Stem Progenitor Cells Research Articles

Long-term combination therapy with metformin and oxymetholone in a Fanconi anemia mouse model.

Fanconi anemia (FA) is a disease caused by defective deoxyribonucleic acid (DNA) repair that manifests as bone marrow failure, cancer predisposition, and developmental defects. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wildtype controls with either MET alone, OXM alone, MET+OXM, or placebo diet from age 3weeks to 18months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p=.01) and hemoglobin levels (p<.05). In addition, the percentage of quiescent hematopoietic stem cell (HSC) (LSK [Lin-Sca+c-Kit+]) was significantly increased (p=.001) by long-term treatment with MET alone. The combination of metformin and oxymetholone did not result in a significant synergistic effect in any hematopoietic parameter. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration. We conclude that androgen therapy is not a contraindication to concurrent metformin administration in clinical trials. HIGHLIGHTS: Long-term coadministration of metformin in combination with oxymetholone is well tolerated by Fancd2-/- mice. Hematopoietic stem cell quiescence in mutant mice was enhanced by treatment with metformin alone. Metformin treatment caused a partial normalization of gene expression in the livers of mutant mice.

Stem Cell‐Based Therapies and Tissue Engineering Innovations for Tendinopathy: A Comprehensive Review of Current Strategies and Future Directions

AbstractTendon diseases commonly lead to physical disability, exerting a profound impact on the routine of affected patients. These conditions respond poorly to existing treatments, presenting a substantial challenge for orthopedic scientists. Research into clinical translational therapy has yet to yield highly versatile interventions capable of effectively addressing tendon diseases, including tendinopathy. Stem cell‐based therapies have emerged as a promising avenue for modifying the biological milieu through the secretion of regenerative and immunomodulatory factors. The current review provides an overview of the intricate tendon microenvironment, encompassing various tendon stem progenitor cells within distinct tendon sublocations, gene regulation, and pathways pertinent to tendon development, and the pathology of tendon diseases. Subsequently, the advantages of stem cell‐based therapies are discussed that utilize distinct types of autologous and allogeneic stem cells for tendon regeneration at the translational level. Moreover, this review outlines the challenges, gaps, and future innovations to propose a consolidated stem cell‐based therapy to treat tendinopathy. Finally, regenerative soluble therapies, insoluble bio‐active therapies, along with insoluble bio‐active therapies, and implantable 3D scaffolds for tendon tissue engineering are discussed, thereby presenting a pathway toward enhanced tissue regeneration and engineering.

Abstract PO2-16-04: Proteogenomic characterization of non-luminal A versus luminal A breast cancer tumor cells enriched by laser microdissection

Abstract Introduction: Breast cancer (BC) is classified into four widely-accepted intrinsic subtypes based on PAM (Prediction Analysis of Microarray) 50 gene expression profiles: Luminal A (LumA), Luminal B (LumB), Her2-enriched (Her2) and basal-like (Basal). Recent multi-omic studies of human BC have identified many potential therapeutic biomarkers for these subtypes and have also revealed the extensive molecular heterogeneity of the disease. Patients with LumA tumors generally have better outcomes because these tumors are typically slower-growing and responsive to hormone therapy. However, there is still considerable variation in the clinical outcomes of patients with non-LumA tumors. Here, we strive to understand the proteogenomic characteristics of intrinsically-defined non-LumA tumors in reference to LumA tumors. Methods: A total of 117 retrospectively-collected, untreated primary breast tumor specimens, with a focus on non-LumA subtypes, were selected from the Clinical Breast Care Project and consented using a HIPAA-compliant, IRB-approved protocol. The study cohort had a median patient follow-up of 9.3 years, enabling clinical outcome-related analyses. Immunohistochemistry (IHC) subtyping was used to enrich the cohort with non-LumA subtypes. Breast tumors were embedded in OCT (Optimal Cutting Temperature) compound and processed by laser microdissection (LMD) to enrich for tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor using the illustra triplePrep kit. Paired-end RNA sequencing and whole genome sequencing (WGS) were performed for 117 and 99 tumors, respectively, using the Illumina HiSeq platform. Quantitative global proteomics and phosphoproteomic analyses were performed on 112 and 50 tumors, respectively, using isobaric TMT 6-plex labeling with the “universal reference” strategy. Results: We observed significantly lower stromal, immune and microenvironment scores in non-basal-like tumors, especially the LumA subtype from LMD-processed samples, compared to that of the same tumors but bulk-processed in TCGA-breast cancer study. There was also significantly lower stromal gene expression in LMD LumA compared to TCGA LumA. Unlike a recent report on proteomics clustering of bulk-processed tumors, we did not observe a stromal-enriched cluster, probably because the use of LMD minimized stromal components. Many common patterns of somatic mutations were observed among non-LumA tumors, such as dominant TP53 mutations and 5q deletion, suggesting a potentially common cell-of-origin for non-LumA tumors, such as an ER negative cancer stem cell or progenitor cell for these tumor subtypes. Cell proliferation-associated genes were amplified and proliferation pathways strongly enriched in non-LumA tumors. In addition to this, we identified two distinct phosphoproteomic profiles for relapsed and relapse-free basal-like BC. Moreover, we also identified 17 differentially expressed phosphopeptides that could identify cases of relapsed and relapse-free basal-like BC with a significant difference in progression-free interval of surviving cases. Conclusion: This study provides an integrated proteogenomic characterization of non-LumA vs LumA tumor specimens enriched for cancer cells. We identified many common features of non-LumA tumors and also identified phosphopeptides that could serve as potential biomarkers for less aggressive basal-like BC and possibly guide treatment selections. Disclaimer: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of USUHS, HJF, the DoD or the Departments of the Army, Navy or Air Force or the DOE or PNNL. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Praveen Kumar Raj Kumar, Xiaoying Lin, Tao Liu, Lori Sturtz, Marina Gritsenko, Vladislav Petyuk, Tyler Sagendorf, Brenda Deyarmin, Jianfang Liu, Anupama Praveen-Kumar, Guisong Wang, Jason McDermott, Anil Shukla, Ronald Moore, Matthew Monroe, Bobbie-Jo Webb-Robertson, Jeffrey Hooke, Leigh Fantacone-Campbell, Brad Mostoller, Leonid Kvecher, Jennifer Kane, Jennifer Melley, Stella Somiari, Patrick Soon-Shiong, Richard Smith, Richard Mural, Karin Rodland, Craig Shriver, Albert Kovatich, Hai Hu. Proteogenomic characterization of non-luminal A versus luminal A breast cancer tumor cells enriched by laser microdissection [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-16-04.

Oscillatory control of embryonic development.

Proper embryonic development depends on the timely progression of a genetic program. One of the key mechanisms for achieving precise control of developmental timing is to use gene expression oscillations. In this Review, we examine how gene expression oscillations encode temporal information during vertebrate embryonic development by discussing the gene expression oscillations occurring during somitogenesis, neurogenesis, myogenesis and pancreas development. These oscillations play important but varied physiological functions in different contexts. Oscillations control the period of somite formation during somitogenesis, whereas they regulate the proliferation-to-differentiation switch of stem cells and progenitor cells during neurogenesis, myogenesis and pancreas development. We describe the similarities and differences of the expression pattern in space (i.e. whether oscillations are synchronous or asynchronous across neighboring cells) and in time (i.e. different time scales) of mammalian Hes/zebrafish Her genes and their targets in different tissues. We further summarize experimental evidence for the functional role of their oscillations. Finally, we discuss the outstanding questions for future research.

MicroRNA target homeobox messenger RNA in HIV induced hematopoietic inhibition.

Cytopenias are a common occurrence due to abnormal hematopoiesis persistent in patients suffering from and advancing with HIV/AIDS. In order to develop efficacious therapies against cytopenias, it is necessary to understand the mechanisms by which HIV infection affects the differentiation of hematopoietic stem-progenitor cells (HSPCs), causing hematopoietic inhibition, that leads to hematological disorders. Currently, only the antiretrovirals that are being used to treat HIV infection and indirectly lower the levels of virus replication also co-attenuate cytopenias. The evidence available suggests that this indirect efficacy may not prevail for the lifetime of the infected patients, and the acquired immunodeficiency can overtake the beneficial consequences of decreased virus replication. As cited in this article, we and our colleagues are the first to make a foray into the involvement of microRNAs and their use as potential interventional treatments for the cytopenias that occur with HIV/AIDS. Herein, we progressed further in the direction of the mechanisms of the involvement of homeobox gene regulation to cause cytopenias. We had previously shown that HIV-1 inhibits multi-lineage hematopoiesis of the CD34+ cells using SCID-hu Thy/Liv animals in vivo. Furthermore, we demonstrated that the virus-induced hematopoietic inhibition occurred despite the CD34+ cells being resistant to HIV-1 infection. We set out to search for the specific host factors secreted by CD4+ T-cells that likely participate in the inhibition of hematopoiesis of the HIV infection-resistant CD34+ cells. More recently, we reported the identification of virus-infected CD4+ thymocyte-secreted miRNA-15a and miRNA-24 and that their differential expression following HIV infection causes the indirect inhibition of hematopoiesis. We then hypothesized that the observed miRNA differential expression in the virus-infected T-cells causes the abnormal regulation of homeobox (HOX) gene-encoded transcriptomes in the CD34+ cells, affecting specific MAPK signaling and CD34+ cell fate, thereby disrupting normal hematopoiesis. We present that in HIV infection, miRNA-mediated post-transcriptional dysregulation of HOXB3 mRNA inhibits multi-lineage hematopoiesis, which translates into hematological disorders in virus-infected patients with HIV/AIDS. These observations portend specific microRNA candidates for potential efficacy against the virus-induced cytopenias that are otherwise not treatable by the existing HAART/ART regimens, which are primarily designed and applicable for the attenuation of virus replication.

Extracellular Vesicles Isolated from Equine Adipose-Derived Stromal Stem Cells (ASCs) Mitigate Tunicamycin-Induced ER Stress in Equine Corneal Stromal Stem Cells (CSSCs).

Corneal ulcers, characterized by severe inflammation of the cornea, can lead to serious, debilitating complications and may be vision-threatening for horses. In this study, we aimed to investigate the role of endoplasmic reticulum (ER) stress in corneal stem progenitor cell (CSSC) dysfunction and explore the potential of equine adipose-derived stromal stem cell (ASC)-derived extracellular vesicles (EVs) to improve corneal wound healing. We showed that CSSCs expressed high levels of CD44, CD45, and CD90 surface markers, indicating their stemness. Supplementation of the ER-stress-inducer tunicamycin to CSSCs resulted in reduced proliferative and migratory potential, accumulation of endoplasmic reticulum (ER)-stressed cells in the G0/G1 phase of the cell cycle, increased expression of proinflammatory genes, induced oxidative stress and sustained ER stress, and unfolded protein response (UPR). Importantly, treatment with EVs increased the proliferative activity and number of cells in the G2/Mitosis phase, enhanced migratory ability, suppressed the overexpression of proinflammatory cytokines, and upregulated the anti-inflammatory miRNA-146a-5p, compared to control and/or ER-stressed cells. Additionally, EVs lowered the expression of ER-stress master regulators and effectors (PERK, IRE1, ATF6, and XBP1), increased the number of mitochondria, and reduced the expression of Fis-1 and Parkin, thereby promoting metabolic homeostasis and protecting against apoptosis in equine CSSCs. Our findings demonstrate that MSCs-derived EVs represent an innovative and promising therapeutic strategy for the transfer of bioactive mediators which regulate various cellular and molecular signaling pathways.

Small-molecule α-lipoic acid targets ELK1 to balance human neutrophil and erythrocyte differentiation

BackgroundErythroid and myeloid differentiation disorders are commonly occurred in leukemia. Given that the relationship between erythroid and myeloid lineages is still unclear. To find the co-regulators in erythroid and myeloid differentiation might help to find new target for therapy of myeloid leukemia. In hematopoiesis, ALA (alpha lipoic acid) is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors via splicing factor SF3B1. However, further exploration is needed to determine whether ELK1 is a common regulatory factor for erythroid and myeloid differentiation.MethodsIn vitro culture of isolated CD34+, CMPs (common myeloid progenitors) and CD34+ CD371– HSPCs (hematopoietic stem progenitor cells) were performed to assay the differentiation potential of monocytes, neutrophils, and erythrocytes. Overexpression lentivirus of long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 transduced CD34+ HSPCs were transplanted into NSG mice to assay the human lymphocyte and myeloid differentiation differences 3 months after transplantation. Knocking down of SRSF11, which was high expressed in CD371+GMPs (granulocyte-monocyte progenitors), upregulated by ALA and binding to ELK1-RNA splicing site, was performed to analyze the function in erythroid differentiation derived from CD34+ CD123mid CD38+ CD371– HPCs (hematopoietic progenitor cells). RNA sequencing of L-ELK1 and S-ELK1 overexpressed CD34+ CD123mid CD38+ CD371– HPCs were performed to assay the signals changed by ELK1.ResultsHere, we presented new evidence that ALA promoted erythroid differentiation by targeting the transcription factor ELK1 in CD34+ CD371– hematopoietic stem progenitor cells (HSPCs). Overexpression of either the long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 inhibited erythroid-cell differentiation, but knockdown of ELK1 did not affect erythroid-cell differentiation. RNAseq analysis of CD34+ CD123mid CD38+ CD371– HPCs showed that L-ELK1 upregulated the expression of genes related to neutrophil activity, phosphorylation, and hypoxia signals, while S-ELK1 mainly regulated hypoxia-related signals. However, most of the genes that were upregulated by L-ELK1 were only moderately upregulated by S-ELK1, which might be due to a lack of serum response factor interaction and regulation domains in S-ELK1 compared to L-ELK1. In summary, the differentiation of neutrophils and erythrocytes might need to rely on the dose of L-ELK1 and S-ELK1 to achieve precise regulation via RNA splicing signals at early lineage commitment.ConclusionsALA and ELK1 are found to regulate both human granulopoiesis and erythropoiesis via RNA spliceosome, and ALA-ELK1 signal might be the target of human leukemia therapy.

The bone marrow endothelial progenitor cell response to septic infection.

Early increase in the level of endothelial progenitor cells (EPCs) in the systemic circulation occurs in patients with septic infection/sepsis. The significance and underlying mechanisms of this response remain unclear. This study investigated the bone marrow EPC response in adult mice with septic infection induced by intravenous injection (i.v.) of Escherichia coli. For in vitro experiments, sorted marrow stem/progenitor cells (SPCs) including lineage(lin)-stem cell factor receptor (c-kit)+stem cell antigen-1 (Sca-1)-, lin-c-kit+, and lin- cells were cultured with or without lipopolysaccharides (LPSs) and recombinant murine vascular endothelial growth factor (VEGF) in the absence and presence of anti-Sca-1 crosslinking antibodies. In a separate set of experiments, marrow lin-c-kit+ cells from green fluorescence protein (GFP)+ mice, i.v. challenged with heat-inactivated E. coli or saline for 24h, were subcutaneously implanted in Matrigel plugs for 5 weeks. Marrow lin-c-kit+ cells from Sca-1 knockout (KO) mice challenged with heat-inactivated E. coli for 24h were cultured in the Matrigel medium for 8 weeks. The marrow pool of EPCs bearing the lin-c-kit+Sca-1+VEGF receptor 2 (VEGFR2)+ (LKS VEGFR2+) and LKS CD133+VEGFR2+ surface markers expanded rapidly following septic infection, which was supported by both proliferative activation and phenotypic conversion of marrow stem/progenitor cells. Increase in marrow EPCs and their reprogramming for enhancing angiogenic activity correlated with cell-marked upregulation of Sca-1 expression. Sca-1 was coupled with Ras-related C3 botulinum toxin substrate 2 (Rac2) in signaling the marrow EPC response. Septic infection caused a substantial increase in plasma levels of IFN-γ, VEGF, G-CSF, and SDF-1. The early increase in circulating EPCs was accompanied by their active homing and incorporation into pulmonary microvasculature. These results demonstrate that the marrow EPC response is a critical component of the host defense system. Sca-1 signaling plays a pivotal role in the regulation of EPC response in mice with septic infection.

Stem cell exosomes in the pathophysiology of cardiovascular diseases

This study focused on the current state of the therapeutic potential of extracellular vesicles, which depends on the methods of their isolation and composition and the characteristics of the vesicular and nonvesicular components. Myocardial damage, particularly as a result of acute myocardial infarction, leads to irreversible death of cardiomyocytes and sarcomeres and ultimately to heart failure. The adult heart has limited regenerative capacity; therefore, stimulation of endogenous repair and regenerative potentials using cell therapy has potential. Moreover, the benefit from the injection of stem cells and progenitor cells into the damaged myocardium is mediated by the factors they secrete. In particular, exosomes, nanosized secreted extracellular vesicles of endosomal origin, have become key signaling organelles in intercellular communication and are currently considered key regenerative components of the secretome of stem and progenitor cells. Exosomes released from cardiac embryonic and mesenchymal stem cells, resident stem and progenitor cells (including a specific subgroup of cardiosphere cells), induced pluripotent stem cells, and cardiomyocytes isolated from these cells have cardioprotective, immunomodulatory, and reparative abilities. The use of exosomes in the targeted transport of drugs in lipid-like nanocontainers and extracellular vesicles is another promising area. Because artificial drug carriers, including liposomes and lipid-based nanoparticles, are limited by potential toxicity, immunogenicity, and inability to target specific organs, exosomes hold good promise as potential drug carriers. Compounds can be transported both inside exosomes and on their surface. Secreted extracellular vesicles, particularly exosomes, can be considered a key functional component of the secretome of stem cells and cardiogenic progenitor cells (mesenchymal stem cells, endogenous cardiac progenitor cells, cardiospheres, bone marrow embryonic stem cells, and bone marrow induced pluripotent stem cells). They have demonstrated therapeutic efficacy in preclinical models in the study of cardiovascular pathology.

DDX20 is required for cell-cycle reentry of prospermatogonia and establishment of spermatogonial stem cell pool during testicular development in mice.

RNA-binding proteins (RBPs), as key regulators of mRNA fate, are abundantly expressed in the testis. However, RBPs associated with human male infertility remain largely unknown. Through bioinformatic analyses, we identified 62 such RBPs, including an evolutionarily conserved RBP, DEAD-box helicase 20 (DDX20). Male germ-cell-specific inactivation of Ddx20 at E15.5 caused T1-propsermatogonia (T1-ProSG) to fail to reenter cell cycle during the first week of testicular development in mice. Consequently, neither the foundational spermatogonial stem cell (SSC) pool nor progenitor spermatogonia were ever formed in the knockout testes. Mechanistically, DDX20 functions to control the translation of its target mRNAs, many of which encode cell-cycle-related regulators, by interacting with key components of the translational machinery in prospermatogonia. Our data demonstrate a previously unreported function of DDX20 as a translational regulator of critical cell-cycle-related genes, which is essential for cell-cycle reentry of T1-ProSG and formation of the SSC pool.

179 Stem Progenitor Cell Proliferation in the Brainstem After Spinal Cord Injury: A Potential Target for Therapeutic Intervention Distant From the Site of Injury

INTRODUCTION: Though there has been an increasing number of clinical trials focused on modulating stem/progenitor cell proliferation in the spinal cord after spinal cord injury (SCI), little is known about the acute response of stem/progenitor cells in supraspinal structures. We hypothesized that the signals stimulating stem/progenitor cell proliferation in the spinal cord after SCI activate similar responses in supraspinal regions because pathways injured in the spinal cord have their targets or sources in the brain. METHODS: We employed a mouse model of SCI, bromodeoxyuridine (BrdU) labeling of cells in S-phase, and co-labeling of markers of astrocytes, microglia, glial/oligodendrocyte precursor cells, and neuronal lineages to characterize the proliferative response within the brainstem of C57BL/6J mice 96 hours after a thoracic SCI. The extent of SCI and white matter (WM) spared was quantified by labeling of beta amyloid precursor protein and eriochrome cyanine R staining, respectively. RESULTS: Compared to sham-operated mice, there was a significant increase (3-fold; p &lt; 0.01) in BrdU-labeled cells in the ventral-caudal medulla of injured mice. This response was consistent with the extent of SCI and primarily in WM tracts (57%) and the inferior olive (41%; IO). Co-labeling with cell-specific markers indicated that this response is glial (i.e., NG-2+ glial/oligodendrocyte precursor cells and GFAP+ astrocytes) with the largest increase in the number of BrdU-labeled cells occurring in the IO (6-fold increase; p &lt; 0.01). Notably, there were no BrdU-labeled cells that co-labeled with markers of microglia or neuronal lineages. CONCLUSIONS: Our results demonstrate a stem/progenitor cell response within days of SCI that occurs distant from the injury site, notably in brainstem structures involved in motor learning and coordination. These findings highlight an acute response of glial/oligodendrocyte precursor cells in the brainstem as a potential target for future studies and therapeutic intervention after SCI.

AAV6 mediated Gsx1 expression in neural stem progenitor cells promotes neurogenesis and restores locomotor function after contusion spinal cord injury

Genomic screened homeobox 1 (Gsx1 or Gsh1) is a neurogenic transcription factor required for the generation of excitatory and inhibitory interneurons during spinal cord development. In the adult, lentivirus (LV) mediated Gsx1 expression promotes neural regeneration and functional locomotor recovery in a mouse model of lateral hemisection spinal cord injury (SCI). The LV delivery method is clinically unsafe due to insertional mutations to the host DNA. In addition, the most common clinical case of SCI is contusion/compression. In this study, we identify that adeno-associated virus serotype 6 (AAV6) preferentially infects neural stem/progenitor cells (NSPCs) in the injured spinal cord. Using a rat model of contusion SCI, we demonstrate that AAV6 mediated Gsx1 expression promotes neurogenesis, increases the number of neuroblasts/immature neurons, restores excitatory/inhibitory neuron balance and serotonergic neuronal activity through the lesion core, and promotes locomotor functional recovery. Our findings support that AAV6 preferentially targets NSPCs for gene delivery and confirmed Gsx1 efficacy in clinically relevant rat model of contusion SCI.

Adult Neurogenesis of Teleost Fish Determines High Neuronal Plasticity and Regeneration.

Studying the properties of neural stem progenitor cells (NSPCs) in a fish model will provide new information about the organization of neurogenic niches containing embryonic and adult neural stem cells, reflecting their development, origin cell lines and proliferative dynamics. Currently, the molecular signatures of these populations in homeostasis and repair in the vertebrate forebrain are being intensively studied. Outside the telencephalon, the regenerative plasticity of NSPCs and their biological significance have not yet been practically studied. The impressive capacity of juvenile salmon to regenerate brain suggests that most NSPCs are likely multipotent, as they are capable of replacing virtually all cell lineages lost during injury, including neuroepithelial cells, radial glia, oligodendrocytes, and neurons. However, the unique regenerative profile of individual cell phenotypes in the diverse niches of brain stem cells remains unclear. Various types of neuronal precursors, as previously shown, are contained in sufficient numbers in different parts of the brain in juvenile Pacific salmon. This review article aims to provide an update on NSPCs in the brain of common models of zebrafish and other fish species, including Pacific salmon, and the involvement of these cells in homeostatic brain growth as well as reparative processes during the postraumatic period. Additionally, new data are presented on the participation of astrocytic glia in the functioning of neural circuits and animal behavior. Thus, from a molecular aspect, zebrafish radial glia cells are seen to be similar to mammalian astrocytes, and can therefore also be referred to as astroglia. However, a question exists as to if zebrafish astroglia cells interact functionally with neurons, in a similar way to their mammalian counterparts. Future studies of this fish will complement those on rodents and provide important information about the cellular and physiological processes underlying astroglial function that modulate neural activity and behavior in animals.

Abstract 251: MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis

Abstract To establish and maintain functional and properly formed tissues, cells must integrate cues from their environment into fate decisions. External cues—from other cells and the extracellular matrix for instance—must be met with appropriate responses to ensure cell fate decisions that control lineage plasticity and favor proper development, maintenance, or regeneration of tissues while suppressing neoplastic transformation. MCAM (CD146; MUC18; S-endo-1; and Gicerin) is an integral membrane glycoprotein whose misexpression is associated with a variety of epithelial cancers, including breast cancer. However, the mechanisms by which MCAM conveys extracellular cues into malignant cellular outputs is not well understood. Our observation that MCAM is normally expressed in transplantable fetal and adult mammary stem cells (MaSCs) with multilineage potential, suggested it could control mammary cell state plasticity in both neoplastic and normal mammary tissue. Using a plastic, mouse mammary tumor cell line derived from the MMTV-PyMT mouse (Py230) that has been reported to exhibit mammary stem cell and luminal progenitor features, we tested the role of Mcam in controlling the tumorigenicity and lineage/cell-state relationships of mammary tumor cells. We found that Mcam knockdown in Py230 cells increases STAT3 expression, activation, and downstream target transcription, leading to a shift in the predominant cell state away from a luminal progenitor state toward more differentiated alveolar and basal phenotypes. Mechanistically, these changes involve altered activity of casein kinase II (CK2) against multiple substrates including STAT3 and PTEN. This effect may be mediated by direct coupling and sequestration of CK2 to various substrates via the cytoplasmic tail of MCAM. We have also found that inhibition of Ck2 or Stat3 reversed the transcriptional state change elicited by Mcam knockdown. In grafted tumors in vivo, this state switch correlates with reduced overall tumorigenicity and attenuated expression of Sox10, a gatekeeper of neural crest-like invasive features. In the normal gland, we hypothesize that this activity regulates multilineage stem cell potential upon transplantation and multilineage regenerative potential across lactation/involution cycles as we found reciprocal expression patterns for MCAM and STAT3 in alveolar mammary epithelial cells. These data point to an integral role for MCAM in lineage plasticity of normal mammary epithelial cells that is co-opted during transformation to potentiate tumor cell plasticity, progression, and evasion of targeted cancer therapies. Citation Format: Brooke L. Gates, Ozlen Balcioglu, David W. Freeman, Berhane M. Hagos, Benjamin T. Spike. MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis [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 251.

Abstract 1733: SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia

Abstract The SET-NUP214 (S/N) fusion gene, which results from either cryptic t(9:9) (q34;q34) or del(9) (q34.11q34.13), is found in a very narrow spectrum of patients diagnosed with Acute Myeloid Leukemia (AML) or T-cell acute lymphoblastic leukemia (T-ALL). AML patients with S/N fusion were reported to have relatively poor prognostic outcomes despite receiving myeloablative conditioning. While previously, the fusion has been described to overrepresent HOX cluster genes, the underlying mechanism of gene regulation that contributes to the pathology remains elusive due to the rare incidence of the disease. The current study used the MEGAL (ACC719, DSMZ) cell line, identified as the solo megakaryocytic AML line expressing the S/N-fusion, for determining genome-wide DNA methylation (DNAm) and gene expression using 935K Infinium MethylationEPIC v2.0 BeadChip array and RNA-sequencing, respectively. The differential DNAm and expression data were analyzed and compared to the hematopoietic stem progenitor cells (HSPC) or normal megakaryocytes (MK). The ChIP-sequencing data from the S/N positive T-ALL cell line LOUCY has been used to represent the coincidence of regulatory chromatin marks and differentially methylated CpG (mC) sites. We observed n=1934 genes upregulated and n=2396 downregulated in MEGAL, compared to both HSPC and MK. The direction of expression changes between MEGAL and HSPC and MEGAL and MK showed a high correlation (r=0.92, p&amp;lt; 0.01). Per the previous literature, we observed an overrepresentation of HOX genes (n=19) in MEGAL. Based on DNAm profiling, we noticed a propensity of hypermethylation in MEGAL across the promoters (93%±0.6), bodies (93%±0.8), and intergenic (85%±0.3) regions, compared to HSPC/MK. Next, we integrated the mC and expression at the promoters and identified that the majority (75%±3) of the genes belong to the hyper-down (n=2563 against HPSC; n=1907 against MK) or hyper-up (n=965 against HPSC; n=923 against MK) clusters. We further investigated the HOXB (HOXB7, HOXB8, HOXB9) and HOXC (HOXC10, HOXC12, HOXC13, HOXC13-AS, HOXC4, HOXC5, HOC6, HOXC8, HOXC9, HOXC-AS1, HOXC-AS2, and HOXC-AS3) cluster genes, where hypermethylated CpGs mainly were concentrated at the CpG islands and overlapped with active promoter (H3K4me3, H3K4me1) signatures or enhancer-like (CTCF bound) elements. We also observed frequent overlaps between mC and H3K36me3 across the observed genes. The differentially expressed genes in this AML subtype were enriched for the hippo-signaling pathway, calcium signaling, focal adhesion, and PI3-Akt pathways. Collectively, our data showed that S/N favors genome-wide hypermethylation, which, in conjunction with the cis-regulatory elements, induces overrepresentation of the HOX cluster genes. These novel epigenetic vulnerabilities are worth pursuing for investigating the cellular function and pathophysiology of the disease. Citation Format: Samrat Roy Choudhury, Akhilesh Kaushal. SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia [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 1733.

Abstract 6836: Single-cell RNA sequencing of FUS::DDIT3 myxoid/round cell liposarcoma delineates mechanisms of oncogenic progression and immune escape

Abstract Myxoid/Round Cell Liposarcoma (MRCL) is an understudied soft tissue sarcoma that typically affects young adults and accounts for 30% of all liposarcomas. MRCL is characterized by oncogenic DDIT3 fusions, with FUS::DDIT3 being present in over 90% of cases. However, the cellular origins and oncogenic mechanisms of MRCL remain incompletely defined. Here we report oncogene transcription, and tumor-immune interactions in a cohort of 5 patient tumors with matched normal tissue for which we sequenced the transcriptomes of approximately 45,000 single cells using 10X Genomics, and full-length transcripts in approximately 20,000 single cells via Oxford Nanopore in 2 of these patients. Our results reveal two distinct cancer cell populations: Early Cancer Cells (ECC) and Advanced Cancer Cells (ACC). These cells appear to derive from adipose stem progenitor cells (ASPC) and fall along an ASPC→ECC→ACC pseudo-time axis. Along this axis, a distinct pattern emerges where oncogenes tend to be upregulated and tumor suppressor genes (TSG) tend to be downregulated and these appear to be mutually exclusive. Comparing ECC to ASPC we see increased expression of HIPPO and PI3K pathway oncogenes, FGFR2 and IGF1R RTKs, and downregulation of the TSG CDKN2A and CDKN1A. ACC show upregulation of hedgehog, ErbB, WNT, HIPPO, PI3K, and MAPK signaling pathways compared to ECC. Full-length transcript sequencing at the single-cell level shows a consistent expression of the FUS::DDIT3 fusion transcript in both ECC and ACC populations, confirming that these populations are cancer cells. Furthermore, MRCL displays several immune escape mechanisms, including poor immune infiltration and a marked decline of HLA Class I expression along the pseudo-time axis from ECC to ACC, suggesting a complex interplay between oncogenic signaling and immune evasion. These results suggest that MRCL likely originates from ASPC and expression of FUS:DDIT3 oncoprotein induces early HIPPO, PI3K, FGFR2, and insulin signaling pathway expression in ECC with further hedgehog, ErbB, MAPK, and WNT pathway activation in ACC. This progressive activation in oncogenic signaling is associated with immune evasion. This is the first single-cell RNA seq analysis to detect the presence of the FUS::DDIT3 fusion in ECC and ACC significantly advancing our understanding of the mechanisms of MRCL oncogenesis, immune escape, and identifies potential novel therapeutic targets. Citation Format: Rodrigo Gularte-Mérida, Evan Seffar, George Li, Young-Mi Kim, Narasimhan P. Agaram, Nicholas Socci, Francisco Sanchez-Vega, Nikolaus Schultz, Samuel Singer. Single-cell RNA sequencing of FUS::DDIT3 myxoid/round cell liposarcoma delineates mechanisms of oncogenic progression and immune escape [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 6836.

Abstract 3044: Novel agents with efficacy against germline mutant RUNX1 familial platelet disorder with myeloid malignancy (FPD-MM)

Abstract RUNX1 is a master-transcriptional regulator involved in normal and malignant hematopoiesis. Germline, mono-allelic, missense mutations in RUNX1 cause large deletions or truncations that are mostly loss-of-function mutations. They cause familial platelet disorder (RUNX1-FPD), which evolves into myeloid malignancy (FPD-MM), e.g., MDS or AML. Although curative in some patients, allogeneic stem cell transplantation from matched, un-related donors carries the risk of GVHD and relapse in FPD-MM. Thus, there is a need for novel therapies to revert FPD-MM back to FPD. LINCS1000-CMap analysis, of the RUNX1 knockdown RNA-Seq signature revealed homoharringtonine (HHT or omacetaxine) and the anthelmintic fenbendazole (analog of mebendazole (MB)) as the top expression mimickers. In our current studies, we demonstrate that treatment with HHT or MB induced significantly greater loss of viability in seven patient-derived samples of FPD-MM vs RUNX1-FPD. Analyses conducted on BMA cells harvested longitudinally from the same patient at the RUNX1-FPD versus FPD-MM stage demonstrated greater chromatin accessibility in FPD-MM cells along with increased mRNA expressions of MYB, MECOM and BCL2. Following HHT treatment, scRNA-Seq analysis demonstrated a marked reduction in the HSC population, but concomitant increase in the macrophage population in FPD-MM. This was associated with positive enrichment of apoptosis signaling and TP53 targets gene-sets, while negatively enriching those of c-Myc targets. CyTOF analysis demonstrated that HHT treatment reduced c-Myc, EVI1, MCL1, BFL1 and CDK6 expression in phenotypically characterized FPD-MM stem-progenitor cells. Utilizing an FPD-MM sample harboring Runx1 K194N, also detected in other pedigree members with FPD or FPD-MM, we established the first ever cell line (GMR-AML1) expressing germline mtRUNX1. GMR-AML1 cells exhibited high surface expression of CD117 (c-KIT), CD123 (IL-3R) and CD33. GMR-AML1 cells in which Runx1 was knocked-out by CRISPR-gRNA were outcompeted in vitro and in vivo, by cells that retained Runx1. Treatment with HHT or MB induced loss of viability in GMR-AML1 cells (LD50: 40 or 330 nM, respectively). HHT also caused concordant decline in ATAC-Seq and RNA-Seq peaks in ribosomal genes involved in protein translation. Treatment with MB inhibited cell cycle and protein translation. CyTOF analysis revealed that MB treatment also depleted CDK6, RUNX1, c-Myc and EVI1 in phenotypically defined FPD-MM stem cells. Finally, in the GMR-AML1 cell xenograft, treatment with omacetaxine or MB significantly reduced AML burden and improved overall survival of NSG mice. These preclinical findings highlight the molecular features associated with progression of RUNX1-FPD to FPD-MM and highlight HHT or MB as effective against cellular models of FPD-MM. Citation Format: Christopher P. Mill, Warren C. Fiskus, Courtney D. DiNardo, Patrick K. Reville, John A. Davis, Christine E. Birdwell, Kaberi Das, Hanxi Hou, Koichi Takahashi, Sanam Loghavi, Joseph D. Khoury, Kapil Bhalla. Novel agents with efficacy against germline mutant RUNX1 familial platelet disorder with myeloid malignancy (FPD-MM) [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 3044.

The effect of thyme essential oil and endothelial progenitor stem cells on lipopolysaccharide-induced sepsis in C57BL/6 mice.

Sepsis is a potentially fatal syndrome related to severe systemic inflammation developed by infection. Despite different antimicrobial therapies, morbidity and mortality rates remain high. Herbs along with cell therapy have been introduced as a promising option to improve the symptoms of sepsis. The present study aimed to evaluate the therapeutic effect of simultaneous administration of thyme essential oil (TEO) and endothelial progenitor stem cells (EPCs) on lipopolysaccharide (LPS)-induced sepsis in C57BL/6 mice. Sepsis was induced in C57Bl/6J mice by intraperitoneal injection of LPS, followed 2h later by an intravenous injection of EPCs or oral administration of TEO or simultaneous administration of TEO and EPCs. After 10 days, the complete blood cell, renal and liver factors, serum levels of inflammatory cytokines, and angiogenic factors were measured. Simultaneous treatment with EPCs and TEO significantly increased the survival of mice with sepsis and modulated the inflammatory response by reducing the serum levels of pro-inflammatory cytokines. Moreover, this treatment significantly reduced the level of white blood cells and neutrophils and increased the number of red blood cells, the percentage of hematocrit, and hemoglobin. The combination of TEO with EPCs decreased organ injuries and was assessed by lower levels of the liver enzymes alanine aminotransferase and aspartate aminotransferase compared to the sepsis group. Administration of EPCs and TEO also significantly improved angiogenic factors, lung function, and toll-like receptor 4 expression. EPCs in combination with TEO increase survival in the LPS-induced sepsis mice model by acting on several targets. Thus, the combination of TEO with EPCs can be a feasible approach for the future clinical treatment and control of sepsis.

Histopathological changes in the spleen of rats exposed to N,N-dimethylhydrazine with the following protective input of Au/Ag/Fe

Colon cancer is one of the leading causes of cancer-related deaths worldwide. Despite the growing number of studies on nanoparticles of different metals, there is still a lack of reliable information about their ability to work together as a composition and their antitumor effects. It has been established that the regulation of genes encoding proteins of the Bcl-2 family changes in cancer. Bcl-2 family proteins can be a potential target in cancer diagnosis and have a prognostic value in treating cancer with chemotherapeutic drugs. The tumor microenvironment includes myeloid suppressor cells, tumor-associated neutrophils, and tumor-associated macrophages that promote cancer progression and are derived from splenic hematopoietic stem cells and progenitor cells. The work aimed to ascertain histological changes and expression of Bcl-2 family proteins in the spleen of rats with N,N-dimethylhydrazine-induced carcinogenesis followed by the protective administration of Au/Ag/Fe nanometal composition. The study was conducted on 72 outbred white male rats. Colon adenocarcinoma was modelled by N,N-dimethylhydrazine hydrochloride administration for 30 weeks. Animals received an aqueous dispersion of Au/Ag/Fe nanoparticles intragastrically once a day for 21 days at a dose of 0.842 mg Ag/0.0526 mg Fe/1.625 μg Au per 1 kg of rat body weight. Paraffin sections of the spleen were stained with hematoxylin and eosin. For immunohistochemical analysis, sections were stained with rabbit monoclonal antibodies to Bcl-2. Under conditions of 30-week exposure to N,N-dimethylhydrazine hydrochloride, blood circulation disorders, stasis, thrombosis, reduction in the size of white pulp lymphatic nodules and loss of their zonation were histologically proved in the rat's spleen. The development of sclerotic processes in the organ was noted. It was indicated that using metal nanoparticles under N,N-dimethylhydrazine hydrochloride-induced colon carcinogenesis leads to less evidenced morphological manifestations of structural changes in the spleen. Immunohistochemically, the expression of Bcl-2 protein in the white pulp of the spleen of animals subjected to correction was lower than in animals without the corrective effect of the composition of nanoparticle metals. Administration of the Au/Ag/Fe metal nanoparticle composition to intact white rats does not cause pathological changes in the spleen morphology. The structural components of the red and white pulp match the typical histological structure of the organ.

ArCH: improving the performance of clonal hematopoiesis variant calling and interpretation.

The acquisition of somatic mutations in hematopoietic stem and progenitor stem cells with resultant clonal expansion, termed clonal hematopoiesis (CH), is associated with increased risk of hematologic malignancies and other adverse outcomes. CH is generally present at low allelic fractions, but clonal expansion and acquisition of additional mutations leads to hematologic cancers in a small proportion of individuals. With high depth and high sensitivity sequencing, CH can be detected in most adults and its clonal trajectory mapped over time. However, accurate CH variant calling is challenging due to the difficulty in distinguishing low frequency CH mutations from sequencing artifacts. The lack of well-validated bioinformatic pipelines for CH calling may contribute to lack of reproducibility in studies of CH. Here, we developed ArCH, an Artifact filtering Clonal Hematopoiesis variant calling pipeline for detecting single nucleotide variants and short insertions/deletions by combining the output of four variant calling tools and filtering based on variant characteristics and sequencing error rate estimation. ArCH is an end-to-end cloud-based pipeline optimized to accept a variety of inputs with customizable parameters adaptable to multiple sequencing technologies, research questions, and datasets. Using deep targeted sequencing data generated from six acute myeloid leukemia patient tumor: normal dilutions, 31 blood samples with orthogonal validation, and 26 blood samples with technical replicates, we show that ArCH improves the sensitivity and positive predictive value of CH variant detection at low allele frequencies compared to standard application of commonly used variant calling approaches. The code for this workflow is available at: https://github.com/kbolton-lab/ArCH.