed circulation. PDL was then performed in the INS1Cre mice 2 weeks after parabiosis, which was one week after establishment of the stable blood chimera. The contribution of red cells from ROSA-mTmG mice to beta-cells in INS1Cre mouse was evaluated based on red fluorescence, while cell fusion was evaluated by the presence of green fluorescence in beta-cells. We did not detect any red or green insulin+ cells in the INS1Cre mice, suggesting that there was no contribution of BMCs to the newly formed beta-cells, either by direct differentiation, or by cell fusion. Thus, the contribution of BMCs to beta-cells in the inflamed pancreas should be minimal, if any.

Peritransplantation Glucocorticoid Haploidentical Stem Cell Transplantation Is a Promising Strategy for AML Patients With High Leukemic Burden: Comparison With Transplantations Using Other Donor Types

Using a murine haploidentical bone marrow transplantation (BMT) model, we recently showed that peritransplantation administration of glucocorticoid (GC) redistributed donor T cells from the gastrointestinal tract to bone marrow, which resulted in a significant reduction of graft-versus-host disease (GVHD) while promoting graft-versus-leukemia effects. Furthermore, in a retrospective clinical study of patients with acute myelogenous leukemia (AML) undergoing transplantation in non-remission, we also showed that haploidentical stem cell transplantation (haplo-SCT) using peritransplantation GC administration led to a significantly lower relapse rate and better overall survival rate compared with haplo-SCT using post-transplantation cyclophosphamide. In the present study, using the same dataset of patients undergoing GC haplo-SCT, we retrospectively compared with patients with AML undergoing transplantation in non-remission using 3 other donor types: matched sibling donor (MSD), matched unrelated donor (MUD), and umbilical cord blood (UCB). For GC haplo-SCT, 44 patients underwent peripheral blood stem cell transplantation in a single center (Hyogo College of Medicine), with the conditioning treatment consisting of fludarabine, melphalan, anti-thymocyte globulin (2.5 mg/kg), and TBI 3 Gy. Methylprednisolone was given from the start of conditioning treatment, and the GVHD prophylaxis consisted of tacrolimus and methylprednisolone (1 mg/kg). The transplantation outcomes were compared with data of 1889 patients undergoing MSD-SCT (n = 449), MUD-BMT (n = 493), or UCB transplantation (UCBT) (n = 947) in non-CR, which were extracted from the Transplant Registry Unified Management Program data, the largest data registry in Japan. For donor engraftment, significantly faster neutrophil and platelet engraftment was achieved with GC haplo-SCT compared with allo-SCT using the 3 other donor types. Neutrophil engraftment was achieved at a median of 10 days for GC haplo-SCT, and 20 days for MSD-, MUD-, and UCB-transplants. Platelet engraftment was achieved at a median of 19.5 days for GC haplo-SCT, 42 days for MSD-SCT and MUD-BMT, and 43 days for UCBT, respectively. The incidence of grade II-IV acute GVHD was lower after allo-SCTs using MSD (hazard ratio [HR] = 0.465, P = .003), MUD (HR = 0.524, P = .010), and UCB (HR = 0.647, P = .067) compared with GC haplo-SCT. There was no significant difference in the incidence of chronic GVHD between GC haplo-SCT and allo-SCT using the other 3 donor types. Regarding relapse, GC haplo-SCT was associated with a significantly lower risk compared with MSD-SCT (P < .001) or MUD-BMT (P = .004). GC haplo-SCT tended to have a lower risk compared with UCBT (P = .063). Especially, all the 43 evaluable GC haplo-SCT recipients achieved CR after transplantation, whereas 23.9%, 22.8%, and 27.0% of patients who underwent MSD-SCT, MUD-BMT, and UCBT could not achieve CR after transplantation, respectively. Regarding non-relapse mortality, GC haplo-SCT was associated with a significantly higher risk compared with MUD-BMT (P = .014), and tended to have a higher risk compared with MSD-SCT (P = .061). There was no significant difference between GC haplo-SCT and UCBT (P = .600). Allo-SCTs using MSD (HR = 2.548, P < .001), MUD (HR = 2.134, P = .005), and UCB (HR = 2.376, P = .001) lead to significantly higher overall mortality compared with GC haplo-SCT; the adjusted overall survival at 3 years was 19.8% for MSD, 26.1% for MUD, 28.0% for UCB, and 65.1% for GC haplo. Thus GC haplo-SCT is a promising treatment option for patients with AML with a high leukemic burden.

A method of lower and upper solutions for control problems and application to a model of bone marrow transplantation

A method of lower and upper solutions for control problems and application to a model of bone marrow transplantation

Toll-like receptor-2 in cardiomyocytes and macrophages mediates isoproterenol-induced cardiac inflammation and remodeling.

Heart failure (HF) is the leading cause of morbidity and mortality worldwide. Activation of the innate immune system initiates an inflammatory response during cardiac remodeling induced by isoproterenol (ISO). Here, we investigated whether Toll-like receptor-2 (TLR2) mediates ISO-induced inflammation, hypertrophy, and fibrosis. TLR2 was found to be increased in the heart tissues of mouse with HF under ISO challenge. Further, cardiomyocytes and macrophages were identified as the main cellular sources of the increased TLR2 levels in the model under ISO stimulation. The effect of TLR2 deficiency on ISO-induced cardiac remodeling was determined using TLR2 knockout mice and bone marrow transplantation models. In vitro studies involving ISO-treated cultured cardiomyocytes and macrophages showed that TLR2 knockdown significantly decreased ISO-induced cell inflammation and remodeling via MAPKs/NF-κB signaling. Mechanistically, ISO significantly increased the TLR2-MyD88 interaction in the above cells in a TLR1-dependent manner. Finally, DAMPs, such as HSP70 and fibronectin 1 (FN1), were found to be released from the cells under ISO stimulation, which further activated TLR1/2-Myd88 signaling and subsequently activated pro-inflammatory cytokine expression and cardiac remodeling. In summary, our findings suggest that TLR2 may be a target for the alleviation of chronic adrenergic stimulation-associated HF. In addition, this paper points out the possibility of TLR2 as a new target for heart failure under ISO stimulation.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Iron Overload and Iron-Induced Microbiome Changes May Affect Lymphoid Levels Post Bone Marrow Transplant and Graft-Versus-Host Disease Survival in Mice

Introduction: Bone Marrow Transplant (BMT) represents the only cure for many malignant and non-malignant hematopoietic disorders. This treatment is often accompanied by multiple red blood cell transfusions, which can lead to iron overload. Iron overload is associated with poorer outcomes following BMT, such as increased risk of infections or Graft-versus-Host Disease (GVHD); however, it is not yet clear whether this correlation is caused by iron itself or is simply confounded by patients with more severe disease requiring more transfusions. Previous studies using mouse models, have shown that transfusional iron overload reduces the population of beneficial bacteria in the gut microbiome or decreases the production of microbiome-produced metabolites (i.e. indoles) that have been associated with BMT outcomes and GVHD risk. Here, we developed a reductionist mouse model of iron overload and BMT to investigate the effect of iron and iron-induced changes to gut microbiota and their metabolites on BMT outcomes and GVHD survival. Methods: Female Balb/C mice were infused twice a week, for two weeks, with a total of 50 mg of iron dextran or saline followed by lethal irradiation (9 Gy). Cohorts of mice (n=10 per group) received 1x10^10 T cell-depleted bone marrow cells with or without 5x10^6 T cells from UBC-GFP C57Bl6 mice to establish allogenic BMT and GHVD models, respectively. Mice were then monitored daily for survival with a formal severity score evaluated twice a week. Analysis of donor red blood cells, platelets, and white blood cells (neutrophils, B and T cells) from peripheral blood was also performed once a week to check for engraftment. To examine the impact of iron-induced microbiota changes, fecal microbiome transplant (FMT) was performed in mice receiving BMT. Briefly, mice were treated with an antibiotic cocktail (ampicillin 1 g/L, kanamycin 1 g/L, metronidazole 1g/L, vancomycin 0.5g/L) by gavage 2 days before the start of FMT. Feces from control and iron overloaded mice were collected freshly and weighed. Stool pellets were then resuspended in PBS (100 mg feces/ml PBS), vortexed and then spun down at 800g for 3 minutes. Mice received 100ul of supernatant by gavage. Additional cohorts of mice were administrated with indole-3 proprionic acid (IPA) in the drinking water at a concentration of 0.1 mg/ml prior to irradiation throughout the experiment and monitored for survival after receipt of allogeneic T cells to induce GVHD. All mice received enrofloxacin (10mg/kg) starting on the day of BMT and for the following 3 days by intraperitoneal injection. Results: There was a significant reduction in overall lymphoid (CD45+ CD172a- CD11b-/low GFP+), B (CD45+ CD172a- CD11b-/low CD19+ GFP+), and T (CD45+ CD172a- CD11b-/low CD3+ GFP+) cells in iron overloaded mice compared to control mice (p<0.0001 at day +28) by flow cytometry analysis in peripheral blood after BMT. Although FMT from control feces into iron overloaded mice did not affect lymphoid numbers, FMT from iron overloaded feces into control mice reduced the number of donor lymphoid and B cells (p<0.05 at day +28; Figure 1A). Iron overload reduced survival compared to control mice in the GHVD mouse model (p<0.05 Mantel-Cox test). Administration of IPA in iron overloaded mice did not significantly affect overall survival (Figure 1B). Conclusions: Our results show that iron overload negatively impacts lymphocyte engraftment in a mouse model of BMT. Furthermore, iron overload also reduces survival in mice with GVHD. Iron overload was previously shown to reduce indole levels in mice. Although we did not observe a significant impact of exogenous indole administration on GVHD survival, FMT from iron overloaded into control mice reduced levels of lymphoid and B cells after BMT implicating gut microbiota in this outcome. Taken together, these results suggest that iron overload is an important factor that negatively affects BMT outcomes. The extent to which iron-induced gut microbiota changes are involved in the mechanisms responsible for this effect requires further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The DNA-Sensor AIM2 Promotes BCR-Activated B Cells in Chronic Graft-Versus-Host-Disease

Chronic graft-versus-host-disease (cGVHD) remains a common and poorly understood cause of late non-relapse morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HCT). T cells and IgG-producing B cells mediate cGVHD development and perpetuation, but molecular mechanisms driving immune pathology require further study. We have shown that B cells from cGVHD patients are B Cell Receptor (BCR)-activated and primed for survival. In a mouse model of bone-marrow transplantation (BMT) with cGVHD manifestations, BCR signaling is promoted by alloantigens and BAFF (Jia et al., Blood 2021). BCR signaling is a viable therapeutic target in cGVHD, but other targetable pathways that promote pathological BCR-activated B cells remain largely unknown. Our single cell RNA-sequencing study of B cells from the peripheral blood (PB) of HCT patients identified 'Absent in Melanoma 2' (AIM2) as a prime candidate for further study (ASH abstract 2019, Poe et al., manuscript submitted). AIM2 was a signature gene in a BCR-activated subset after HCT, and AIM2 transcripts were significantly increased in a pre-germinal center (GC) B cell subset in cGVHD vs. no cGVHD patients (adjusted p=0.049). AIM2 incites inflammasome-mediated pyroptotic cell death in myeloid cells, but the function of AIM2 in B cells is largely unknown. AIM2 is expressed by CD27+ antigen (Ag)-experienced B cells in healthy individuals (HI) (Svensson et al., PlosOne 2017) and emerging evidence suggests that AIM2 promotes antibody production (Yang et al., Signal Transduct Target Ther 2021). We hypothesize that AIM2 promotes pathological BCR-activated B cells and anti-host IgG production in cGVHD. Using intracellular flow cytometry (IFC) and PB samples from HI (n=27), we first confirmed that the AIM2 protein is mainly expressed by CD27+ Ag-experienced B cells. Specifically, 88% and 99% of the IgD+ pre- and IgD- post-GC CD27+ B cells expressed AIM2, respectively. In HCT, we found a significantly higher proportion of AIM2+ cells in the circulating CD27+ IgD- post-GC B cells from cGVHD patients (n=7) vs. no cGVHD patients (n=5) (p=0.02). We also found significant overexpression of AIM2 transcripts in B cells from mice with cGVHD manifestations vs. no cGVHD mice (n=5/group, p=0.03, Figure 1A). To determine if Ag-activation together with other extrinsic factors typically encountered by B cells after HCT induced AIM2 expression by purified CD27- B cells from HI, we used IFC. The proportion of AIM2+ CD27- B cells increased after BCR +/- Toll-like receptor 7 or 9 stimulation only when IFNγ was present. Next, we studied the function of AIM2 in B cells. Unlike myeloid cells, CD27+ B cells from HI did not undergo pyroptotic cell death after stimulation with double stranded DNA. This lack of pyroptosis was not due to a deficiency in the inflammasome components and B cells had the capacity to undergo pyroptosis after exposure to the NLRP1/3 activator, nigericin. To assess effects of AIM2 on specific IgG production, we used AIM2 KO mice in a hapten-carrier immunization model (NP-KLH + alum i.p.). AIM2-KO mice had larger splenic GC vs. WT controls at day 8 post-immunization (p<0.0001). GC enlargement paradoxically associated with lower anti-NP IgG levels (p=0.03). Finally, having previously shown that BM+Spl recipients have a significantly higher proportion of GL7+ BCR-activated blood B cells (Jia et al., Blood 2021), we asked whether AIM2 promoted GL7+ B cells in cGVHD. To address this, we used AIM2-floxed mice, kindly supplied by Genentech, to breed AIM2fl/fl Mb1Cre/+ conditional KO mice (AIM2-CKO), deficient in AIM2 only in B cells. We then used these AIM2-CKO vs. AIM2fl/fl Mb1+/+ control littermates mice which are AIM2-sufficient as BMT donors. At day 24 post-BMT, recipients of BM+Spl from AIM2-CKO mice had a significantly lower percentage of mature (CD19+ CD93-) GL7+ B cells in PB vs. recipients of BM+Spl from the AIM2-sufficient littermates (n=10/group, p=0.004, Figure 1B). This was not due to baseline B cell differences in donor CKO mice. These results are being confirmed at later time points in ongoing, additional experiments. Our data show that AIM2 promotes IgG-producing B cells and circulating GL7+ B cells, suggesting that AIM2 plays a novel inflammasome-independent role in cGVHD B cell pathology. We are actively investigating how AIM2 effects alloantibody production and cGVHD development, so that therapeutic targets can be identified. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Isocitrate Dehydrogenase 2 Mutation Drives Bone Marrow Macrophage Dysfunction without a Complete Block in Hematopoietic Differentiation

Myelodysplastic syndromes (MDS) represent a broad category of myeloid malignancies characterized by myeloid skewed hematopoiesis, blood cytopenias and increased risk of transformation to leukemia. While not common, isocitrate dehydrogenase 2 (IDH2) mutations are represented in clonal hematopoiesis and in MDS, are found in patients with higher International Prognosis Scoring System (IPSS) disease scores and confer a higher risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite which disrupts hematopoiesis. However, the mechanisms that induces clonal progression and transformation remains incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutation on mature myeloid cell functions and the bone marrow microenvironment (BMME) remains to be elucidated. Using RAW264.7 cells, a monocyte/macrophage-like murine cell line, we retrovirally infected cells with human wt IDH2 or the R172K IDH2 mutant, a common IDH2 mutation. After confirming a 30-fold increase of 2HG (p<.005) and observing evidence of hypermethylation in the IDH2 mutant RAW cells, we tested several key functions of bone marrow macrophages: ability to engulf apoptotic cells (efferocytosis), erythropoietic support, and cytokine production. After labeled apoptotic neutrophils were fed as efferocytic targets to RAW cells at a 10:1 ratio for 6 hours, we observed a 10% decrease of efferocytosis in IDH2 mutants (p < 0.05). Next, we cocultured RAW cells with primary murine late-stage erythroid progenitors and quantified their capacity to form colonies (CFU-E) in the presence of erythropoietin. Compared to wt, IDH2 mutant RAW cells inhibited CFU-E formation by 25% (p < 0.0075). Cytokine array on cell culture supernatant from mutant RAW cells had elevated proinflammatory chemokines/cytokines (CCL5, CXCL10, TNF-a) and decreased cytokines related to inflammation resolution and efferocytosis (GDF-15, Gas6, Endostatin, CCL2). To test these findings further in vivo we generated a murine bone marrow transplant model with retrovirally infected hematopoietic stem cells (HSC: lineage-, c-Kit+, Sca-1+) containing the human wt or R172K IDH2 mutation with an mCherry reporter and transplanted into CD45.1 congenic mice (10 mutant, 10 wt, 1:1 male:female). Mutant IDH2 HSCs demonstrated better engraftment (85-90%) compared to wt (70-80%) and also had higher levels of mCherry positivity within CD45.2+ cells (80-90% mutant vs 40-60% in wt). Longitudinal blood draws showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes in IDH2 mutants by 16 weeks post-transplant. In contrast to current literature, IDH2 mutant cells did not exhibit a complete block in myeloid cell differentiation, as the mutant clone gave rise to mature monocytes, macrophages, and neutrophils. We also harvested bone marrow at 16 weeks post transplantation. Compared to wt IDH2, the mutant IDH2 bone marrow cells exhibited myeloid skewing hematopoiesis and a 3-fold increase in bone marrow monocytes (CD45+, Ly6G-, Ly6C++, F4/80+, p < 0.00001). In the IDH2 mutant bone marrow cells, myeloid progenitors were also expanded: common myeloid progenitors (CMPs: 4-fold increase p < 0.001, CD45+, lineage-, c-Kit+, Sca-1-, CD34+, CD16/32-), and granulocytic monocytic precursors (GMPs: 3-fold increase p < 0.0001, CD45+, c-Kit+, Sca-1-, CD34+, CD16/32+). We also observed a previously unreported loss of long-term hematopoietic stem cells (LT-HSC: 13.5-fold reduction, p < 0.00001, CD45+, lineage-, c-Kit+, Sca-1+, Flt3-, CD48-, CD150+). When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow derived macrophages fed normal apoptotic neutrophils at a 10:1 ratio for 3 or 24 hours. In summary, we identify defects in efferocytic function and cytokine production in monocytes and macrophages derived from IDH2 mutant hematopoietic stem and progenitor cells. We have previously shown that defects in the efferocytic function of bone marrow macrophages induce increases in inflammatory signals and contribute to skewing of hematopoiesis. Since these macrophage dysfunctions have not previously been attributed to IDH2 single mutation, they represent a potential novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and transformation to leukemia.

Type 2 but Not Type 1 Calreticulin Mutants Exhibit Loss of Chaperone Function Due to C-Terminal Structural Interference, and Bind to Wild Type Calreticulin in a Dominant Negative Manner

Calreticulin (CALR) mutations are the second most common genetic driver of myeloproliferative neoplasms (MPNs). Most CALR mutations are classified as either type 1 (52 bp deletion; del52) or type 2 (5 bp insertion; ins5). Both lead to the generation of an identical mutant C-terminus, which is required to transform cells through aberrant binding to and activation of the thrombopoietin receptor MPL. Despite their identical C-termini and shared mechanism of transformation, patients with type 1 and type 2 CALR mutations display significant clinical and prognostic differences. The mechanisms underlying these divergent clinical phenotypes remain incompletely understood. Though classified as gain-of-function mutations, we previously found that both CALRdel52 and CALRins5 mutants display differential losses-of-function; CALRdel52 loses its ability to bind calcium (ASH abstract #147859, 2021), while CALRins5 loses its function as a molecular chaperone. We demonstrated that loss of CALRins5 chaperone function leads to activation of the ATF6 arm of the unfolded protein response (ASH abstract #3588, 2021), and that this signaling culminates in ATF6-mediated up-regulation of BCL-xL, which renders CALRins5 cells dependent upon this pathway for survival However, these studies did not address the molecular mechanism by which CALRins5 loses chaperone function. To dissect this, we performed structural modeling studies and found that the mutant C-terminal tail of the CALRins5 but not the CALRdel52 protein exhibits structural proximity to the globular N domain of the protein, which is critical for CALR chaperone function. We thus hypothesized that the observed loss of chaperone function may be due to structural interference by the CALRins5 C-terminus. To test this, we created a series of CALRins5 truncation variants in which we progressively trimmed back the mutant C-terminal tail. We found that such trimming restored chaperone function to the CALRins5 protein, suggesting that indeed the loss of chaperone function is due to interference from the mutant C-terminal tail. Because CALR mutations are heterozygous in patients, we next asked why the wild type copy of CALR does not compensate for the loss of chaperone function in CALRins5-expressing cells. Though it has been shown that CALRdel52 does not bind to CALRwt, this has never been tested for CALRins5. We thus hypothesized that CALRins5 may bind to CALRwt in a dominant negative manner, leading to complete loss of CALR-mediated chaperone function in CALRins5-expressing cells. To test this, we performed co-immunoprecipitation assays and found that CALRins5 does indeed bind to CALRwt, while, consistent with previous findings, CALRdel52 and CALRwt do not interact. To determine whether this binding was in fact dominant negative, we performed an in vitro chaperone assay and found that CALRwt chaperone function is suppressed when in the presence of CALRins5. This suggests that CALRins5 exhibits a dominant negative effect over CALRwt chaperone function. Finally, we sought to determine if this loss in chaperone ability was important for CALRins5 cell survival. To test this, we utilized the chemical chaperone tauroursodeoxycholic acid (TUDCA) to restore ER chaperone capacity. We found that CALRins5 but not CALRdel52 or CALRwt cell lines and patient samples displayed decreased viability upon TUDCA treatment, suggesting chaperone loss is necessary for survival of CALRins5 cells. Importantly, TUDCA has shown immense clinical benefit for diseases characterized by molecular chaperone dysfunction (e.g., neurodegenerative diseases). To test whether this benefit translates to MPNs, we utilized our mutant CALR-driven bone marrow transplantation model. We found that CALRins5 mice treated with TUDCA displayed significantly decreased platelet counts compared to those treated with vehicle alone, while TUDCA had no effect on CALRdel52-driven disease. Taken together, we have dissected the mechanism by which CALRins5 proteins lose chaperone ability, as well as demonstrate that CALRins5 proteins exhibit a dominant negative effect over CALRwt chaperone function. Finally, we reveal TUDCA as a potential therapeutic strategy for CALRins5 patients that has immediate clinical tractability.

Total Marrow Irradiation Reduces Organ Damage in a Bone Marrow Transplant Model of Sickle Cell Disease

Sickle cell disease (SCD) is associated with significant organ pathology. Currently, the only curative option is bone marrow transplantation (BMT) after myeloablative conditioning. However, significant organ damage from myeloablative total body irradiation (TBI) remains one of the major side effects of BMT. To reduce exposure to vital organs, but efficient enough to maintain engraftment, we developed 3D image guided-total marrow irradiation (TMI) for targeted delivery to the bone marrow (BM) and spleen of humanized BERK sickle mice while sparing the vital organs. We hypothesized that TMI will prevent organ damage compared to that induced by TBI during BMT. Sickle mice were treated with: [i] TBI (4:4), 4 Gy to the BM & 4 Gy to the whole body; [ii] TMI (4:0), 4 Gy to BM, 0 Gy to the body, and [iii] TMI (8:2), 8 Gy to BM & 2 Gy to the whole body. After 24h of irradiation, SS mice were transplanted with 5 million donor BM cells from HbAA BERK mice expressing normal human hemoglobin A (n=3/group). Thirty days after BMT mice were euthanized, and organs were necropsied for histopathology. Formalin-fixed paraffin-embedded sections of the lung, liver, kidney, spleen, and bone marrow (femur & tibia) were stained with H&E and Prussian blue (iron deposits). Morphologic findings of vascular congestion, inflammatory infiltrate, and iron deposits were scored from '0' for no lesion up to '6' for severe lesions occupying 90-100% of the field (Manci et al., Blood 2006). Tubular brush border thickness was assessed on periodic acid Schiff's (PAS)-hematoxylin-stained kidney sections using a 0-4 grading scale: no changes ='0'; lesion area <25%=1; 25%-50% = 2; >50% = 3 and 100% = 4 (Kasztan et al. J Am Soc Nephrol. 2017). We observed significantly increased vascular congestion in the liver (p=0.003), and spleen (p=0.013), and inflammatory infiltrates in the lungs (p=0.031) of TBI-treated mice compared to TMI (8:2). Total body irradiation has been shown to induce inflammation with granulocyte infiltration into tissues in association with endothelial damage, vascular dilatation, and congestion. Moreover, increase iron content in BM and serum and disrupted iron metabolism is another known sequala of total body irradiation, which contributes to hemosiderin formation in tissues. We found significant increase in iron deposits in TBI (4:4) vs TMI (8:2) in liver (p=0.001), spleen (p=0.014), and kidney (p=0.043). TBI (4:4) led to more renal tubular lesions (p=0.003) marked by a significant loss in the tubular brush border on PAS-stained kidney sections compared to TMI (8:2). Thus, TMI shows lesser damaging effect on vital organs compared to TBI, while TMI (8:2) showed donor chimerism similar to TBI on days 7 and 14 but higher than TMI (4:0) treated mice. However, the engraftment failed (less than 10% chimerism) in all groups by day 30. To improve engraftment, we transplanted 10- and 25-million donor HbAA BERK BM cells into TMI (4:0) and TMI (8:2) treated sickle mice. TMI (8:2) showed sustained donor chimerism but TMI (4:0) did not. Histopathological analysis 90 days post-BMT showed that TMI (8:2) treatment (n=3) results in significantly lesser organ pathology Vs untreated sickle mice (n=2), as evinced by reduction in: vascular congestion (lung, p=0.014; liver, p=0.014; spleen p= 0.014), inflammatory infiltrate (lung, p= 0.031; liver p= 0.005), liver infarctions (p=0.003) and liver and kidney iron deposits (p=0.041 & 0.021, respectively). Untreated mice showed a significant loss in the tubular brush border compared to TMI (8:2) treatment (p=0.008). BM in TMI (8:2) (cellularity~70%) showed improvement in cellular differentiation and topography in comparison to the untreated (cellularity ~90% ) that were packed with minimal fat spaces, thin trabeculae, and marked erythroid hyperplasia. These data suggest recovery from sickle cell phenotype with TMI-8:2 without significant organ pathology. Additionally, toluidine blue stained skin sections showed that the number (~45 Vs ~15) and % degranulating mast cells (~85 % Vs 50 %) were significantly lower in TMI (8:2) Vs untreated sickle mice (p<0.05), suggesting that TMI (8:2) treatment and successful engraftment of healthy donor cells may reduce inflammation and pain phenotype. We show that dose escalated TMI (8:2) is well tolerated with reduced organ pathology. Therefore, TMI has the potential to improve BMT-related outcomes without an adverse effect on organ damage.

AML-060 A Truncated Nucleus Isoform of FGFR1 Drives the Transformation of Leukemia Stem Cells in Leukemogenesis

Myeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated in the cytoplasm as a result of chromosome translocations. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated. In this study, we used the mouse bone marrow transduction and transplantation model to demonstrate the leukemic transformation ability of this tnFGFR1 and identified its critical downstream targets, FLT3 and KIT, in mouse and acute myeloid leukemia (AML) patient samples. Stem cell transduction and transplantation in syngeneic mice was used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and RPPA and RNA-Seq were used to examine the related signaling pathways and regulated target genes. We now show that tnFGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells. These leukemia cells show a mixed immunophenotype with a B-cell B220+Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. tnFGFR1 induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. De novo human AML also expresses tnFGFR1, which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but sensitive to the FLT3 inhibitor quizartinib (AC220), suggesting an alternative approach for the treatment of SCLL. This study demonstrates a novel model for the transformation of hematopoietic stem cells by chimeric FGFR1 kinases with the combined effects of direct protein activation by the full-length kinases and transcriptional regulation by tnFGFR1. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile.

Development and characterization of a preclinical total marrow irradiation conditioning-based bone marrow transplant model for sickle cell disease

Sickle cell disease (SCD) is a serious global health problem, and currently, the only curative option is hematopoietic stem cell transplant (HCT). However, myeloablative total body irradiation (TBI)-based HCT is associated with high mortality/morbidity in SCD patients. Therefore, reduced-intensity (2–4 Gy) total body radiation (TBI) is currently used as a conditioning regimen resulting in mixed chimerism with the rescue of the SCD disease characteristic features. However, donor chimerism gradually reduces in a few years, resulting in a relapse of the SCD features, and organ toxicities remained the primary concern for long-term survivors. Targeted marrow irradiation (TMI) is a novel technique developed to deliver radiation to the desired target while sparing vital organs and is successfully used for HCT in refractory/relapsed patients with leukemia. However, it is unknown if TMI will be an effective treatment for a hematological disorder like SCD without adverse effects seen on TBI. Therefore, we examined preclinical feasibility to determine the tolerated dose escalation, its impact on donor engraftment, and reduction in organ damage using our recently developed TMI in the humanized homozygous Berkley SCD mouse model (SS). We show that dose-escalated TMI (8:2) (8 Gy to the bone marrow and 2 Gy to the rest of the body) is tolerated with reduced organ pathology compared with TBI (4:4)-treated mice. Furthermore, with increased SCD control (AA) mice (25 million) donor BM cells, TMI (8:2)-treated mice show successful long-term engraftment while engraftment failed in TBI (2:2)-treated mice. We further evaluated the benefit of dose-escalated TMI and donor cell engraftment in alleviating SCD features. The donor engraftment in SCD mice completely rescues SCD disease features including recovery in RBCs, hematocrit, platelets, and reduced reticulocytes. Moreover, two-photon microscopy imaging of skull BM of transplanted SCD mice shows reduced vessel density and leakiness compared to untreated control SCD mice, indicating vascular recovery post-BMT.

Melatonin Improves Ischemia-Induced Circulation Recovery Impairment in Mice with Streptozotocin-Induced Diabetes by Improving the Endothelial Progenitor Cells Functioning.

Patients with diabetes mellitus tend to develop ischemia-related complications and have compromised endothelial progenitor cell (EPC) function. Melatonin protects against ischemic injury, possibly via EPC modulation. We investigated whether melatonin pretreatment could restore EPC function impairment and improve circulation recovery in a diabetic critical limb ischemia mouse model. Under 25 mM high-glucose medium in vitro, EPC proliferation, nitric oxide production, tube formation, and endothelial nitric oxide synthase (eNOS) phosphorylation were significantly suppressed. Hyperglycemia promoted EPC senescence and apoptosis as well as increased reactive oxygen species (ROS) production. Melatonin treatment reversed the harmful effects of hyperglycemia on EPC through adenosine monophosphate–activated protein kinase-related mechanisms to increase eNOS phosphorylation and heme oxygenase-1 expression. In an in-vivo study, after a 4-week surgical induction of hindlimb ischemia, mice with streptozotocin (STZ)-induced diabetes showed significant reductions in new vessel formation, tissue reperfusion, and EPC mobilization in ischemic hindlimbs compared to non-diabetic mice. Mice with STZ-induced diabetes that received melatonin treatment (10 mg/kg/day, intraperitoneal) had significantly improved blood perfusion ratios of ischemic to non-ischemic limb, EPC mobilization, and densities of capillaries. In addition, a murine bone marrow transplantation model to support these findings demonstrated that melatonin stimulated bone marrow-originated EPCs to differentiate into vascular endothelial cells in femoral ligation-induced ischemic muscles. In summary, this study suggests that melatonin treatment augments EPC function along with neovascularization in response to ischemia in diabetic mice. We illustrated the protective effects of melatonin on EPC H2O2 production, senescence, and migration through melatonin receptors and modulating eNOS, AMPK, and HO-1 activities at the cellular level. Thus, melatonin might be used to treat the impairment of EPC mobilization and circulation recuperation in response to ischemic injury caused by chronic hyperglycemia. Additional studies are needed to elucidate the applicability of the results in humans.

Neutrophils as regulators of macrophage-induced inflammation in a setting of allogeneic bone marrow transplantation.

SummaryClinical data reveal that patients with allogeneic hematopoietic stem cell transplantation (HSCT) are vulnerable to infection and prone to developing severe sepsis, which greatly compromises the success of transplantation, indicating a dysregulation of inflammatory immune response in this clinical setting. Here, by using a mouse model of haploidentical bone marrow transplantation (haplo-BMT), we found that uncontrolled macrophage inflammation underlies the pathogenesis of both LPS- and E.coli-induced sepsis in recipient animals with graft-versus-host disease (GVHD). Deficient neutrophil maturation in GVHD mice post-haplo-BMT diminished modulation of macrophage-induced inflammation, which was mechanistically dependent on MMP9-mediated activation of TGF-β1. Accordingly, adoptive transfer of mature neutrophils purified from wild-type donor mice inhibited both sterile and infectious sepsis in GVHD mice post-haplo-BMT. Together, our findings identify a novel mature neutrophil-dependent regulation of macrophage inflammatory response in a haplo-BMT setting and provide useful clues for developing clinical strategies for patients suffering from post-HSCT sepsis.

Abstract 3617: METTL16 drives leukemogenesis and maintains leukemia stem cell self-renewal via reprogramming BCAA metabolism

Abstract Background: As the most prevalent internal decorations in mammalian mRNA, N6-methyladenosine (m6A) has been reported to be involved in many physiological and pathological processes, including acute myeloid leukemia (AML). METTL3 and METTL14, the well-recognized m6A methyltransferase complex, contribute to AML. METTL16 is a recently identified m6A methyltransferase that has been reported to deposit m6A in a few targets. While, unlike METTL3/14, the biological functions of METTL16 are largely unknown. Here, we explored the function and mechanism of METTL16 in AML pathogenesis and evaluated its therapeutic potential for AML treatment. Methods: We performed CRISPR-Cas9 screen to evaluate the dependency of METTL16 in AML cells. We created METTL16 knockout (KO) cells and conditional KO mice to evaluate its role in leukemogenesis and normal hematopoiesis. We employed bone marrow transplantation (BMT), xenograft, and AML patient-derived xenograft (PDX) models to determine its role in AML development and progression. To identify the targets of METTL16, we performed m6A-seq and RNA-seq, followed m6A-qPCR, CLIP-qPCR, in vitro methyltransferase assays and RNA stability assays. To examine the effect of METTL16 on branched chain amino acid (BCAA) metabolism, we performed metabolic profiling with 13C, 15N-leucine. Results: CRISPR-Cas9 screen showed METTL16 is one of the most essential genes for the survival of AMLs. The AML cells display more robust dependency on METTL16 than METTL3/14. We found METTL16 is highly expressed in AML patients compared to healthy controls. METTL16 KO significantly inhibited AML cell proliferation, promoted cell apoptosis and myeloid differentiation in vitro, which could be totally reversed by forced expression of wild-type METTL16, but not catalytic-dead mutant. METTL16 depletion dramatically inhibited AML progression and prolonged survival of recipient mice in the BMT, xenograft and PDX models. In addition, METTL16 is highly expressed in LSCs contrast to leukemic bulk cells and METTL16 KO significantly attenuates LSC self-renewal in vitro and in vivo. By contrast, the role of METLL16 is largely spared in normal hematopoietic cells. Via integrated analysis of m6A-seq data and RNA-seq data, we identified two bona fide targets of METTL16, BCAT1 and BCAT2, which encode two critical BCAA transaminases in BCAA biosynthesis pathway. METTL16 promotes the expression of BCAT1 and BCAT2 via an m6A dependent manner. Metabolomics with 13C, 15N-leucine tracing showed that METTL16 KO results in suppressed pools of TCA cycle intermediates, some non-essential amino acids and nucleotides. Conclusion: We uncover a tumor-promoting role of METTL16 in AML and LSC self-renewal via reprogramming BCAA metabolism, in which METTL16 functions as an m6A methyltransferase to regulate expression of BCAT1 and BCAT2. Our data suggest that METTL16 is an attractive target for AML therapy. Citation Format: Li Han, Lei Dong, Keith Leung, Zhicong Zhao, Yangchan Li, Ying Qing, Jianhuang Xue, Chao Shen, Zhenhua Chen, Lei Gao, Kitty Wang, Keren Zhou, Wei Li, Brandon Tan, Zheng Zhang, Xi Qin, Rui Su, Xiaolan Deng, Jianjun Chen. METTL16 drives leukemogenesis and maintains leukemia stem cell self-renewal via reprogramming BCAA metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3617.