Hematopoietic Stem Cell Population Research Articles

Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow.

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors (MkPs) are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) mainly in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in adult mice. Single-cell colony assay and single-cell transplantation showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average 8 megakaryocytes per colony) than did previously reported MkPs. Single-cell RNA sequencing supported that HSCs give rise to MkPs through MgPs along a Mk differentiation pathway. Single-cell reverse transcription polymerase chain reaction (RT-PCR) analysis showed that MgPs expressed Mk-related genes, but were transcriptionally heterogenous. Clonal culture of HSCs suggested that MgPs are not direct progeny of HSCs. We propose a differentiation model in which HSCs give rise to MgPs which then give rise to MkPs, supporting a classic model in which Mk-lineage commitment takes place at a late stage of differentiation.

Abstract 4215: Single-cell proteomic characterization of the tumor microenvironment in humanized NOG-EXL patient-derived xenograft (PDX) models

Abstract Introduction: CD34+ hematopoietic stem cell (HSC) derived humanized mice engineered to express human cytokine transgenes are promising models for the evaluation of novel immunotherapeutic treatments. While flow cytometry-based analyses have confirmed the presence of many human immune cell populations within humanized mice bearing patient-derived tumor xenografts (PDX), comprehensive high-dimensional immunophenotyping of the tumor microenvironment (TME) of these models remains limited. Beyond this, the relative influence of PDX vs. donor CD34+ HSC populations on TME composition is poorly understood. Here, we characterize the quantity and diversity of immune cell subpopulations in the TME, bone marrow, and spleen of humanized NOG-EXL (huNOG-EXL) mice using Cytometry by Time-Of-Flight (CyTOF) data with > 60 features. Methods: We performed CyTOF analysis on 250 dissociated samples from primary tumors (n = 15; n = 6 head and neck cancer, n = 6 ovarian cancer, n = 3 renal cell carcinoma), as well as xenograft (n = 59), spleen (n = 88), and bone marrow (n = 88) samples from huNOG-EXL mice (Taconic). Each sample was profiled with two partially overlapping antibody panels; an innate immunity-focused panel (40 features), and an adaptive immunity-focused panel (40 features). Results: CyTOF data were collected from over 10 million live immune cells for each of the adaptive and innate antibody panels. Unbiased clustering of single-cell marker expression revealed clusters corresponding to all major lymphoid and myeloid cell types. Therapeutically relevant cell states were identified including exhausted CD8+ T cells marked by PD-1 and Tim-3 expression, and PD-L1+ macrophages. Hierarchical clustering of bone marrow and spleen samples suggested that systemically, immune composition was not driven by tumor-intrinsic factors alone with many samples clustering on the basis of CD34+ stem cell donor ID. Conversely, clustering PDX samples revealed strong consistency in xenografts originating from the same primary tumor sample regardless of the chosen stem cell donor. The immune composition of primary tumor samples showed greater similarity with matched PDX samples versus unmatched PDX samples, further supporting that tumor-intrinsic factors drive immune composition in xenografts. Conclusions: We provide a high-resolution characterization of the immune phenotypes present in huNOG-EXL PDX models across three cancer types. These data captured substantial heterogeneity in the immune composition of humanized xenograft samples, and we demonstrate that immune composition in humanized xenografts is strongly driven by tumor-intrinsic features. Overall, this resource data supports the use of these models for immuno-oncology studies with future work focused on functionally validating specific immune cell populations of interest. Citation Format: Daniel Stueckmann, Jalna Meens, Sally Zhang, Shirley Hui, Sujana Sivapatham, Sephane Chevrier, Jennifer Pfeil, Lisa Martin, Maria Komisarenko, Philip Dube, Susan Prendeville, Hartland Jackson, Antonio Finelli, Gary Bader, Bernd Bodenmiller, Keith A. Lawson, Laurie Ailles. Single-cell proteomic characterization of the tumor microenvironment in humanized NOG-EXL patient-derived xenograft (PDX) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4215.

Phenotypic Analysis of Hematopoietic Stem and Progenitor Cell Populations in Acute Myeloid Leukemia Based on Spectral Flow Cytometry, a 20-Color Panel, and Unsupervised Learning Algorithms.

The analysis of hematopoietic stem and progenitor cell populations (HSPCs) is fundamental in the understanding of normal hematopoiesis as well as in the management of malignant diseases, such as leukemias, and in their diagnosis and follow-up, particularly the measurement of treatment efficiency with the detection of measurable residual disease (MRD). In this study, I designed a 20-color flow cytometry panel tailored for the comprehensive analysis of HSPCs using a spectral cytometer. My investigation encompassed the examination of forty-six samples derived from both normal human bone marrows (BMs) and patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) along with those subjected to chemotherapy and BM transplantation. By comparing my findings to those obtained through conventional flow cytometric analyses utilizing multiple tubes, I demonstrate that my innovative 20-color approach enables a more in-depth exploration of HSPC subpopulations and the detection of MRD with at least comparable sensitivity. Furthermore, leveraging advanced analytical tools such as t-SNE and FlowSOM learning algorithms, I conduct extensive cross-sample comparisons with two-dimensional gating approaches. My results underscore the efficacy of these two methods as powerful unsupervised alternatives for manual HSPC subpopulation analysis. I expect that in the future, complex multi-dimensional flow cytometric data analyses, such as those employed in this study, will be increasingly used in hematologic diagnostics.

β-glucans from Agaricus bisporus mushroom products drive Trained Immunity.

Macrofungi, such as edible mushrooms, have been used as a valuable medical resource for millennia as a result of their antibacterial and immuno-modulatory components. Mushrooms contain dietary fibers known as β-glucans, a class of polysaccharides previously linked to the induction of Trained Immunity. However, little is known about the ability of mushroom-derived β-glucans to induce Trained Immunity. Using various powdered forms of the white button mushroom (Agaricus bisporus), we found that mouse macrophages pre-treated with whole mushroom powder (WMP) displayed enhanced responses to restimulation with TLR ligands, being particularly sensitive to Toll-like receptor (TLR)-2 stimulation using synthetic lipopeptides. This trained response was modest compared to training observed with yeast-derived β-glucans and correlated with the amount of available β-glucans in the WMP. Enriching for β-glucans content using either a simulated in-vitro digestion or chemical fractionation retained and boosted the trained response with WMP, respectively. Importantly, both WMP and digested-WMP preparations retained β-glucans as identified by nuclear magnetic resonance analysis and both displayed the capacity to train human monocytes and enhanced responses to restimulation. To determine if dietary incorporation of mushroom products can lead to Trained Immunity in myeloid cells in vivo, mice were given a regimen of WMP by oral gavage prior to sacrifice. Flow cytometric analysis of bone-marrow progenitors indicated alterations in hematopoietic stem and progenitor cells population dynamics, with shift toward myeloid-committed multi-potent progenitor cells. Mature bone marrow-derived macrophages derived from these mice displayed enhanced responses to restimulation, again particularly sensitive to TLR2. Taken together, these data demonstrate that β-glucans from common macrofungi can train innate immune cells and could point to novel ways of delivering bio-available β-glucans for education of the innate immune system.

Disruption of placental ACKR3 impairs growth and hematopoietic development of offspring.

ACKR3 scavenges and degrades the stem cell recruiting chemokine CXCL12, which is essential for proper embryonic and, in particular, haematopoietic development. Here, we demonstrate strong expression of ACKR3 on trophoblasts. Using a maternally administered pharmacological blocker and Cre-mediated genetic approaches, we demonstrate that trophoblast ACKR3 is essential for preventing movement of CXCL12 from the mother to the embryo, with elevated plasma CXCL12 levels being detected in embryos from ACKR3-blocker-treated mothers. Mice born to mothers treated with the blocker are lighter and shorter than those born to vehicle-treated mothers and, in addition, display profound anaemia associated with a markedly reduced bone marrow haematopoietic stem cell population. Importantly, although the haematopoietic abnormalities are corrected as mice age, our studies reveal a postnatal window during which offspring of ACKR3-blocker-treated mice are unable to mount effective inflammatory responses to inflammatory/infectious stimuli. Overall, these data demonstrate that ACKR3 is essential for preventing CXCL12 transfer from mother to embryo and for ensuring properly regulated CXCL12 control over the development of the haematopoietic system.

Autophagy prevents graft failure during murine graft-versus-host disease

AbstractAutophagy is an intracellular survival process that has established roles in the long-term survival and function of hematopoietic stem cells (HSC). We investigated the contribution of autophagy to HSC fitness during allogeneic transplantation and graft-versus-host disease (GVHD). We demonstrate in vitro that both tumor necrosis factor and IL-1β, major components of GVHD cytokine storm, synergistically promote autophagy in both HSC and their more mature hematopoietic progenitor cells (HPC). In vivo we demonstrate that autophagy is increased in donor HSC and HPC during GVHD. Competitive transplant experiments demonstrated that autophagy-deficient cells display reduced capacity to reconstitute the hematopoietic system compared to wild-type counterparts. In a major histocompatibility complex-mismatched model of GVHD and associated cytokine dysregulation, we demonstrate that autophagy-deficient HSC and progenitors fail to establish durable hematopoiesis, leading to primary graft failure and universal transplant related mortality. Using several different models, we confirm that autophagy activity is increased in early progenitor and HSC populations in the presence of T-cell–derived inflammatory cytokines and that these HSC populations require autophagy to survive. Thus, autophagy serves as a key survival mechanism in HSC and progenitor populations after allogeneic stem cell transplant and may represent a therapeutic target to prevent graft failure during GVHD.

3D-bioprinted bone scaffolds incorporating SR1 nanoparticles enhance blood vessel regeneration in rat calvarial defects

The inherent limitations of bone grafting in the treatment of critical-sized bone defects have led to a growing demand for bone repair implants. Three-dimensional (3D) bioprinting has emerged as a promising manufacturing technique for implants, offering flexibility in their structural design and the use of applicable materials. Although numerous 3D-bioprinted bone scaffolds have been developed to enhance osteogenesis, angiogenesis remains a challenge. Angiogenesis is crucial for successful bone healing because the process forms blood vessels to deliver essential nutrients and oxygen. Endothelial progenitor cells (EPCs) play a pivotal role in the early stages of vascularization. These cells, capable of differentiating into endothelial cells (ECs), are recruited from the bone marrow to the injured area during the healing process. CD34+ cells, a subset of EPCs, have gained attention because of their neovascularization potential and ability to contribute to bone regeneration. The incorporation of CD34+ cell-enhancing factors into 3D-printed bone scaffolds may facilitate successful bone healing in critical defects. StemRegenin-1 (SR1), a molecule that promotes CD34+ cell expansion, has shown promising results in increasing CD34+ hematopoietic stem and progenitor cell populations. This study aimed to investigate the sustained release of SR1 from a collagen-based scaffold integrated with mesoporous silica nanoparticles (MSNs) to promote angiogenesis and enhance bone healing. The sustained release of SR1 from the collagen scaffold is hypothesized to promote angiogenesis, thereby facilitating bone repair. In vitro studies have demonstrated the angiogenic potential of SR1; however, further in vivo investigations are required to establish its clinical efficacy. This study contributes to the development of novel therapies targeting CD34+ cells and demonstrates the potential of SR1 as a promising agent for promoting angiogenesis and enhancing bone healing in critical defects.

Distinct Cellular, Transcriptional, and Mechanistic Properties Determine HSC Fate during Emergency Granulopoiesis

Emergency granulopoiesis (EG) is the process responsible for the rapid and enhanced production of granulocytes during acute infections. Traditionally, EG was understood as a cellular mechanism mostly initiated and supported by myeloid progenitors, however, recent data suggest that hematopoietic stem cells (HSCs) might also participate in the process. To investigate whether and how HSCs contribute to the initial steps of EG, we performed scRNA-seq and ATAC-seq analysis of sorted murine HSCs 4 hours after in vivo lipopolysaccharide (LPS) administration, mimicking an acute bacterial infection. Strikingly, we observed radical transcriptional changes between HSCs isolated from PBS control and LPS treated mice, mostly marked by alterations in HSC lineage bias. We identified a steady state lymphoid-biased subpopulation of HSCs, marked by expression of Procr (CD201). Following LPS stimulation, the CD201 expression was lost and this population was transcriptionally rewired to a myeloid-biased HSC population. Accordingly, ATACseq data corroborated opening of myeloid-bias loci upon LPS administration rather than steady-state lymphoid-biased loci. Further, we confirmed the loss of CD201 expression in HSCs by flow cytometry in mice after LPS and G-CSF treatment, as well as Candida albicans infection, suggesting that the loss of CD201 expression is a general event during EG induced by different stimuli. Interestingly, the CD201 downregulation in HSCs was independent of the master regulator of EG C/EBPβ, while we observed the contribution of the TLR4-MyD88 signaling axis. Next, we functionally validated our scRNA-seq results in vitro and in vivo. When cultivated under myeloid differentiation conditions, the CD201- HSCs gave rise to mature granulocytes, whereas CD201+ HSCs remained rather immature. In transplantation settings, CD201+ HSCs showed increased engraftment and preferential bias to produce lymphoid cells, while CD201- HSC showed decreased engraftment ability and a bias towards myeloid production. Surprisingly, LPS challenge of BM chimeras transplanted with lymphoid-biased CD201+ HSCs led to EG response marked by rapid loss of lymphoid-biased multipotent progenitors (MPPs) and the expansion of myeloid-biased MPPs, thus confirming the lympho-myeloid switch observed in the scRNA-seq data. Interestingly, the treatment of WT mice with CD201 blocking antibody was able to partially impair the lympho-myeloid switch, suggesting that CD201 plays an active role in HSCs during EG. Mechanistically, we observed that both CD201+ and CD201- HSCs rely on different signaling pathways under EG. While CD201+ HSCs express higher levels of TLR4 and have higher activation of NF-κB signaling upon in vitro LPS stimulation, the CD201- HSCs express higher levels of G-CSF-R and have higher activation of pSTAT3 signaling upon G-CSF-stimulation. Moreover, while the LIP isoform of C/EBPβ, important for cell proliferation, is present in both CD201+ and CD201- HSCs, the LAP/LAP* isoform, important for myeloid differentiation, is present only in the CD201- HSCs fraction. Altogether, our data suggest that EG is supported by a population of HSCs which upon pathogen sensing undergo a radical transcriptional rewiring that promotes their myeloid output. Initially, the pathogen is directly sensed by TLR4 on the surface of a steady state lymphoid-biased CD201+ HSCs, causing a rapid activation of the downstream NF-κB signaling pathway. Subsequently, the lymphoid-myeloid transcriptional switch, marked by the loss of CD201 expression, occurs and EG is then supported by myeloid-biased CD201- HSCs. CD201- HSCs respond to the infection in an indirect manner through G-CSF-R on their surface and exhibit enhanced pSTAT3 activation and elevated LAP/LAP* C/EBPβ isoform, cellular mechanisms known to promote myeloid differentiation and granulocytic production. In conclusion, the switch from CD201+ to CD201- HSCs facilitates both fast and sustained EG leading to the supply of new granulocytes to fight the infection. This detailed understanding of the distinct cellular, transcriptional, and mechanistic properties that determine HSC fate during emergency granulopoiesis opens new venues to potentially modulate granulocytic production in certain clinical conditions such as sepsis. This work was partially supported by a GACR 22-18300S, GAUK 327722, and IMG institutional funding RVO 68378050.

Decline in Cardiolipin in Hematopoietic Stem Cell during Aging Alters Their Regenerative Potential

Hematopoietic Stem Cells (HSCs) are known for their regenerative potential. This property allowed the use of HSC in bone marrow transplantation to treat hematological disorders such as leukemia. However, aging influences these characteristics of HSCs. The major hallmarks of aging are limited self-renewal and reconstitution capacity resulting in HSC exhaustion in aged individuals. Mitochondrial metabolism and activity are active drivers of HSC fate decisions and data from our lab shows that mitochondria in aged HSCs have increased sphericity with a polarized mitochondrial network and a lower mitochondrial membrane potential. An important aspect of mitochondrial functions is the lipid composition of their membranes. A lipid trafficking assay showed an atypical pattern of lipid incorporation by mitochondria in aged HSCs suggesting that mitochondrial lipids become abnormal upon aging. The overall content of Cardiolipin (CL) which is a signature mitochondrial lipid, found exclusively in the inner mitochondrial membrane was also reduced during aging. This was done with flow cytometry using a CL marker NAO (Non-Acridine Orange). Tafazzin encoded by the gene TAZ is crucial for remodeling CL into its mature form which is required for maintaining mitochondrial functions. Mutations in the TAZ gene have been known to cause a reduction in the synthesis of mature CL which is often associated with HSCs functional defect. Western Blot analysis showed a considerable decrease in Taz protein expression in aged stem and progenitor cells (HSPC) compared to young HSPC. To understand the effect of reduced Taz expression on HSC functions, we used a doxycycline inducible, sh-RNA mediated TAZ knock-down (KD) mouse model. Bone marrow analysis indicates that the frequency of HSC and multipotent progenitors decreases significantly in 6-month-old TAZ KD mice compared to wild type (WT) mice. Our data shows that following 5FU induction TAZ KD mice have a significant reduction in the multipotent progenitor and HSC population on day 10 and day 21 post 5FU, indicating TAZ KD decrease the regenerative potential of HSCs under stress conditions. Further pointing towards the significance of cardiolipin in HSC regeneration. TAZ KD mice also have abnormal peripheral blood recovery post 5FU mainly affecting the platelets. We next examined the repopulation potential of HSCs isolated from TAZ KD mice using serial competitive transplantation assays. While primary transplanted mice of TAZ KD HSCs exhibited similar donor-cell chimerism compared to mice transplanted with WT HSCs, about 50-60%, secondary recipient of TAZ KD cells had significantly lower donor-cell chimerism than secondary recipients of WT cells. Further, donor-derived HSC frequency in the bone marrow of recipients of TAZ KD HSCs 24 weeks following engraftment was also significantly lower than recipients of WT HSCs. Hence, Taz-deficiency causes a defect in HSC self-renewal under regenerative conditions. We next evaluated whether restoring cardiolipin levels in mid-aged HSCs could rescue their functions. For this, mid-aged HSC were incubated for 3 days under conditions that maintain HSC functions in vitro with Alcar (Acetyl-L-Carnitine), which promotes CL production. In lipid incorporation assay, Alcar supplementation restored mitochondrial membrane potential, mitochondrial organization and lipid incorporation of mid-aged HSCs. In competitive transplant assay, while mid-aged WT HSC treated with vehicle gave rise to a myeloid-bias graft, Alcar supplemented mid-aged WT HSC gave rise to a more balanced graft with increased ratio lymphoid to myeloid cells in the peripheral blood 16 weeks following transplantation, albeit the rescue was partial. This study indicates that HSC mitochondrial membrane lipids become abnormal with aging which causes a decline in HSC function and highlights the critical role of mitochondrial cardiolipin in HSC functions. It implies that metabolite supplementation is a viable option for restoring age-related HSC functional defects.

Myelodysplastic Syndrome Hematopoietic Stem Cells Can Undergo Differentiation Trajectory Rewiring to Escape Venetoclax Vulnerability

Myelodysplastic syndromes (MDS) are incurable neoplasms that arise from early hematopoietic stem cells (HSCs). The standard of care for MDS is hypomethylating agent (HMA) therapy. However, the disease invariably fails these agents, resulting in fatal cytopenias or progression to acute myeloid leukemia (AML) with a dismal prognosis. We previously showed that the cellular hierarchy of MDS HSCs predicts the mechanisms of progression after HMA failure and can guide the design or choice of specific therapeutic approaches targeting these cells (Ganan-Gomez et al., Nature Medicine 2022). MDS HSCs in one of two differentiation states, long-term HSCs or lymphoid-primed multipotent progenitors, have a “common myeloid progenitor (CMP) pattern” or “granulocytic-monocytic progenitor (GMP) pattern” of differentiation. HSCs in these 2 differentiation states persist throughout the disease course and, driven by the recurrent activation of BCL2- or NF-κB-mediated survival pathways, respectively, expand at disease progression. In our preclinical studies, pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden, which suggests that only patients with “CMP pattern” MDS may benefit from venetoclax-based therapies. Accordingly, patients with “CMP pattern” MDS, whose disease progressed after frontline HMA failure, enrolled in clinical trials of venetoclax-based therapy (NCT04160052, NCT04550442, or NCT04655755; n=28: 12 “CMP pattern” MDS and 16 “GMP pattern” MDS) had a shorter cumulative time to complete remission (P=0.012) and a longer relapse-free survival duration (14.0 months vs 5.2 months) than those with “GMP pattern” MDS did. Consistent with the hypothesis that venetoclax-based therapy selectively targets different HSC populations after HMA therapy failure, bone marrow (BM) HSC frequencies were significantly decreased at the time of remission after venetoclax-based therapy in “CMP pattern” MDS patients, but not in those with “GMP pattern” MDS. These results explain why “GMP pattern” MDS are resistant to venetoclax therapy. However, 8 of the 12 patients with “CMP pattern” MDS relapsed during venetoclax treatment and/or progressed to AML after an initial remission. In 2 patients with “CMP pattern” MDS, sequential flow cytometry and next generation sequencing analyses showed that the HSC hierarchy switched to “GMP pattern” MDS before venetoclax failure. This immunophenotypic hierarchical change was associated with the detection or expansion of STAG2- or RUNX1-mutant clones with isolated trisomy 8. To evaluate whether the immunophenotypic switch resulted from the distinct differentiation potential of early HSCs or from the aberrant expression of cell surface markers, we performed sequential single-cell RNA sequencing analyses of BM mononuclear cells isolated from the 2 “CMP pattern” MDS patients whose disease changed hierarchy before venetoclax failure. Our analyses revealed that after undergoing significant depletion during disease remission, HSCs expanded at therapy failure. Differential expression analysis showed that the expansion of STAG2- or RUNX1-mutant clones in the HSC compartment not only rewired MDS HSCs' differentiation state towards a granulo-monocytic-biased trajectory but also changed HSCs' survival dependency from BCL2-mediated anti-apoptotic pathways to TNFa-induced pro-survival NF-κB signaling, thus enabling HSCs to evade the cytotoxic effects of venetoclax (Fig. 1). The anti-apoptotic gene MCL1, a direct transcriptional target of NF-κB, was also significantly upregulated in HSCs at the time of progression, which suggests that the combination of venetoclax with an MCL1 inhibitor can overcome secondary failure to venetoclax. Together, these results reveal a novel mechanism of venetoclax resistance. HSCs under venetoclax therapy undergo survival pressure, which results in the expansion or acquisition of clones carrying specific mutations that change HSCs' survival dependencies (Fig. 2). Our study suggests that MDS patients receiving venetoclax-based therapy should be monitored at the molecular level and enrolled in clinical trials of other targeted agents before their disease undergoes clonal evolution and HSC hierarchical rewiring.

The Effect of SCD-1 Inhibition on Human Hematopoietic Stem Cell Mitochondrial Metabolism, Cell Proliferation, and Differentiation Potential

Hematopoietic stem cells (HSC) give rise to all blood lineages and sustain the production of blood cells throughout life. Due to their inherently high regenerative potential, HSC are used in a variety of clinical settings, including bone marrow transplantation (BMT) directly to cure a variety of hematologic and oncologic disorders often with gene therapy. During regeneration, HSC are activated into cycle. For this, HSC undergo drastic mitochondrial and metabolic remodeling to meet the bioenergetic and biosynthetic needs of activated HSC. A growing body of evidence indicates that HSC sustain injury during activation. This remodeling is important for optimal HSC function but is permanently changed after HSC activating stress. It is thus important to identify the metabolic needs of activated HSC to improve HSC functions for therapeutic purposes. In both murine and human HSC, numerous metabolic enzymes get upregulated during activation, including those involved in de novo lipid synthesis. This metabolism is poorly understood in human HSC. Our overarching question is to understand the metabolic needs of HSC within the human system. Stearoyl-co-A-desaturase 1 (SCD-1), an enzyme responsible for conversion of stearic to oleic acid within the de novo lipid synthesis pathway, is suspected to play a role in metabolic reprogramming after stress. Using CD34+ mobilized human peripheral blood, we analyzed the effect of SCD-1 inhibition on HSC and progenitor proliferation, mitochondrial metabolism, and lineage differentiation potential. Cells were cultured either in vehicle control conditions or with SCD-1 inhibitor (SCDi). Subsequently, cells were counted and analyzed by flow cytometry on Days 4-5 and 10-15. More primitive or stem cell markers were utilized on Days 0 and 5 whereas differentiation markers were utilized on Day 10-15. Both tetramethylrhodamine-ethyl ester dye (TMRE) and MitoSOX reagents were used to assess mitochondrial membrane potential and mitochondrial ROS, respectively. Our results demonstrate that the size of the HSC population generated by SCDi-treated CD34+ cells within 5 days of culture was similar to vehicle treated cells and was composed of similar proportion of CD34+CD38+, CD34+CD38-, and CD38+CD34- cells, indicating that SCD-1 inhibition does not alter HSC expansion under these culture conditions. However, TMRE levels were lower in all HSC populations whereas mitoSOX levels were unchanged by SCD-1 inhibition compared to vehicle. In differentiation conditions, SCDi cultures were composed of a larger proportion of myeloid progenitors and a smaller proportion of erythroid cells after 10-15 days, compared to vehicle-treated cultures. These findings suggest that de novo lipid synthesis is necessary for HSC differentiation but dispensable for proliferation/expansion in vitro. We then used a xenotransplant model to assess the effect of SCDi on HSC regenerative potential in vivo. CD34+ mobilized human peripheral blood cells were cultured for 3 days under conditions that maintain HSC functions in vitro and transplanted into immunodeficient, sublethally irradiated NSG mice. Peripheral blood of xenotransplanted mice was analyzed monthly for human cell chimerism and mature blood lineage potential from 1 to 6 months post-transplant. Bone marrow analysis was performed at the 6 month time point as well, assessing for both HSC markers and lineage markers. Mice that received SCDi-treated cells exhibited human cell chimerism at a level similar to mice that were transplanted with vehicle treated cells, at about 3 to 30%. However, SCDi-treated cells gave rise to a lymphoid-biased graft, in particular T cells, both in the peripheral blood and in the bone marrow of xenotransplanted mice, compared to a more balanced myeloid-lymphoid graft from vehicle treated cells. In conclusion, these findings suggest that de novo lipid synthesis is critically important for HSC lineage fate and balanced differentiation in vitro and in vivo. This is especially clinically relevant as this work may implicate a possible therapeutic target as improving de novo lipid synthesis may aid in patients who suffer from persistent cytopenias after BMT and graft failure.

CD90-Targeted Cocal-Pseudotyped Lentivirus As a Robust Platform for Human HSC Gene Therapy

Ex vivo gene delivery and editing in hematopoietic stem and progenitor cells (HSPCs) provide a promising approach for the treatment of many chronic conditions such as hemoglobinopathies, lysosomal storage diseases, and HIV. However, such approaches require highly specialized biomedical infrastructure limiting its accessibility to most patients who could benefit. The development of a targeted in vivo approach to HSPC gene delivery/editing is therefore necessary for the broadened use of gene therapies. The CD34 +CD90 + subset of HSPCs represents a highly enriched population for both short-term and long-term hematopoietic lineage repopulation capacity as seen in the non-human primate (NHP) transplant model. Furthermore, ex vivo gene editing of solely CD34 +CD90 + Human and NHP hematopoietic stem cells (HSCs) was sufficient to produce therapeutic thresholds for sickle cell phenotypic amelioration after transplantation. Thus, the development of novel gene therapy vectors targeting the CD34 +CD90 + HSC population is crucial for more accessible and robust gene therapy platforms. Here we report the development of a novel CD90-targeted Cocal pseudotyped lentivirus system capable of targeted transduction of HSPCs in vitro and in vivo. Additionally, we compare transduction efficiency and specificity of this new technology with the more routinely used VSVG targeting methodology. To produce targeted lentivirus, native targeting of VSVG or Cocal envelope to the LDL-Receptor (LDLR) was knocked out by amino acid substitution. An anti-CD90 single-chain variable fragment (scFv) was introduced onto the mutated capsids using CD8 hinge and ICAM transmembrane domains. Mutagenesis of Cocal envelope LDLR binding motif was performed by Gibson cloning and confirmed via nanopore sequencing. Successful mutated viral production was assessed using hydrodynamic titration methods followed by functional analysis of transduction on CD90 + cell lines. In vitro transduction specificity and efficiency assays were performed on Human GCSF-mobilized CD34 +enriched apheresis samples and analyzed by flow cytometry. Humanized NBSGW mice were treated with targeted-lentiviral particles via intravenous (IV) and intraosseous (IO) injection. Mice were necropsied 4-8 weeks post injection. Transgene expression was analyzed by flow cytometry or luminescence imaging and confirmed by PCR on human-CD34 + FACS-sorted cells from necropsied mice. CD90-Targeted Cocal-pseudotyped lentivirus (Cocal-CD90) transduced CD34 +CD90 + HSCs 2.12-fold (p=.017) more robustly than CD90-targeted VSVG-pseudotyped lentivirus (VSVG-CD90) in vitro. Cocal-CD90 virus also showed significantly greater on-target transduction specificity of CD34 +CD90 + HSCs (p=.0008) compared to untargeted wild-type Cocal-pseudotyped lentivirus (Cocal-WT); but similar specificity to VSVG-CD90 virus (p=.118). Unlike VSVG-CD90 virus, we did not observe decreased transduction efficiency of CD34 +CD90 + HSCs using Cocal-CD90 virus in comparison to Cocal-WT virus. IV and IO injection of both CD90-targeted viruses in humanized mice resulted in transduction of immobilized human HSPCs (CD34 +CD38 -CD133 +) in the bone marrow. Transduced cells maintained multilineage differentiation capacity as seen by transgene expression of T-cell, B-cell, and myeloid compartments in the bone marrow, spleen, and peripheral blood at necropsy. As observed in vitro, Cocal-CD90 treated mice consistently displayed more robust transgene expression than VSVG-CD90 treated mice. Importantly, transduction of off-target organs (e.g., liver and lungs) post-IV injection with targeted virus was not detected by luminescence imaging during 8 weeks of follow-up or at necropsy. We provide the first evidence of targeted engineering of a knockout Cocal envelope using the attenuation of native LDLR tropism coupled with a CD90-scFv fusogen protein. Cocal-CD90 virus has increased specificity for CD90 + transduction compared to Cocal-WT lentivirus, without lowering transduction efficiency of on-target cells. Furthermore, in comparison to targeted VSVG engineering, Cocal engineered virus does not decrease transduction efficiency of HSCs in vitro or in vivo. Coupled with Cocal lentiviruses' resistance to human serum inactivation and low immunogenicity, targeted Cocal viral vectors are more suitable for future in vivo gene delivery platforms than their VSVG counterparts.

Chronic TNF in the Aging Microenvironment Exacerbates TET2-loss-of-Function Myeloid Expansion

Introduction: Age-associated TET2 somatic mutations impart an intrinsic hematopoietic stem cell (HSC) advantage and contribute to the phenomenon of clonal hematopoiesis of indeterminate potential (CHIP). Individuals with TET2-mutantCHIP have a higher risk of developing myeloid neoplasms and other age-related conditions, including heart, lung, liver, kidney and infectious disease, and have increased risk of all-cause mortality. Despite its role in unhealthy aging, the extrinsic mechanisms driving TET2-mutant CHIP clonal expansion remain unclear. We previously showed an environment containing TNF favours TET2-mutant HSC expansion in vitro. We therefore postulated that age-related increases in TNF also provide an advantage to HSC and progeny with TET2-mutations in vivo. Methods: All human and mouse studies met ethics approval. C57Bl/6 background wildtype (WT), TNF-α knockout ( TNF-/-), Tet2 hematopoietic knockout (Vav1-iCre-mediated; Tet2-/-) and floxed control mice ( Tet2f/f) mice were obtained from the Jackson Laboratory. Young (6-mo [n=9]) and old (18-22 mo [n=9]) WT mice, and old TNF-/- mice (n=7) were subjected to nonirradiative myeloablation with busulfan, and retro-orbital injections of 8x10 6 cells/ml T-cell-depleted bone marrow (BM), equally harvested from 4-mo-old WT CD45.1 (n=5) and Tet2-/- CD45.2 donor mice (n=5). Flow cytometry was used to confirm and monitor engraftment. Mice were harvested 8 weeks post-transplant for analysis of HSC, progenitor, monocyte and neutrophil populations, and cytokine analyses. Consenting human research participants diagnosed with rheumatoid arthritis (RA) were recruited from the Greater Hamilton Area (Ontario, Canada) from 2016-2018. Blood draws occurred prior to any immunomodulatory treatment (baseline), and at 3- and 6-months following treatment with Adalimumab (Humira®), an anti-TNF agent. CHIP status was determined with our successful 48-gene, targeted, Ion-Torrent based sequencing approach to isolated genomic DNA from PBMC. Confirmation of calls and increased sensitivity to detect clones with VAF < 0.02 employed our established single molecule molecular inversion probes (smMIP)-targeted genomic capture technique and high-depth (47,500X avg. coverage) paired-end sequencing, employing high-stringency filters for error-suppression. Statistical analyses were performed in GraphPad Prism V9.2 or R 4.1.2. Results: 1. Mixed BM chimeric mice of WT and TNF -/- genotypes reconstituted with WT CD45.1 + and Tet2 -/- CD45.2 + HSC showed that age-associated increases in TNF significantly increased by 2- to 2.5-fold the proportions of HSC in old recipient mice, with myeloid lineage skewing (2- to 2.5-fold more granulocyte-monocyte, monocyte-dendritic and common monocyte progenitor cells in BM, and dramatic expansion of Tet2-/- monocytes and neutrophils in blood). This aberrant myelomonocytic advantage was mitigated in old TNF -/- recipient mice, suggesting that TNF signalling in the BM is essential for Tet2-mutant myeloid expansion. 2. Age-associated TNF predisposed Tet2 -/- HSC to the development of Ly6C high inflammatory monocytes, including increased intracellular and serum TNF levels, further exacerbating an inflammatory environment in favor of Tet2-mutantexpansion. 3. Examination of human RA patients (n=7; avg. age 57y) with serial PBMC sampling revealed one patient with CHIP driven by CBL and PPM1D variants, with a 32% reduction in clone size between 3 and 6 months of anti-TNF therapy (adalimumab). Two additional variants were detected at baseline in this RA patient, TP53 R196* (VAF = 1.34%) and ASXL1 P689L (VAF = 3.13%), but these were not detected at 3- and 6-months post-treatment even with high-depth, error suppression sequencing. Two additional patients had detectible low-level CH clones prior to anti-TNF treatment - one with a STAG2 R1033Q variant at baseline (VAF = 1.65%) and the other with a TET2 baseline E798K variant (VAF = 1.68%) - but not detected during anti-TNF treatment. Conclusions: We present novel findings that TNF in the aging BM environment has a causal role in driving TET2-mutant CHIP in vivo, and the first examination of the impact of TNF blockade on CH dynamics in humans, suggesting promise for reducing clonal burden. Larger, prospective studies are required to confirm these findings and to determine if inflammatory cytokine blockade may improve CHIP-comorbid conditions and myeloid cancer risk.

Pre-Clinical Developmental Hematopoiesis Demonstrates the Heterogeneity of KMT2A-Rearranged Infant Acute Lymphoblastic Leukemic Populations

KMT2A-rearranged (KMT2A-r) infant acute lymphoblastic leukemia (ALL) has a poor prognosis with increased risk of relapse when diagnosed at younger ages. Understanding the origin of these leukemic cells is crucial to comprehend disease development and prognosis differences. The unclear delineation of the identity of early blood populations in humans is a challenge in defining the cell type(s) driving this infant ALL. Recent pre-clinical studies revealed that definitive hematopoietic stem cells (HSCs) have minimal contribution to embryonic hematopoiesis, while independently arising embryonic hematopoietic progenitors (HPCs) that possess more limited self-renewal and/or multilineage differentiation capacities, are both necessary and sufficient to sustain the developing embryo. Here, we aim to leverage pre-clinical knowledge on the markers that distinguish HSCs from HPCs to investigate the potential contribution of HPCs in driving prenatal and infant leukemias. We defined transcriptional signatures from developmental HSC and HPC populations using thirteen single cell RNA sequencing (scRNA-seq) datasets from zebrafish, eight datasets generated by our research group and five from independent published studies. We then employed a neural network approach to define the orthologous cell type signatures most likely correlating with human healthy and blast cells from publicly available data of 18 KMT2A-r infant ALL patients. In accordance with previous literature, HSC and lymphomyeloid progenitor signatures are both highly represented in the leukemic blast populations. Additionally, we found subpopulation differences in gene expression within leukemic blasts cells including lymphoid progenitor, granulocytic, and T cell signatures, possibly suggesting heterogeneity in either their cell type of origin or developmental differentiation potential. Our findings support the ability to use orthologous scRNA-seq to decipher new understanding on the role of HSCs and HPCs in hematological malignances affecting infants and children.

TMI-Based Dose-Escalated Bone Marrow Transplantation Can Help Preserve the Bone Marrow Microenvironment and Reduce Cellular Senescence in Old Mice

Introduction:Senescence is one of the hallmarks of aging and age-related conditions, including but not limited to, inflammaging, frailty, and various age-related diseases. It has been well-established that radiation and chemotherapy can accelerate senescence. While myeloablative conditioning-based allogeneic hematopoietic cell transplant (HCT) is a curative treatment for patients with leukemia. The dose of total body irradiation (TBI) conditioning has to be reduced in older patients to reduce potential high treatment-related mortality, which may still cause accelerated senescence in aged individuals, resulting in poor survival outcomes. We initiated the testing of a novel total marrow irradiation (TMI) based myeloablative conditioning in older patients to enhance the anti-leukemia effect and overall survival. We hypothesize that this new radiation preconditioning strategy may also reduce radiation-induced senescence in aged individuals compared to TBI to reduce treatment-associated side effects and improve the outcomes of allogeneic HCT. We tested this hypothesis in the present study and further investigated the mechanisms of the effects of TMI-based myeloablative and dose-escalated bone marrow transplantation (BMT) on old mice by examining the changes in expression of various senescence markers and senescence-associated secretory phenotype (SASP) in the bone marrow (BM) and selected organs. Method: The radiation treatment regimens were: TMI (12:4), TMI (16:4) and TBI (12:12), and TBI (16:16), were developed as described (Zuro et al., 2021); i.e. (12:4) means the radiation dose to the BM was 12 Gy and the dose to the organs was 4 Gy. The young (8 weeks old) and aged mice (90 weeks old) of host C57BL/6j (JAX000664, CD45.2) were irradiated in 2 fractions 24h apart, and 25 million whole BM cells from donor (JAX002014, CD45.1) were transplanted by i.v. at 24 h after irradiation. For the hematopoietic stem and progenitor cell (HSPC) population study, more mature hematopoietic progenitors (Lin − Sca-1 −c-Kit + or LK cells) and HSPCs (Lin − Sca-1 + c-Kit + or LSK cells) were examined in BM by flow analysis. Cultured bone marrow mesenchymal stem cells (MSCs) were analyzed for the gene expression of the senescence marker CDNK1a (or p21) and SASP factors MMP13 and CXCL12 by qPCR. For senescence study in organs such as the liver, gut, and spleen, senescence-associated beta-galactosidase (SA-β-gal) staining was performed and expression of the CDNK2a (or p16) was analyzed by qPCR. Results: TMI or TBI treatment with young donor BMT repopulated the BM HSPC population without significant bone damage in aged mice (data not shown). While the BM LK and LSK populations significantly reduced in aged mice after TMI or TBI with young donor BMT compared to aged control mice ( Fig 1A), these populations were not different from young mice groups ( Fig 1B). In addition, TMI or TBI treatment with young donor BMT reduced MSC senescence. Cultured MSCs analysis showed the reduction of p21, MMP13, and CXCL12 expression ( Fig 1C-E) after TMI or TBI with young donor BMT in aged compared to aged control without radiation and BMT. However, while TMI treatment and BMT reduced senescence in the organs, mice receiving TBI treatment and BMT showed a higher senescence burden. In the liver, the aged control mice showed a significant increase in SA-β-gal staining, which was decreased in TMI (12:4) and TMI (16:4) mice ( Fig 1F). In contrast, this effect was not observed in aged mice after TBI (12:12) or (16:16) ( Fig 1G). Moreover, the senescence marker p16 expression in the liver and gut was reduced only in TMI (12:4) and TMI (16:4), not in TBI (12:12) or (16:16) ( Fig 1H, I), whereas the p16 expression in the hematopoietic tissue, spleen was decreased in all treatment groups TMI vs. TBI ( Fig 1J). Conclusion: TMI-based dose escalates with young donor BMT in aged mice preserved BM microenvironment and reduced senescence of organs while TBI treatment causes senescence of the organs. Young donor BMT, which combined with TMI or TBI treatment, preserved bone structure and reduced senescence of HSPC and MSC. However, TMI treatment decreased the SA-β-gal staining and p16 expression significantly in organs such as the liver and gut, but not by TBI treatment. This study showed the senescence reduction in both bone marrow and organs after young donor BMT and TMI treatment. For further study, we have a plan to study the mechanism and signaling pathway of senescence reduction.

TAF1, the Largest Subunit of Tfiid, Is Dispensable for Adult Hematopoiesis

Temporal and spatial control of gene expression is central to normal hematopoietic stem cell (HSC) biology. Many sequence-specific transcription factors have been identified as key regulators of HSC fate decision, while the function of general transcription factors in HSC behavior is poorly understood. We previously reported that TAF1, the largest subunit of the TFIID complex, plays a critical role in AML1-ETO driven acute myeloid leukemogenesis. To evaluate the function of TAF1 in normal fetal and adult hematopoiesis, we generated TAF1 conditional knockout (CKO) mice and identified an essential role of TAF1 in fetal erythropoiesis. Surprisingly, deletion of TAF1 in adult mice was not lethal to hematopoiesis; rather, we observed a marked expansion of hematopoietic stem and progenitor cell (HSPC) populations, with increased self-renewal and impaired differentiation capacity in these TAF1- null cells. Consistently, we found that TAF1-null HSPCs failed to up-regulate key differentiation-associated genes when induced to differentiate in vitro. Using a variety of genome-wide assays and biochemical approaches, we found that TAF1 loss not only disrupted TFIID complex formation and chromatin recruitment, but also reduced promoter accessibility, impaired RNA Polymerase II promoter recruitment and activity, as well as its promoter-proximal pausing mechanism. Thus, the effect of TAF1 loss on normal adult hematopoiesis primarily relates to the inability to upregulate genes involved in differentiation, while HSC self-renewal and the expression of self-renewal genes proceeds relatively normally.

DNMT3A R882H Exhibits Greater Inflammatory Potential Than R882C in Primary Hematopoietic Stem and Progenitor Cell Knock-in Model and Population Data

Clonal hematopoiesis (CH) is an age-associated phenomenon which is known to increase the risk for hematologic malignancy and cardiovascular disease. The most commonly mutated gene in CH is de novo DNA methyltransferase DNMT3A. R882H and R882C are the most common hotspot mutations in this gene, and these mutations are commonly grouped together in interpretation of clinical data even though in vitro experiments suggest divergence in their biochemical consequences. Here, we used CRISPR knock-in of primary hematopoietic stem and progenitor cells (HSPCs) along with population data from the UK Biobank (UKB) to evaluate the extent to which R882H and R882C differentially predispose to inflammatory phenotypes. Using two homology donor (HD) templates with distinct self-cleaving fluorescent tags, we used FACS to isolate homozygous R882H- or R882C-mutant cells from six unique samples of cord blood HSPCs. We subsequently cultured these isogeneic mutant cells and their mock-electroporated wild-type (WT) controls in monocyte differentiation media and evaluated their transcriptomes via bulk RNA sequencing (RNAseq) and production of secreted cytokines in a Luminex-based assay. Analysis of transcripts using differential expression for repeated measures (Dream) revealed that, when compared to WT, the mutants demonstrated many shared differentially expressed genes (DEGs) but also exhibited many DEGs unique to each variant. Furthermore, gene set enrichment analysis (GSEA) of Hallmark pathways revealed common enrichment of cell cycle related pathways in both variants, but found that R882H displayed greater enrichment of inflammation-related pathways than either WT or R882C (Figure 1A). Rank-order GO and KEGG pathway enrichment comparison of R882H vs R882C showed further evidence of bias in inflammation-related pathways among the most upregulated gene sets (e.g., GO:0019882, Antigen Processing & Presentation and KEGG:hsa04145, Phagosome). IL-6 is significantly regulated by R882 genotype in our model; levels of IL-6 for R882H (13,065 ± 2,446; mean ± s.e. in pg/mL) but not R882C (10,340 ± 2,446) were significantly different than WT (6,281 ± 1,730; p = 0.014). We next sought to better understand if this biased inflammatory priming would also be observed for heterozygous mutations and across different cell types, so using mutant and WT HD templates we performed single cell RNAseq on CRISPR'd heterozygous mutants and knock-in WT controls. Comparing R882H to R882C in this context demonstrated upregulation of numerous inflammation-related genes, including CXCL8 (IL-8), S100A8, and S100A9. GSEA of Hallmark pathways revealed enrichment of inflammatory pathways (e.g., Inflammatory Response & TNFA Signaling via NFKB) in monocytic cells but also in progenitor cells. We then investigated whether these transcriptional differences between R882H and R882C might be due to divergent patterns of DNA (hypo)methylation. Employing 5-letter next-generation sequencing (A, C, G, T, methyl-C), we compared the methylation landscape of three isogeneic donors with homozygous R882H, R882C, or mock-electroporated WT genotypes. As expected, comparison of either mutant to WT showed a global hypomethylation. Comparison of R882H to R882C revealed no systematic bias in methylation of gene promoters but a pervasive hypomethylation of gene bodies. A gene set of the 50 most upregulated genes from our single cell RNAseq experiment was significantly enriched in rank order analysis of R882H vs R882C hypomethylation of gene bodies (NES = 1.47, p = 0.036) but not promoters (NES = -0.86, p = 0.71). Finally, we asked if there were differences in patient phenotypes for those with R882H or R882C clonal hematopoiesis. Here, we used our previously published UKB clonal hematopoiesis cohort (Vlasschaert et al. 2023, Blood). In Cox proportional hazards models controlling for basic demographics (N = 451K) or demographics plus cardiovascular-relevant covariates (N = 390K), we found that R882H but not R882C was associated with significantly greater incidence of heart failure and of a composite measure consisting of death or coronary artery disease (Figure 1B). In conclusion, our study found evidence in experimental models and patient data that DNMT3A R882H may pose a greater risk for inflammatory phenotypes than R882C.

Phenotypic HSPC Rescue By RNA Lipid Nanoparticles in a Murine Model of Fanconi Anemia

Background: Fanconi Anemia (FA) is a recessively heritable multisystem disorder that manifests with bone marrow failure in early childhood. Current gene therapy trials for FA rely on transplantation of ex vivo transduced autologous hematopoietic stem cells (HSCs). This approach is limited by poor mobilization efficiency and a depleted HSC pool in FA patients. The recent use of mRNA encapsulated within lipid nanoparticles (LNP mRNA) for cancer immunotherapy and vaccines has created an interest in adapting mRNA delivery for protein replacement therapies. Phase 1/2 trials have demonstrated both safety and efficacy for in vivo treatment of multiple monogenic metabolic disorders. We hypothesized that LNP Fancc could be developed as an in vivo protein replacement therapy for FA. Methods: Custom mRNA transcripts modified with N1-methylpseudouridine and packaged into LNPs (diameter 80 ± 5nm; PDI 0.01), their performance was tested ex vivo and in vivo. LNP mRNA were delivered either intrafemorally or by tail vein injection into Fancc -/- mice, a mouse model of FA or Ai14 mice, a Cre-sensitive reporter mouse model. We also tested LNP performance using ex vivo expanded hematopoietic stem and progenitor cell (HSPC) populations and fresh CD150 +CD48 -Lin -Sca1 +ckit + (long-term hematopoietic stem cells; LT-HSC) cells obtained from wildtype (WT, Fancc +/+) and knockout (KO, Fancc -/-) mice. LNP formulations delivered mRNA transcripts of the fluorescent reporter mCherry (LNP mCherry), cre-recombinase (LNP Cre), luciferase bioluminescent reporter gene (LNP Luc) or a truncated murine FANCC gene (LNP Fancc). Results: To characterize access to the bone marrow compartment in vivo, we delivered LNP mCherry or LNP Cre via systemic (intravenous) and direct (intraosseous) routes to healthy WT and Ai14 mice. Using LNP Cre, we achieve up to 92.6% ± 5% tdTomato reporter expression in Lin -Sca1 +ckit + (LSK) cells 5 days following intraosseous delivery. Consistent with reports by others, we demonstrate lower average transfection rate of 24.8% ± 15.9% fluorescent positivity rates in LSK cells after intraosseous LNP mCherry delivery, and 9.5% ± 6.7% after intravenous delivery. mCherry fluorescent signal was detectable by FACS for up to 72 hours following LNP mCherry injection. We reasoned that meaningful FA HSC correction would require more extended protein expression. Circular mRNA shows improved biostability and durability of expression. Comparing both linear and circular mRNA performance, we observed that circularized LNP Luc is expressed for up to 7 days post ex vivo exposure in LT-HSC populations as compared to 3 days when using linear LNP Luc. Functionally, ex vivo LNP Fancc treatment improves proliferation rates and Mitomycin C (MMC) resistance in colony forming unit (CFU) assays of Fancc -/-HSPC. Here, LNP Fancc treated HSPC demonstrate improved MMC survival rates reaching 37.2% ± 10.5% up from 16.1% ± 8.2%. Ex vivo expanded Fancc -/- HSPC demonstrate an even greater benefit from LNP Fancc treatment, with CFU survival rates increasing to 62.7% from a baseline of 3.3%. Finally, we demonstrate improved repopulation of LNP Fancc treated HSPC 2 weeks after intravenous delivery to myeloablated recipients, measuring up to 73.9% donor chimerism after treatment with circular LNP Fancc compared to 50% among untreated (no LNP) controls. Conclusion: Our studies show that LNP Fancc can rescue Fancc -/- HSPC in vitro, and ongoing experiments reveal improved repopulation in vivo. Along with evidence for efficient in vivo delivery to the bone marrow, our data support the use of LNP mRNA as a potential in vivo treatment of bone marrow failure in FA patients.

Exploring Aging Dependent Adaptations of Splicing Factor Mutations in Clonal Hematopoiesis

Introduction: It has been shown that leukemia-associated mutations can be detected in the blood of healthy individuals and their clonal expansion in hematopoietic stem and progenitor cell (HSPC) populations is referred to as Clonal Hematopoiesis (CH). In one study, a population analysis of a cohort of 17,182 patients performed by Bick et al. [PMID7944936] selected without prior screening for hematological malignancies, indicated that advancing age is directly associated with an increase in CH. When the mutant clone accounts for 4% of the nucleated blood cells, it is then referred to as Clonal Hematopoiesis of Indeterminant Potential (CHIP). CHIP has been classified by the World Health Organization as a premalignant state and risk criteria for neoplastic development. CHIP is highly associated with adverse outcomes including auto-immune or auto-inflammatory conditions, cardiovascular disease, cytopenia, cancers in general, and other hematological neoplasms. The associated hematopoietic neoplasms include myelodysplastic syndromes (MDS), myeloproliferative disorders (MPD), and acute myeloid leukemia (AML). Population studies reveal splicing factor mutations to be among the most common mutations in CHIP and are highly correlated with age and show a high hazard ratio for risk of developing secondary neoplasms including AML. RNA splicing factors U2AF1, SRSF2, and SF3B1 make up core components of the spliceosome and are critical to prevent aberrant splicing. Mutations in these factors have widespread effects implicating a multitude of cellular processes. There are several established mutant splicing factor mouse models that can be utilized; however, many employ non-specific or leaky Cre drivers. Methods: To avoid non-specific and leaky Cre activation during aging, we used a Cre/lox-mediated U2af1(Q157R) conditional knock-in mouse model that does not contain an internal Cre driver. This mouse has a floxed wild-type U2af1 cDNA insert that expresses wild-type U2AF1 under normal circumstances. However, upon the exogenous addition of recombinant Cre the U2af1 cDNA is excised and the U2af1(Q157R) splicing factor mutation in exon 6 is expressed instead. This mouse model also possesses lox-stop-lox-YFP, leading to expression of YFP along with mutant U2AF1(Q157R) when Cre is introduced. We then isolated bone marrow from 6 week (young) and 6 months (aged) floxed Q157R/YFP mice and treated lineage-depleted bone marrow with recombinant Cre protein for two hours, followed by transplantation into mildly conditioned 6 week (young) and 18 month (old) wild-type mice. The percentage of YFP in the peripheral blood is quantified every 3 weeks. Full analyses, including RNA-seq, of the bone marrow progenitor populations after 3 months is in progress. Results and Discussion: Using the previously mentioned bone marrow transplant model our work shows that when a U2AF1 mutation is introduced into young and old recipient mice, there is significant preferential outgrowth when the donor mice are aged (Figure 1). This is indicated by an increase in the ratio of YFP, which represents the U2AF1(Q157R) mutant, to CD45.1 events, which represent an internal engraftment control. This would suggest that the expansion of U2AF1(Q157R) splicing mutant cells seen in clinical data may be the result of intrinsic factors from the aging cell population. Comprehensive analysis of the effects of alternative splicing in this model is ongoing, however, these data provide support for further investigation of the intrinsic effects of splicing factor mutations in an aged context.

ABL001 (asciminib) Efficiently Targets Transplantable BCR::ABL1 Lymphoid Blast Crisis in the Scl-Tta-p210-BCR::ABL1 Mouse Model

Objectives: This study aimed at investigating the therapeutic efficacy of ABL001 in the ScltTA-p210-BCR::ABL1 transgenic mouse model. ABL001 (asciminib) is a novel allosteric inhibitor of ABL kinase demonstrating its particular efficacy against BCR::ABL1 mutations that emerge during treatment with traditional TKIs. However, the activity and tolerability of ABL001 in a primary mouse model of BCR::ABL1 disease remain unknown. In addition, resistance mutations against ABL001 were observed in the myristoyl pocket of BCR::ABL1, but their emergence has not been analyzed in BCR::ABL1+ murine hematopoietic cells. Importantly, leukemic stem cells (LSCs) expressing BCR::ABL1 have developed mechanisms to evade eradication by tyrosine kinase inhibitors, underscoring the necessity for alternative therapeutic approaches. Methods: 5-fluorouracil- (5-FU-) treated bone marrow (BM) cells harvested from FVB/N-tg(ScltTA)(tetO- p210-BCR::ABL1) double-transgenic (dtg) donor mice (CD45.1+) were transplanted into lethally irradiated recipient mice (CD45.2+). One week after transplantation, tetracycline (tet) was removed from the drinking water to induce Bcr-Abl1 expression. Following an additional week, mice were treated daily with ABL001 or vehicle control by oral gavage (30 mg/kg body weight). Therapeutic effects were assessed using flow cytometry (peripheral blood, spleen, and BM), histological examination, RNA analysis and DNA sequencing of the myristoyl pocket of BCR::ABL1. Secondary transplants using splenocytes were performed to analyze whether ABL001 treatment was able to suppress BCR::ABL1-positive repopulating malignant stem cells. Additionally, we assessed potential ABL001-resistant subclones after secondary transplantation by subjecting the mice to further ABL001 treatment, modeling clinically relevant mutations in BCR::ABL1-positive disease. Results: Within 3 weeks after tet withdrawal, the transplanted mice developed an aggressive B-lymphoblastic p210-BCR::ABL1-positive disease, mimicking lymphoid blast crisis, as described previously (Koschmieder et al., Blood, 2005). Interestingly, ABL001 treatment significantly reduced this BCR::ABL1-driven phenotype as indicated by suppression of malignant B cells and restoration of normal hematopoiesis. ABL001 also significantly improved survival compared to the vehicle control group (p=0.026). Histological analysis confirmed the alleviation of the BCR::ABL1-driven phenotype in spleen and BM. Furthermore, ABL001 treatment significantly reduced BCR::ABL1 transcripts to background levels, demonstrating its ability to suppress BCR::ABL1-induced disease in the BM. The efficacy of ABL001 in reducing the malignant clone correlated with normalization of spleen weight. Essentially, ABL001 treatment normalized the phenotypic long-term repopulating hematopoietic stem cell (LT-HSC) population in the BM, suggesting restoration of the stem cell pool and targeting malignant LT-HSCs. This was supported by secondary transplantation experiments, showing that several mice receiving ABL001-treated splenocytes failed to develop leukemia, while all mice who received vehicle-treated cells did. Moreover, none of the secondary transplanted mice who were treated with ABL001 developed leukemia. Interestingly, Sanger sequencing of the BCR::ABL1 myristoyl pocket region demonstrated wildtype sequences and the absence of resistance mutations. Conclusion: Our study demonstrates the efficacy of ABL001 in ameliorating p210-BCR::ABL1-induced lymphoid blast crisis by specifically targeting BCR::ABL1-positive malignant stem cells and restoring normal hematopoiesis in a transgenic mouse model. These promising findings highlight the potential of ABL001 as an alternative therapeutic strategy for first-line treatment of BCR::ABL1-driven leukemia, including B-lymphoblastic blast crisis. Furthermore, since TKI treatment alone has not proven successful in patients with lymphoid blast crisis, this now opens new avenues for the in vivo-testing of novel combination treatments of ABL001 with chemotherapy or other targeting agents.