Stem Cell Fraction Research Articles

7574 Diabetes Stem Cells: A Common Target for Abnormal Insulin Secretion and Insulin Resistance

Abstract Disclosure: H. Kojima: None. M. Katagi: None. J. Okano: None. T. Nakagawa: None. Diabetes is based on chronic inflammation and cannot be cured using only drugs that lower blood glucose levels. A culprit was lurking there that was hindering healing. We discovered abnormal hematopoietic stem cells that caused persistent chronic inflammation in the bone marrow of diabetic model animals. The real culprit is born after several days of hyperglycemia, never dies, and each time glycemic control deteriorates, it moves its progeny in large numbers throughout the body, fusing with unique cells in various organs and producing TNF-α from the organ cells. We have named these worst stem cells, which resemble cancer stem cells, "diabetes stem cells," and attempted to induce complete remission in diabetic model animals by eliminating them with drugs. In this study, we used streptozotocin (STZ)-induced insulin-deficient diabetes model mice. "Diabetes stem cells" were included in the CD106-positive short-term hematopoietic stem cell fraction, in which progenitor cells that play an important role in the regeneration of the thymus and peripheral tissues, and were characterized by abnormal expression of proinsulin and TNF-α. Furthermore, like cancer cells, the expression of histone deacetylases (HDACs) increased, and once the cells were born, they would not disappear. Therefore, we aimed to eliminate diabetes by administering an HDAC inhibitor and subcutaneously injecting insulin pellets to temporarily correct blood glucose levels. We treated diabetic mice with HDAC inhibitor for 8 weeks, along with several weeks of insulin therapy. Insulin aims to prevent the formation of new abnormal cells in the bone marrow and to prevent the inhibition of pancreatic islet wasting due to the forced insulin secretion caused by hyperglycemia. The combination therapy allowed the beta cells to regenerate and produce enough insulin to normalize blood glucose levels and maintain them even after treatment was discontinued. It also solved the mystery of how the abnormal cell population evades attacks from the T cells. In STZ diabetes, the supply of normal CD106-positive progenitor cells from the bone marrow for thymic regeneration was completely blocked, resulting in marked thymic atrophy. As a result, the thymus, which had lost its progenitor cells, was unable to establish an immune surveillance system to eliminate mutated cells throughout the body. In other words, the combination therapy completely broke the vicious cycle caused by atrophies in both the islet and the thymus. Moreover, when the thymus was surgically removed along with the adventitia to prevent regeneration, diabetes remission was completely inhibited, supporting the importance of thymic normalization. Diabetes is a hematopoietic stem cell disease accompanied by thymic dysfunction, and the removal of "diabetes stem cells" paves the way for the cure of diabetes. Presentation: 6/2/2024

Distinctive field effects of smoking and lung cancer case-control status on bronchial basal cell growth and signaling

RationalBasal cells (BCs) are bronchial progenitor/stem cells that can regenerate injured airway that, in smokers, may undergo malignant transformation. As a model for early stages of lung carcinogenesis, we set out to characterize cytologically normal BC outgrowths from never-smokers and ever-smokers without cancers (controls), as well as from the normal epithelial “field” of ever-smokers with anatomically remote cancers, including lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) (cases).MethodsPrimary BCs were cultured and expanded from endobronchial brushings taken remote from the site of clinical or visible lesions/tumors. Donor subgroups were tested for growth, morphology, and underlying molecular features by qRT-PCR, RNAseq, flow cytometry, immunofluorescence, and immunoblot.Results(a) the BC population includes epithelial cell adhesion molecule (EpCAM) positive and negative cell subsets; (b) smoking reduced overall BC proliferation corresponding with a 2.6-fold reduction in the EpCAMpos/ITGA6 pos/CD24pos stem cell fraction; (c) LUSC donor cells demonstrated up to 2.8-fold increase in dysmorphic BCs; and (d) cells procured from LUAD patients displayed increased proliferation and S-phase cell cycle fractions. These differences corresponded with: (i) disparate NOTCH1/NOTCH2 transcript expression and altered expression of potential downstream (ii) E-cadherin (CDH1), tumor protein-63 (TP63), secretoglobin family 1a member 1 (SCGB1A1), and Hairy/enhancer-of-split related with YRPW motif 1 (HEY1); and (iii) reduced EPCAM and increased NK2 homeobox-1 (NKX2-1) mRNA expression in LUAD donor BCs.ConclusionsThese and other findings demonstrate impacts of donor age, smoking, and lung cancer case-control status on BC phenotypic and molecular traits and may suggest Notch signaling pathway deregulation during early human lung cancer pathogenesis.

In-Vitro Proliferation and Enrichment of CD34+ Autologous Haematopoietic Stem Cells, Obtained from Patients with End-Stage Left Ventricular Failure

With a rising global prevalence of end-stage heart failure and a limited availability of cardiac transplantation programmes or left ventricular assist devices in many parts of the world, there is a renewed interest in potential alternative treatment options.Along with other groups, we have previously demonstrated the beneficial effect of intracoronary autologous bone marrow derived peripheral haemopoietic stem cell transplantation, to improve myocardial contractility in end-stage heart failure.We now assess the ability of these cells to expand in numbers in-vitro, to achieve enrichment of stem cell counts, using standard cell culture methods and flow- cytometric analysis.We demonstrate that with in-vitro culturing (within 3 passages post-harvest), the CD34+ stem cell fraction increases in standard culture media, across multiple samples. However, optimal culture conditions to achieve near pure stem cell populations with rapid cell proliferation, still needs to be defined.

Acute myeloid leukemia stem cell signature gene EMP1 is not an eligible therapeutic target.

Relapse in pediatric acute myeloid leukemia (pedAML) patients is known to be associated with residual leukemic stem cells (LSC). We have previously shown that epithelial membrane protein 1 (EMP1) is significantly overexpressed in LSC compared to hematological stem cell fractions. EMP1 was also documented as part of the 17-gene stemness score and a 6-membrane protein gene score, both correlating high EMP1 expression with worse overall survival. However, its potential as a therapeutic target in pedAML is still unexplored. Association analyses of EMP1 expression with clinical and molecular AML characteristics were performed. Expression of EMP1 was evaluated in pedAML and cord blood samples. Expression in normal blood cells and tissues was evaluated by flow cytometry and immunohistochemistry, respectively. In silico analyses showed variable mRNA expression of EMP1 in multiple pedAML datasets, and a significant correlation between high EMP1 transcript levels and the presence of inv(16). Flow cytometry showed overexpression of EMP1 in pedAML samples, as well as expression in normal blood subsets. Importantly, immunohistochemistry revealed EMP1 expression in multiple normal tissues. Although EMP1 presents as an interesting membrane-associated target in pedAML, its abundant expression in normal blood cells and tissues will impede it from further exploration as a therapeutic target. EMP1 is highly expressed in multiple cancer types, but expression in acute myeloid leukemia (AML) and normal tissues is unexplored. As EMP1 is investigated in other cancer types, expression in normal tissues and blood cells is relevant in predicting the success of EMP1-targeted therapies. In this study, we showed expression of EMP1 in multiple tissues, predicting high on-target off-tumor toxicity, which will warn other researchers of possible toxicities when generating EMP1-targeted therapy. Finally, we showed that high EMP1 expression is associated with better overall survival of pediatric AML patients, reducing the need for EMP1-targeted therapy.

Abstract A113: Extracellular vesicles derived from enriched ovarian cancer stem cell fractions promote stem like properties in the more vulnerable tumor cell populations

Abstract Ovarian cancer stem cells (CSCs) are thought to serve as seed cells for recurrent drug resistant disease. Extracellular vesicles (EVs) derived from CSCs are known to promote a more favorable tumor microenvironment. However, there is limited research focused on the impact of EV exchange between tumor cells. We hypothesized that CSC enriched tumor cell fractions could confer their stem like properties to chemo-sensitive non-CSCs via EVs. To test our hypothesis, we utilized established ovarian cancer (OvCa) cell lines (SKOV3, A2780, and UWB1.289 olaparib sensitive (U-OlaSen) and resistant (U-OlaRes)) and high grade serous ovarian cancer (HGSOC) patient derived organoid (PDO) lines. Small EVs were isolated via ultra-filtration and centrifugation, validated, and quantified. EVs from CSC enriched cultures, PARP inhibitor resistant lines, or drug treated (olaparib or carboplatin) lines were cultured with treatment naïve or treatment sensitive lines for defined time points. The endpoints included assessment of changes in ALDH activity (ALDEFLUOR™ Kit) and cell viability (Annexin V staining) using flow cytometry. In addition, relative levels of Enhancer of Zeste Homologue 2 (EZH2), Histone 3 lysine 27 trimethylation (H3k27me3), Disrupted Meiotic cDNA recombinase 1 (DMC1), phosphorylated and non-phosphorylated CHK1 were determined by western blotting. Drug resistance was determined post exposure of the more sensitive cells to EVs derived from the more resistant cells by treatment by either carboplatin or olaparib. At least 3 independent experiments were completed for each endpoint. Statistical analysis was performed by GraphPad Prism and p < 0.05 was considered as statistically significant. Exposure of small EVs (2.5 ug/ml) derived from CSC enriched cultures or drug resistant cell fractions to the more sensitive tumor cell populations resulted in a marked increase in ALDH activity, indicating an increase in CSC markers. This increase was concurrent with an EV induced increase in the relative levels of EZH2, DMC1, total and phosphorylated CHK1 compared to controls. EVs derived from tumor cells treated with carboplatin or olaparib promoted resistance in the more sensitive tumor cell cultures further implicating the ability to exchange resistance properties. Similar results were observed in response to EVs from known drug resistant human HGSOC organoids on U-OlaSen cells. In summary, these findings provide evidence that EVs from CSC enriched fractions can confer CSC like properties via canonical and non-canonical signaling to the more sensitive tumor cells. We conclude that in addition to the concept that tumor cells can promote drug resistance by diluting cytotoxics among neighboring tumor cells, the more resistant CSCs can promote stem like activity in neighboring drug sensitive tumor cells allowing them to overcome anticancer drug treatment and ensuring their survival and adaptation. Citation Format: Venkatesh Pooladanda, Shaan Kumar, Yusuke Matoba, Maryam Azimi Mohammadabadi, Dominique T. Zarrella, Ursula A. Winter, Sarah J. Hill, Hyungsoon Im, Bo R. Rueda. Extracellular vesicles derived from enriched ovarian cancer stem cell fractions promote stem like properties in the more vulnerable tumor cell populations [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A113.

Ex vivo culture resting time impacts transplantation outcomes of genome-edited human hematopoietic stem and progenitor cells in xenograft mouse models

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease.

CD133-Dependent Activation of Phosphoinositide 3-Kinase /AKT/Mammalian Target of Rapamycin Signaling in Melanoma Progression and Drug Resistance.

Melanoma frequently harbors genetic alterations in key molecules leading to the aberrant activation of PI3K and its downstream pathways. Although the role of PI3K/AKT/mTOR in melanoma progression and drug resistance is well documented, targeting the PI3K/AKT/mTOR pathway showed less efficiency in clinical trials than might have been expected, since the suppression of the PI3K/mTOR signaling pathway-induced feedback loops is mostly associated with the activation of compensatory pathways such as MAPK/MEK/ERK. Consequently, the development of intrinsic and acquired resistance can occur. As a solid tumor, melanoma is notorious for its heterogeneity. This can be expressed in the form of genetically divergent subpopulations including a small fraction of cancer stem-like cells (CSCs) and non-cancer stem cells (non-CSCs) that make the most of the tumor mass. Like other CSCs, melanoma stem-like cells (MSCs) are characterized by their unique cell surface proteins/stemness markers and aberrant signaling pathways. In addition to its function as a robust marker for stemness properties, CD133 is crucial for the maintenance of stemness properties and drug resistance. Herein, the role of CD133-dependent activation of PI3K/mTOR in the regulation of melanoma progression, drug resistance, and recurrence is reviewed.

Mechanisms of Melanoma Progression and Treatment Resistance: Role of Cancer Stem-like Cells.

Melanoma is the third most common type of skin cancer, characterized by its heterogeneity and propensity to metastasize to distant organs. Melanoma is a heterogeneous tumor, composed of genetically divergent subpopulations, including a small fraction of melanoma-initiating cancer stem-like cells (CSCs) and many non-cancer stem cells (non-CSCs). CSCs are characterized by their unique surface proteins associated with aberrant signaling pathways with a causal or consequential relationship with tumor progression, drug resistance, and recurrence. Melanomas also harbor significant alterations in functional genes (BRAF, CDKN2A, NRAS, TP53, and NF1). Of these, the most common are the BRAF and NRAS oncogenes, with 50% of melanomas demonstrating the BRAF mutation (BRAFV600E). While the successful targeting of BRAFV600E does improve overall survival, the long-term efficacy of available therapeutic options is limited due to adverse side effects and reduced clinical efficacy. Additionally, drug resistance develops rapidly via mechanisms involving fast feedback re-activation of MAPK signaling pathways. This article updates information relevant to the mechanisms of melanoma progression and resistance and particularly the mechanistic role of CSCs in melanoma progression, drug resistance, and recurrence.

Evaluation of the Precision of Kinetic Stem Cell (KSC) Counting for Specific Quantification of Human Mesenchymal Stem Cells in Heterogeneous Tissue Cell Preparations.

Kinetic stem cell (KSC) counting is a recently introduced first technology for quantifying tissue stem cells in vertebrate organ and tissue cell preparations. Previously, effective quantification of the fraction or dosage of tissue stem cells had been largely lacking in stem cell science and medicine. A general method for the quantification of tissue stem cells will accelerate progress in both of these disciplines as well as related industries like drug development. Triplicate samples of human oral alveolar bone cell preparations, which contain mesenchymal stem cells (MSCs), were used to estimate the precision of KSC counting analyses conducted at three independent sites. A high degree of intra-site precision was found, with coefficients of variation for determinations of MSC-specific fractions of 8.9% (p < 0.003), 13% (p < 0.006), and 25% (p < 0.02). The estimates of inter-site precision, 11% (p < 0.0001) and 26% (p < 0.0001), also indicated a high level of precision. Results are also presented to show the ability of KSC counting to define cell subtype-specific kinetics factors responsible for changes in the stem cell fraction during cell culture. The presented findings support the continued development of KSC counting as a new tool for advancing stem cell science and medicine.

Variegate expression of Cre recombinase in hematopoietic cells in CD11c-cre transgenic mice

In this study, we performed an in-depth analysis of Cre expression in the widely used CD11c-Cre transgenic mice generated by the group of Boris Reizis. In contrast to previous observation, using the highly sensitive Rosa-26-floxed-tdTomato reporter mouse line, we show variegated expression of Cre in multiple hematopoietic linage cells starting in hematopoietic stem cells. Indeed, we found that in the CD11c-Cre driver mice, Cre is expressed in cDC linage cells and pDC starting from the myeloid dendritic cell precursor, as expected, but also in a substantial fraction of hematopoietic stem cells and common lymphoid progenitors and, consequently, in >50% of all leukocytes. Hence, this study indicates that the reporter mice used to characterize Cre expression in Cre-driver mice should be selected with caution and considering the sensitivity of the reporter system. This study also suggests that the interpretation of some reports using this CD11c-Cre transgenic mice may need to be re-considered based on a careful evaluation of the cell type-specificity of Cre-mediated in their model.

Stem-Cell Enriched Cellular Hierarchy of TP53 Mutant Acute Myeloid Leukemia Is Vulnerable to Targeted Protein Degradation of c-MYC

Deregulation of MYC genes occurs in up to 70% of all human cancers and is associated with hallmarks of cancer including mitochondrial and ribosomal biogenesis, cell cycle progression, and metabolic abnormalities. TP53 regulates MYC while MYC suppresses TP53, suggesting counteracting negative feedback loops. Therefore, MYC or its function can be activated when TP53 is not functional. TP53 mutations occur in 30% of relapsed/refractory acute myeloid leukemias (AMLs) patients' survival is dismal, and there are no effective therapies for these patients. Compared to TP53 wild-type (TP53wt), TP53 mutant (TP53mut) AMLs have lower percentages and numbers of leukemia blasts with increased immature CD34+ cells and resistance to chemo- or molecularly targeted therapies. However, the exact cellular hierarchy of TP53mut AML has not been elucidated. We observed significantly increased MYC mRNA levels in (TP53mut), as compared to TP53wt AML, and also increased levels in TP53mut versus TP53wt AML leukemia stem cell (LSC) fractions. This finding was confirmed in a dataset from the Munich Leukemia Laboratory (N = 732). We found significantly upregulated MYC pathways in TP53mut compared to TP53wt AML LSC. We confirmed the increased MYC mRNA levels at the protein level in TP53mut AML by single-cell mass cytometry (CyTOF). To dissect the cellular hierarchy in TP53mut AML, we performed single-cell RNA sequencing of 32 BM samples from healthy donors (N = 3), newly diagnosed, high-risk TP53wt (N = 7) and TP53mut (N = 22) AML patients, with 6,685, 10,687 and 10,687 cells from normal, TP53wt and TP53mut AML bone marrow (BM) cells, respectively. We found highly enriched HSC-like cells and reduced progenitor- and GMP-like cells in TP53mut AML compared to normal BM (NBM) and TP53wt AML samples. We overlayed MYC expression levels on the mapping of cellular components and found higher MYC levels in HSC-like cells in TP53mut AML compared to HSC and HSC-like cells in NBM and TP53wt AML samples, suggesting enrichment of immature HSC-like cells and increased activity of MYC in TP53mut AML LSCs ( Fig. A). To target c-MYC and MYC signaling, we utilized GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC protein (Nishida, ASH 2022). CyTOF confirmed the presence of much increased c-MYC protein levels in primary CD34+ AML than in CD34+ NBM hematopoietic stem cells (HSCs). Notably, CD34+ AML cells showed much greater sensitivity to GT19715 compared to CD34+ NBM cells. Data suggest on-target activity of GT19715 against c-MYC in LSCs with a therapeutic window between LSCs vs. NBM HSCs. Intriguingly, c-MYC protein levels are higher in CD34+CD38+ than in CD34+CD38- AML cells, suggesting that c-MYC drives the proliferation of AML progenitor cells differentiating from quiescent LSCs. Consequently, CD34+CD38+ AML progenitor cells exhibited greater sensitivity to GT19715 compared to CD34+CD38- LSCs. Using paired, TP53 null HL-60 GT19715-sensitive and -resistant cells generated through chronic exposure to GT19715, we interrogated the impact of GT19715 on metabolic changes. GT19715 induced pronounced reductions in basal and maximal oxygen consumption rates (OCRs) in GT19715-sensitive HL-60 cells. GT19715 reduced both basal, glutamine- and glucose-dependent extracellular acidification rates (ECARs) in GT19715-sensitive cells while no inhibition in OCRs or ECARs was observed in GT19715-resistant cells. GT19715 severely inhibited ECARs even after adding glucose to GT19715-sensitive cells, suggesting irreversible inhibition of glycolysis as one of the mechanisms of action of GT19715. GT19715 profoundly reduced AML blasts in TP53mut AML samples (N = 3) ( Fig. B), and in a very aggressive patient-derived xenograft (PDX) TP53mut AML model established from a patient with TP53 p.Y220C and p.P151A mutations along with MECOM rearrangement and K/NRAS mutations. In humanized Crbn I391V mice, where the Crbn-mediated protein degradation is operational, GT19715 only reduced WBC counts along with minimal body weight loss. GT19715 but did not reduce total mouse BM CD45+ cells, suggesting favorable toxicity profiles of GT19715. In conclusion, TP53mut AML comprised highly enriched LSC populations compared to TP53wt AML and targeting of c-MYC protein is highly effective in TP53mut AML in vitro and in vivo with a therapeutic window between AML LSC and normal hematopoietic cells.

Intracellular IL-23 Receptor (IL-23R) Is a Regulator of Mitotic Spindle and Centrosome Formation and Is Essential for AML Viability

IL-23 receptor (IL-23R) is a heterodimeric cytokine cell surface receptor that is traditionally expressed on T cells. The cytokine, IL-23, is secreted by dendritic cells and macrophages in response to inflammatory stimuli. Here, we identify a novel function and localization for IL-23R in which intracellular IL-23R is necessary for AML viability and acts as a critical regulator of the mitotic spindle. We analyzed gene ontologies that were upregulated in AML samples compared to normal hematopoietic cells. Amongst upregulated gene ontologies, the mitotic spindle ontology was enriched in AML samples. Further analysis of all protein coding genes and their correlation to the mitotic spindle ontology surprisingly returned IL-23R as a top hit. Given that IL-23R is not known to be expressed in AML, we confirmed the presence of IL-23R protein by immunoblotting in 14 of 20 primary AML patient samples, while it was undetectable in bulk (n=5) and CD34+ sorted (n=3) normal hematopoietic cells. Consistent with previous investigations, we confirmed cell surface localization of IL-23R in double negative T cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were present on the cell surface of AML cells. Instead, IL-23R was detected intracellularly in the cytoplasm and nucleus of AML cell lines, as well as primary AML cells (including the stem cell fraction). We demonstrated intracellular localization using flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. To ensure we were detecting genuine IL-23R protein, we probed for IL-23R using 4 different antibodies targeting 4 different epitopes of the receptor whilst using 4 methods of detection (immunoblotting, flow cytometry, confocal microscopy, and immunoprecipitation). We also demonstrated intracellular localization of the IL-23R heterodimer subunit, IL12Rβ1, which is known to bind the IL-23R subunit to form the fully functional IL-23 receptor. To elucidate the function of intracellular IL-23R, we performed BioID mass spectrometry to identify proteins that interact with IL-23R. Compared to controls, we identified 61 proteins that preferentially interacted with IL-23R. 36 of those 61 proteins are known cytoplasmic or nuclear localized proteins. Pathway analysis of those interacting proteins identified the mitotic spindle as a top pathway corroborating with our bioinformatics analysis. Proximity Ligation Assay (PLA) and confocal microscopy verified that endogenous IL-23R protein interacted with mitotic spindle associated proteins, NUMA, TMEM201, TACC1, and BAG6 in OCI-AML2 cells and primary AML samples. Consistent with our PLA results, we demonstrated IL-23R co-localized with the mitotic spindle and centrosomes in AML cell lines and primary patient samples. Knockdown and knockout of IL-23R in AML cells led to the dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and spindle orientation errors. Knockdown of IL-23R reduced cell proliferation and viability in OCI-AML2, TEX, K562, NB4, and U937 cells compared to non-targeting controls. IL-23R knockdown also reduced the engraftment efficiency of TEX leukemia cells in murine bone marrow. Subsequent knockdowns of IL-23R in primary AML cells demonstrated decreased clonogenic growth and reduced engraftment into the marrow of immune deficient mice. In contrast, knockdown of IL-23R in normal human cord blood cells did not impair their engraftment into murine bone marrow. Finally, we analyzed hematopoietic cells and stem cells in IL-23R -/- mice and found that knockout of IL-23R did not decrease blood counts or the abundance and function of normal hematopoietic cells. In summary, we discovered a novel intracellular localization and function for IL-23R in AML. IL-23R regulates mitotic spindle and centrosome formation and is necessary for AML cell viability. We have thus identified a new biological function for IL-23R and a potential therapeutic target for AML.

Quantification of the Culture Stability of Stem Cell Fractions from Oral-Derived, Human Mesenchymal Stem Cell Preparations: A Significant Step toward the Clinical Translation of Cell Therapies.

A continuing limitation and major challenge in the development and utilization of predictable stem cell therapies (SCTs) is the determination of the optimal dosages of stem cells. Herein, we report the quantification of stem cell fractions (SCF) of human mesenchymal stem cell (MSC) preparations derived from oral tissues. A novel computational methodology, kinetic stem cell (KSC) counting, was used to quantify the SCF and specific cell culture kinetics of stem cells in oral alveolar bone-derived MSC (aBMSCs) from eight patients. These analyses established, for the first time, that the SCF within these heterogeneous, mixed-cell populations differs significantly among donors, ranging from 7% to 77% (ANOVA p < 0.0001). Both the initial SCF of aBMSC preparations and changes in the level of the SCF with serial culture over time showed a high degree of inter-donor variation. Hence, it was revealed that the stability of the SCF of human aBMSC preparations during serial cell culture shows inter-donor variation, with some patient preparations exhibiting sufficient stability to support the long-term net expansion of stem cells. These findings provide important insights for the clinical-scale expansion and biomanufacturing of MSCs, which can facilitate establishing more effective and predictable outcomes in clinical trials and treatments employing SCT.

The Genomic and Transcriptomic Landscape of Myeloid Sarcoma and Associated Acute Myeloid Leukemia

Background: Myeloid sarcoma (MS) is a distinct form of acute myeloid leukemia (AML) found in ~10% of patients, in which mass-forming myeloid blasts proliferate in extramedullary sites. MS may present as an isolated lesion as the primary manifestation of disease, co-occur with AML, or, more commonly, arise in the setting of AML relapse. It is associated with poor outcomes, and treatment options are sparse. While increased knowledge of AML and its biology have improved the care and survival of AML patients, those with MS continue to have limited successful treatment options, as MS has typically not been included in AML genetic and genomic profiling efforts, and patients are excluded from clinical trials. Leukemia cutis (LC) is a form of extramedullary AML characterized by cutaneous involvement, often with an infiltrative and non-mass forming pattern of growth. Still, historically the literature has not made a clear distinction between MS and LC. Although recent small pilot studies have suggested differences in AML-associated mutations between medullary AML and MS, knowledge of the complete molecular and cellular composition of MS and comprehensive comparison with AML is lacking. While molecular characterization of the extramedullary disease site is encouraged in the updated clinical recommendations, no definitive guidance exists for changes in the management and care of AML patients with MS, given the substantial gap in our current knowledge and understanding of extramedullary AML. Methods: Here, we performed RNA-Seq (n=96), single-cell (sc) RNA-Seq (n=33), and mutation profiling using either whole exome sequencing (WES, n=6) or a 409-gene targeted sequencing panel (n=48) of MS or LC patient samples and bone marrow (BM) aspirates from AML patients with and without extramedullary disease to present the first comprehensive characterization of MS and LC. Results: Our WES data showed thatMS and LC evolve independently in the extramedullary site from concurrent medullary disease with distinct dominant clones, characterized by higher median tumor mutation burden (1.66 vs. 064 mutations per megabase, respectively; p=0.0059) and increased copy number aberrations, which both validated in an MS Tet2 HR mouse model. Notably, we observed location-specific molecular evolution in patients with multiple concurrent MS sites (Figure 1A) and patients with recurring MS in WES and targeted panel data. While targetable mutations (i.e., IDH1/2 mutations and FLT3-ITD/TKD) occur, the inter-site variability of molecular aberrations may explain the short-lived responses of AML patients with MS to IDH/FLT3 inhibitors. Transcriptional profile differences between MS and associated medullary disease included up-regulation of extracellular matrix organization and cell-substrate adhesion and down-regulation of inflammatory response pathways in both bulk and scRNA-seq data (Figure 1B). Further, involved BM malignant cells of MS patients compared to patients without MS up-regulated specific pathways, including interferon-gamma response observed in both bulk and scRNA-seq, as well as chromatin organization and splicing. We further revealed that MS patients without overt BM involvement show remodeling of the BM immune microenvironment, with increased fractions of hematopoietic stem and progenitor cells that down-regulate HLA class II genes. Comparing LC and MS in the skin revealed distinct differences in their expression profiles, including high expression of genes involved in cell migration and immune response in LC, suggesting LC and MS represent distinct disease entities. Lastly, we demonstrated high expression of BCL2 in extramedullary disease (p=0.0074) and presented initial evidence that treatment with BH3-mimetics may be beneficial for the treatment of isolated MS. Conclusions: Myeloid sarcoma shows distinct molecular evolution and microenvironmental composition compared to medullary disease, including location-specific evolution necessitating personalized management consideration. Leukemia cutis is distinct from MS tumorous lesions and should be considered a separate entity. High BCL2 expression in extramedullary disease might represent a promising targetable vulnerability in patients with isolated MS.

Abstract PR01: Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells

Abstract Most patients with acute myeloid leukemia (AML) initially achieve clinical remission after chemotherapy but eventually die from disease relapse as they succumb to chemotherapy-resistant AML cells, including the rare fraction of leukemia stem cells (LSC) that persist and often reinitiate a more aggressive disease. While eliminating LSCs has become a desirable strategy to achieve durable AML remission, much remains unknown about LSC vulnerabilities and the specific cellular programs that enable their disease activity. Spurred by increasing evidence that disruptions in proteome homeostasis or “proteostasis” impair stem cell integrity, we investigated how acute myeloid leukemogenesis is impacted by the inositol-requiring enzyme-1 alpha (IRE1a), the most conserved enzyme of the endoplasmic reticulum unfolded protein response that resolves proteotoxic stress in eukaryotic cells. Activated IRE1a catalyzes an unconventional splicing of the XBP1U mRNA to generate an XBP1S transcript that encodes the transcription factor XBP1 (X-box binding protein-1), the main effector of IRE1a-transduced signals. Using transcriptomics and genomics to profile the activation and function of IRE1a in primary human AML samples and murine AML models, we identified the IRE1a-XBP1 axis as a cell-intrinsic brake against pro-myeloid leukemogenic programs in hematopoietic stem and progenitor cells (HSPC). Notably, compared to blast cells from the same AML patient, LSCs expressed significantly reduced levels of the genes encoding IRE1a (ERN1) and its spliced product XBP1S suggesting that downregulation of IRE1a-XBP1 activity promotes LSC activity and acute myeloid leukemogenesis. Consistent with this hypothesis, genetic induction of XBP1S-encoded XBP1 in patient-derived AML cells suppressed their LSC transcriptional program, impaired their clonogenic capacity, and limited their ability to cause AML in immunodeficient animals. By contrast, genetically engineered IRE1a or XBP1 insufficiency enriched the LSC signature in HSPCs and cooperated with the myeloproliferative oncogene FLT3-ITD to initiate a lethal AML disease in mice. Integrated RNA-Seq and ChIP-Seq of XBP1 target genes in HSPCs revealed a mechanism in which IRE1a-induced XBP1 functions as a potent transcriptional repressor of signature pro-LSC gene programs, exemplified by the Wnt/beta-catenin pathway. Accordingly, we defined an independently prognostic “18-gene” signature of XBP1-repressed beta-catenin target genes that when highly expressed correlated with poor survival outcomes in multiple cohorts of AML patients. Taken together, our findings uncover a novel anti-leukemia function for IRE1a-XBP1 signaling that restrains acute myeloid leukemogenesis in HSPCs and suggest that this pathway is likely subverted in LSCs to facilitate AML initiation and progression. Our results further suggest that IRE1a activation is a vulnerability in AML stem cells that can be harnessed therapeutically to achieve durable disease remission. Citation Format: Brendan M Barton, Francheska Son, Akanksha Verma, Laurie Glimcher, Stanley Adoro. Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr PR01.

Bone marrow endosteal stem cells dictate active osteogenesis and aggressive tumorigenesis

The bone marrow contains various populations of skeletal stem cells (SSCs) in the stromal compartment, which are important regulators of bone formation. It is well-described that leptin receptor (LepR)+ perivascular stromal cells provide a major source of bone-forming osteoblasts in adult and aged bone marrow. However, the identity of SSCs in young bone marrow and how they coordinate active bone formation remains unclear. Here we show that bone marrow endosteal SSCs are defined by fibroblast growth factor receptor 3 (Fgfr3) and osteoblast-chondrocyte transitional (OCT) identities with some characteristics of bone osteoblasts and chondrocytes. These Fgfr3-creER-marked endosteal stromal cells contribute to a stem cell fraction in young stages, which is later replaced by Lepr-cre-marked stromal cells in adult stages. Further, Fgfr3+ endosteal stromal cells give rise to aggressive osteosarcoma-like lesions upon loss of p53 tumor suppressor through unregulated self-renewal and aberrant osteogenic fates. Therefore, Fgfr3+ endosteal SSCs are abundant in young bone marrow and provide a robust source of osteoblasts, contributing to both normal and aberrant osteogenesis.

Neuronal-Hematopoietic Cell Fusion in Diabetic Neuropathy.

Diabetic neuropathy is a major complication of diabetes mellitus that occurs during the early stages of the disease. Many pathogenic mechanisms are related and induced by hyperglycemia. However, even if these factors improve, diabetic neuropathy cannot go into remission and progresses slowly. Furthermore, diabetic neuropathy often progresses even with proper glycemic control. Recently, bone marrow-derived cells (BMDCs) were reported to be involved in the pathogenesis of diabetic neuropathy. BMDCs expressing proinsulin and TNFα migrate to the dorsal root ganglion and fuse with neurons, and this neuronal-hematopoietic cell fusion induces neuronal dysfunction and apoptosis. The CD106-positive lineage-sca1+c-kit+ (LSK) stem cell fraction in the bone marrow is strongly involved in cell fusion with neurons, leading to diabetic neuropathy. Surprisingly, when CD106-positive LSK stem cells obtained from diabetic mice were transplanted into nondiabetic mice, they fused with dorsal root ganglion neurons and induced neuropathy in non-hyperglycemic normal mice. The transplanted CD106-positive LSK fraction inherited the trait even after transplantation; this "progeny effect" may explain the irreversibility of diabetic neuropathy and is a significant finding for determining the target of radical treatments and provides new directions for developing therapeutic methods for diabetic neuropathy.

RNA splicing factor Rbm25 underlies heterogeneous preleukemic clonal expansion in mice

AbstractClonal expansion sets the stage for cancer genesis by allowing for the accumulation of molecular alterations. Although genetic mutations such as Tet2 that induce clonal expansion and malignancy have been identified, these mutations are also frequently found in healthy individuals. Here, we tracked preleukemic clonal expansion using genetic barcoding in an inducible Tet2 knockout mouse model and found that only a small fraction of hematopoietic stem cells (HSCs) expanded excessively upon Tet2 knockout. These overexpanded HSCs expressed significantly lower levels of genes associated with leukemia and RNA splicing than nonoverexpanded Tet2 knockout HSCs. Knocking down Rbm25, an identified RNA splicing factor, accelerated the expansion of Tet2-knockout hematopoietic cells in vitro and in vivo. Our data suggest that mutations of an epigenetic factor Tet2 induce variability in the expression of an RNA splicing factor Rbm25, which subsequently drives heterogeneous preleukemic clonal expansion. This heterogeneous clonal expansion could contribute to the variable disease risks across individuals.

Proteasome inhibitor bortezomib stabilizes and activates p53 in hematopoietic stem/progenitors and double-negative T cells in vivo

We have previously shown that proteasome inhibitor bortezomib stabilizes p53 in stem and progenitor cells within gastrointestinal tissues. Here, we characterize the effect of bortezomib treatment on primary and secondary lymphoid tissues in mice. We find that bortezomib stabilizes p53 in significant fractions of hematopoietic stem and progenitor cells in the bone marrow, including common lymphoid and myeloid progenitors, granulocyte-monocyte progenitors, and dendritic cell progenitors. The stabilization of p53 is also observed in multipotent progenitors and hematopoietic stem cells, albeit at lower frequencies. In the thymus, bortezomib stabilizes p53 in CD4-CD8- T cells. Although there is less p53 stabilization in secondary lymphoid organs, cells in the germinal center of the spleen and Peyer's patch accumulate p53 in response to bortezomib. Bortezomib induces the upregulation of p53 target genes and p53 dependent/independent apoptosis in the bone marrow and thymus, suggesting that cells in these organs are robustly affected by proteasome inhibition. Comparative analysis of cell percentages in the bone marrow indicates expanded stem and multipotent progenitor pools in p53R172H mutant mice compared with p53 wild-type mice, suggesting a critical role for p53 in regulating the development and maturation of hematopoietic cells in the bone marrow. We propose that progenitors along the hematopoietic differentiation pathway express relatively high levels of p53 protein, which under steady-state conditions is constantly degraded by Mdm2 E3 ligase; however, these cells rapidly respond to stress to regulate stem cell renewal and consequently maintain the genomic integrity of hematopoietic stem/progenitor cell populations.

Longitudinal single-cell profiling of chemotherapy response in acute myeloid leukemia

Acute myeloid leukemia may be characterized by a fraction of leukemia stem cells (LSCs) that sustain disease propagation eventually leading to relapse. Yet, the contribution of LSCs to early therapy resistance and AML regeneration remains controversial. We prospectively identify LSCs in AML patients and xenografts by single-cell RNA sequencing coupled with functional validation by a microRNA-126 reporter enriching for LSCs. Through nucleophosmin 1 (NPM1) mutation calling or chromosomal monosomy detection in single-cell transcriptomes, we discriminate LSCs from regenerating hematopoiesis, and assess their longitudinal response to chemotherapy. Chemotherapy induced a generalized inflammatory and senescence-associated response. Moreover, we observe heterogeneity within progenitor AML cells, some of which proliferate and differentiate with expression of oxidative-phosphorylation (OxPhos) signatures, while others are OxPhos (low) miR-126 (high) and display enforced stemness and quiescence features. miR-126 (high) LSCs are enriched at diagnosis in chemotherapy-refractory AML and at relapse, and their transcriptional signature robustly stratifies patients for survival in large AML cohorts.