Engraftment Potential Research Articles

Mesenchymal Stem Cells Derived from Human Urine-Derived iPSCs Exhibit Low Immunogenicity and Reduced Immunomodulatory Profile.

Human-induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) represent a promising and renewable cell source for therapeutic applications. A systematic evaluation of the immunological properties and engraftment potential of iMSCs generated from urine-derived iPSCs is lacking, which has impeded their broader application. In this study, we differentiated urine-derived iPSCs into iMSCs and assessed their fundamental MSC characteristics, immunogenicity, immunomodulatory capacity and in vivo engraftment. Compared to umbilical cord-derived MSCs (UCMSCs), iMSCs demonstrated an enhanced proliferative capacity, a higher level of regenerative gene expression, and lower immunogenicity, strengthening resistance to apoptosis induced by allogeneic peripheral blood mononuclear cells (PBMCs) and the NK-92 cell line. In addition, iMSCs exhibited a diminished ability to inhibit T cell proliferation and activation compared with UCMSCs. Transcriptomic analyses further revealed the decreased expression of immune regulatory factors in iMSCs. After transfusion into mouse models, iMSCs engrafted in the lungs, liver, and spleen and exhibited the ability to migrate to tumor tissues. Our results indicated that iMSCs generated from urine-derived iPSCs have a significant replicative capacity, low immunogenicity and unique immunomodulatory properties, and hence offer obvious advantages in immune privilege and allogenic therapeutic application prospects.

Sphingosine-1 phosphate receptor 1 (S1PR1) expression maintains stemness of acute myeloid leukemia stem cells

Acute myeloid leukemia (AML) arises from leukemia stem cells (LSCs) and is maintained by cells which have acquired features of stemness. We compared transcription profiles of AML cells with/without stem cell features defined as in vitro clonogenicity and serial engraftment in immune-deficient mice xenograft model. We used multi-parameter flow cytometry (MPFC) to separate CD34+ bone marrow-derived leukemia cells into sphingosine-1 phosphate receptor 1 (S1PR1)+ and S1PR1- fractions. Cells in the S1PR1+ fraction demonstrated significantly higher clonogenicity and higher engraftment potential compared with those in the S1PR1- fraction. In contrast, CD34+ bone marrow cells from normal samples showed reduced clonogenicity in the S1PR1+ fraction compared with the S1PR1- fraction. Inhibition of S1PR1 expression in an AML cell line reduced the colony-forming potential of KG1 cells. Transcriptomic analyses and rescue experiments indicated PI3K/AKT pathway and MYBL2 are downstream mediators of S1PR1-associated stemness. These findings implicate S1PR1 as a functional biomarker of LSCs and suggest its potential as a therapeutic target in AML treatment.

An in vitro model of acute horizontal basal cell activation reveals gene regulatory networks underlying the nascent activation phase

While horizontal basal cells (HBCs) make minor contributions to olfactory epithelium (OE) regeneration during homeostatic conditions, they possess a potent, latent capacity to activate and subsequently regenerate the OE following severe injury. Activation requires, and is mediated by, the downregulation of the transcription factor (TF) TP63. In this paper, we describe the cellular processes that drive the nascent stages of HBC activation. The compound phorbol 12-myristate 13-acetate (PMA) induces a rapid loss in TP63 protein and rapid enrichment of HOPX and the nuclear translocation of RELA, previously identified as components of HBC activation. Using bulk RNA sequencing (RNA-seq), we find that PMA-treated HBCs pass through various stages of activation identifiable by transcriptional regulatory signatures that mimic stages identified invivo. These temporal stages are associated with varying degrees of engraftment and differentiation potential in transplantation assays. Together, these data show that our invitro HBC activation system models physiologically relevant features of invivo HBC activation and identifies new candidates for mechanistic testing.

Bioengineered MSCCxcr2 transdifferentiated keratinocyte-like cell-derived organoid potentiates skin regeneration through ERK1/2 and STAT3 signaling in diabetic wound

Skin regeneration is severely compromised in diabetic foot ulcers. Allogeneic mesenchymal stem cell (MSC) transplantation is limited due to the poor engraftment, mitogenic, and differentiation potential in the harsh wound microenvironment. Thus, to improve the efficacy of cell therapy, the chemokine receptor Cxcr2 was overexpressed in MSCs (MSCCxcr2). CXCL2/CXCR2 axis induction led to the enhanced proliferation of MSCs through the activation of STAT3 and ERK1/2 signaling. Transcriptional upregulation of FGFR2IIIb (KGF Receptor) promoter by the activated STAT3 and ERK1/2 suggested trans-differentiation of MSCs into keratinocytes. These stable MSCCxcr2 in 2D and 3D (spheroid) cell cultures efficiently transdifferentiated into keratinocyte-like cells (KLCs). An in vivo therapeutic potential of MSCCxcr2 transplantation and its keratinocyte-specific cell fate was observed by accelerated skin tissue regeneration in an excisional splinting wound healing murine model of streptozotocin-induced type 1 diabetes. Finally, 3D skin organoids generated using MSCCxcr2-derived KLCs upon grafting in a relatively avascular and non-healing wounds of type 2 diabetic db/db transgenic old mice resulted in a significant enhancement in the rate of wound closure by increased epithelialization (epidermal layer) and endothelialization (dermal layer). Our findings emphasize the therapeutic role of the CXCL2/CXCR2 axis in inducing trans-differentiation of the MSCs toward KLCs through the activation of ERK1/2 and STAT3 signaling and enhanced skin regeneration potential of 3D organoids grafting in chronic diabetic wounds.

HES6knockdown in human hematopoietic precursor cells reduces their in vivo engraftment potential and their capacity to differentiate into erythroid cells, B cells, T cells and plasmacytoid dendritic cells.

Hematopoiesis is driven by molecular mechanisms that induce differentiation and proliferation of hematopoietic stem cells and their progeny. This involves the activity of various transcription factors, such as members of the Hairy/Enhancer of Split (HES) family, and important roles for both HES1 and HES4 have been shown in normal and malignant hematopoiesis. Here, we investigated the role of HES6 in human hematopoiesis using in vitro and in vivo models. Using bulk and single-cell RNA-sequencing data, we show that HES6 is expressed during erythroid/megakaryocyte and plasmacytoid dendritic cell development, as well as in multipotent precursors and at specific stages of T- and B-cell development following pre-B-cell receptor and pre-T-cell receptor signaling, respectively. Consistently, knockdown of HES6 in cord blood-derived hematopoietic precursors in well-defined in vitro differentiation assays resulted in reduced differentiation of human hematopoietic precursors towards megakaryocytes, erythrocytes, plasmacytoid dendritic cells, B cells and T cells. In addition, HES6 knockdown hematopoietic stem and progenitor cells displayed reduced colony-forming unit capacity in vitro and impaired potential to reconstitute hematopoiesis in vivo in a competitive transplantation assay. We demonstrate that loss of HES6 expression has an impact on cell cycle progression during erythroid differentiation and provide evidence for potential downstream target genes that affect these perturbations. Thus, our study provides new insights into the role of HES6 in human hematopoiesis.

Generation of complex bone marrow organoids from human induced pluripotent stem cells

The human bone marrow (BM) niche sustains hematopoiesis throughout life. We present a method for generating complex BM-like organoids (BMOs) from human induced pluripotent stem cells (iPSCs). BMOs consist of key cell types that self-organize into spatially defined three-dimensional structures mimicking cellular, structural and molecular characteristics of the hematopoietic microenvironment. Functional properties of BMOs include the presence of an in vivo-like vascular network, the presence of multipotent mesenchymal stem/progenitor cells, the support of neutrophil differentiation and responsiveness to inflammatory stimuli. Single-cell RNA sequencing revealed a heterocellular composition including the presence of a hematopoietic stem/progenitor (HSPC) cluster expressing genes of fetal HSCs. BMO-derived HSPCs also exhibited lymphoid potential and a subset demonstrated transient engraftment potential upon xenotransplantation in mice. We show that the BMOs could enable the modeling of hematopoietic developmental aspects and inborn errors of hematopoiesis, as shown for human VPS45 deficiency. Thus, iPSC-derived BMOs serve as a physiologically relevant in vitro model of the human BM microenvironment to study hematopoietic development and BM diseases.

In vivo PSC differentiation as a platform to identify factors for improving the engraftability of cultured muscle stem cells.

Producing an adequate number of muscle stem cells (MuSCs) with robust regenerative potential is essential for the successful cell therapy of muscle-wasting disorders. We have recently developed a method to produce skeletal myogenic cells with exceptional engraftability and expandability through an in vivo pluripotent stem cell (PSC) differentiation approach. We have subsequently mapped engraftment and gene expression and found that leukemia inhibitory factor receptor (Lifr) expression is positively correlated with engraftability. We therefore investigated the effect of LIF, the endogenous ligand of LIFR, on cultured MuSCs and examined their engraftment potential. We found that LIF-treated MuSCs exhibited elevated expression of PAX7, formed larger colonies from single cells, and favored the retention of PAX7+ "reserve cells" upon myogenic differentiation. This suggested that LIF promoted the maintenance of cultured MuSCs at a stem cell stage. Moreover, LIF enhanced the engraftment capability of MuSCs that had been expanded in vitro for 12 days by 5-fold and increased the number of MuSCs that repopulated the stem cell pool post-transplantation. These results thereby demonstrated the effectiveness of our in vivo PSC differentiation platform to identify positive regulators of the engraftability of cultured MuSCs.

Activating p53 abolishes self-renewal of quiescent leukaemic stem cells in residual CML disease

Whilst it is recognised that targeting self-renewal is an effective way to functionally impair the quiescent leukaemic stem cells (LSC) that persist as residual disease in chronic myeloid leukaemia (CML), developing therapeutic strategies to achieve this have proved challenging. We demonstrate that the regulatory programmes of quiescent LSC in chronic phase CML are similar to that of embryonic stem cells, pointing to a role for wild type p53 in LSC self-renewal. In support of this, increasing p53 activity in primitive CML cells using an MDM2 inhibitor in combination with a tyrosine kinase inhibitor resulted in reduced CFC outputs and engraftment potential, followed by loss of multilineage priming potential and LSC exhaustion when combination treatment was discontinued. Our work provides evidence that targeting LSC self-renewal is exploitable in the clinic to irreversibly impair quiescent LSC function in CML residual disease – with the potential to enable more CML patients to discontinue therapy and remain in therapy-free remission.

BCL11B Modulates Hemogenic Endothelium-Derived Multipotent Hematopoietic Progenitors and Enhances Myeloid and Lymphoid Pathways from Human iPSCs

Induced pluripotent stem cell (iPSC) derived human hematopoietic stem cells (HSCs) have revolutionized the field of regenerative medicine and provided a promising platform for advancing cell-based therapies, disease modeling, and drug discovery in the context of hematological disorders and beyond. However, efficient derivation of functional iPSC-derived HSCs with in vivo engraftment and multilineage potential remains challenging. The discovery of the hemogenic endothelium (HE) stage in iPSC culture opens possibilities to define molecular determinants for blood development from the endothelium and provides a prospective platform for the de novo generation of HSCs. BCL11B encodes a transcription factor required for T-cell commitment and is known to play important role in immune and aortic cell function. Recently, BCL11B expressionwas observed in lymphoid-myeloid clusters and HE from human iPSCs (hPSCs). However, nothing is known regarding the role of BCL11B in human hematopoiesis. While expression levels of BCL11B have been tracked in human NK cells, direct evidence showing that BCL11B actively drives NK cell maturation is lacking. Here, we genetically engineered hiPSCs with inducible overexpression of BCL11B via CRISPR/Cas9-mediated gene knock-in and investigated the temporal role of this transcription factor during hematopoietic differentiation. We demonstrated that BCL11B upregulation during the mesodermal stage of development enhances arterial specification from hiPSCs differentiated in chemically defined conditions. The boost in arterial programming by BCL11B was associated with enhanced HE formation and a DLL4 +CXCR4 + arterial phenotype (KDR +CD144 +DLL4 +CXCR +CD73 -CD43 -). We also demonstrated that enhancing definitive hematopoiesis with lympho-myeloid potential could be achieved through modulation of BCL11B expression. Transient BCL11B overexpression enhanced the production of multipotent hematopoietic progenitors (CD34 +CD43 +CD45 +CD235a -CD41a -) and multipotent colony-forming cells (CFCs) in iPSC differentiation culture. Flow cytometry analysis of day 10 cultures showed that early BCL11B expression yielded a 3-fold increase in the arterial hemogenic endothelial (AHE) and a 2.5-fold increase in the putative HSC population. In combination with conventional HSC markers (CD34 +CD38 -CD45RA -CD43 +CD90 +), we also observed the upregulation of the HSC-specific marker CD49f (ITGA6) from dox-treated iBCL11B-iPSCs. CD49f + cells are highly efficient in generating long-term multilineage grafts compared to CD49f - cells. Additionally, our results demonstrated that BCL11B overexpression significantly promoted NK cell generation under chemically defined culture conditions. The resulting iBCL11B-hiPSC-derived NK cells exhibited various mature NK-specific markers and displayed effective cellular cytotoxicity, with more rapid killing kinetics compared to NK cells derived from no-dox-treated iBCL11B-iPSCs. We conclude that BCL11B-enforced activation during early mesoderm induction yields an increased population of hematopoietic progenitors with lymphoid and myeloid lineage potential, serving as an improved off-the-shelf cell source for targeted cancer immunotherapy.

Epitope Edited Hematopoietic Stem Cells Allow Immune-Based In Vivo Selection of Genome-Engineered Cells

Hematopoietic stem/progenitor cell (HSPC) transplantation (HSCT) offers curative options for patients affected by conditions for which the substitution of endogenous hematopoiesis with genetically corrected or healthy donor-derived cells can halt the pathogenic process. Nonetheless, the short and long-term adverse effects of genotoxic conditioning remain a substantial barrier to a wider application of HSCT and gene therapy. HSCT survivors suffer from life-long irreversible consequences, such as chronic graft-versus-host-disease (GvHD), infertility, susceptibility to viral infections, organ damage (including renal, liver and lung) and increased risk of secondary malignancies. Whereas monoclonal and toxin-conjugated antibodies (Abs) targeting HSPCs have been proposed as an alternative to chemo/radiotherapy, the mechanism of action and pharmacokinetics of these agents hamper their safe clinical use due to the risk of on target killing of transplanted HSPCs. To avoid this, the cell product is not administered at peak myeloablation to allow for washout of the conditioning agent, further impairing engraftment and therapeutic potential. Here, we show that precise editing of the targeted epitope in HSPCs can endow hematopoietic lineages with selective resistance to monoclonal Abs (mAbs) without affecting stem cell function. This strategy allows improved myeloablation - which can continue after HSCT - and progressive enrichment of gene-modified cells. We have identified amino-acid changes in the extracellular domain of KIT (CD117) that abrogate the binding of therapeutic mAbs without affecting surface expression, ligand affinity, kinase phosphorylation, downstream signaling, transcriptional and proliferative response (Fig.A). We exploited adenine base editing (BE) to introduce these mutations with high efficiency (~80%) in CD34+ HSPCs, with preservation of long-term repopulating and multilineage differentiation capacity in primary and secondary recipient mice. To ensure full preservation of KIT functionality, we performed growth analysis, RNA sequencing, and phospho-proteomic analyses of epitope-edited HSPCs stimulated with KIT ligand (stem cell factor, SCF) and observed no difference in the proliferation, transcriptional, and phosphorylation response compared to controls. In vitro differentiation of edited HSPCs toward myeloid, macrophage, classical dendritic, granulocytic and megakaryocytic lineages was similar to controls and did not result in counterselection of edited cells. Lineage-differentiated cells showed preserved functionality, including: reactive oxygen species production by myeloid cells, E.coli phagocytosis by macrophages, M1/M2-like macrophage polarization, phospho-flow profiling of IL4-, PMA/ionomycin-, GM-CSF-, IFN type-I-, IL-6- and LPS-stimulated myeloid cells, HLA class-II/CD86 upregulation by DCs, induction of granulocyte NETosis and generation of hyperdiploid megakaryocytes. By plating edited HSPCs with increasing concentrations of the mAb, we observed preserved expansion of edited HSPCs in response to SCF while control cells were inhibited in a dose-dependent manner. To improve HSPCs therapies for hemoglobinopathies, such as sickle cell disease, we multiplexed BE of the KIT epitope and two BCL11A erythroid enhancer motifs. We obtained editing efficiencies comparable to single editing and the erythroid progeny of triple-edited cells showed upregulation of HbF, as expected (~40%). In a competitive xeno-transplantation setting, with co-injection of multiplex BE and AAVS1-BE control CD34+ cells, mice treated with KIT mAb showed a 2-fold co-enrichment of both KIT and BCL11A editing when compared to mock treated controls, confirming the possibility to select gene-modified cells up to therapeutic thresholds. Similarly, mAb conditioning combined with epitope-edited grafts allowed human hematopoietic substitution in mice pre-humanized with unedited CD34+ HSPCs (89% substitution with 7.3-fold higher absolute cell counts, Fig.B), compared to AAVS1-edited controls that failed to engraft. We envision a new paradigm for non-genotoxic conditioning based on transplantation of epitope engineered HSPCs resistant to immunotherapeutic agents to achieve engraftment and in vivo selection of gene corrected HSPC with substantial advantages over alternatives in terms of safety and tolerability.

Polyamines Are Required for AML LSC Function through Their Role in Regulating eIF5A Dependent Protein Synthesis

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapies to fully eradicate disease initiating leukemia stem cells (LSCs) (Shlush et al., 2017). Many studies have demonstrated that LSCs have unique metabolic properties compared to AML blasts and normal hematopoietic stem and progenitor cells (HSPCs) (Jones et al., 2021). In this study we sought to quantify metabolite levels in LSCs compared to HSPCs using an unbiased metabolites screen. To examine metabolite levels in enriched LSCs and HSPCs we used mass spectrometry-based metabolomics analysis on enriched LSCs from 18 AML patients and HSPCs from 5 normal bone marrow (BM) specimens. Pathway analysis showed that arginine metabolism and biosynthesis were the most significantly enriched pathways in LSCs compared to HSPCs. Clinical studies have shown limited efficacy of targeting arginine levels using arginine degrading enzymes; therefore, we sought to target pathways downstream of arginine. To identify the pathways that arginine metabolism supports in LSCs, we used stable isotope labeled tracing analysis which showed arginine was metabolized through the urea cycle into polyamines in LSCs. Interestingly, the polyamine spermidine was the most significantly enriched metabolite in LSCs compared to HSPCs in our metabolomics analysis. Moreover, expression of SAT1, the enzyme catalyzing the export of polyamines from the cells, was decreased in AML compared to normal BM. Based on these observations, we sought to determine if the polyamine pathway represents a metabolic vulnerability that can be exploited to target LSCs. Polyamines are cationic compounds which have been previously shown to be important for the growth of various cancers but their role in AML LSCs has yet to be determined. To investigate if polyamines are essential for LSC survival and function, we treated LSCs and HSPCs with N 1, N 11-diethylnorspermine (DENSpm), a polyamine analog causing polyamine depletion through increased SAT1 expression and subsequent cellular export. DENSpm treatment decreased the viability of LSCs in 10 out of 12 of patients tested but had no impact on HSPCs viability. Further, treatment with DENSpm resulted in decreased engraftment potential of 5 AML specimens but did not alter the engraftment of HSPCs from 3 normal BM specimens, suggesting that targeting polyamines represents a promising approach to kill LSCs while sparing HSPCs. We then investigated the mechanisms by which polyamines are essential in LSCs. Using RNA-sequencing analysis on enriched LSCs from 5 AML patients and HSPCs from 3 normal BM specimens we observed an enrichment in myeloid differentiation signatures in DENSpm-treated LSCs but not in DENSpm-treated HSPCs. In line with this, expression of myeloid markers CD11b and CD15 were increased in patient-derived xenograft models engrafted with DENSpm-treated LSCs. As we did not observe such dysregulation in mice engrafted with DENSpm-treated HSPCs, these data suggest polyamine depletion promotes differentiation of LSCs but not HSPCs. Our transcriptomic analysis also unveiled decreased expression of genes involved in protein synthesis in DENSpm-treated LSCs but not in normal HSPCs. Protein synthesis and its regulation are critical for AML survival (Chen et al., 2019; Messling et al., 2022) but the role of polyamines in protein synthesis in LSCs has not been explored. Polyamines stimulate translation notably by serving as precursors for the hypusination of eukaryotic translation initiation factor 5A (eIF5A) (Park et al., 2010). Accordingly, DENSpm led to decreased hypusination of eIF5A in AML cells. This was associated with reduced protein synthesis level in AML cell lines and LSCs but not in normal HSPCs. Strikingly, eIF5A hypusination, protein synthesis and viability were rescued upon co-treatment with spermidine. Moreover, knockdown of eIF5A using siRNA resulted in impaired colony forming potential of AML and altered engraftment in patient-derived xenograft models. Altogether, these results suggest that DENSpm alters AML cell and LSC viability and function at least in part through decreased eIF5A-mediated protein translation. Altogether, our data demonstrates that depletion of polyamines decreased LSC function while sparing normal HSPCs. Mechanistically, our data suggest that LSC targeting is accomplished through decreased eIF5A-mediated synthesis of proteins crucial for LSCs.

Maternal Obesity Causes Sex Specific Long-Term Hematopoietic and Metabolic Defects

Maternal obesity is increasingly common and can negatively impact offspring health. Children born to obese mothers are at higher risk of developing diseases involved with metabolic health (e.g., type 2 diabetes). Many of these diseases are associated with abnormalities within the hematopoietic system such as atypical immune profiles or hematopoiesis. Interestingly, disease incidence and/or severity is often dependent on the offspring's gender. This suggests that there may be sex-specific reprogramming of hematopoietic stem and progenitor cells (HSPCs) during immune cell development. This study aims to test the hypothesis that offspring of obese mice exhibit sex-differences in HSPC function that affect offspring's metabolic health. To test this, C57BL/6 (CD45.2+) dams were fed standard chow (2018SX, Envigo, IN) or western-style diet (TD.88137, Envigo, IN) for four weeks prior to mating, through pregnancy, and the lactating period. At postnatal day 21, HSPCs (Lineage negative, Sca1 positive, cKit positive cells) were flow-sorted from bone marrow (BM) of offspring from dams fed chow (Con) or western-style diet (MatOb) and used for competitive transplant (Boy/J BM competitor cells; CD45.1+) into same-sex lethally irradiated C57BL/6 x Boy/J F1 (CD45.1+CD45.2+) recipient mice (8-10 weeks of age) or for RNA-sequencing analysis. Secondary transplants were performed to look at long-term engraftment potential of HSPCs. Body weight, body adiposity, fasting insulin, glucose tolerance test (GTT), and pancreatic β and α-cell area were assessed on male and female recipients of both primary and secondary transplants. Compared to mice that received male Con HSPCs, recipient mice that received male MatOb HSPCs had significantly lower donor chimerism in the blood and BM post-secondary transplantation. This was associated with a significant reduction in donor hematopoietic stem cell (HSC) numbers in the BM 16 weeks post-secondary transplant, indicating male MatOb HSPCs had decreased long-term engraftment capability due to an exhaustion of the HSC pool. This reduction in HSPC engraftment seen between male MatOb versus Con HSPCs was not seen when using HSPCs from female offspring. Next, metabolic parameters of our recipient mice was examined. Sixteen weeks after primary transplant, there were no differences detected in body weight and adiposity in the Con and MatOb HSPC recipient mice. Compared to sex-matched controls, we found that recipients transplanted with male MatOb HSPCs developed glucose intolerance, increased fasting serum insulin concentrations, and decreased β-cell area, supporting the presence of impaired glucose metabolism due to a combination of insulin resistance and lower β-cell mass. There was no difference in recipients of HSPCs from female Con and female MatOb pups. RNA-seq of BM HSPCs at time of transplant (p21) from Con or MatOb revealed 100 differentially expressed genes (DEG) in male MatOb HSPCs and 1788 DEG in female MatOb HSPCs. There were more and higher magnitude of differential gene expression in female mice than male, despite the long-term phenotypic differences being evident in males but not females, and the majority of genes changed in males were also altered in females. This suggests that the gene programs responsible for the observed phenotypes may be altered by maternal obesity in both male and female offspring, but that female offspring have a transcriptomic profile that confers protection on the HSPC population. One of the most striking findings from the transcriptomic analysis was a marked enrichment in gene programs associated with immune response, interferon signaling, and cytokine production in female MatOb offspring compared to sex matched controls that is not seen in male MatOb offspring. Pathway analysis identified upregulation of programs involving both IFNγ and IL-10 in the female MatOb HSPCs when compared to sex-mated Con HSPCs which correlated with an increase in IL-10 concentrations in the serum of p21 female MatOb mice. These pathways were downregulated or unaltered in males with no change in systemic IL-10 concentration.Our results demonstrate that maternal obesity results in sex-specific changes in offspring HSPC function and a novel role HSPCs may play in regulating glucose metabolism in the offspring of obese dams.

Circadian Rhythms Metabolically Regulate Bone Marrow Retained Primitive Hematopoietic Stem Cell Size and Function

Most primitive hematopoietic stem cells (pHSC; LSK SLAM CD34 - EPCR +) are bone marrow (BM) retained in a quiescent non motile state, and are the only chemotherapy resistant HSC which requires their rapid exit from homeostatic quiescence on demand to prevent lethal hematology failure. Circadian rhythms induce daily release of progenitor cells and low levels of rarely cycling pHSCs to replenish the blood and lymphatics with new maturing cells. Metabolically, pHSCs are characterized by their dependence on glycolysis, with low mitochondrial activity and membrane potential (MMP), associated with higher competitive repopulation potential. However, if their metabolism is influenced by circadian cues is currently unknown. We report here that pHSCs at night are more glycolytic with higher surface expression of the glucose transporter Glut1, and higher glucose uptake. Repeated administration of the glucose analog, 2-deoxyglucose (2-DG), during daylight mimicked the night phenotype and enhanced HSC engraftment potential. However, nocturnal administration of 2-DG did not further improve glucose uptake nor HSC engraftment potential, suggesting glycolytic saturation. To further understand these metabolic oscillations of glucose uptake, we investigated the circadian, transcriptional signatures by single pHSC RNA-seq analysis. We found higher expression of mitophagy-related genes at night. Genes regulating mitochondrial function, were also significantly down regulated at night leading to low MMP in pHSC. While mitochondrial ROS levels were higher, cytoplasmic ROS levels were significantly reduced in pHSC at night. Importantly, we further found at night, low-functioning pHSC mitochondria to also undergo higher levels of fission as assessed by increased expression of the active mitochondrial fission marker, phospho-Drp1 (S616). To better understand the molecular pathways mediating daily circadian metabolic reprogramming between glycolysis and mitochondrial activity, we monitored our gene set enrichment analysis. We found the Wnt/b-catennin and HIF-1a signaling pathways to be significantly increased at night in pHSC. Active Wnt signaling, leads to enhanced b-catenin localization to the nucleus which is enhanced by downstream effector of Wnt activation, FOXM1. We observed higher total protein expression of FOXM1 at night. While its nuclear and cytoplasmic localization were reduced, we observed significantly higher FOXM1 levels in the mitochondria. FOXM1 localization to the energy powerhouse has been shown to inhibit mitochondrial activity. Blocking mitochondrial recruitment of FOXM1 with a specific canonical Wnt inhibitor at night led to upregulation of MMP, reversing mitochondrial metabolic responses in pHSC. Finally, daily genetic changes between night Wnt, HIF-1a and glycolysis, versus daylight GSK3b, m-TOR signaling and enhanced mitochondrial activity in BM retained pHSC, also led to cell size changes. We documented reduced quiescent pHSC size at night associated with their increased BM maintenance and higher repopulation potential. While larger sized pHSCs in the morning during the time of blood replenishment, increased their migration and development potential, for their rapid exit from quiescence on demand. Our study reveals daily circadian cues dynamically determine Wnt-mediated FOXM1 bidirectional shuttling between the nucleus and the mitochondria, leading to metabolic switching of pHSCs to maintain their cellular fitness. Our data define key signaling pathways controlling pHSC quiescence and diverse circadian metabolic activities for accumulated ROS and old mitochondria clearance and turnover, keeping them ready for rapid activation in alarm situations.

Generation of a C57BL/6J mouse strain expressing the CD45.1 epitope to improve hematopoietic stem cell engraftment and adoptive cell transfer experiments.

Adoptive cell transfer between genetically identical hosts relies on the use of a congenic marker to distinguish the donor cells from the host cells. CD45, a glycoprotein expressed by all hematopoietic cells, is one of the main congenic markers used because its two isoforms, CD45.1 and CD45.2, can be discriminated by flow cytometry. As a consequence, C57BL/6J (B6; CD45.2) and B6.SJL-Ptprca Pepcb/BoyJ (B6.SJL; CD45.1) mice are widely used in adoptive cell transfer experiments, under the presumption that they differ only at the CD45 (Ptprc) locus. However, recent studies have identified genetic variations between these congenic strains and have notably highlighted a differential expression of cathepsin E (CTSE). The B6.SJL mouse presents a number of functional differences in hematopoietic stem cell engraftment potential and immune cell numbers compared with the B6 mouse. In this study, we showed that B6 and B6.SJL mice also differ in their CD8+ T cell compartment and CD8+ T cell responses to viral infection. We identified Ctse as the most differentially expressed gene between CD8+ T cells of B6 and B6.SJL and demonstrated that the differences reported between these two mouse strains are not due to CTSE. Finally, using CRISPR-Cas9 genome editing, we generated a CD45.1-expressing B6 mouse by inserting one nucleotide mutation (A904G) leading to an amino acid change (K302E) in the Ptprc gene of the B6 mouse. We showed that this new B6-Ptprcem(K302E)Jmar/J mouse resolves the experimental biases reported between the B6 and B6.SJL mouse lines and should thus represent the new gold standard for adoptive cell transfer experiments in B6.

Human Induced Pluripotent Stem Cell-Derived Bone Marrow Organoids to Model Hematopoietic Development

The human bone marrow niche plays an essential role in supporting and regulating hematopoiesis throughout life and inherited bone marrow failure syndromes are often linked to dysfunction of the niche. Recently, methods for generating induced pluripotent stem cell (iPSC)-derived hematopoietic organoids (Motazedian et al., 2020) and bone marrow organoids (Khan et al., 2022) have been developed. We recently presented our approach to generate a model of a complex human bone marrow microenvironment in vitro (ASH annual meeting 2022 ). Here, we characterize our model in greater detail and analyse functionality of bone marrow organoid (BMO)-derived HSPCs. We devised a feeder- and serum-free protocol to generate a complex human iPSC-derived BMO within three weeks. In comparison to the protocol by Khan et al., we incorporated a sequential combination of Wnt activation and Activin/Nodal inhibition to induce mesoderm formation and patterning. We replicated the method with five different iPSC lines, including fibroblast- and PBMC-derived iPSCs, as well as iPSCs generated from renal epithelial cells isolated from urine samples. Using confocal and two-photon microscopy we visualized the spatial architecture of differentiated BMOs and detected CD45 + cells embedded into a network of CD31 + vascular cells and CD271 + stromal cells. PDGFRβ expressing pericytes were enwrapping the vascular cells. Moreover, stromal cells expressed CXCL12, Leptin receptor and Nestin. Electron microscopy, HE staining of organoid sections and 3D surface rendering showed lumen-forming vascular structures containing hematopoietic cells. To further elucidate the cell type composition, we performed single-cell RNA sequencing (scRNA-seq) of differentiated BMOs. Clustering of the scRNA-seq data by marker gene expression yielded three main populations: Cells in the endothelial cluster expressed canonical genes of arterial ECs. In the mesenchymal cluster we could distinguish pericytes, vascular smooth muscle cells and osteochondrogenic precursors. In the hematopoietic compartment, we identified cells expressing molecular signatures of granulocytes, monocytes and their progenitors, but also megakaryocytic-like cells and lymphoid progenitors. Remarkably, a cluster of cells expressed genes of fetal HSCs. This suggests that BMOs may recapitulate definitive hematopoiesis. To test T-cell differentiation potential in vitro, we sorted CD34 + HSPCs from dissociated BMOs and differentiated them within an artificial thymic organoid system (ATO). After three weeks we detected CD45 +CD4 +CD8 + cells, some of them expressing CD3 +TCRαβ +. Thus, we postulate that T-cells phenotypically resembling conventional T-cells emerge from BMO-derived HSPCs. To assess functional properties of BMO-derived HSPCs in vivo, we transplanted sorted CD34 + HSPCs into immunodeficient NSG mice and are currently analysing their engraftment potential. In summary, we provide a novel bone marrow niche model to study hematopoietic development in a complex three-dimensional organoid derived from human iPSCs.

Metabolic Programming of Hematopoietic Stem Cell Function By Prenatal Folate

Hematopoietic stem cells (HSCs) are programmed by metabolic changes resulting in alterations to HSC function. Here, we discovered that varying prenatal folate status metabolically programs HSCs during development and affects adult HSC self-renewal and engraftment potential into adulthood. Folate status varies greatly in the global population based on nutritional intake, common genetic polymorphisms, and widespread supplementation. Folate-derived one-carbon metabolism (OCM) regulates cellular methylation, de novo nucleotide biosynthesis and mitochondrial metabolism, processes critical to HSC function and establishment. Despite immense therapeutic potential, there is limited understanding of mechanisms driving HSC developmental programming. We hypothesize that varying prenatal folate status affects risk for hematologic dysfunction by developmentally programming HSCs. To test how prenatal folate modulated HSC function, female mice were assigned to one of three experimental diets to model population-wide folate consumption: 0mg/kg (deficient, FD), 2mg/kg (control, FC) and 8mg/kg (supplemented, FS). Profiling of fetal liver (FL) hematopoietic cells revealed that FD diet caused expansion of long term (LT)-HSCs that was propagated across the hematopoietic hierarchy, including myeloid and lymphoid progenitors and mature myeloid cells. In contrast, FS decreased FL cell output. We next determined if these changes were driven by metabolic activity. FD fetal HSPCs exhibited higher mitochondrial membrane potential and lower glycolytic activity, whereas FS fetal HSPCs displayed both higher glycolytic activity and oxygen consumption despite impaired production of downstream hematopoietic cells. Together, these data suggest that HSPC metabolic programming initiates during fetal development and is folate-dose dependent. To determine if prenatal folate programs hematopoiesis into adulthood, we quantified bone marrow (BM) hematopoietic cells in adult offspring after weaning onto standard chow. Surprisingly, FD expanded lymphoid progenitors in adult offspring with no impact on mature lymphoid cell output. In contrast, FS diet expanded myeloid and lymphoid progenitors in the BM as well as downstream mature cells, including monocytes and B-cells. To determine if these effects were HSC cell-intrinsic, we performed competitive transplantation assays of LT-HSCs isolated from adult offspring. FD offspring HSCs showed worse engraftment by chimerism across all peripheral blood (PB) populations compared to FC. In comparison, FS offspring HSCs showed enhanced total cellular output by donor cells/uL across all PB mature lineages except platelets. Secondary whole BM transplantation revealed that FS HSCs retained enhanced total cellular output and superior engraftment compared to FC, whereas FD HSCs demonstrated complete exhaustion. These data indicate that adult HSC function is programmed by prenatal folate status. To investigate molecular alterations driving metabolic programming of adult hematopoiesis by prenatal folate, we performed single-cell RNA sequencing (Scseq) and metabolomics on adult offspring HSPCs. Scseq analysis revealed most differential gene expression (DE) in HSC clusters, with less DE in progenitors. Re-clustering of all HSCs revealed distinct heterogeneity within this population, where both FD and FS HSCs were identified as distinct clusters compared to FC. Metabolomic profiling further suggested that FD HSCs exhibited signs of mitochondrial dysfunction through, 1) accumulating glucose and lactate suggesting reliance on glycolysis and, 2) accumulating succinate and glutamine suggesting complex II impairment and inadequate utilization of mitochondrial metabolism. Surprisingly, the same changes associated with mitochondrial dysfunction in FD HSCs persisted downstream into lymphoid-biased multipotent progenitors. In contrast, limited metabolic changes were observed in FS HSCs, suggesting alternative pathways for sustained programming. These changes indicate that FD and FS offspring have distinct metabolic programming. Together, our data suggest that prenatal folate status programs hematopoietic stem cell function into adulthood and is driven by alterations to metabolic activity. Ongoing studies examine how prenatal folate status affects the adult hematological and immune response to challenge.

Assessment of Transduction of CD34+ Human Hematopoietic Stem Cells from Patients with Severe Hemophilia-Α with Lentiviral Vector Carrying a High Expression FVIII Transgene (CD68-ET3-LV)

Background: Alternative strategies are needed for the large number of people with hemophilia who are ineligible for AAV based gene therapy due to age or high levels of anti-AAV neutralizing antibodies. High inter-individual variability of expression and ill sustained factor levels are also challenges with AAV based gene therapy for hemophilia A, in particular. We have developed a third-generation lentiviral vector mediated hematopoietic stem cell-based gene therapy for hemophilia A. (Doering et al Human Gene Therapy 2018; 29: 1183-1201) This vector (CD68-ET3-LV) has a high expression FVIII transgene with a CD68 promoter targeting expression in monocytic cells predominantly. We report here successful transduction of severe hemophilia A patient derived human hematopoietic stem cells with this vector. Methods: Mobilized peripheral blood stem cells were collected by apheresis from three patients with severe hemophilia A without inhibitors who also received prophylactic clotting factor replacement therapy during this period. CD34+ hematopoietic stem cells (HSCs) were enriched on the CliniMACS Plus® system (Miltenyi Biotec, Bergish Gladbach, Germany) using the CliniMACS CD34® reagent system. Purified HSC were then transduced with this CD68-ET3-LV as follows - HSCs cultured on retronectin coated surfaces in xenofree media with cytokines were exposed to clinical grade CD68-ET3-LV vector in two ways - a double transduction protocol without any enhancer and a single transduction with an enhancer. After completion of this step, the product was assessed for viability and vector copy number (VCN) by trypan blue labelling and Q-PCR performed on genomic DNA from CFU cells, respectively. Transduced HSCs were also assessed for their engraftment potential. CD68-ET3-LV vector transduced HSC (1x10 6) were transplanted into NBSGW mice via tail vein injection. Engraftment was assessed at 16 weeks after transplantation. This protocol was approved by the Institutional Review Board of the Christian Medical College, Vellore, India. Results: G-CSF (10 ug/kg/day) based peripheral blood stem cell mobilization was well tolerated by all participants. The apheresis procedures which were done with plasma FVIII levels in the normal range after prophylactic replacement therapy were unremarkable. The total collection of mobilized CD34+ cells from the three donors was 268±80.5x10 6 CD34+ cells. An aliquot of 2x10 6 CD34+ cells/ml was used in the transduction experiments. The data on viability and vector copy number post transduction shown in the tables 1 and 2 confirm that viability was not affected by the manipulation of these HSCs in both double and single transduction methods. The vector copy number (VCN) in the genomic DNA obtained from CFU cells collected from CD34+ HSCs cultured in MethoCult H4434™ (Stem Cell Technologies™, Vancouver, Canada) varied from 0.62 to 1.21 in double transduction (n=3) and nearly doubled in the single transduction method with an enhancer to VCNs of 1.5 and 2.4 in two samples tested. Transplantation studies in NBSGW mice showed successful engraftment of human CD34+ HSCs with a mean engraftment of 80.99 ± 4.2% of CD45+ multilineage hematopoietic cells in the bone marrow 16 weeks after transplantation. Conclusion: To the best of our knowledge, this is the first report of transduction of mobilized peripheral blood CD34+ HSCs from patients with severe hemophilia A using a clinical grade lentiviral vector with a high expression FVIII transgene. These data establish feasibility and safety of the procedure. The transduction protocol showed good efficiency with very high viability, significant VCN and good engraftment of these gene modified human HSCs in a mouse model. These protocols are currently being evaluated in a first in human phase 1 clinical trial of gene therapy for severe hemophilia A without inhibitors.

Pathobiology and Targeting of CD38 in Cutaneous T-Cell Lymphoma

Introduction: Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature CD4+ T-cells that primarily affects the skin. Despite a wide variety of topical and systemic therapies for patients, CTCL remains difficult to treat. Because of its high likelihood of relapse and resistance, new therapeutic approaches are needed. We recently showed strong CD38 expression on neoplastic T-cells from patients with mature T-cell neoplasms, including CTCL. In this study, we evaluated i) the role of CD38 in CTCL pathogenesis ii) strategies to enhance CD38 expression in CTCL and iii) tested efficacy of combination therapy in vivo. Methods: Skin and peripheral blood samples from patients and healthy donors were evaluated for CD38 expression by immunohistochemistry (N=7), microarray (N=82), single-cell RNA-sequencing (N=11), and flow cytometry (N=14). To investigate the role of CD38 in CTCL, we generated CD38 knockout cell lines (H9, HH, and HuT-78) using CRISPR/Cas9-mediated genomic deletion. The CD38 wild-type (CD38 WT) and knockout (CD38 KO) cells were purified, cultured, and used for downstream functional analysis. We evaluated the lines for growth, metabolic capacities (XFe24 Seahorse assay), and engraftment potential in vivo. For in vivo therapeutic studies, we transduced firefly-luciferase gene into H9 CD38 WT and CD38 KO cells, and engrafted them intravenously into immunodeficient NOD Rag -/-γc -/- mice. Mice were treated with anti-CD38 antibody daratumumab or IgG control and disease progression was monitored over time using an in vivo imaging system. For combination studies, we evaluated CD38 expression by flow cytometry in H9 cells treated with increasing doses of HDAC inhibitor, panobinostat (5nM, 10nM, 25nM) for 24, 48, and 72 hours. Next, we tested co-treatment with panobinostat and daratumumab in vivo in mice engrafted with H9 CD38 WT cells with four treatment groups (vehicle/IgG, panobinostat/IgG, vehicle/daratumumab, and panobinostat/daratumumab). Results: Patient skin biopsies showed increased CD38 expression at both protein and RNA levels (log fold change 4.8; p<0.0001 by Mann-Whitney test). Across nine days of tracking under standard cell culture conditions, CD38 KO CTCL cells showed minimal growth differences compared to CD38 WT (p=0.22 by linear regression). However, CD38 KO CTCL cells showed significantly increased basal and maximum oxygen consumption rates (OCR) via seahorse assay when compared to CD38 WT cells (basal OCR 2.26 fold increase, p<0.0001; max OCR 1.83 fold increase, p<0.0001 by Mann-Whitney test). Using IVIS luminescent imaging, we found significantly increased in vivo growth of CD38 KO CTCL cells compared to CD38 WT cells (CD38 WT=2.2e8 photons/sec average total flux, N=7; CD38 KO=1e9 photons/sec, N=5; p=0.003 by Mann-Whitney test). These data suggest that the loss of CD38 in CTCL cells can accelerate the development of tumors in mice. In order to study the effectiveness of a targeted drug against CD38 in CTCL, we used anti-CD38 antibody daratumumab in our H9 xenograft model. Daratumumab treatment had a significant therapeutic effect on CD38 WT tumors compared to isotype control, reducing tumor burden by 84% (daratumumab average total flux=1.4e7 photons/sec, N=4; IgG average total flux=9.0e7 photons/sec, N=3; p=0.0002). Additionally, we explored methods to increase CD38 expression on CTCL cells. We found that panobinostat significantly increased CD38 expression on H9 CTCL cells in a dose dependent manner across multiple time points (max 85% increase in CD38 expression with 25nM panobinostat vs. DMSO at 72 hours; p<0.0001 by 2way ANOVA). In vivo testing demonstrated a statistically significant improved survival benefit in mice treated with the combination of panobinostat and daratumumab compared to mice that received daratumumab alone (vehicle median survival 23 days, N=4; daratumumab alone median survival 32 days N=4; panobinostat alone median survival 27.5 days, N=4; combination median survival 39 days, N=4; p=0.01 by log-rank test). The prolonged average survival benefit amounted to approximately one quarter of their entire overall lifespan. Conclusion: Our studies demonstrate strong evidence for further study into how CD38 regulates the growth and survival of CTCL cells. Our data also provide preliminary evidence for the clinical usefulness of combination therapies that increase CD38 expression to enhance tumor cell immunotherapeutic targeting.

A Novel CD64 CAR-T Therapy for the Treatment of Monocytic Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) is a difficult to treat, life threatening malignancy with overall survival remaining extremely poor, approximately 15% at 5 years. Recent advances in therapeutic approaches for AML have significantly improved outcomes, most notably venetoclax and azacitidine which induces a remission in approximately 70% of patients. Despite this, the majority of patients ultimately relapse and succumb to their disease. We and others have previously shown that more monocytic (M5 by FAB classification) AML cells demonstrate increased resistance to venetoclax therapy and that relapse after venetoclax and azacitidine treatment shows a monocytic phenotype in approximately 30% of patients (Pei, et. al. Cancer Discovery, 2020). These patients experience decreased overall survival compared to patients with non-M5 AML. Furthermore, we have demonstrated that AML with a dominant monocytic phenotype can arise from a unique subclass of leukemia stem cells (LSCs) that we have termed monocytic-LSCs (m-LSCs) due to their immunophenotype, including expression of CD64. m-LSCs are highly resistant to venetoclax-based therapies (Pei, et. al., Cancer Discovery, 2023). As venetoclax is increasingly utilized in AML treatment regimens, it is essential to identify methods of targeting this m-LSC subtype to prevent and treat relapsed and refractory disease. Methods: To more effectively target monocytic AML, we have developed a novel chimeric antigen receptor (CAR) T cell therapy targeting CD64, an antigen commonly expressed in monocytic cells and strongly expressed in the m-LSC population. We generated two second-generation CAR constructs targeting CD64, one with CD28 and the other with 41BB costimulatory domains. We expressed these CARs in healthy donor human T cells via lentiviral transduction and tested their function in vitro and in vivo. Results: We demonstrate that CD64 CAR-T cells effectively treat monocytic AML using pre-clinical models. CD64 CAR-T cells eliminate CD64+ leukemia cell lines in vitro in a dose dependent manner with an effector to target (E:T) ratio of 1:4 and higher eliminating 100% of Molm14 leukemia cells at 18 hours compared to 0-10% in mock T-cell controls. We also show that CD64 CAR-T cells eliminate established Molm14 leukemia in vivo, with 100% of CAR-T treated mice surviving over 50 days with no evidence of leukemia versus less than 30 days survival with high tumor burden in mock treated mice (p<0.01, Figure 1). We find that CD64 CAR-T cells with CD28 verses 41BB costimulatory domains are similarly efficacious against monocytic AML, though we find more prolonged persistence of CD64-41BBz CAR-T cells in mice with long term leukemia free survival. Furthermore, we demonstrate that CD64 CAR-T cells kill CD64+ primary patient derived AML cells in ex vivo co-culture, resulting in a 60-80% reduction in viable AML cells at 18 hours across 1:1 to 1:16 E:T ratios compared to mock T cell controls. We further show that CD64 CAR-T cells target m-LSCs and reduce the engraftment potential of primary AML cells in mice with a nearly 9-fold reduction in leukemia burden in CAR-T treated versus control mice. Finally, preliminary experiments with AML patient derived xenograft models further support the in vivo efficacy of CD64 CAR-T cell treatment. Conclusions: Our data provide promising preclinical evidence for the efficacy of a novel CD64 CAR-T therapy in the treatment of monocytic AML. We propose that using this regimen to augment AML therapy is a potentially powerful means to eradicate m-LSCs, which are inherently resistant to venetoclax, and thereby increase remission duration. This concept serves as the basis for an upcoming clinical trial of CD64-directed CAR-T cells for CD64 expressing AML. Figure 1: CD64 CAR-T cells eliminate AML in in vivo murine leukemia models. A) NSG mice leukemia models of GFP expressing Molm14, a CD64+ human leukemia cell line, experience leukemia clearance within 3 days after treatment with CD64-28z CAR-T cells, as shown by decreased bioluminescent signal in images of CAR-T treated mice. Mock transduced human T-cells from the same human donor serve as a control. B) Survival curves demonstrate 100% survival of CAR-T treated mice over the experiment (p<0.01).

A Necroptosis Inhibitor Functions As a Ferroptosis Inducer in Drug-Resistant Myeloid Malignancies

Introduction: Acute myeloid leukemia (AML) is associated with poor prognosis, especially in elderly patients unfit for intensive chemotherapy. AML chemotherapy agents, including venetoclax, a BH3-mimetic, are designed to induce cell death through apoptosis. Unfortunately, AML cells eventually develop a resistance to BCL-2-dependent apoptosis which causes clinical disease recurrence. Alternative cell death pathways like ferroptosis and necroptosis are lytic forms of cell death mediated by plasma membrane lipid oxidation or MLKL phosphorylation (p-MLKL), respectively, both of which disrupt plasma membrane integrity. Because apoptosis and ferroptosis involve different molecular pathways, ferroptosis-inducing therapies provide an opportunity to overcome therapy resistance in AML patients. Here, we show how necroptosis inhibitor targeting basal necroptosis signal selectively induces ferroptosis in venetoclax-resistant AML cells causing cell death. Methods: We established venetoclax-resistant myeloid cell lines by exposing cell lines to increasing doses of venetoclax. We evaluated resistance to venetoclax-induced apoptosis by measuring IC50 after Annexin-V/PI staining. We screened venetoclax-resistant myeloid cell lines and primary AML cells (n=77) for basal activation of necroptosis by pMLKL or phosphorylated RIP3 (pRIP3) western blotting. Xenotransplantation was performed to evaluate the bone marrow engraftment potentials of basal activated necroptosis primary AMLs. Necrosome inhibitor (Zharp-99) was tested to estimate IC50 for cell death induction in myeloid cell lines after Annexin-V/PI staining. Ferroptosis inhibitors (ferrostatin-1 and lipoxstatin-1) and apoptosis inhibitor (zVAD-FMK) were used to determine which death pathway was induced by necroptosis inhibitors in AML. Results: We established venetoclax-resistant myeloid cell lines (THP1-VR and HL60-VR), which showed a significant shift in IC50 for venetoclax-induced apoptosis compared to parent cell lines. Venetoclax-resistant cell lines acquired an upregulation of known anti-apoptotic proteins; BCL-2, BCL-XL, and MCL-1. Venetoclax-resistant cell lines upregulated pRIP3 signal, a known necroptosis driver, by western blot. Necrosome screening of primary AML samples revealed 73 AMLs (94%) showed basal necroptosis signal confirmed by pMLKL with heterogeneous intensities (Figure. A). Xenotransplantation confirmed engraftment of basal necroptosis-activated primary AMLs is achievable and with retention of necroptosis signal at engraftment. The basal necroptosis signal in venetoclax-resistant cell lines and primary AML samples drove us to evaluate necroptosis inhibitor Zharp-99 (RIP3K inhibitor) IC50 for venetoclax-resistant cell lines. Compared to parent cell lines, venetoclax-resistant cell lines became susceptible to cell death by necrosome inhibition. Surprisingly, the cell death induced by Zharp-99 was not from apoptosis but through ferroptosis, confirmed by the venetoclax-resistant myeloid cells being rescued from cell death by ferroptosis inhibitor (ferrostatin-1 and lipoxstatin-1) (Figure B). Conclusion: Basal activation of necrosome is observed in venetoclax-resistant myeloid cell lines and primary AML patient samples. We found necroptosis inhibitor Zharp-99 promoted cell death in necrosome-activated venetoclax-resistant cell lines. Our data suggest necroptosis inhibition targeting RIP3K activity has a novel function of inducing ferroptosis in myeloid malignancies resistant to apoptosis. RIP3K inhibitor, as a novel function of ferroptosis inducer in apoptosis-resistant myeloid cell lines, opens a new therapy opportunity to treat apoptosis resistance in AML patients. We plan to investigate in vivo effects of necrosome inhibitors using our xenotransplant model.