Multipotent Progenitor Research Articles

P1018 Diyushengbai tablets alleviate azathioprine-induced leukopenia in patients with Crohn’s Disease

Abstract Background Azathiopurine (AZA) is the classic immunosuppressant for crohn’s disease (CD) and plays a critical role in induction and maintenance of remission, facilitation of mucosal healing, and prevention of postoperative recurrence. The incidence of azathiopurine-induced leukopenia (AIL) in Asians is high, posing a serious challenge for clinical medication. Diyushengbai tablets are synthetic traditional Chinese medicine, widely used to prevent and treat leukopenia caused by chemotherapy. It remains unclear about its role in treating AIL. Ziyuglycoside II is the key ingredient of Diyushengbai tablet. The mechanism of Ziyuglycoside II in preventing AIL is yet unclear. Therefore, the objectives of this study are to explore the effects of Diyushengbai tablets in the treatment of AIL and the possible mechanism of Ziyuglycoside II in the prevention of AIL. Methods CD patients with AIL were prospectively enrolled. The leukocyte counts of CD patients before, four weeks, and eight weeks after taking Diyushengbai tablets were collected. AIL mouse models were established by intragastric administration of AZA (10mg/kg) treated with or without Ziyuglycoside II (5mg/kg and 10mg/kg). The leukocyte counts of mice were measured at weeks 0, 1, 2, and 3. The bone marrow cells of mice were extracted for flow cytometry. Results 21 CD patients with AIL were enrolled in this study. The leukocyte counts before, four weeks, and eight weeks after treatment were 2.96±0.42×109/L, 3.48±0.73×109/L, and 4.26±1.85×109/L, respectively. Diyushengbai tablets increased the leukocyte counts at week four (P=0.004) and week eight (P<0.001), respectively. They maintained the leukocyte counts no less than 3.0×109/L in 76.19% (16/21) and 85.71% (18/21) of patients after four-week and eight-week treatment, respectively. They also prevented more than 95% (20/21) of patients from withholding AZA in those who developed AIL during the eight-week following-up. The decrease of leukocyte counts was significantly lower in the Ziyuglycoside II-treatment group compared with the AIL group. Ziyuglycoside II treatment increased the number of hematopoietic stem and progenitor cells (HSPCs) including long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs), and multipotent progenitor cells (MPPs). Conclusion Diyushengbai tablets can be used to treat AIL. They increase the leukocyte counts and prevent most CD patients with AIL from withholding AZA. Ziyuglycoside II increases the leukocyte counts and prevents AIL by stimulates the proliferation and differentiation of HSPCs. These findings highlight a promising role of Diyushengbai tablets in treating AIL.

NR2F6 regulates stem cell hematopoiesis and myelopoiesis in mice.

Nuclear receptors regulate hematopoietic stem cells (HSCs) and peripheral immune cells in mice and humans. The nuclear orphan receptor NR2F6 (EAR-2) has been shown to control murine hematopoiesis. Still, detailed analysis of the distinct stem cell, myeloid, and lymphoid progenitors in the bone marrow in a genetic loss of function model remains pending. In this study, we found that adult germline Nr2f6-deficient mice contained increased percentages of total long-term and short-term HSCs, as well as a subpopulation within the lineage-biased multipotent progenitor (MPP3) cells. The loss of NR2F6 thus led to an increase in the percentage of LSK+ cells. Following the differentiation from the common myeloid progenitors (CMP), the granulocyte-monocyte progenitors (GMP) were decreased, while monocyte-dendritic progenitors (MDP) were increased in Nr2f6-deficient bone marrow. Within the pre-conventional dendritic progenitors (pre-cDCs), the subpopulation of pre-cDC2s was reduced in the bone marrow of Nr2f6-deficient mice. We did not observe differences in the development of common lymphoid progenitor populations. Our findings contrast previous studies but underscore the role of NR2F6 in regulating gene expression levels during mouse bone marrow hematopoiesis and myelopoiesis.

Polarity and migration of cranial and cardiac neural crest cells: underlying molecular mechanisms and disease implications.

The Neural Crest cells are multipotent progenitor cells formed at the neural plate border that differentiate and give rise to a wide range of cell types and organs. Directional migration of NC cells and their correct positioning at target sites are essential during embryonic development, and defects in these processes results in congenital diseases. The NC migration begins with the epithelial-mesenchymal transition and extracellular matrix remodeling. The main cellular mechanisms that sustain this migration include contact inhibition of locomotion, co-attraction, chemotaxis and mechanical cues from the surrounding environment, all regulated by proteins that orchestrate cell polarity and motility. In this review we highlight the molecular mechanisms involved in neural crest cell migration and polarity, focusing on the role of small GTPases, Heterotrimeric G proteins and planar cell polarity complex. Here, we also discuss different congenital diseases caused by altered NC cell migration.

Driver mutations in myeloid and lymphoid cells point to multipotent progenitor origin of diverse histiocytic neoplasms

Driver mutations in myeloid and lymphoid cells point to multipotent progenitor origin of diverse histiocytic neoplasms

Population dynamics modeling reveals that myeloid bias involves both HSC differentiation and progenitor proliferation biases.

Aging and chronic inflammation are associated with overabundant myeloid-primed multipotent progenitors (MPPs) amongst hematopoietic stem and progenitor cells (HSPCs). While HSC differentiation bias has been considered a primary cause of myeloid bias, whether it is sufficient has not been quantitatively evaluated. Here, we analyzed bone marrow data from the IκB- (Nfkbia+/-Nfkbib-/-Nfkbie-/-) mouse model of inflammation with elevated NFκB activity, which shows increased myeloid-biased MPPs. We interpreted these data with differential equations models of population dynamics to identify alterations of HSPC proliferation and differentiation rates. This analysis revealed that short-term (ST) HSC differentiation bias alone is likely insufficient to account for the increase in myeloid-biased MPPs. To explore additional mechanisms, we used single-cell RNA sequencing (scRNA-seq) measurements of IκB- and wild-type HSPCs to track the continuous differentiation-trajectories from HSCs to erythrocyte/megakaryocyte, myeloid, and lymphoid primed progenitors. Fitting a partial differential equations model of population dynamics to these data revealed not only less lymphoid-fate specification amongst HSCs, but also increased expansion of early myeloid-primed progenitors. Differentially expressed genes along the differentiation-trajectories supported increased proliferation amongst these progenitors. These findings were conserved when wild-type HSPCs were transplanted into IκB- recipients, indicating that an inflamed bone marrow microenvironment is a sufficient driver. We then applied our analysis pipeline to scRNA-seq measurements of HSPCs isolated from aged mice, as well as human myeloid neoplasm patients. These analyses identified the same myeloid-primed progenitor expansion as in the IκB- models, suggesting that it is a common feature across different settings of myeloid bias.

Oncogene activated human breast luminal progenitors contribute basally located myoepithelial cells

BackgroundBasal-like breast cancer originates in luminal progenitors, frequently with an altered PI3K pathway, and focally in close association with genetically altered myoepithelial cells at the site of tumor initiation. The exact trajectory behind this bi-lineage phenomenon remains poorly understood.Methods and resultsHere we used a breast cancer relevant transduction protocol including hTERT, shp16, shp53, and PIK3CAH1047R to immortalize FACS isolated luminal cells, and we identified a candidate multipotent progenitor. Specifically, we identified a keratin 23 (K23)+/ALDH1A3+/CALML5− ductal-like progenitor with the potential to differentiate into CALML5+ lobular-like cells. We found that the apparent luminal phenotype of these oncogene transduced progenitors was metastable giving rise to basal-like cells dependent on culture conditions. In 3D organoid culture and upon transplantation to mice the bipotent progenitor cell line organized into a bi-layered acinus-like structure reminiscent of that of the normal breast gland.ConclusionsThese findings provide proof of principle that progenitors within the human breast luminal epithelial compartment may serve as a source of correctly positioned myoepithelial cells. This may prove useful in assessing the role of myoepithelial cells in breast tumor progression.

Alteration of skin fibroblast steady state contributes to healing outcomes.

Fibroblasts display complex functions associated with distinct gene expression profiles that influence matrix production and cell communications and the autonomy of tissue development and repair. Thrombospondin-2 (TSP-2), produced by fibroblasts, is a potent angiogenesis inhibitor and negatively associated with tissue repair. Single-cell (sc) sequencing analysis on WT and TSP2KO skin fibroblasts demonstrate distinct cell heterogeneity. Specifically, we found an enrichment of Sox10+ multipotent progenitor cells, identified as Schwann precursor cells, in TSP2KO fibroblasts, while fibrosis-related subpopulations decreased. Immunostaining of tissue and cells validated the increase of this Sox10+ population in KO fibroblasts. Furthermore, in silico analysis suggested enhanced pro-survival signaling, including WNT, TGF-β, and PDGF-β, alongside a reduced BMP4 response. Additionally, the creation of two TSP2KO NIH3T3 cell lines using the CRISPR/Cas9 technique allowed functional and signaling validation in a less complex system. Moreover, KO 3T3 cells exhibited enhanced migration and proliferation, with elevated levels of pro-regenerative molecules including TGF-β3 and Wnt4, and enrichment of nuclear β-catenin. These functional and molecular alterations likely contribute to improved healing and increased neurogenesis in TSP2-deficient wounds. Overall, our findings describe the heterogeneity of dermal fibroblasts and identify pro-regenerative features of TSP2KO fibroblasts.

Siwu decoction mitigates radiation-induced immune senescence by attenuating hematopoietic damage

BackgroundTo investigate the long term effects of ionizing radiation (IR) on hematopoietic stem/progenitor cells (HSPCs), immune tissues and cells, and the effects of Siwu decoction (SWD) on immune senescence mice.MethodsC57BL/6 J mice were exposed to 6.0 Gy 60Co γ irradiation. After 8-weeks of IR, SWD (5, 10, 20 g/kg/d) was administered for 30 days. The changes of HSPCs in bone marrow (BM) and T, B type lymphocyte and natural killer (NK) cells in spleen were detected by flow cytometry. The changes of peripheral blood cells were also examined. Hematoxylin–eosin staining were used to detect the pathological lesions of hippocampus, spleen and thymus tissues. Absolute mouse telomere length quantification qPCR assay kit was used to measure the telomere length of BM cells. The expression of factors associated with inflammation and aging such as p16, β-galactosidase in spleen, thymus and BM was determined.ResultsAdministration of SWD could increase the proportion of LSK (Lin−, Sca-1 + , c-Kit−), multipotent progenitor cells and multipotent progenitor cells and decrease the proportion of common myeloid progenitors and granulocyte–macrophage progenitors in BM. The proportion of B cells and NK cells in spleen and the content of white blood cells, red blood cells, hemoglobin, lymphocytes and eosinophils in peripheral blood were increased, at the same time, the proportion of neutrophils and monocytes was reduced by SWD. The pathological lesions of hippocampus, spleen and thymus were improved. The expression of p16 and β-galactosidase in spleen, thymus and BM was reduced and shortening of the telomere of BM cells was inhibited after administration. In addition, SWD could reduce the content of Janus activated kinase (JAK) 1, JAK2 and signal transducer and activator of transcription 3 (STAT3) in BM and spleen.ConclusionsSWD could slow down IR-induced immune senescence by improving hematopoietic and immunologic injury. SWD might reduce the inflammation level of BM hematopoietic microenvironment by acting on JAK/STAT signaling pathway, while the immune damage of mice was improved by affecting the differentiation of HSPCs. The remission of hematopoietic and immunologic senescence was further demonstrated at the overall level.Graphical

Delineation of erythropoietic intermediates by flow cytometry

Erythropoiesis occurs through specification from multipotent progenitors to erythroid restricted potential, expansion of erythroid progenitors, and terminal maturation of precursors to red blood cells. Acute anemia can induce changes at multiple stages of erythropoiesis, thus delineation and comparison of intermediates is critical to understanding this regulation. Historically, erythropoietic intermediates have been defined by functional colony forming assays (progenitors) or microscopy (precursors). While these sensitive single cell techniques have allowed detailed studies of the erythron, they do not allow for prospectively identifying and isolating live cells for experimental analyses. This has fueled development of flow cytometric criteria for analyzing the erythron from many different research groups for both the human and mouse systems. With these data, models of the immunophenotypic continuum of the erythron can be generated progressing from the earliest erythroid specific progenitors through late erythroblasts revealing remarkable conservation between human and murine cells. Recent data have also uncovered issues with previous classification schemes of erythromyeloid progenitors that are particularly problematic for erythroid progenitors. Applying these flow cytometric tools requires consideration of gating on a continuum in a reproducible fashion, fragments of macrophages caused by tissue dissociation on a proportion of erythropoietic cells, and ultimately application in anemia where signaling may impact the range of expression of specific immunophenotyping markers.

A timed epigenetic switch balances T and ILC lineage proportions in the thymus.

How multipotent progenitors give rise to multiple cell types in defined numbers is a central question in developmental biology. Epigenetic switches, acting at single gene loci, can generate extended delays in the activation of lineage-specifying genes and impact lineage decisions and cell type output. Here, we analyzed a timed epigenetic switch controlling expression of mouse Bcl11b, a transcription factor that drives T-cell commitment, but only after a multi-day delay. To investigate roles for this delay in controlling lineage decision making, we analyzed progenitors with a deletion in a distal Bcl11b enhancer, which extends this delay by ∼3 days. Strikingly, delaying Bcl11b activation reduces T-cell output but enhances innate lymphoid cell (ILC) generation in the thymus by redirecting uncommitted progenitors to the ILC lineages. Mechanistically, delaying Bcl11b activation promoted ILC redirection by enabling upregulation of the ILC-specifying transcription factor PLZF. Despite the upregulation of PLZF, committed ILC progenitors could subsequently express Bcl11b, which is also needed for type 2 ILC differentiation. These results show that epigenetic switches can control the activation timing and order of lineage-specifying genes to modulate cell type numbers and proportions.

Morphogenesis and regeneration share a conserved core transition cell state program that controls lung epithelial cell fate.

Transitional cell states are at the crossroads of crucial developmental and regenerative events, yet little is known about how these states emerge and influence outcomes. The alveolar and airway epithelia arise from distal lung multipotent progenitors, which undergo cell fate transitions to form these distinct compartments. The identification and impact of cell states in the developing lung are poorly understood. Here, we identified a population of Icam1/Nkx2-1 epithelial progenitors harboring a transitional state program remarkably conserved in humans and mice during lung morphogenesis and regeneration. Lineage-tracing and functional analyses reveal their role as progenitors to both airways and alveolar cells and the requirement of this transitional program to make distal lung progenitors competent to undergo airway cell fate specification. The identification of a common progenitor cell state in vastly distinct processes suggests a unified program reiteratively regulating outcomes in development and regeneration.

Impact of tumor on the cell cycle and differentiation of hematopoietic stem cells

Today, there is a theory that proliferative potential of hematopoietic stem cells is depleted, and the balance of committed precursor cells shifts towards suppressors during the development of cancer. However, differentiation of hematopoietic stem cells can vary depending on the tumor type, localization, and microenvironment specifics. The study aimed to assess the impact of tumors of various origins on the CD34+ hematopoietic stem cells (n = 10). Assessment of the cell cycle and cell differentiation via both direct contact with the tumor and exchanging humoral factors only in transwells was conducted by flow cytometry. In the co-culture with К562, the number of hematopoietic stem cells being in their synthesis phase was 2.1%, while in the control it was 11.2% (p = 0.01); in the co-culture with SK-mel37, the number of hematopoietic stem cells being in the G2‒M cell cycle phase was reduced to 0.3% (p < 0.05). 1301 and К562 directed the hematopoietic stem cell differentiation towards granulocyte-macrophage precursor cells (p < 0.05), while 1301 and SK-mel37 directed it towards common multipotent progenitor cells. It is interesting that the number of pluripotent hematopoietic stem cells significantly increased (2-fold) compared to control after incubation with К562 in transwells (24.17% and 10.19%, respectively). Thus, properties of hematopoietic stem cells can vary depending on both tumor type and the way of interacting with these cells.

Aging negatively affects postoperative recovery of biomechanical strength through decreased recruitment of Scx+/Sox9+ enthesis-related progenitors after rotator cuff repair in rats

BackgroundAlthough older adult patients have a higher retear rate after rotator cuff (RC) repair, the influence of aging on the healing process remains unclear. During mouse enthesis development, a multipotent progenitor co-expressing scleraxis (Scx) and SRY-box 9 (Sox9) contributes to enthesis formation. Scx+/Sox9+ cells may act as enthesis progenitors even during postnatal healing, and their number decreases with maturation. However, the pathophysiology of decreased RC healing ability due to aging remains unclear. We aimed to evaluate the effects of aging on tendon-to-bone healing after surgical RC repair in rats through biomechanical and histological analyses of Scx+ and Sox9+ progenitors in a ScxGFP transgenic rat model. MethodsThis was a controlled laboratory study. Adult Sprague-Dawley rats (n = 111) underwent unilateral surgery for supraspinatus tendon repair immediately after transection. The rats were divided into aged (95 weeks old) and control (12 weeks old) groups. The effects of aging were assessed using biomechanical tests at 6 weeks postoperatively and histologically at 2 and 6 weeks postoperatively. ScxGFP transgenic rats were used for the immunohistochemical assessment of Scx- and Sox9-expressing progenitors during the repair process. Sox9, Scx, and tenomodulin expression was assessed using real-time reverse transcription polymerase chain reaction. ResultsIn the biomechanical test at 6 weeks postoperatively, the aged group had lower ultimate load to failure (Control: 20.4 ± 6.1 N, Aged: 14.9 ± 6.6 N, P = 0.007), stiffness (Control: 16.1 ± 6.2 N/mm, Aged: 12.6 ± 5.5 N/mm, P = 0.023), and ultimate stress to failure (Control: 6.0 ± 3.4 N/mm2, Aged: 3.4 ± 2.5 N/mm2, P < 0.001) than the control group. The total histological score of the aged group was lower than that of the control group at 6 weeks postoperatively (Control: 8.8 ± 0.8, Aged: 5.8 ± 0.4, P = 0.029). Immunohistochemistry tests showed that the percentages of Sox9+ (Control: 6.6 ± 1.1, Aged: 2.0 ± 1.0, P = 0.029) and Scx+/Sox9+ (Control: 3.6 ± 0.8, Aged: 1.1 ± 0.6, P = 0.029) cells at the reparative site were lower in the aged group than in the control group at 2 weeks postoperatively. ConclusionIn a rat RC tendon-to-bone healing model, the decreased recruitment of Scx+/Sox9+ enthesis-related progenitor cells in the early phase may be associated with delayed reparative tissue remodeling in aging animals. These findings encourage the development of therapeutic strategies that biologically promote healing and reduce postoperative retears in older adult patients.

Zinc Ameliorates High Pi and Ca-Mediated Osteogenic Differentiation of Mesenchymal Stem Cells.

Zinc is the second most abundant trace element in the human body, stored mainly in the bones. Zinc is required for bone growth and homeostasis and is also a crucial cofactor for numerous proteins that play key roles in maintaining microstructural integrity and bone remodeling. Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent progenitors found in the bone marrow stroma and can differentiate along multiple lineage pathways. In this study, we investigated the effect of zinc on the osteogenic differentiation of BMSCs. We stimulated the osteogenic differentiation of BMSCs with high phosphate and Ca-containing osteogenic medium (PiCa) in the presence or absence of zinc. We followed calcification by measuring ECM mineralization, the Ca content of the ECM, mRNA, and the protein expression of the osteo-chondrogenic transcription factor RUNX2 and SOX9 and its targets OCN and ALP. Zinc dose-dependently abolished PiCa-induced ECM mineralization and decreased the expression of RUNX2, SOX9, OCN, and ALP. Serum albumin did not alter the inhibitory effect of zinc on BMSC mineralization. Our further analysis with the zinc-chelator TPEN and ZnCl2 confirmed the specific inhibitory effect of free zinc ions on BMSC mineralization. Zinc inhibited phosphate uptake and PiCa-induced upregulation of the sodium-dependent phosphate cotransporters (PiT-1 and PiT-2). Zinc attenuated the PiCa-induced increase in ROS production. Taken together, these data suggest that zinc inhibits PiCa-induced BMSC calcification by regulating phosphate uptake and ROS production.

A developmental biliary lineage program cooperates with Wnt activation to promote cell proliferation in hepatoblastoma

Cancers evolve not only through the acquisition and clonal transmission of somatic mutations but also by epigenetic mechanisms that modify cell phenotype. Here, we use histology-guided and spatial transcriptomics to characterize hepatoblastoma, a childhood liver cancer that exhibits significant histologic and proliferative heterogeneity despite clonal activating mutations in the Wnt/β-catenin pathway. Highly proliferative regions with embryonal histology show high expression of Wnt target genes, the embryonic biliary transcription factor SOX4, and striking focal expression of the growth factor FGF19. In patient-derived tumoroids with constitutive Wnt activation, FGF19 is a required growth signal for FGF19-negative cells. Indeed, some tumoroids contain subsets of cells that endogenously express FGF19, downstream of Wnt/β-catenin and SOX4. Thus, the embryonic biliary lineage program cooperates with stabilized nuclear β-catenin, inducing FGF19 as a paracrine growth signal that promotes tumor cell proliferation, together with active Wnt signaling. In this pediatric cancer presumed to originate from a multipotent hepatobiliary progenitor, lineage-driven heterogeneity results in a functional growth advantage, a non-genetic mechanism whereby developmental lineage programs influence tumor evolution.

Abstract 4137935: Adipogenesis in Bone Marrow Niche under Cardiac Stress Worsens Cardiac Function

Background: We have recently reported that repetitive cardiac decompensation with multimorbidity often experienced by patients with heart failure (HF) is attributed to epigenetic modifications of hematopoietic stem cells (HSCs) in the bone marrow (BM). HF reprogrammed HSCs differentiation and altered tissue macrophage homeostasis. These findings demonstrate that the BM can carry an innate immune memory of cardiac stress that may exacerbate HF and predispose other organs to pathology. Aims: Because the stemness of HSCs is mainly regulated by mesenchymal stromal cells (MSCs) in the BM niche, we investigated phenotypic alterations of MSCs under cardiac stress. Methods&amp;Results: Transcriptome analysis of MSCs showed preferential differentiation toward adipocytes in murine pressure overload models. In vitro assays and histological BM sections also support this finding. Furthermore, single-cell RNA sequencing of MSCs demonstrated that the percentage of adipocyte-primed MSCs increased in proportion to the severity of cardiac dysfunction, and also correlated with the frequency of myeloid-lineage progenitor cells. To investigate the influence of adipo-lineage MSCs on HSCs, we conducted BM transplantation supplemented with MSCs from control or HF mice. Recipient mice transplanted with HF-MSCs showed significant increases in myeloid-biased multipotent progenitors in BM and myeloid cells in peripheral blood. Additionally, the number of proinflammatory cardiac macrophages was significantly increased in the HF-MSCs group, promoting cardiac fibrosis and dysfunction. Conclusions: Our results demonstrated that the BM niche could perceive cardiac stress in the form of adipocytic skewing of MSCs in the setting of HF, which changed the differentiation behavior in HSCs and ultimately led to further deterioration of cardiac function through the impaired differentiation of circulating monocytes into cardiac macrophages. Therefore, suppressing the adipocytic differentiation of MSCs could have a novel therapeutic potential to avoid repeated HF events.

CSIG-24. BRAIN MICROENVIRONMENT-DEPENDENT NEURODEVELOPMENTAL LINEAGE PLASTICITY PROMOTES ADAPTATION TO ONCOGENE INHIBITION IN MALIGNANT GLIOMAS

Abstract Phenotypic plasticity drives non-genetic tumor adaptation in response to various microenvironmental and therapeutic pressures, consequently promoting tumor heterogeneity and progression. In malignant gliomas, the intrinsic and extrinsic mechanisms underlying phenotypic lineage plasticity and its contribution to drug resistance remain unclear. The epidermal growth factor receptor (EGFR) is frequently altered in glioma and serves as a critical regulator of normal radial glia (RG) cells – multipotent progenitors that give rise to cortical neurons and glia in the developing brain. Here, through interrogation of a large panel of diverse glioblastoma (GBM) patient-derived gliomaspheres and orthotopic xenografts, we find that enrichment of RG-like cells is significantly correlated with responses to pharmacological ablation of EGFR, highlighting EGFR as a crucial lineage survival factor for a population of malignant RG-like cells in GBM. Single-cell RNA sequencing and quantitative proteomics reveal that adaptation to EGFR inhibition is linked to a temporal contraction in RG-like cells and concomitant increase in lineage-restricted neuronal and oligodendrocyte progenitor (NPC/OPC)-like states exclusively in the orthotopic brain environment. This lineage transition is coupled to heightened RAS signaling gene expression programs and sustained activation of MAPK signaling, despite robust and durable inhibition of EGFR activation. Furthermore, constitutively active MEK – but not AKT – is sufficient to rescue tumor proliferation under EGFRi, suggesting that the brain microenvironment modulates RAS-MAPK oncogenic signaling to drive lineage transitions following EGFR blockade. Collectively, these results highlight a critical link between oncogenic signaling and neurodevelopmental lineage plasticity in malignant gliomas, presenting a compelling therapeutic opportunity to block tumor cell state transitions for more durable tumor responses in the native glioma tumor microenvironment.

STEM-14. INTEGRATED ANALYSIS OF GLIOBLASTOMA PROGENITOR SUBTYPES REVEALS A TUMOR-DERIVED STEM CELL POPULATION WITH NEURAL AND VASCULAR POTENTIAL

Abstract Glioblastoma (GBM) exhibits a vast heterogeneity in cell types and states. Recent research highlights the reactivation of neurodevelopmental programs in GBM, contributing to this heterogeneity and, ultimately, therapy resistance. To characterize the landscape of progenitor cell types in human primary GBM, we compiled a meta-atlas of single-cell transcriptomes from seven published studies. Using a novel bioinformatic method for gene program analysis, we derived GBM gene expression meta-modules represented across multiple tumors. Our analyses validated previously described GBM gene programs and revealed novel tumor progenitor subtypes. Among these subtypes, we identified a tumor-derived stem cell population exhibiting a mixed vascular identity and transcriptional programs reminiscent of neural stem cells, which we identified as a putative neurovascular progenitor (NVP). To our knowledge, this population has not been previously described in GBM. We validated the existence of NVPs in situ using immunofluorescence from primary patient GBM samples. We then performed an enrichment for NVPs using fluorescence-activated cell sorting direct from patient tumors, followed by single-cell RNA sequencing. Examination of data from traditional model systems, including gliomaspheres and mouse GBM models, confirmed the preservation of NVP identity across systems. In vivo analysis of NVPs in multiple mouse models of GBM elucidate the influence of NVPs on GBM cell type composition, highlighting their role as multipotent progenitors. Finally, we utilized single-cell lineage barcoding to trace the progeny of patient-derived NVP cells in a novel organoid transplantation model, revealing NVP differentiation into vascular-like and neural-like cells. These data indicate that NVP cells play crucial roles in tumor plasticity and may represent a valuable therapeutic target for future exploration.

Parallel Immunotranscriptomic and Immunohistochemistry Enhance Cluster Discrimination in Acute Myelogenous Leukemia

Background: Acute myeloid leukemia (AML) demonstrates cell differentiation stages emphasizing the role for hematopoietic maturation arrest during leukemogenesis. Indeed, less differentiated AML stages retain enhanced sensitivity to hypomethylating agents (HMA) plus BCL-2 inhibitor, a phenomenon less observed in monocytic skewed cluster, for which menin inhibitors represent promising intervention. Deconvolution of bulk AML transcriptome allows for better understanding of disease pathogenesis. However, the role of “Immunotranscriptome analysis [i.e. CD34, CD117, HLADR, CD33, CD38, CD11b, BCL2 Gene Expression (GE)]” in identifying maturation arrest remains uncharacterized. In this study, we seek to harmonize marrow immunohistochemistry (IHC) and bulk AML immunotranscriptomic to better discriminate cluster designation. Methods: After IRB approval, 49 AML cases were included for analysis. 6 clusters were designed based on marrow IHC [i.e., Hematopoietic Stem Cell (HSC)-like, Multipotent Progenitor (MPP)-like, Common Myeloid Progenitor (CMP)-like, Granulocytic Monocytic (GMP)-like, Monocyte Progenitor (MP)-like, Granulocyte Progenitor (GP)-like]. Differential immunotranscriptomic for individual GE [i.e. CD34, CD33, CD117 etc.] was examined in our “founder” IHC cohort. Next, discrimination analysis [DA] to detect cluster “membership” was obtained by integrating individual Immunotranscriptome expression into categorical IHC clustering. Descriptive statistics, ANOVA and discrimination procedure were performed with SAS software. Results: Mean age was 65 years (range, 25-92). 22(51%) of cases were male. 19(44%), 22(51%), 2(5%), and 1(2%) were white, African Ancestry (AA), Hispanic and Asians. ELN22 risk was available in 5/49(10.2%), 11/49 (22.4%), 33/49 (67.3%) of favorable, intermediate and adverse groups. 7/49(14.2%), 3/49(6.1%), 10/49(20.4%), 7/49(14.2%), 16/49(33%), and 6/49(12.2%) were HSC, GMP, MP, GP, CMP and MPP-like. CD34; p=0.003, HLA-DR; p=0.01, CD11b; p=0.03 were differentially expressed among clusters. “Stringent” MDS mutations (mut) [i.e. splicing, ASXL1, cohesin, BCOR etc.] were observed in higher proportion of CMP-like cluster (60% vs 21.8%, p=0.01). 7/13 (88%) vs 1/13(12%) of cases with and without MP-like cluster definition harbored KM2TA abnormality, p=&amp;lt;0.0001. By adding GE to our “founder” IHC clustering, 6/6 (100%, p=&amp;lt;0.0001) of pt were allocated in HSC-like disease, 3/3 (100%, p=0.0001) to GMP-like, 6/9 (66.3%. p=0.33) to MP-like, 6/6 (100%, p=&amp;lt;0.0001) GP-like, 2/13 (15.3%) CMP-like and 3/3 (100%, p=0.0001) MPP-like. Conclusions: Our analysis demonstrates that leukemia clusters can be identified based on IHC. GE facilitates robust cluster “membership” for all groups except MP-like and CMP-like. While KM2TA and MDS like mutations were observed significantly higher in MP-like and CMP-like diseases, it is possible that complex epigenetic rewiring [i.e. GE changes induced by KM2TA and MDS mutations] alter Immunotranscriptome ability to enhance cluster designation. Validation of our founder cohort findings is needed.

Linker Histone Regulates the Myeloid Versus Lymphoid Bifurcation of Multipotent Hematopoietic Stem and Progenitors

Linker Histone Regulates the Myeloid Versus Lymphoid Bifurcation of Multipotent Hematopoietic Stem and Progenitors