Progenitor Lineage Research Articles

Neuronal lineage tracing from progenitors in human cortical organoids reveals mechanisms of neuronal production, diversity, and disease

The contribution of progenitor subtypes to generating the billions of neurons produced during human cortical neurogenesis is not well understood. We developed the cortical organoid lineage-tracing (COR-LT) system for human cortical organoids. Differential fluorescent reporter activation in distinct progenitor cells leads to permanent reporter expression, enabling the progenitor cell lineage of neurons to be determined. Surprisingly, nearly all excitatory neurons produced in cortical organoids were generated indirectly from intermediate progenitor cells. Additionally, neurons of different progenitor lineages were transcriptionally distinct. Isogenic lines made from an autistic individual with and without a likely pathogenic CTNNB1 variant demonstrated that the variant substantially altered the proportion of neurons derived from specific progenitor cell lineages, as well as the lineage-specific transcriptional profiles of these neurons, suggesting a pathogenic mechanism for this mutation. These results suggest individual progenitor subtypes play roles in generating the diverse neurons of the human cerebral cortex.

CAR T-cell therapy in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive leukemic malignancy that affects myeloid lineage progenitors. Relapsed or refractory AML patients continue to have poor prognoses, necessitating the development of novel therapy alternatives. Adoptive T-cell therapy with chimeric antigen receptors (CARs) is an intriguing possibility in the field of leukemia treatment. Chimeric antigen receptor T-cell therapy is now being tested in clinical trials (mostly in phase I and phase II) using AML targets including CD33, CD123, and CLL-1. Preliminary data showed promising results. However, due to the cellular and molecular heterogeneity of AML and the co-expression of some AML targets on hematopoietic stem cells, these clinical investigations have shown substantial "on-target off-tumor" toxicities, indicating that more research is required. In this review, the latest significant breakthroughs in AML CAR T cell therapy are presented. Furthermore, the limitations of CAR T-cell technology and future directions to overcome these challenges are discussed.

Deciphering Transcriptomic Variations in Hematopoietic Lineages: HSCs, EBs, and MKs.

In the realm of hematopoiesis, hematopoietic stem cells (HSCs) serve as pivotal entities responsible for generating various blood cell types, initiating both the myeloid and lymphoid branches within the hematopoietic lineage. This intricate process is marked by genetic variations that underscore the crucial role of genes in regulating cellular functions and interactions. Recognizing the significance of genetic factors in this context, this article delves into a genetic perspective, aiming to unravel the biological factors that govern the transition from one cell's fate to another within the hematopoietic system. To gain deeper insights into the genetic traits of three distinct blood cell types-HSCs, erythroblasts (EBs), and megakaryocytes (MKs)-we conducted a comprehensive transcriptomic analysis. Leveraging diverse hematopoietic cell datasets from healthy individuals, sourced from The BLUEPRINT consortium, our investigation targeted the identification of genetic variants responsible for changes in gene expression levels and epigenetic modifications across the entire human genome in each of these cell types. The total number of normalized expressed transcripts includes 14,233 novel trinity lncRNAs, 13,749 mRNAs, and 3092 lncRNAs. This scrutiny revealed a total of 31,074 transcripts, with a notable revelation that 14,233 of them were previously unidentified or novel lncRNAs, highlighting a substantial reservoir of genetic information yet to be explored. Examining their expression across distinct lineages further unveiled 2845 differentially expressed (DE) mRNAs and 354 DE long noncoding RNAs (lncRNAs) notably enriched among the three distinct blood cell types: HSCs, EBs, and MKs. Our investigation extended beyond mRNA to focus on the dynamic expression of lncRNAs, revealing a well-defined pattern that played a significant role in regulating differentiation and cell-fate specification. This coordination of lncRNA dynamics extended to aberrations in both mRNA and lncRNA transcriptomes within HSCs, EBs, and MKs. We specifically characterized lncRNAs with preferential expression in HSCs, as well as in various downstream differentiated lineage progenitors of EBs and MKs, providing a comprehensive perspective on lncRNAs in human hematopoietic cells. Notably, the expression of lncRNAs exhibited substantial cell-to-cell variation, a phenomenon discernible only through single-cell analysis. The comparative analysis undertaken in this study provides valuable insights into the distinctive genetic signatures guiding the differentiation of these crucial hematopoietic cell types.

Segregated basal ganglia output pathways correspond to genetically divergent neuronal subclasses.

The basal ganglia control multiple sensorimotor behaviors though anatomically segregated and topographically organized subcircuits with outputs to specific downstream circuits. However, it is unclear how the anatomical organization of basal ganglia output circuits relates to the molecular diversity of cell types. Here, we demonstrate that the major output nucleus of the basal ganglia, the substantia nigra pars reticulata (SNr) is comprised of transcriptomically distinct subclasses that reflect its distinct progenitor lineages. We show that these subclasses are topographically organized within SNr, project to distinct targets in the midbrain and hindbrain, and receive inputs from different striatal subregions. Finally, we show that these mouse subclasses are also identifiable in human SNr neurons, suggesting that the genetic organization of SNr is evolutionarily conserved. These findings provide a unifying logic for how the developmental specification of diverse SNr neurons relates to the anatomical organization of basal ganglia circuits controlling specialized downstream brain regions.

Lin-CD117+CD34+FcεRI+ progenitor cells are increased in chronic spontaneous urticaria and predict clinical responsiveness to anti-IgE therapy.

Chronic spontaneous urticaria (CSU) is a common, debilitating skin disorder characterized by recurring episodes of raised, itchy and sometimes painful wheals lasting longer than 6 weeks. CSU is mediated by mast cells which are absent from peripheral blood. However, lineage-CD34hiCD117int/hiFcεRI+ cells in blood have previously been shown to represent a mast cell precursor. We enumerated FcεRI-, FcεRI+ and FcεRIhi lineage-CD34+CD117+ cells using flow cytometry in blood of patients with CSU (n = 55), including 12 patients receiving omalizumab and 43 not receiving omalizumab (n = 43). Twenty-two control samples were studied. Disease control and patient response to omalizumab was evaluated using the urticaria control test. We performed single-cell RNA sequencing (scRNA-Seq) on lineage-CD34hiCD117hi blood cells from a subset of patients with CSU (n = 8) and healthy controls (n = 4). CSU patients had more lineage-CD34+CD117+FcεRI+ blood cells than controls. Lineage-CD34+CD117+FcεRI+ cells were significantly higher in patients with CSU who had an objective clinical response to omalizumab when compared to patients who had poor disease control 90 days after initiation of omalizumab. scRNA-Seq revealed that lineage-CD34+CD117+FcεRI+ cells contained both lymphoid and myeloid progenitor lineages, with omalizumab responsive patients having proportionally more myeloid progenitors. The myeloid progenitor lineage contained small numbers of true mast cell precursors along with more immature FcεRI- and FcεRI+ myeloid progenitors. Increased blood CD34+CD117+FcεRI+ cells may reflect enhanced bone marrow egress in the setting of CSU. High expression of these cells strongly predicts better clinical responses to the anti-IgE therapy, omalizumab.

The Endosomal Sorting Complex, ESCRT, has diverse roles in blood progenitor maintenance, lineage choice and immune response.

Most hematological malignancies are associated with reduced expression of one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT). However, the roles of ESCRT in stem cell and progenitor maintenance are not resolved. Parsing signaling pathways in relation to the canonical role of ESCRT poses a challenge. The Drosophila hematopoietic organ, the larval lymph gland, provides a path to dissect the roles of cellular trafficking pathways such as ESCRT in blood development and maintenance. Drosophila has 13 core ESCRT components. Knockdown of individual ESCRTs showed that only Vps28 and Vp36 were required in all lymph gland progenitors. Using the well-conserved ESCRT-II complex as an example of the range of phenotypes seen upon ESCRT depletion, we show that ESCRTs have cell-autonomous as well as non-autonomous roles in progenitor maintenance and differentiation. ESCRT depletion also sensitized posterior lobe progenitors to respond to immunogenic wasp infestation. We also identify key heterotypic roles for ESCRT in position-dependent control of Notch activation to suppress crystal cell differentiation. Our study shows that the cargo sorting machinery determines the identity of progenitors and their adaptability to the dynamic microenvironment. These mechanisms for control of cell fate may tailor developmental diversity in multiple contexts.

Vascular restoration through local delivery of angiogenic factors stimulates bone regeneration in critical size defects

Critical size bone defects represent a significant challenge worldwide, often leading to persistent pain and physical disability that profoundly impact patients’ quality of life and mental well-being. To address the intricate and complex repair processes involved in these defects, we performed single-cell RNA sequencing and revealed notable shifts in cellular populations within regenerative tissue. Specifically, we observed a decrease in progenitor lineage cells and endothelial cells, coupled with an increase in fibrotic lineage cells and pro-inflammatory cells within regenerative tissue. Furthermore, our analysis of differentially expressed genes and associated signaling pathway at the single-cell level highlighted impaired angiogenesis as a central pathway in critical size bone defects, notably influenced by reduction of Spp1 and Cxcl12 expression. This deficiency was particularly pronounced in progenitor lineage cells and myeloid lineage cells, underscoring its significance in the regeneration process. In response to these findings, we developed an innovative approach to enhance bone regeneration in critical size bone defects. Our fabrication process involves the integration of electrospun PCL fibers with electrosprayed PLGA microspheres carrying Spp1 and Cxcl12. This design allows for the gradual release of Spp1 and Cxcl12 in vitro and in vivo. To evaluate the efficacy of our approach, we locally applied PCL scaffolds loaded with Spp1 and Cxcl12 in a murine model of critical size bone defects. Our results demonstrated restored angiogenesis, accelerated bone regeneration, alleviated pain responses and improved mobility in treated mice.

Adhesion GPCR ADGRE2 Maintains Proteostasis to Promote Progression in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous hematologic malignancy. In elderly patients, AML incidence is high and has a poor prognosis due to a lack of effective therapies. G protein-coupled receptors (GPCR) play integral roles in physiologic processes and human diseases. Particularly, one third of adhesion GPCRs, the second largest group of GPCRs, are highly expressed in hematopoietic stem and progenitor cells or lineage cells. Here, we investigate the role of adhesion GPCRs in AML and whether they could be harnessed as antileukemia targets. Systematic screening of the impact of adhesion GPCRs on AML functionality by bioinformatic and functional analyses revealed high expression of ADGRE2 in AML, particularly in leukemic stem cells, which is associated with poor patient outcomes. Silencing ADGRE2 not only exerts antileukemic effects in AML cell lines and cells derived from patients with AML in vitro, but also delays AML progression in xenograft models in vivo. Mechanistically, ADGRE2 activates phospholipase Cβ/protein kinase C/MEK/ERK signaling to enhance the expression of AP1 and transcriptionally drive the expression of DUSP1, a protein phosphatase. DUSP1 dephosphorylates Ser16 in the J-domain of the co-chaperone DNAJB1, which facilitates the DNAJB1-HSP70 interaction and maintenance of proteostasis in AML. Finally, combined inhibition of MEK, AP1, and DUSP1 exhibits robust therapeutic efficacy in AML xenograft mouse models. Collectively, this study deciphers the roles and mechanisms of ADGRE2 in AML and provides a promising therapeutic strategy for treating AML. Significance: Increased expression of the adhesion GPCR member ADGRE2 in AML supports leukemia stem cell self-renewal and leukemogenesis by modulating proteostasis via an MEK/AP1/DUSP1 axis, which can be targeted to suppress AML progression.

Abstract 363: Genetic or pharmacological inactivation of CREBBP sensitizes B-cell acute lymphoblastic leukemia to ferroptotic cell death upon BCL2 inhibition

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological malignancy of B lineage progenitors. It remains a leading cause of death in childhood, while outcomes in adults are dismal. There is therefore a need to better understand drivers of high-risk B-ALL and to develop novel therapeutic approaches targeting these challenging patient cohorts. Loss-of-function mutations affecting CREBBP are recurrent second-hit mutations across multiple genetic subtypes of B-ALL and are associated with adverse features, including high-risk genetic subtypes and persistent measurable residual disease. In addition, they have been mechanistically associated with chemoresistance and are more frequently found in relapse. We sought to identify novel treatment options for CREBBP-mutated high-risk B-ALL. CREBBP-mutated isogenic human B-ALL cell lines were genome-engineered to provide a platform for synthetic lethal drug screening. We subjected these cell lines to a targeted drug screen focused on clinically-actionable drugs in classes that have either been implicated or hypothesized to show differential sensitivity patterns in published models of B cell lymphoma and other CREBBP-mutated malignancies. Unexpectedly, CREBBP-mutated cells were not differentially sensitive to traditional cytotoxic chemotherapy, and paradoxically showed a degree of sensitization to the glucocorticoid Dexamethasone, used in current ALL induction regimens. As anticipated, and validating our screen design, inhibitors of the CREBBP paralogue EP300 (the CREBBP/EP300-specific bromodomain inhibitor Inobrodib and the CREBBP/EP300 acetylase inhibitor A485) exhibited synthetic lethality, consistent with previous reports in B-cell lymphoma. The most potent hit was the BCL2 inhibitor Venetoclax, which we show acts through a non-canonical, but BCL2-dependent mechanism resulting in ferroptotic programmed cell death. CREBBP-mutated cell lines were transcriptionally and functionally characterized, revealing underlying differences in cell-cycle, metabolism and response to oxidative stress. Acquisition of resistance to Venetoclax further dysregulated these pathways and resulted in a transcriptionally-convergent state. Lastly, we demonstrate that small-molecule inhibition of CREBBP sensitizes B-ALL cells, regardless of genotype, to Venetoclax-induced ferroptosis in vitro and in vivo, providing a potential novel drug combination for broader clinical translation in B-ALL. In summary, we have identified a number of actionable compounds that specifically target CREBBP-mutated high-risk B-ALL, demonstrate a novel mechanism-of-action for the BCL2 inhibitor Venetoclax in B-ALL and propose CREBBP-inhibitors and Venetoclax as a novel treatment combination for B-ALL across genotypes. Citation Format: Alicia Garcia Gimenez, Jonathan Ditcham, Dhoyazan M. Azazi, Eshwar Meduri, Ryan Asby, Nathalie Sakakini, Cecile K. Lopez, Nisha Narayan, Jaana Bagri, Tumas Beinortas, Shuchi Agrawal Singh, George Giotopoulos, Michael P. Murphy, Sarah J. Horton, Brian J. Huntly, Simon E. Richardson. Genetic or pharmacological inactivation of CREBBP sensitizes B-cell acute lymphoblastic leukemia to ferroptotic cell death upon BCL2 inhibition [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 363.

An arms-race against resistance: leukemic stem cells and lineage plasticity.

Acute myeloid leukemia (AML) therapy is undergoing rapid development, but primary and acquired resistance to therapy complicates the prospect of a durable cure. Recent functional and single-cell multi-omics approaches have greatly expanded our knowledge of the diversity of lineage trajectories in AML settings. AML cells range from undifferentiated stem-like cells to more differentiated myeloid or megakaryocyte/erythroid cells. Current clinically relevant drugs predominantly target the myeloid progenitor lineage, while monocyte- or stem cell-like states can evade current AML treatment and may be targeted in the future with lineage-specific inhibitors. The extent of aberrant lineage plasticity upon therapeutic pressure in AML cells in conjunction with hijacking of normal differentiation pathways is still a poorly understood topic. Insights into the mechanisms of lineage plasticity of AML stem cells could identify both therapy-specific and cross-drug resistance pathways and reveal novel strategies to overcome them.

Embryonic temporal-spatial delineation of excitatory spinal V3 interneuron diversity

Spinal neural circuits that execute movement are composed of cardinal classes of neurons that emerged from distinct progenitor lineages. Each cardinal class contains multiple neuronal subtypes characterized by distinct molecular, anatomical, and physiological characteristics. Through a focus on the excitatory V3 interneuron class, here we demonstrate that interneuron subtype diversity is delineated through a combination of neurogenesis timing and final laminar settling position. We have revealed that early-born and late-born embryonic V3 temporal classes further diversify into subclasses with spatially and molecularly discrete identities. While neurogenesis timing accounts for V3 morphological diversification, laminar settling position accounts for electrophysiological profiles distinguishing V3 subtypes within the same temporal classes. Furthermore, V3 interneuron subtypes display independent behavioral recruitment patterns demonstrating a functional modularity underlying V3 interneuron diversity. These studies provide a framework for how early embryonic temporal and spatial mechanisms combine to delineate spinal interneuron classes into molecularly, anatomically, and functionally relevant subtypes in adults.

PELI2 regulates early B-cell progenitor differentiation and related leukemia via the IL-7R expression.

Little is known about the transition mechanisms that govern early lymphoid lineage progenitors from common lymphoid progenitors (CLP). Pellino2 (PELI2) is a newly discovered E3 ubiquitin ligase, which plays important roles in inflammation and the immune system. However, the physiological and molecular roles of PELI2 in the differentiation of immune cells are largely unknown. Here, by using a conditional knockout mouse model, we demonstrated that PELI2 is required for early B-cell development and stressed hematopoiesis. PELI2 interacted with and stabilized PU.1 via K63-polyubiquitination to regulate IL-7R expression. The defects of B-cell development induced by PELI2 deletion were restored by overexpression of PU.1. Similarly, PELI2 promoted TCF3 protein stability via K63-polyubiquitination to regulate IL-7R expression, which is required for the proliferation of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. These results underscore the significance of PELI2 in both normal B lymphopoiesis and malignant B-cell acute lymphoblastic leukemia via the regulation of IL-7R expression, providing a potential therapeutic approach for BCP-ALL.

Targeting Endothelial PERK-DLL4 Axis to Enhance Hematopoietic Stem Cell and Lymphoid Progenitor Regeneration

Despite our current information on hundreds of genes implicated in hematopoietic stem cells (HSC) biology, major gaps still exist in our understanding of HSC physiology and their regeneration mechanism under disease and stress conditions, hindering the development of strategies to enhance HSC regeneration. HSCs reside in a tightly controlled bone marrow HSC niche that regulates and maintains hematopoietic homeostasis. HSC niche is composed of a heterogeneous population of specialized stroma cells. Among these, the endothelial cells and the perivascular stroma cells provide critical pro-hematopoietic factors to support HSC pool and committed lineage progenitors particularly lymphoid progenitors. In addition, evidence suggests that endothelium-expressing Notch ligand may promote Notch-dependent long-term HSC proliferation. Recent single cell transcriptome profiling revealed that Delta-like 4 (DLL4) is expressed by the vascular endothelial cells at a higher level than other Notch ligands while its expression down-regulates in response to myeloablation. However, the molecular mechanism regulating the dynamic DLL4 expression during hematopoietic regeneration in response to myeloablative stimuli is unknown. Unfolded protein response (UPR) (also known as endoplasmic reticulum stress, ER stress) has been increasingly recognized as crucial for HSC maintenance. During hematopoietic regeneration, UPR is critical for meeting the increased demand for protein synthesis required for rapid cellular proliferation and for adapting to pathological conditions such as increased levels of reactive oxygen species following irradiation or chemotherapy. Unlike HSCs, how HSC niche endothelial cells deal with redox imbalance and higher requirements of vessel regeneration to regulate stress hematopoietic regeneration is not clear. In this study, using genetically engineered mouse models, we examined the dynamic expression and function of PERK, one arm of UPR, and its potential downstream target, Dll4, during the stress hematopoiesis triggered by transplantation. We found that PERK is activated in the mouse bone marrow endothelial cells following irradiation. Ablation of endothelial Perk in mice (referred as Perk iΔEC mice) promoted post-transplantation immediate recovery of blood cells derived from wild type donor mice and enhanced HSC and lymphoid progenitor regeneration with the expansion of B lineage progenitors including pre-proB, pro-B, and pre-B cells. In addition, by whole-mount immunostaining and intravital microscopy, we found that endothelial Perk deletion restrained disorganized angiogenesis and vascular leakage provoked by irradiation. Although irradiation itself had no effect on endothelial Dll4, Perk ablation markedly increased endothelial Dll4 expression. We found that Dll4 is essential for the post-irradiation HSC and lymphoid progenitor rejuvenation as ablation of endothelial Dll4 in mice (referred as Dll4 iΔEC mice) led to impaired short-term and long-term hematopoietic regeneration and markedly decreased wild type donor derived peripheral B cell and lymphoid progenitor recovery. We found that endothelial Dll4 is required for HSC quiescence maintenance, HSC niche retention and HSC self-renewal. Further, ablation of endothelial Dll4 led to disorganized bone marrow angiogenesis and increased marrow vascular leakage. Finally, we showed that deletion of Dll4 in Perk iΔEC mice completely abrogated the increased numbers of HSC and lymphoid progenitors mediated by Perk deletion, suggesting that up-regulation of endothelial Dll4 is likely responsible for Perk ablation-mediated enhanced HSC and lymphoid progenitor regeneration. Taken together, our results revealed a novel regulatory mechanism by which endothelial Dll4 expression is suppressed by PERK of UPR during transplantation while ablating Perk increases Dll4 expression. Endothelial DLL4 in the bone marrow endothelial cells is critical for supporting vascular integrity and myeloablation-induced HSC and lymphoid progenitor regeneration. Our findings suggest that targeting endothelial PERK-DLL4 axis could be a viable option to improve post-irradiation regeneration of HSC and lymphoid progenitors.

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.

A Mouse Model of Zhu-Tokita-Takenouchi-Kim Syndrome Reveals Indispensable Son Functions in Myeloid-Lymphoid Lineage Balance and Terminal Differentiation

SON is a DNA- and RNA-binding protein mainly localized in nuclear speckles. Compromised SON function leads to aberrant and alternative RNA splicing, particularly for transcripts bearing weak splice sites. SON also binds to DNA and suppresses H3K4me3 modifications at transcription start sites by inhibiting the MLL1/2 methyltransferase complex. More recent studies suggest that SON serves as the core of nuclear speckles, and SON is required for the association between nuclear speckles and p53 target genes to boost RNA expression. Therefore, SON governs the expression of a myriad of target genes by regulating transcription and RNA splicing as well as nuclear speckle assembly, yet exerts its effects on specific sets of genes. However, the role of SON in organ development and hematopoiesis remains poorly understood. Recently, we identified that heterozygous loss-of-function (LoF) mutations in the SON gene cause a neurodevelopmental disorder with multi-organ anomalies in human patients. This syndrome has been designated as a novel rare disease named Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome. The ZTTK SON-Shine Foundation, a family-initiated non-profit organization, was launched, and through this foundation, we work with families to characterize clinical symptoms associated with SON LoF. Among the clinical symptoms observed in individuals with ZTTK syndrome, a noticeable feature is a wide range of hematological abnormalities, including bone marrow failure, high mean corpuscular volume (MCV), polycythemia, severe anemia, and thrombosis. Furthermore, immunoglobulin deficiency, poor responses to vaccines, and frequent infections were reported, implicating SON LoF in various hematopoietic abnormalities. To study how heterozygous LoF of SON affects organ development and causes clinical features associated with ZTTK syndrome, we created a mouse model of Son haploinsufficiency ( Son +/-). While complete knockout of Son ( Son -/-) led to embryonic lethality, Son +/- mice were viable and recapitulated many clinical features of human ZTTK syndrome patients. The Son +/- mice showed severe growth retardation, signs of cognitive impairment, skeletal abnormalities, kidney agenesis/hypoplasia, and, importantly, hematopoietic abnormalities, such as high MCV, low lymphocyte counts, and immunoglobulin deficiency. To examine the hematopoietic features found in ZTTK syndrome patients are due to SON LoF, we further examined hematopoiesis of our Son +/- mice. We found that Son +/- fetal liver as well as adult mouse bone marrow contain a reduced size of the early-stage hematopoietic stem and progenitor cell (HSPC) pool (marked as Lin-Sca1+cKit+: LSK). Analysis of LSK subpopulations revealed that Son +/- mice have myeloid-lymphoid lineage imbalance at the stage of multipotent progenitor (MPP), with increased megakaryocyte/erythroid lineage-biased MPPs (MPP2) while lymphoid lineage-biased MPPs (MPP4) are decreased. In addition, erythroid terminal differentiation and B cell maturation were impaired in Son +/- mice. Single-cell RNA-sequencing of mouse bone marrow Lin-cKit+ cells and transcriptome-based clustering also revealed that Son haploinsufficiency leads to a reduction in lymphoid lineage-committed progenitor cells and decreased expression of lymphoid lineage/B cell development genes, while erythroid lineage-promoting genes were aberrantly activated in myeloid lineage progenitors. Deep bulk RNA-sequencing and alternative splicing analysis identified that that Son haploinsufficiency caused alternative splicing of chromatin modifiers genes and epigenetic regulator genes. Furthermore, from our ATAC-sequencing analysis, we found that Son haploinsufficiency causes decreased chromatin accessibility in the gene loci critical for lymphoid lineage specification. Taken together, our Son +/- mouse model revealed that the normal level of Son is required to achieve the sufficient expression levels of the genes directing lymphoid lineage specification, B cell lineage development, and erythroid terminal differentiation. Our study demonstrates the indispensable roles of SON in multiple stages of hematopoiesis and provides valuable insight into the clinical significance of abnormal SON levels in hematopoietic diseases.

Identification of vulnerabilities for targeting BCL-2 family members in T-Cell Acute Lymphoblastic Leukemia

T-Cell acute lymphoblastic leukemia (T-ALL) is a disease caused by the malignant transformation of T-cell lineage progenitors. With the use of intensive chemotherapies survival rates have improved, but outcomes particularly of relapsed patients remain poor. In addition, currently used intensive chemotherapies are associated with high rates of treatment-related morbidity and mortality, emphasizing the need to develop new and improved therapies. The intrinsic apoptosis pathway, one of the key pathways controlling cell death, is dysregulated in many cancers and contributes to leukemogenesis and treatment failure. The main steps of this pathway are controlled by proteins of the B-cell lymphoma (BCL-2) family at the outer mitochondrial membrane. Hence, targeting this pathway by BH3-mimetics has emerged as an effective new treatment strategy in different cancers. Venetoclax is a selective BCL-2 inhibitor and is successfully used in CLL and AML, but heterogeneous sensitivity to venetoclax has been described in ALL and inhibitors of other BCL-2 family members including BCL-XL-selective A-1331852 and MCL-1 selective AZD5991 have been developed. Moreover, a dual inhibitor of the anti-apoptotic BCL-2 family members BCL-2 and BCL-XL (AZD4320) with its dendrimer conjugate (AZD0466) has recently shown anti-tumor activity in hematologic cancer models with manageable toxicity. The aims of this study are to evaluate susceptibilities of T-ALL to inhibitors of BCL-2 family proteins, to identify markers of response, to elucidate mechanisms of action and to assess combination effects. First, we analyzed the anti-leukemia activities of inhibitors targeting BCL-2 (venetoclax), BCL-XL (A-1331852) and MCL-1 (AZD5991) and of the dual BCL-2/BCL-XL inhibitor AZD4320 in T-ALL cell lines (N=6) and a series of patient-derived xenograft (PDX) samples (N=8). Inhibition of MCL-1 alone was not effective in the cell lines tested (EC50 >1 µM), but heterogeneous sensitivity was found in PDX samples (EC50 <1µM in 3/8 samples). In cell lines, sensitivity for BCL-2 inhibition was exclusively found in a cell line with an early T-cell precursor ALL phenotype (Loucy). Similar to cell lines, we found insensitivity for BCL-2 inhibition in all PDX samples tested (N=8). For inhibition of BCL-XL, we found heterogeneous sensitivities with 4/6 cell lines and 5/8 PDX samples showing an EC 50 <1 µM. Interestingly, BH3-profiling confirmed BCL-2 dependence in Loucy and BCL-XL dependence in the other cell lines. Importantly, dual inhibition of BCL-2 and BCL-XL showed clear responses in cell lines and PDX samples (median EC 50s 796.1 nM and 721.6 nM). Notably, sensitivity was associated with priming of the BH3-peptide BAD (r s=0.89, p=0.03), providing a marker of response. We next sought to elucidate the molecular mechanisms by which different BH3-mimetics induce cell death in T-ALL. Analyzing protein interactions of BCL-2 family members by co-immunoprecipitation studies, we found that AZD4230 acts on-target by releasing BIM from BCL-2 and BCL-XL, but with compensatory increased protein complexes of BIM and MCL-1. Accordingly, combined inhibition of BCL-2 and BCL-XL with inhibition of MCL-1 leads to release of BIM from all three anti-apoptotic proteins enabling downstream apoptosis signaling. These findings suggest that combined inhibition of BCL-2 and BCL-XL together with MCL-1 may provide synergistic benefits. To further validate these data, we applied dynamic BH3-profiling and determined dependencies of T-ALL samples on BCL-2 family members in response to BCL-2/BCL-XL inhibition. Here, we found that the MCL-1 dependence strongly increases upon BCL-2/BCL-XL inhibition, suggesting MCL-1 as a target for combined inhibition. Based on these results, we performed dose-response matrix analyses to test the effects of combined BCL-2/BCL-XL and MCL-1 inhibition. Most importantly, combined inhibition showed synergism in all cell lines and PDX samples tested (Bliss scores >10), including leukemias being insensitive to single compounds. Taken together, we found vulnerabilities of T-ALL to BCL-2 family inhibition, particularly to dual BCL-2/BCL-XL inhibition by AZD4320, and we demonstrated on-target activities. Using BH3-profiling, we identified BAD-priming as a marker of response for AZD4320 and MCL-1 dependence as a resistance mechanism that can be targeted by combination treatment, suggesting further clinical evaluation.

Sickle Cell Trait As a Risk Factor for Leukemia: A Nationwide Study

INTRODUCTION: Leukemia is a cancer of blood-forming cells. It can arise from cells of lymphoid or myeloid lineage. It can arise from genetic mutations in tumor suppressor genes, proto-oncogenes or both. It affects over two million people worldwide. A combination of genetic factors and extrinsic factors are believed to play a role. Sickle cell disease (SCD), an inherited disorder leading to the formation of abnormal hemoglobin (HbS) and red blood cell sickling, has been shown to increase the risk for leukemia, presumably due to an increased bone marrow turnover rate. We aimed to explore if sickle cell trait (SCT), when people inherit only one of the two sickle cell genes, is associated with an increased risk for leukemia. METHODS: In this case-control study, we evaluated an association between leukemia (and its subtypes) and SCT in a nationwide cohort of hospitalized patients included in the National Inpatient Sample (NIS) 2016-2019 database. The inclusion criteria for cases were patients who had any of the following ICD-10-CM codes for leukemia: C91 (lymphoid leukemia), C92 (myeloid leukemia), C93 (monocytic leukemia), C94 (other leukemia of specified cell type), and C95 (leukemia of unspecified cell type). The exclusion criteria were Down syndrome (Q90), Fanconi anemia (D61.09), ataxia-telangiectasia (G11.3), Bloom syndrome (Q82), myelodysplastic syndrome (D46), lymphoma (C81, 85), and solid malignancies (C00-75) as these diseases and/or their treatments are known to increase the risk for leukemia independently of SCT (ICD-10-CM D57.3). The controls included patients in the NIS database who do not have the ICD-10-CM codes for leukemia and meet the exclusion criteria applied to the cases. For each year, we used a 1:4 propensity score matching routine. Our match variables were sex, the six race categories used by NIS, and 11 age groups. Four perfect matches (controls) were identified for each case (‘any leukemia’ diagnosis). Patient characteristics were compared using Pearson chi-squared test. Odds ratios (ORs) were calculated for ‘any leukemia’ diagnosis, as well as for each subtype of leukemia. Five multivariate logistic regression models were used to examine the relationship between the leukemia subtypes and SCT while controlling for age group, sex, and race. RESULTS: We identified 181,676 cases and 726,704 controls. The mean age of subjects was 59 years. The majority of subjects (56%) were male. A greater proportion of patients with SCT were found among cases (0.12%) compared to controls (0.08%) (p < 0.0001). The OR for ‘any leukemia’ was 1.48 (95% CI 1.27-1.73, p < 0.0001). When the association between SCT and specific subtypes of leukemia was evaluated, it was statistically significant for myeloid and monocytic leukemias, OR 1.74 (95% CI 1.40 - 2.17) and 2.20 (95% CI 1.09 - 4.42) correspondingly. CONCLUSION: Our case-control study revealed statistically significantly higher odds of SCT among patients with any leukemia (and specifically myeloid and monocytic leukemias) as compared to controls which can be postulated to the common myeloid progenitor lineage of erythrocytes. Our results suggest that, similarly to SCD, SCT although a benign condition can increase the risk for leukemia. To better understand this association, future prospective studies are needed for the mapping of individual risk factors and examining the HbS% levels in SCT carriers who go on to develop leukemia.

New Insights into Hematopoietic Redefinition and New Paradigms in Hematopoietic Hierarchy Roadmap

The definition of hematopoietic progenitor cells (HPCs) has not been standardized. Surface markers are insufficient to capture the entire differentiation stages of progenitor cells and reflect the dynamic differentiation mechanisms underlying hematopoiesis. Deciphering the hematopoiesis mechanism that underlies progenitor cells definition and fate decision are critical to understanding fundamental questions of hematopoietic lineage commitment. Here, we redefined the entire spectrum of adult hematopoietic progenitors, and recovered the critical transcript factors (TFs) in lineages fate decision, and reaffirmed the lineage commitment model of HPCs by a comprehensive transcriptome. The progenitor lineages were accurately redefined with unique makers and transcription factors by unsupervised dimensionality reduction and clustering, which subverted the conventional definition of HPCs（Table 1）. The expression characteristics of TFs, such as GATA1, KLF1, PRDM1, and MAFB, presented enormous capacity to define the entire spectrum of HPCs. The transcriptional atlas showed that original hematopoietic progenitors were simultaneously primed into lymphoid, neutrophils, and megakaryocytic-erythroid progenitor cell types in the initial differentiation stage and thereafter matured into different hematopoietic lineages (Figure 1). The common intermediate progenitor stage was not observed among myeloid lineages, indicating the absence of a common myeloid progenitor during the lineage commitment. Our transcriptional atlas revealed the major fate decision factors in each branch among hematopoietic hierarchy formation (Table 1). Several fate decision factors were identified, including GATA2, TESPA1, and MYB of myeloid progenitors and HOXA9, HOPX, and TSC22D1 of lymphoid progenitors. Through the sequential activation/deactivation of TFs, synergism with key receptor genes, and signal activation, the original progenitor cells were primed and differentiated into various progenitors. The expression and activation characteristics of fate decision TFs faithfully reflect the mechanisms underlying hematopoietic lineage commitment. A hematopoietic hierarchy roadmap was reaffirmed. Our reaffirmed hematopoietic hierarchy validated that the megakaryocytic-erythroid lineage process is continuous, while the lymphoid lineage process is more likely a discrete stepwise progression with distinct lineage-committed populations during hematopoiesis, opening a new perspective on hematopoiesis (Figure 1). Our study revealed that the original progenitors of basophils, eosinophils, and neutrophils differed. Plasma progenitor cells were first identified and defined, and the rearrangements of IGHV, IGKV, and IGLV genes were already existed and occurred during the progenitor state of plasma cells. In summary, our transcriptional atlas of HPCs provides an accurate definition, hierarchy, and biological insights into hematopoiesis and lineage commitment. Our study opens up numerous possibilities for precise and controlled progenitor cell differentiation, facilitating research development in regenerative medicine and disease treatment.

DNMT3A-Mutated Stem and Progenitor Cells Contribute to Altered T Cell Activation in Clonal Hematopoiesis

Clonal hematopoiesis is characterized by an expansion of blood cells derived from a single mutated hematopoietic stem cell (HSC). When this expansion is driven by a leukemia-associated mutation that exceeds a variant allele frequency (VAF) of 2%, it is termed clonal hematopoiesis of indeterminate potential (CHIP). Approximately half of CHIP cases are driven by mutations in the DNA methyltransferase gene DNMT3A. In patients with CHIP, greater clonal expansion is associated with an increased risk of cardiovascular diseases and hematologic malignancies. While DNMT3A impairment has been shown to alter inflammatory signaling in HSCs and myeloid cells, less is known about the impact of DNMT3A-mutated CHIP on T cell biology. Here, we leverage a cohort of human CHIP samples to assess how the expansion of DNMT3A-mutated cells affects gene expression across blood cell types and generate a new mouse model to assess antigen-specific immune cell interactions. We find evidence that the accumulation of DNMT3A-mutated stem and progenitor cells can cause T cell dysfunction. We first identified CHIP in a cohort of 191 sternum bone marrow samples collected during heart surgery. We detected CHIP in 45 samples (23.6%), including 22 samples with mutated DNMT3A (48.9% of CHIP samples). To mitigate confounding factors that might be introduced by comparing CHIP to healthy individuals, we used ten DNMT3A-mutated CHIP samples for this study, splitting them into a low-VAF (<10%, three samples) and high-VAF (>10%, seven samples) cohorts. We performed single-cell RNA sequencing on these 10 samples, generating high-quality data for 95,245 cells. We clustered and annotated 33 cell types according to the expression of canonical marker genes, representing the full spectrum of hematopoietic progenitors to myeloid, erythroid and lymphoid lineages. To evaluate how greater DNMT3A clonal expansion affects immune cell states, we performed differential gene expression analysis in the high-VAF compared to the low-VAF cohort with DESeq2 using a pseudobulk approach followed by gene set enrichment analysis using Hallmark pathways. We found that TGF-beta signaling, known for its immunosuppressive role, is upregulated in hematopoietic stem and progenitor cells (HSPCs), myeloid and T cell populations in the high-VAF compared to the low-VAF cohort. Both interferon alpha and gamma signaling pathways are consistently downregulated in HSPCs and multiple myeloid cell types as well as T cells. TNF-alpha signaling is upregulated in HSPCs and myeloid cells, consistent with recent observations linking TNF-alpha to the survival and proliferation of HSPCs and myeloid cells. Overall, greater expansion of DNMT3A-mutated cells is associated with variable gene expression changes in progenitor and myeloid cell types, whereas gene expression changes in T cells are consistent with diminished activation (Figure 1). To evaluate mechanisms contributing to altered T cell activation in DNMT3A-mutated CHIP, we combined our Dnmt3a-fl-R878H/+ inducible mouse model with the ovalbumin (OVA) trackable antigen system. Endogenously expressed OVA is processed into a peptide that is loaded onto MHCI and recognized by transgenic CD8+ T cells from OT-I mice. We crossed the Dnmt3a-fl-R878H/+ mouse with a mouse expressing OVA under the actin promoter, resulting in Dnmt3a-mutated mice with constitutive OVA expression. To interrogate antigen-specific T cell responses, we co-cultured Dnmt3a-mutated OVA+ lineage negative (Lin-) stem and progenitor cells with CD8+ T cells from OT-I mice. By measuring T cell proliferation using CFSE dilution, we observed an increase in T cell proliferation when CD8+ T cells were co-cultured with Dnmt3a-mutated OVA+ Lin- cells compared to wildtype OVA+ Lin- cells (Figure 2). These results suggest that Lin- Dnmt3a-mutated cells have an increased capacity for antigen-specific T cell activation. In conclusion, our studies demonstrate that greater expansion of DNMT3A-mutated CHIP is associated with reduced expression of activation signatures in T cells. In apparent contrast, Dnmt3a-mutated mouse cells have an increased capacity for antigen-specific T cell stimulation. Since chronic antigenic stimulation causes T cell exhaustion, our findings are consistent with a model in which the expansion of DNMT3A-mutated progenitor and myeloid cells causes chronic stimulation and dysfunction of T cells.

Differential DNA damage repair and PARP inhibitor vulnerability of the mammary epithelial lineages

It is widely assumed that all normal somatic cells can equally perform homologous recombination (HR) and non-homologous end joining in the DNA damage response (DDR). Here, we show that the DDR in normal mammary gland inherently depends on the epithelial cell lineage identity. Bioinformatics, post-irradiation DNA damage repair kinetics, and clonogenic assays demonstrated luminal lineage exhibiting a more pronounced DDR and HR repair compared to the basal lineage. Consequently, basal progenitors were far more sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis) in both mouse and human mammary epithelium. Furthermore, PARPi sensitivity of murine and human breast cancer cell lines as well as patient-derived xenografts correlated with their molecular resemblance to the mammary progenitor lineages. Thus, mammary epithelial cells are intrinsically divergent in their DNA damage repair capacity and PARPi vulnerability, potentially influencing the clinical utility of this targeted therapy.