Human Hematopoietic Progenitor Research Articles

Targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells invivo using the engineered AVID adenovirus vector platform

Targeted delivery and cell-type-specific expression of gene-editing proteins in various cell types invivo represent major challenges for all viral and non-viral delivery platforms developed to date. Here, we describe the development and analysis of artificial vectors for intravascular delivery (AVIDs), an engineered adenovirus-based gene delivery platform that allows for highly targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells (HSPCs) invivo after intravenous vector administration. Due to a set of refined structural modifications, intravenous administration of AVIDs did not trigger cytokine storm, hepatotoxicity, or thrombocytopenia. Single intravenous administration of AVIDs to humanized mice, grafted with human CD34+ cells, led to up to 20% transduction of CD34+CD38-CD45RA- HSPC subsets in the bone marrow. Importantly, targeted invivo transduction of CD34+CD38-CD45RA-CD90-CD49f+ subsets, highly enriched for human hematopoietic stem cells (HSCs), reached up to 19%, which represented a 1,900-fold selectivity in gene delivery to HSC-enriched over lineage-committed CD34-negative cell populations. Because the AVID platform allows for regulated, cell-type-specific expression of gene-editing technologies as well as expression of immunomodulatory proteins to ensure persistence of corrected HSCs invivo, the HSC-targeted AVID platform may enable development of curative therapies through invivo gene correction in human HSCs after a single intravenous administration.

Human Hematopoietic Stem Cells Have Altered Mitochondrial Activity after Stem Cell Transplantation

Hematopoietic stem cells (HSCs) give rise to the entire blood system. With their high regenerative potential, they are sometimes the only curative option for many malignancies. The success of HSCT depends on both the quantity of HSCs and on their ability to produce life-long blood. However, their regenerative potential is not unlimited. Transplanted HSCs sustain injury that alters their subsequent ability to generate blood cells. This leads to ineffective hematopoiesis and can ultimately lead to graft failure. Injury to HSCs is also a risk factor for secondary neoplasms, including leukemia. Our goal is to elucidate the mechanisms behind post-transplant HSC decline. Mitochondria are critically important for HSC function (Filippi Exp Hematol 2021). During regeneration of the bone marrow, HSCs become activated and undergo mitochondrial reprogramming, including reorganization of the mitochondrial network and enhanced mitochondrial activity (Filippi et al Blood 2019, Ito et al Nat Rev Mol Cell Biol 2014). However, after transplantation, HSCs accumulate mitochondria that are abnormal in organization and activity. These abnormal mitochondria lead to decline of HSC function and ineffective hematopoiesis (Hinge et al Cell Stem Cell 2020). While these findings have been replicated in murine models, it has not been studied within the human system. Our hypothesis is that after transplantation, human HSCs have altered mitochondria leading to ineffective hematopoiesis. We examined changes in HSC function using post-transplant samples of patient's bone marrow and a xenotransplant system (human CD34+ peripheral blood stem cells transplanted into sublethally irradiated immunodeficient NOD/SCID/IL-2 receptor-γ null mice). We first measured mitochondrial membrane potential using tetramethyl rhodamine ethyl ester (TMRE) dye and analyzed via flow cytometry. Three to six months after transplant in the xenotransplant model, we found that the mitochondrial membrane potential of huCD34+CD38-CD90+ HSCs was similar to the non-transplanted HSCs. However, huCD34+CD38+ progenitors exhibited a drastic 3-fold increase in mitochondrial membrane potential in comparison to the non-transplanted, huCD34+CD38+ progenitor cells. The mitochondrial membrane potential of CD34+CD38+ progenitors from transplanted bone marrow patient samples was also higher than non-transplanted CD34+CD38+bone marrow control cells. To understand if alterations in mitochondrial activity affect HSC differentiation, we placed xenotransplanted HSCs into in vitro culture. Post-transplanted huCD34+ cells failed to expand within 6 days of in vitro culture (serum-free culture with self-renewing cytokines, SCF+TPO+FLT3) compared to huCD34+ control. In subsequent differentiation assays, post-transplanted huCD34+ cultures gave rise to myeloid cells only. This contrasts with control huCD34+ cultures that produced myeloid, erythroid, and megakaryocytic cells. Interestingly, if the post-transplanted HSCs were given serum and cytokines for differentiation after only 3 days in self-renewing conditions, the cultured cells expanded similarly to non-transplanted HSCs and generated myeloid, erythroid, and megakaryocytic cells. This suggests that transplanted HSCs have decreased proliferation and differentiation ability in comparison to non-transplanted HSCs and respond differently to cytokines in vitro. This could account for ineffective hematopoiesis observed in some post-transplant patients. In conclusion, this study indicates that both mitochondrial activity and in-vitro growth change in HSCs after transplantation, despite re-establishing quiescence. This indicates that human hematopoietic stem and progenitor cells incur injury after transplantation. These findings are clinically important as the changes in mitochondrial activity in HSCs may play a role in post-transplant hematopoietic stem cell decline. Future single cell RNA sequencing analyses will provide mechanistic information regarding post-transplant changes in metabolism and potential targets for improving HSC function in transplant patients.

Loss of Metabolic Control Beyond the Promyelocyte Stage Resolves Myeloid Maturation Arrest in Reticular Dysgenesis

Reticular Dysgenesis (RD) is a particularly grave syndromic form of severe combined immunodeficiency. The hematopoietic phenotype manifests in defective myeloid and lymphoid development with profound neutropenia and lymphopenia. RD is caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2), which catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) in the mitochondrial intermembrane space. ADP is then phosphorylated to adenosine triphosphate (ATP) during the OXPHOS process. While there are abundant other sources of APD, AK2 constitutes the only mitochondrial enzyme that contributes to AMP “recycling” by phosphorylation. Accordingly, AK2-deficient myeloid cells have been shown to exhibit increased AMP levels 1. Beyond this observation, the cellular basis for the combined hematopoietic defect in RD remains elusive. Using a CRISPR/Cas9 AK2 biallelic knock-out model in human hematopoietic stem and progenitor cells (HSPCs), we have previously shown that AK2-/- HSPCs recapitulate RD myelopoiesis defects in vitro. AK2-/- HSPCs exhibit a severe proliferative defect that becomes apparent at the myelocyte (MC) and neutrophil (NP) stages, while the promyelocyte (PM) stage appears relatively spared, consistent with observations in RD patients. Our prior metabolomic analysis showed that AK2-/- MCs not only have increased levels of AMP but up of 20-fold increased levels of IMP derived by converting excess AMP to IMP conversion via AMP deaminases (AMPDs). In addition, we have shown that beginning at the MC stage, AK2 -/- cells exhibit an increase in NADH:NAD+ ratio, consistent with reductive stress and decreased aspartate levels. Our new data revealed that expressing the aspartate transporter GLAST and/or the water-forming NADH oxidase LbNOX in AK2-/- HSPCs partially rescues differentiation but not the proliferation defect. It raises the possibility that reductive stress and aspartate deficiency compromise protein synthesis necessary for maturation, but are separate from the proliferative defect related to high IMP and AMP levels. There were no metabolomic abnormalities noted at the PM stage. PMs have a higher proliferative index than MCs and NPs. This raises the question why the most metabolically active stage is phenotypically spared by AK2 deficiency. To better understand why the metabolic abnormalities associated with RD selectively affect differentiation beyond the promyelocyte stage, we investigated key regulators of cellular metabolism and proliferation, i.e., AMPK, ACC1 and S6, a target of mTOR signaling, at PM, MC and NP stages. We found activation of AMPK, ACC1 and S6 only at the PM stage, while AMPK, ACC1 and S6 protein expression at MC and NP stages was virtually undetectable. Interestingly, AK2-/- PMs exhibited notably lower ATP synthesis rates compared to controls, while ATP synthesis in AK2-/- MCs and NPs was significantly higher than in controls of corresponding stages, paralleling the accumulation of AMP and IMP noted earlier. These findings raise the possibility that tight metabolic control at the promyelocyte stage prevents AMP levels from rising by curtailing OXPHOS metabolism in AK2-/- PMs. In contrast, lack of metabolic control beyond the PM stage permits unopposed OXPHOS activity, thereby allowing AMP and IMP levels to rise to toxic levels that interfere with proliferation. In our ongoing work, we are testing whether pharmacologically redirecting metabolism from OXPHOS to glycolysis with UK5099, could rescue AK2-/- myelopoiesis defects. UK5099 is a potent inhibitor of the mitochondrial pyruvate carrier, which facilitates pyruvate transport across the mitochondrial inner membrane to fuel the TCA cycle. Additionally, we developed a hematopoietic-specific and inducible Cre-mediated Ak2 knock-out mouse model, which recapitulates the profound neutropenia and lymphopenia observed in RD patients. This mouse model will allow us to corroborate our in vitro observations and to investigate if the same mechanistic underpinnings also apply to the lymphoid lineage. Rissone A, Weinacht KG, la Marca G, et al. Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress. J Exp Med. Jul 27 2015;212(8):1185-202. doi:10.1084/jem.20141286

BloodChIP Xtra: an expanded database of comparative genome-wide transcription factor binding and gene-expression profiles in healthy human stem/progenitor subsets and leukemic cells.

The BloodChIP Xtra database (http://bloodchipXtra.vafaeelab.com/) facilitates genome-wide exploration and visualization of transcription factor (TF) occupancy and chromatin configuration in rare primary human hematopoietic stem (HSC-MPP) and progenitor (CMP, GMP, MEP) cells and acute myeloid leukemia (AML) cell lines (KG-1, ME-1, Kasumi1, TSU-1621-MT), along with chromatin accessibility and gene expression data from these and primary patient AMLs. BloodChIP Xtra features significantly more datasets than our earlier database BloodChIP (two primary cell types and two cell lines). Improved methodologies for determining TF occupancy and chromatin accessibility have led to increased availability of data for rare primary cell types across the spectrum of healthy and AML hematopoiesis. However, there is a continuing need for these data to be integrated in an easily accessible manner for gene-based queries and use in downstream applications. Here, we provide a user-friendly database based around genome-wide binding profiles of key hematopoietic TFs and histone marks in healthy stem/progenitor cell types. These are compared with binding profiles and chromatin accessibility derived from primary and cell line AML and integrated with expression data from corresponding cell types. All queries can be exported to construct TF-gene and protein-protein networks and evaluate the association of genes with specific cellular processes.

Prostaglandin E2 as transduction enhancer affects competitive engraftment of human hematopoietic stem and progenitor cells

Exvivo gene therapy (GT) is a promising treatment for inherited genetic diseases. An ideal transduction protocol should determine high gene marking in long-term self-renewing hematopoietic stem cells (HSCs), preserving their repopulation potential during invitro manipulation. In the context of the improvement of a clinically applicable transduction protocol, we tested prostaglandin E2 (PGE2) as a transduction enhancer (TE). The addition of PGE2 shortly before transduction of human CD34+ cells determined a significant transduction increase in the invitro cell progeny paralleled by a significant reduction of their clonogenic potential. This effect increased with the duration of PGE2 exposure and correlated with an increase of CXCR4 expression. Blockage of CXCR4 with AMD3100 (plerixafor, Mozobil) did not affect transduction efficiency but partially rescued CD34+ clonogenic impairment invitro. Once transplanted invivo in a competitive repopulation assay, human CD34+ cells transduced with PGE2 contributed significantly less than cells transduced with a standard protocol to the repopulation of recipient mice, indicating a relative repopulation disadvantage of the PGE2-treated CD34+ cells and a counter-selection for the PGE2-treated cell progeny invivo. In conclusion, our data indicate the need for risk/benefit evaluations in the use of PGE2 as a TE for clinical protocols of GT.

Non-Viral Episomal Vector Mediates Efficient Gene Transfer of the β-Globin Gene into K562 and Human Haematopoietic Progenitor Cells.

β-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPβ-globin for the physiological β-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the β-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the β-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce β-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPβ-globin provides the basis for the development of therapeutic EV for gene therapy of β-thalassemias.

Genotoxic effects of base and prime editing in human hematopoietic stem cells

Base and prime editors (BEs and PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses and genotoxicity. Here, we compared state-of-the-art BEs and PEs and Cas9 in human hematopoietic stem and progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes and on-target and genome-wide genotoxicity. BEs and PEs induced detrimental transcriptional responses that reduced editing efficiency and hematopoietic repopulation in xenotransplants and also generated DNA double-strand breaks and genotoxic byproducts, including deletions and translocations, at a lower frequency than Cas9. These effects were strongest for cytidine BEs due to suboptimal inhibition of base excision repair and were mitigated by tailoring delivery timing and editor expression through optimized mRNA design. However, BEs altered the mutational landscape of hematopoietic stem and progenitor cells across the genome by increasing the load and relative proportions of nucleotide variants. These findings raise concerns about the genotoxicity of BEs and PEs and warrant further investigation in view of their clinical application.

Fluorinated Silane-Modified Filtroporation Devices Enable Gene Knockout in Human Hematopoietic Stem and Progenitor Cells.

Intracellular delivery technologies that are cost-effective, non-cytotoxic, efficient, and cargo-agnostic are needed to enable the manufacturing of cell-based therapies as well as gene manipulation for research applications. Current technologies capable of delivering large cargoes, such as plasmids and CRISPR-Cas9 ribonucleoproteins (RNPs), are plagued with high costs and/or cytotoxicity and often require substantial specialized equipment and reagents, which may not be available in resource-limited settings. Here, we report an intracellular delivery technology that can be assembled from materials available in most research laboratories, thus democratizing access to intracellular delivery for researchers and clinicians in low-resource areas of the world. These filtroporation devices permeabilize cells by pulling them through the pores of a cell culture insert by the application of vacuum available in biosafety cabinets. In a format that costs less than $10 in materials per experiment, we demonstrate the delivery of fluorescently labeled dextran, expression plasmids, and RNPs for gene knockout to Jurkat cells and human CD34+ hematopoietic stem and progenitor cell populations with delivery efficiencies of up to 40% for RNP knockout and viabilities of >80%. We show that functionalizing the surfaces of the filters with fluorinated silane moieties further enhances the delivery efficiency. These devices are capable of processing 500,000 to 4 million cells per experiment, and when combined with a 3D-printed vacuum application chamber, this throughput can be straightforwardly increased 6-12-fold in parallel experiments.

Stabilization of the human cytomegalovirus UL136p33 reactivation determinant overcomes the requirement for UL135 for replication in hematopoietic cells.

Human cytomegalovirus (HCMV) is a beta herpesvirus that persists indefinitely in the human host through a latent infection. The polycistronic UL133-UL138 gene locus of HCMV encodes genes regulating latency and reactivation. While UL138 is pro-latency, restricting virus replication in CD34+ hematopoietic progenitor cells (HPCs), UL135 overcomes this restriction and is required for reactivation. By contrast, UL136 is expressed with later kinetics and encodes multiple proteins with differential roles in latency and reactivation. Like UL135, the largest UL136 isoform, UL136p33, is required for reactivation from latency in HPCs; viruses failing to express either protein are unresponsive to reactivation stimuli. Furthermore, UL136p33 is unstable, and its instability is important for the establishment of latency, and sufficient accumulation of UL136p33 is a checkpoint for reactivation. We hypothesized that stabilizing UL136p33 might overcome the requirement of UL135 for replication. We generated recombinant viruses lacking UL135 that expressed a stabilized variant of UL136p33. Stabilizing UL136p33 did not impact the replication of the UL135 mutant virus in fibroblasts. However, in the context of infection in HPCs, stabilization of UL136p33 strikingly compensated for the loss of UL135, resulting in increased replication in CD34+ HPCs and in humanized NOD-scid IL2Rγcnull (huNSG) mice. This finding suggests that while UL135 is essential for replication in HPCs, it functions largely at steps preceding the accumulation of UL136p33, and that stabilized expression of UL136p33 largely overcomes the requirement for UL135. Taken together, our genetic evidence indicates an epistatic relationship between UL136p33 and UL135, whereby UL135 may initiate events early in reactivation that drive the accumulation of UL136p33 to a threshold required for productive reactivation. IMPORTANCE Human cytomegalovirus (HCMV) is one of nine human herpesviruses and a significant human pathogen. While HCMV establishes a lifelong latent infection that is typically asymptomatic in healthy individuals, its reactivation from latency can have devastating consequences in the immunocompromised. Defining viral genes important in the establishment of or reactivation from latency is important to defining the molecular basis of latent and replicative states and in controlling infection and CMV disease. Here we define a genetic relationship between two viral genes in controlling virus reactivation from latency using primary human hematopoietic progenitor cells and humanized mouse models.

An in vitro model of human hematopoiesis identifies a regulatory role for the aryl hydrocarbon receptor

AbstractIn vitro models to study simultaneous development of different human immune cells and hematopoietic lineages are lacking. We identified and characterized, using single-cell methods, an in vitro stromal cell–free culture system of human hematopoietic stem and progenitor cell (HSPC) differentiation that allows concurrent development of multiple immune cell lineages. The aryl hydrocarbon receptor (AHR) is a ligand–activated transcription factor influencing many biological processes in diverse cell types. Using this in vitro model, we found that AHR activation by the highly specific AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, drives differentiation of human umbilical cord blood–derived CD34+ HSPCs toward monocytes and granulocytes with a significant decrease in lymphoid and megakaryocyte lineage specification that may lead to reduced immune competence. To our knowledge, we also discovered for the first time, using single-cell modalities, that AHR activation decreased the expression of BCL11A and IRF8 in progenitor cells, which are critical genes involved in hematopoietic lineage specification processes at both transcriptomic and protein levels. Our in vitro model of hematopoiesis, coupled with single-cell tools, therefore allows for a better understanding of the role played by AHR in modulating hematopoietic differentiation.

Base editors dissect genetic variants in human hematopoietic cells on a large scale

In a recent study, Martin-Rufino and colleagues combined massively parallel base editing in primary human hematopoietic stem and progenitor cells (HSPCs) with functional and single-cell transcriptomic readouts. A series of proof-of-principle experiments highlight the breadth of applications made possible with this approach, which range from gene therapy and immunotherapy, to characterizing single nucleotide variants.

A Microwell Device for the Efficient Generation of Arrays of Microtissues and Humanized Bone Marrow Micro-Ossicles

(1) Background: There are no high-throughput microtissue platforms for generating bone marrow micro-ossicles. Herein, we describe a method for the assembly of arrays of microtissues from bone marrow stromal cells (BMSC) in vitro and their maturation into bone marrow micro-ossicles in vivo. (2) Methods: Discs with arrays of 50 microwells were used to assemble microtissues from 3 × 105 BMSCs each on a nylon mesh carrier. Microtissues were cultured in chondrogenic induction medium followed by hypertrophic medium in an attempt to drive endochondral ossification, and then they were implanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, where they were remodeled into bone marrow micro-ossicles. Mice were transplanted with 105 human umbilical cord blood CD34+ cells. (3) Results: Micro-ossicles contained more human CD45+ cells, but fewer human CD34+ progenitor cells than mouse marrow. Human hematopoietic progenitor cells cycle rapidly at non-physiological rates in mouse marrow, and reduced CD34+ cell content in micro-ossicles is consistent with the notion that the humanized niche better controls progenitor cell cycling. (4) Conclusions: Assembling microtissues in microwells, linked by a nylon membrane carrier, provides an elegant method to manufacture and handle arrays of microtissues with bone organ-like properties. More generally, this approach and platform could aid bridging the gap between in vitro microtissue manipulation and in vivo microtissue implantation.

Analysis of Immunophenotypic Changes during Ex Vivo Human Erythropoiesis and Its Application in the Study of Normal and Defective Erythropoiesis.

Erythropoiesis is a highly regulated process and undergoes several genotypic and phenotypic changes during differentiation. The phenotypic changes can be evaluated using a combination of cell surface markers expressed at different cellular stages of erythropoiesis using FACS. However, limited studies are available on the in-depth phenotypic characterization of progenitors from human adult hematopoietic stem and progenitor cells (HSPCs) to red blood cells. Therefore, using a set of designed marker panels, in the current study we have kinetically characterized the hematopoietic, erythroid progenitors, and terminally differentiated erythroblasts ex vivo. Furthermore, the progenitor stages were explored for expression of CD117, CD31, CD41a, CD133, and CD45, along with known key markers CD36, CD71, CD105, and GPA. Additionally, we used these marker panels to study the stage-specific phenotypic changes regulated by the epigenetic regulator; Nuclear receptor binding SET Domain protein 1 (NSD1) during erythropoiesis and to study ineffective erythropoiesis in myelodysplastic syndrome (MDS) and pure red cell aplasia (PRCA) patients. Our immunophenotyping strategy can be used to sort and study erythroid-primed hematopoietic and erythroid precursors at specified time points and to study diseases resulting from erythroid dyspoiesis. Overall, the current study explores the in-depth kinetics of phenotypic changes occurring during human erythropoiesis and applies this strategy to study normal and defective erythropoiesis.

Abstract A56: TP53 mutant human AMLs stem cells rely on the proteasome for self-renewal

Abstract AML with TP53 mutations (TP53Mut) is a poor-risk AML subtype that is largely insensitive to chemotherapy, targeted agents, and hematopoietic stem cell transplantation. TP53Muts are seen in approximately 10-20% of AML and confer a particularly poor prognosis with 1–2-year survival rates to 0-10%. Self-renewal is the defining feature of leukemia stem cells (LSCs) and is central to leukemia progression and relapse. Mutant p53 protein enhances self-renewal in murine models of AML and in human hematopoietic progenitors. In solid tumors, analysis of a variety of TP53Muts defined the proteasome pathway as a common transcriptional target and mediator of oncogenesis among these mutations. Notably, the proteasome degrades I kappa B which activates NF kappa B, which enforces AML self-renewal. Proteasome inhibitors have shown some efficacy in AML clinical trials but have not been well-evaluated in TP53Mut patients. We hypothesize that the proteasome is mediator of self-renewal in TP53Mut AML. To define the transcriptional features of TP53Mut AML, we analyzed the gene expression data in the BEAT AML dataset of primary human AML samples (Tyner, et al. 2018). Relative to TP53WT (n=377 samples), the TP53 mutant samples (n=36) were enriched in every NF kappa B and proteasome gene-set queried. Next, we analyzed a single cell RNA seq dataset of human AML samples (van Galen et al. 2019) and found that the LSC compartment of TP53Mut samples (n=3) strongly upregulated every proteasome gene set we queried (relative to TP53WT samples (n=13)). A recent in vitro screen identified a panel of small molecule inhibitors that reduced viability in TP53-deleted cell lines (Nechiporuk et al. 2019). We tested the activity of these inhibitors in primary human TP53Mut samples. Four of these inhibitors were effective in reducing colony forming capacity or reduced LSC frequency in TP53Mut samples (n=9). We used CyTOF to assess intracellular signaling pathways in LSCs and found that the inhibitors that were most effective in abolishing colony formation and reducing LSC frequency also attenuated NF kappa B levels in TP53Mut LSCs. Accordingly, we found that knockdown of mutant TP53 reduced NF kappa B levels in Kasumi cells (a TP53R248Q AML cell line). Next, we treated a panel of primary human TP53Mut AML samples with the pan-proteasome inhibitors, carfilzomib and bortezomib (FDA approved for lymphoid malignancies) and an immunoproteasome inhibitor, PR957 (in clinical trials for autoimmune conditions). Each drug independently reduced NF kappa B and led to a significant reduction of primary and secondary colony formation. These data suggest that the proteasome/NF kappa B pathway may be an important determinant of self-renewal function in TP53Mut AML and proteasome inhibitors may target the LSCs of this treatment-refractory AML subtype. Citation Format: Marie Lue Antony, Yoonkyu Lee, Klara E Noble-Orcutt, Zohar Sachs. TP53 mutant human AMLs stem cells rely on the proteasome for self-renewal [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A56.

Abstract A54: Polyamines are required for AML LSC function through their role in regulating eIF5A dependent protein synthesis

Abstract 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 (Jones et al., 2021). However, the metabolite profiles of human LSCs and hematopoietic stem and progenitor cells (HSPCs) have not yet been directly compared. 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 9 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. Stable isotope labeled tracing analysis showed arginine was metabolized through the urea cycle into polyamines in human 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 N1,N11-diethylnorspermine (DENSpm), a polyamine analog causing polyamine depletion through increased cellular export. DENSpm treatment decreased the viability of LSCs and AML blasts in 10 out of 12 of patients tested but had no impact on HSPC viability. Further, treatment with DENSpm resulted in decreased engraftment potential of the 4 AML specimens examined in immune deficient mice but did not alter the engraftment of normal HSPCs, suggesting that targeting polyamines represents a promising approach to kill LSCs while sparing HSPCs. We then investigated the mechanisms regulated by polyamines in LSCs. Using RNA-sequencing analysis on enriched LSCs from 5 AML patients and HSPCs from 3 normal BM specimens we observed decreased expression of genes involved in protein synthesis in DENSpm-treated LSCs but not 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 translation 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 reduced protein synthesis level in MOLM13 AML cell line and primary AML samples. Strikingly, protein synthesis and viability were rescued upon co-treatment with spermidine, suggesting that DENSpm induces AML cells death 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 polyamine regulated protein synthesis. Citation Format: Vincent Rondeau, Cristiana O’Brien, Jacob M Berman, Duhan Yendi, Tianyi Ling, Aleksandra Bourdine, Aarushi Gupta, Jonathan St-Germain, Rachel Culp-Hill, Julie A Reisz, Soheil Jahangiri-Tazehkand, Anastasia Tikhonova, Andrea Arruda, Mark D Minden, Angelo D’Alessandro, Brian Raught, Courtney L Jones. Polyamines are required for AML LSC function through their role in regulating eIF5A dependent protein synthesis [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A54.

Massively parallel base editing to map variant effects in human hematopoiesis

Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.

Physical biomarkers for human hematopoietic stem and progenitor cells

Adhesion of hematopoietic stem and progenitor cells (HSPCs) to the bone marrow niche plays critical roles in the maintenance of the most primitive HSPCs. The interactions of HSPC−niche interactions are clinically relevant in acute myeloid leukemia (AML), because (i) leukemia-initiating cells adhered to the marrow niche are protected from the cytotoxic effect by chemotherapy and (ii) mobilization of HSPCs from healthy donors' bone marrow is crucial for the effective stem cell transplantation. However, although many clinical agents have been developed for the HSPC mobilization, the effects caused by the extrinsic molecular cues were traditionally evaluated based on phenomenological observations. This review highlights the recent interdisciplinary challenges of hematologists, biophysicists and cell biologists towards the design of defined in vitro niche models and the development of physical biomarkers for quantitative indexing of differential effects of clinical agents on human HSPCs.

Efficient engineering of human and mouse primary cells using peptide-assisted genome editing.

Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing. Unlike electroporation-based methods, PAGE gene editing has low cellular toxicity and shows no significant transcriptional perturbation. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. PAGE provides a broadly generalizable platform for next-generation genome engineering in primary cells.

The Humanized Mouse Model: What Added Value Does It Offer for HIV Research?

In the early 2000s, novel humanized mouse models based on the transplantation of human hematopoietic stem and progenitor cells (HSPCs) into immunocompromised mice were introduced (hu mice). The human HSPCs gave rise to a lymphoid system of human origin. The HIV research community has greatly benefitted from these hu mice. Since human immunodeficiency virus (HIV) type 1 infection results in a high-titer disseminated HIV infection, hu mice have been of great value for all types of HIV research from pathogenesis to novel therapies. Since the first description of this new generation of hu mice, great efforts have been expended to improve humanization by creating other immunodeficient mouse models or supplementing mice with human transgenes to improve human engraftment. Many labs have their own customized hu mouse models, making comparisons quite difficult. Here, we discuss the different hu mouse models in the context of specific research questions in order to define which characteristics should be considered when determining which hu mouse model is appropriate for the question posed. We strongly believe that researchers must first define their research question and then determine whether a hu mouse model exists, allowing the research question to be studied.

Simultaneous inhibition of Sirtuin 3 and cholesterol homeostasis targets acute myeloid leukemia stem cells by perturbing fatty acid β-oxidation and inducing lipotoxicity.

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapeutic regimens to fully eradicate disease initiating leukemia stem cells (LSCs). Previous studies have demonstrated that oxidative phosphorylation (OXPHOS) is an essential process that is targetable in LSCs. Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation, has been shown to regulate OXPHOS in cancer models; however, it has not yet been studied in the context of LSCs. Thus, we sought to identify if SIRT3 is important for LSC function. Using RNAi and a SIRT3 inhibitor (YC8-02), we demonstrate that SIRT3 is a critical target for the survival of primary human LSCs but is not essential for normal human hematopoietic stem and progenitor cell (HSPC) function. To elucidate the molecular mechanisms by which SIRT3 is essential in LSCs we combined transcriptomic, proteomic, and lipidomic approaches, showing that SIRT3 is important for LSC function through the regulation of fatty acid oxidation (FAO) which is required to support oxidative phosphorylation and ATP production in human LSCs. Further, we discovered two approaches to further sensitize LSCs to SIRT3 inhibition. First, we found that LSCs tolerate the toxic effects of fatty acid accumulation induced by SIRT3 inhibition by upregulating cholesterol esterification. Disruption of cholesterol homeostasis sensitizes LSCs to YC8-02 and potentiates LSC cell death. Second, SIRT3 inhibition sensitizes LSCs to BCL-2 inhibitor venetoclax. Together, these findings establish SIRT3 as a regulator of lipid metabolism and potential therapeutic target in primitive AML cells.