Formation Of Progenitor Cells Research Articles

Abnormal epigenetic memory of mesenchymal stem and progenitor cells caused by fetal malnutrition induces hypertension and renal injury in adulthood.

Fetal malnutrition has been reported to induce hypertension and renal injury in adulthood. We hypothesized that this hypertension and renal injury would be associated with abnormal epigenetic memory of stem and progenitor cells contributing to organization in offspring due to fetal malnutrition. We measured blood pressure (BP) for 60 weeks in offspring of pregnant rats fed a normal protein diet (Control), low-protein diet (LP), and LP plus taurine (LPT) in the fetal period. We used western blot analysis to evaluate the expression of αSMA and renin in CD44-positive renal mesenchymal stem cells (MSCs) during differentiation by TGF-β1. We measured kidney label-retaining cells (LRCs) at 11 weeks of age and formation of endothelial progenitor cells (EPCs) at 60 weeks of age from the offspring with fetal malnutrition. Epigenetics of the renal MSCs at 14 weeks were investigated by ATAC-sequence and RNA-sequence analyses. BP was significantly higher in LP than that in Control and LPT after 45-60 weeks of age. Numbers of LRCs and EPC colonies were significantly lower in LP than in Control. Renal MSCs from LP already showed expression of h-caldesmon, αSMA, LXRα, and renin before their differentiation. Epigenetic analyses identified PAR2, Chac1, and Tspan6 genes in the abnormal differentiation of renal MSCs. These findings suggested that epigenetic abnormalities of stem and progenitor cell memory cause hypertension and renal injury that appear in adulthood of offspring with fetal malnutrition.

Ocular pharmacological and biochemical profiles of 6-thioguanine: a drug repurposing study.

The purine analog 6-thioguanine (6TG), an old drug approved in the 60s to treat acute myeloid leukemia (AML), was tested in the diabetic retinopathy (DR) experimental in vivo setting along with a molecular modeling approach. A computational analysis was performed to investigate the interaction of 6TG with MC1R and MC5R. This was confirmed in human umbilical vein endothelial cells (HUVECs) exposed to high glucose (25mM) for 24h. Cell viability in HUVECs exposed to high glucose and treated with 6TG (0.05-0.5-5µM) was performed. To assess tube formation, HUVECs were treated for 24h with 6TG 5µM and AGRP (0.5-1-5µM) or PG20N (0.5-1-5-10µM), which are MC1R and MC5R antagonists, respectively. For the in vivo DR setting, diabetes was induced in C57BL/6J mice through a single streptozotocin (STZ) injection. After 2, 6, and 10weeks, diabetic and control mice received 6TG intravitreally (0.5-1-2.5mg/kg) alone or in combination with AGRP or PG20N. Fluorescein angiography (FA) was performed after 4 and 14weeks after the onset of diabetes. After 14weeks, mice were euthanized, and immunohistochemical analysis was performed to assess retinal levels of CD34, a marker of endothelial progenitor cell formation during neo-angiogenesis. The computational analysis evidenced a more stable binding of 6TG binding at MC5R than MC1R. This was confirmed by the tube formation assay in HUVECs exposed to high glucose. Indeed, the anti-angiogenic activity of 6TG was eradicated by a higher dose of the MC5R antagonist PG20N (10µM) compared to the MC1R antagonist AGRP (5µM). The retinal anti-angiogenic effect of 6TG was evident also in diabetic mice, showing a reduction in retinal vascular alterations by FA analysis. This effect was not observed in diabetic mice receiving 6TG in combination with AGRP or PG20N. Accordingly, retinal CD34 staining was reduced in diabetic mice treated with 6TG. Conversely, it was not decreased in diabetic mice receiving 6TG combined with AGRP or PG20N. 6TG evidenced a marked anti-angiogenic activity in HUVECs exposed to high glucose and in mice with DR. This seems to be mediated by MC1R and MC5R retinal receptors.

Heparin-binding epidermal growth factor and fibroblast growth factor 2 rescue Müller glia-derived progenitor cell formation in microglia- and macrophage-ablated chick retinas.

Recent studies have demonstrated the impact of pro-inflammatory signaling and reactive microglia/macrophages on the formation of Müller glial-derived progenitor cells (MGPCs) in the retina. In chick retina, ablation of microglia/macrophages prevents the formation of MGPCs. Analyses of single-cell RNA-sequencing chick retinal libraries revealed that quiescent and activated microglia/macrophages have a significant impact upon the transcriptomic profile of Müller glia (MG). In damaged monocyte-depleted retinas, MG fail to upregulate genes related to different cell signaling pathways, including those related to Wnt, heparin-binding epidermal growth factor (HBEGF), fibroblast growth factor (FGF) and retinoic acid receptors. Inhibition of GSK3β, to simulate Wnt signaling, failed to rescue the deficit in MGPC formation, whereas application of HBEGF or FGF2 completely rescued the formation of MGPCs in monocyte-depleted retinas. Inhibition of Smad3 or activation of retinoic acid receptors partially rescued the formation of MGPCs in monocyte-depleted retinas. We conclude that signals produced by reactive microglia/macrophages in damaged retinas stimulate MG to upregulate cell signaling through HBEGF, FGF and retinoic acid, and downregulate signaling through TGFβ/Smad3 to promote the reprogramming of MG into proliferating MGPCs.

Tropomyosin 1 Deficiency Facilitates Epithelial-to-Mesenchymal Transitions to Promote Hemogenic Endothelial Specification and Hematopoietic Progenitor Cell Formation

Tropomyosins coat actin filaments and impact many actin-related biology, including signaling and cell morphogenesis. Prior work suggested that TPM1 regulated blood cell formation in vitro, but it was unclear how or when TPM1 affected hematopoiesis. Using gene-edited induced pluripotent stem cell (iPSC)model systems, single cell transcriptomics, and a novel murine model, we aimed to define how and when TPM1 regulated developmental hematopoiesis. We used CRISPR/Cas9 to create TPM1 knockout (KO) iPSC lines in a primitive hematopoiesis system, in which hemogenic endothelial cells spawn hematopoietic progenitor cells (HPC). Western blot showed that TPM1 was highly expressed in iPSCs and endothelial cells but virtually absent in HPCs. TPM1KO enhanced endothelial cell and hematopoietic progenitor cell (HPC) formation without augmenting cell cycle kinetics. RNA sequencing analysis showed that TPM1KO altered epithelial-to-mesenchymal transition (EMT)-related gene expression and functionally enhanced EMT during in vitro hematopoiesis, without generally disrupting normal cell development. Single-cell transcriptomics showed that TPM1 was highly expressed in human and mouse hemogenic endothelium, and abrogated in HPCs, suggesting that TPM1 might also regulate developmental hematopoiesis in vivo similar to our in vitro findings. Indeed, TPM1 expression coincided with EMT-related gene expression during in vivo hematopoiesis (p<1x10-16). To investigate the role of TPM1 in vivo, we used a novel murine TPM1 GeneTrap-Reporter mouse model. By western blot, TPM1 protein was decreased ~50% in peripheral blood. TPM1 GT/GTmice displayed embryonic lethality by E9.5, due to cardiac dysmorphology previously seen in TPM1 knockout mice. TPM1 reporter expression (LacZ) was observed in E9.5 endothelium, temporally consistent with Runx1+/CD31+ hemogenic endothelial cell emergence. Whole-mount imaging of TPM1 GT/+ embryos showed an enhanced percentage (%) of Runx1 + HE at E9.5 and Runx1 +cKit + HPCs at E10.5. Consistent with increased embryonic HPC production, TPM1 GT/+mice showed increased percentages of Lin-Sca1+cKit+ HPCs in bone marrow at 6-8 weeks of age. Complete blood counts in 6-8 weeks-old mice were normal. Conclusions These findings illuminate novel effects of TPM1, a cytoskeletal regulatory molecule, on developmental hematopoiesis. TPM1KO disinhibits EMT to promote hemogenic endothelial specification and HPC production during developmental hematopoiesis, without compromising mature blood cell function. Temporal TPM1 inhibition may offer innovative approaches to enhance blood cell formation in translationally relevant cell culture systems. Our findings may explain genetic associations linking polymorphisms in the TPM1 gene locus with altered human blood traits. More broadly, the radical changes required to form HPCs from endothelial cells during hematopoiesis represent an exciting area of study for actin and tropomyosin biology.

METTL3-Dependent N6-Methyladenosine Modification Programs Human Neural Progenitor Cell Proliferation.

METTL3, a methyltransferase responsible for N6-methyladenosine (m6A) modification, plays key regulatory roles in mammal central neural system (CNS) development. However, the specific epigenetic mechanisms governing human CNS development remain poorly elucidated. Here, we generated small-molecule-assisted shut-off (SMASh)-tagged hESC lines to reduce METTL3 protein levels, and found that METTL3 is not required for human neural progenitor cell (hNPC) formation and neuron differentiation. However, METTL3 deficiency inhibited hNPC proliferation by reducing SLIT2 expression. Mechanistic studies revealed that METTL3 degradation in hNPCs significantly decreased the enrichment of m6A in SLIT2 mRNA, consequently reducing its expression. Our findings reveal a novel functional target (SLIT2) for METTL3 in hNPCs and contribute to a better understanding of m6A-dependent mechanisms in hNPC proliferation.

BCR-ABL promotes hematopoietic stem and progenitor cell formation in embryonic stem cells

Generating hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has been a long-lasting quest in the field of hematopoiesis. Previous studies suggested that enforced expression of BCR-ABL, the unique oncogenic driver of chronic myelogeneous leukemia (CML), in embryonic stem cells (ESCs)-derived hematopoietic cells is sufficient to confer long-term in vivo repopulating potential. To precisely uncover the molecular events regulated by the tyrosine kinase activity of BCR-ABL1 (p210) during the course of hematopoietic differentiation, we engineered a Tet-ON inducible system to modulate its expression in murine ESCs (mESCs). We showed in unique site-directed knock-in ESC model that BCR-ABL expression tightly regulated by doxycycline (dox) controls the formation and the maintenance of immature hematopoietic progenitors. Interestingly, these progenitors can be expanded in vitro for several passages in the presence of dox. Our analysis of cell surface markers and transcriptome compared with wild-type fetal and adult HSCs unraveled a similar molecular signature. Long-term culture initiating cell (LTC-IC) assay confirmed their self-renewal capacities albeit with a differentiation bias toward erythroid and myeloid cells. Collectively, our novel Tet-ON system represents a unique in vitro model to shed lights on ESC-derived hematopoiesis, CML initiation, and maintenance.

Heme-deficient primitive red blood cells induce HSPC ferroptosis by altering iron homeostasis during zebrafish embryogenesis.

The crosstalk between hematopoietic lineages is important for developmental hematopoiesis. However, the role of primitive red blood cells (RBCs) in the formation of definitive hematopoietic stem and progenitor cells (HSPCs) is largely unknown. Primitive RBC deficiencies in mammals always lead to early embryonic lethality, but zebrafish lines with RBC deficiencies can survive to larval stage. By taking advantage of a zebrafish model, we find that the survival of nascent HSPCs is impaired in alas2- or alad-deficient embryos with aberrant heme biosynthesis in RBCs. Heme-deficient primitive RBCs induce ferroptosis of HSPCs through the disruption of iron homeostasis. Mechanistically, heme-deficient primitive RBCs cause blood iron-overload via Slc40a1, and an HSPC iron sensor, Tfr1b, mediates excessive iron absorption. Thus, iron-induced oxidative stress stimulates the lipid peroxidation, which directly leads to HSPC ferroptosis. Anti-ferroptotic treatments efficiently reverse HSPC defects in alas2 or alad mutants. HSPC transplantation assay reveals that the attenuated erythroid reconstitution efficiency may result from the ferroptosis of erythrocyte-biased HSPCs. Together, these results illustrate that heme-deficient primitive RBCs are detrimental to HSPC production and may provide potential implications for iron dysregulation-induced hematological malignancies.

Neurogenic Stem Cell Niche in the Auditory Thalamus: In Vitro Evidence of Neural Stem Cells in the Rat Medial Geniculate Body.

The medial geniculate body (MGB) is a nucleus of the diencephalon representing a relevant segment of the auditory pathway and is part of the metathalamus. It receives afferent information via the inferior brachium of the inferior colliculus and transmits efferent fibers via acoustic radiations to the auditory cortex. Neural stem cells (NSCs) have been detected in certain areas along the auditory pathway. They are of great importance as the induction of an adult stem cell niche might open a regenerative approach to a causal treatment of hearing disorders. Up to now, the existence of NSCs in the MGB has not been determined. Therefore, this study investigated whether the MGB has a neural stem cell potential. For this purpose, cells were extracted from the MGB of PND 8 Sprague-Dawley rats and cultured in a free-floating cell culture assay, which showed mitotic activity and positive staining for stem cell and progenitor markers. In differentiation assays, the markers β-III-tubulin, GFAP, and MBP demonstrated the capacity of single cells to differentiate into neuronal and glial cells. In conclusion, cells from the MGB exhibited the cardinal features of NSCs: self-renewal, the formation of progenitor cells, and differentiation into all neuronal lineage cells. These findings may contribute to a better understanding of the development of the auditory pathway.

NDRG2 regulates the formation of reactive astrocyte-derived progenitor cells via Notch signaling pathway after brain traumatic injury in rats.

In response to traumatic brain injury, a subpopulation of cortical astrocytes is activated, resulting in acquisition of stem cell properties, known as reactive astrocytes-derived progenitor cells (Rad-PCs). However, the underlying mechanisms remain largely unknown during this process. In this study, we examined the role of N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, in Rad-PCs after cortical stab injury in adult rats. Immunohistochemical analysis showed that in the cerebral cortex of normal adult rats, NDRG2 was exclusively expressed in astrocytes. After liu cortical injury, the expression of NDRG2 was significantly elevated around the wound and most cells expressing NDRG2 also expressed GFAP, a reactive astrocyte marker. Importantly, NDRG2-expressing cells were co-labeled with Nestin, a marker for neural stem cells, some of which also expressed cell proliferation marker Ki67. Overexpression of NDRG2 further increased the number of NDRG2/Nestin double-labeling cells around the lesion. In contrast, shRNA knockdown of NDRG2 decreased the number of NDRG2+/Nestin+ cells. Intracerebroventricular administration of stab-injured rats with a Notch antagonist, DAPT, led to a significant decrease in Nestin+/NDRG2+ cells around the injured boundary, but did not affect NDRG2+ cells. Moreover, overexpression or knockdown of NDRG2 led to up- and down-regulation of the expression of Notch intracellular domain NICD and Notch target gene Hes1, respectively. Taken together, these results suggest that NDRG2 may play a role in controlling the formation of Rad-PCs in the cerebral cortex of adult rats following traumatic injury, and that Notch signaling pathway plays a key role in this process.

Hyaluronic acid-based bioink improves the differentiation and network formation of neural progenitor cells.

Introduction: Three-dimensional (3D) bioprinting is a promising technique for the development of neuronal in vitro models because it controls the deposition of materials and cells. Finding a biomaterial that supports neural differentiation in vitro while ensuring compatibility with the technique of 3D bioprinting of a self-standing construct is a challenge. Methods: In this study, gelatin methacryloyl (GelMA), methacrylated alginate (AlgMA), and hyaluronic acid (HA) were examined by exploiting their biocompatibility and tunable mechanical properties to resemble the extracellular matrix (ECM) and to create a suitable material for printing neural progenitor cells (NPCs), supporting their long-term differentiation. NPCs were printed and differentiated for up to 15days, and cell viability and neuronal differentiation markers were assessed throughout the culture. Results and Discussion: This composite biomaterial presented the desired physical properties to mimic the ECM of the brain with high water intake, low stiffness, and slow degradation while allowing the printing of defined structures. The viability rates were maintained at approximately 80% at all time points. However, the levels of β-III tubulin marker increased over time, demonstrating the compatibility of this biomaterial with neuronal cell culture and differentiation. Furthermore, these cells showed increased maturation with corresponding functional properties, which was also demonstrated by the formation of a neuronal network that was observed by recording spontaneous activity via Ca2+ imaging.

Neuroprotective effects of meloxicam on transient brain ischemia in rats: the two faces of anti-inflammatory treatments.

The inflammatory response plays an important role in neuroprotection and regeneration after ischemic insult. The use of non-steroidal anti-inflammatory drugs has been a matter of debate as to whether they have beneficial or detrimental effects. In this context, the effects of the anti-inflammatory agent meloxicam have been scarcely documented after stroke, but its ability to inhibit both cyclooxygenase isoforms (1 and 2) could be a promising strategy to modulate post-ischemic inflammation. This study analyzed the effect of meloxicam in a transient focal cerebral ischemia model in rats, measuring its neuroprotective effect after 48 hours and 7 days of reperfusion and the effects of the treatment on the glial scar and regenerative events such as the generation of new progenitors in the subventricular zone and axonal sprouting at the edge of the damaged area. We show that meloxicam's neuroprotective effects remained after 7 days of reperfusion even if its administration was restricted to the two first days after ischemia. Moreover, meloxicam treatment modulated glial scar reactivity, which matched with an increase in axonal sprouting. However, this treatment decreased the formation of neuronal progenitor cells. This study discusses the dual role of anti-inflammatory treatments after stroke and encourages the careful analysis of both the neuroprotective and the regenerative effects in preclinical studies.

The brain-specific splice variant of the CDC42 GTPase works together with the kinase ACK to downregulate the EGF receptor in promoting neurogenesis

The small GTPase CDC42 plays essential roles in neurogenesis and brain development. Previously, we showed that a CDC42 splice variant that has a ubiquitous tissue distribution specifically stimulates the formation of neural progenitor cells, whereas a brain-specific CDC42 variant, CDC42b, is essential for promoting the transition of neural progenitor cells to neurons. These specific roles of CDC42 and CDC42b in neurogenesis are ascribed to their opposing effects on mTORC1 activity. Specifically, the ubiquitous form of CDC42 stimulates mTORC1 activity and thereby upregulates tissue-specific transcription factors that are essential for neuroprogenitor formation, whereas CDC42b works together with activated CDC42-associated kinase (ACK) to downregulate mTOR expression. Here, we demonstrate that the EGF receptor (EGFR) is an additional and important target of CDC42b and ACK, which is downregulated by their combined actions in promoting neurogenesis. The activation status of the EGFR determines the timing by which neural progenitor cells derived from P19 embryonal carcinoma terminally differentiate into neurons. By promoting EGFR degradation, we found that CDC42b and ACK stimulate autophagy, which protects emerging neurons from apoptosis and helps trigger neural progenitor cells to differentiate into neurons. Moreover, our results reveal that CDC42b is localized in phosphatidylinositol (3,4,5)-triphosphate-enriched microdomains on the plasma membrane, mediated through its polybasic sequence 185KRK187, which is essential for determining its distinct functions. Overall, these findings now highlight a molecular mechanism by which CDC42b and ACK regulate neuronal differentiation and provide new insights into the functional interplay between EGFR degradation and autophagy that occurs during embryonic neurogenesis.

Modelling SHH-Medulloblastoma Using Cerebellar Organoids

Abstract AIMS Medulloblastoma (MB) comprises four main groups (SHH, WNT, group 3 and group 4), with distinct molecular, clinical and demographic features. These differences suggest opportunities for tailored and improved treatment. Despite its promise, translation into targeted therapy has been slow, limited by the lack of laboratory models that recapitulate patient tumours. Sporadic and inherited forms of SHH-MB are frequently due to mutations in the tumour suppressor gene PTCH1. Here, we aim to exploit human induced pluripotent stem cells (hiPSC) and CRISPR/Cas9 gene editing, to target PTCH1 in a clinically relevant context, thereby establishing an innovative model of MB. METHOD CRISPR/Cas9 mutation of PTCH1 in hiPSCs was followed by their differentiation into cerebellar organoids. Mutant organoids were studied for early consequences on cerebellar development and tumorigenesis. RESULTS PTCH1-mutation resulted in enhanced SHH-pathway activity in cerebellar organoids. Homozygous PTCH1-mutation prevented the formation of cerebellar progenitor cells, closely mimicking the effects of high SHH- signalling on neuronal progenitors in early embryonal development. Heterozygous PTCH1-mutant hiPSCs could be differentiated into cerebellar organoids, displayed increased growth rate and expressed oncogenes specific to SHH-MB, resembling preneoplasia and MB stem cells. CONCLUSION These results show that cerebellar organoids are a promising new model to study early oncogenic events in MB. Our model presents opportunities for the discovery of new targets which could benefit patients with a genetic predisposition to MB in particular. Refined knowledge of early drivers will be invaluable to guide drug selection and improve prospects for clinical translation.

Early hematopoietic injury triggered by benzene characterized with inhibition of erythrocyte differentiation involving the mollicutes_RF39-derived citrulline.

Benzene poisoning is a common adverse blood outcome in occupational workers, manifested by hematopoietic dysfunction. However, the specific phenotype and its mechanisms of early hematopoietic toxicity caused by benzene remain unclear. After 15 days of exposure, the WBC levels were not significantly altered in benzene-exposed mice. However, the level of red blood cells (RBC) showed a significant decrease, and it was significantly and negatively correlated with urinary S-phenylmercapturic acid (SPMA). Notably, 5mg/kg benzene exposure significantly inhibited the renewal capacity and the number of colony formation of hematopoietic stem progenitor cells in mice, especially erythrocyte differentiation. These results suggested that the early hematopoietic toxicity phenotype caused by benzene was dominated by inhibition of erythroid differentiation rather than WBC-related inflammation. To further understand the underlying mechanisms of benzene-induced early hematopoietic toxicity, 16S rRNA sequencing and plasma metabolites analysis were conducted to investigate the impact of benzene exposure for 15 days on microbial composition and metabolic profile of mice. We found that short-term benzene exposure induced disturbances in gut microbiota and metabolism. The relative abundance of Mollicutes_RF39 at order levels was significantly reduced in benzene-exposed mice and was strongly correlated with hematopoietic indicators and urinary benzene markers. Interestingly, Mollicutes_RF39 might disturb the levels of eight metabolites, whereas Citrulline was highly linked to Mollicutes_RF39 (r=0.862, P=0.000). Consequently, Mollicutes_RF39-derived Citrulline might be the key regulator of early hematopoietic injury induced by benzene exposure. These findings promote the understanding of early hematotoxicity phenotypes and provide new perspectives on the underlying mechanisms of benzene-induced hematotoxicity.

Abstract P1070: Role Of Vezf1 In Cardiomyocyte Development

Cardiovascular diseases (CVDs) are the leading cause of high morbidity rates and death worldwide. The heart is the first organ that is formed during mammalian development. Progenitor cells that form the cardiovascular system (heart and blood vessels) originate from the mesoderm post gastrulation. To date, there remain considerable gaps in the molecular mechanisms that govern the differentiation of cardiac progenitor cells into a functional heart, knowledge of which will provide advancements in cardiac regenerative medicine. Vascular endothelial zinc finger 1 (Vezf1) is a nuclear protein expressed predominantly in the mesoderm of a developing mouse embryo. A recent study showed that diseased human myocardia had reduced expression of Vezf1 when compared to healthy hearts. However, the function of Vezf1 in heart development is largely uncharacterized and understudied. Using murine embryonic stem cells (mESCs), we sought to investigate the early role(s) of Vezf1 in cardiovascular development. We show that genetic ablation or conditional knockdown of Vezf1 in mESCs impairs cardiomyocyte differentiation, as determined by the absence of contractile activity in these mutants. The absence of cardiomyocytes from Vezf1 mutant cell lines was confirmed using gene expression analysis and immunofluorescence. We observed impaired mesoderm induction in Vezf1 mutants despite external stimulation of Wnt signaling using small molecule inhibitors, suggesting a potential role of Vezf1 in the modulation of genes in the signaling pathway. Indeed, our gene expression analysis and ChIP-qPCR indicate that Vezf1 potentially associates with the promoters of Wnt signaling genes to activate their expression. These studies point towards a model where Vezf1 regulates the formation of cardiac progenitor cells by modulating the Wnt signaling pathway. Our observations suggest that Vezf1 may sit at the center of determining the fate of mesodermal cells into cardiovascular progenitors, the absence of which impairs cardiomyocyte differentiation. This study is the first to delineate the functional role of Vezf1 in the early development of cardiomyocytes and may form the foundation for future cardiac regenerative medicine.

Abstract 5075: PBI-05204, a supercritical CO2 extract of Nerium oleander, suppresses the stemness of glioblastoma stem cells and inhibits the Shh-Gli1-NANOG axis

Abstract Glioblastoma multiforme (GBM) is an aggressive form of primary brain neoplasm that has a poor prognosis and is resistant to conventional treatment. There is a great need to develop new strategies to improve the outcome of this disease. PBI-05204, a supercritical CO2 Nerium oleander extract containing cardiac glycosides, such as oleandrin, has been tested in Phase I and II clinical trials for treatment of solid tumors. The purpose of the present study was to examine the efficacy of PBI-05204 against GBM and to explore potential mechanisms of action. Rapid growth of GBM has been associated with the presence of pluripotent cancer stem cells that have the capacity of self-renewal, proliferation, and formation of cancer progenitor cells. Genome wide expression profiling of Glioblastoma stem cells (GSCs) demonstrates similarities to embryonic stem cells (ESCs) and progenitor cells defining a pattern of hierarchical appearance of Embryonic stem cell markers: NANOG, SOX2, and GFAP. Using cultures of patient derived GSCs: GBM9 expressing WT EGFR and EGFRVIII, and GS28 expressing EGFR WT, we found that PBI-05204 treatment at concentrations of 0.5, 1 and 2.5 μg/ml for 72 hr significantly inhibited the number of tumor spheres by 59.8, 73.2, and 92.3% in GBM9 GSC, and 47.9, 81.6, and 93.4% in GS28 GSC, respectively. The size of the spheres in PBI-05204 (1 μg/ml) treated GBM9 GSC (103.3 ± 17.5 μm) was also significantly smaller than that of the control group (277.5 ± 10.9 μM, p < 0.01). PBI-05204 treatment significantly inhibited protein expressions of ESCs and progenitor cell markers NANOG, SOX2, and GFAP in a conc-dependent manner in GBM9 and GS28 GSCs as determined by Western blot analyses. NANOG is a transcription factor implicated in the pluripotency of ESCs in various cancers including GBM. Previously it has been shown that the Hedge Hog and Gli axis (HH -Gli) is important for GBM growth, stem cell expansion and establishment of an ESC signature. Western blot analysis showed PBI-05204 down-regulated the expression of Gli1 in both GBM9 and GS 28 GSC after 24 hr. of treatment suggesting it inhibits the Shh-Gli1-NANOG axis. Determination of the antitumor efficacy of PBI-05204 in a GSC derived mouse orthotopic tumor model is currently ongoing and the result will be presented at the meeting. Given that the active components of PBI-05204 have the capacity to readily cross the blood-brain barrier, our findings suggest that this novel botanical drug is a viable candidate that should be considered for treatment of GBM due to its ability to inhibit the stemness of GBM independent of highly resistant EGFR/EGFR VIII mark. Citation Format: Sharmistha Chakraborty, Daoyan Wei, Peiying Yang. PBI-05204, a supercritical CO2 extract of Nerium oleander, suppresses the stemness of glioblastoma stem cells and inhibits the Shh-Gli1-NANOG axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5075.

Negative Elongation Factor (NELF) Inhibits Premature Granulocytic Development in Zebrafish.

Gene expression is tightly regulated during hematopoiesis. Recent studies have suggested that RNA polymerase II (Pol II) promoter proximal pausing, a temporary stalling downstream of the promoter region after initiation, plays a critical role in regulating the expression of various genes in metazoans. However, the function of proximal pausing in hematopoietic gene regulation remains largely unknown. The negative elongation factor (NELF) complex is a key factor important for this proximal pausing. Previous studies have suggested that NELF regulates granulocytic differentiation in vitro, but its in vivo function during hematopoiesis remains uncharacterized. Here, we generated the zebrafish mutant for one NELF complex subunit Nelfb using the CRISPR-Cas9 technology. We found that the loss of nelfb selectively induced excessive granulocytic development during primitive and definitive hematopoiesis. The loss of nelfb reduced hematopoietic progenitor cell formation and did not affect erythroid development. Moreover, the accelerated granulocytic differentiation and reduced progenitor cell development could be reversed by inhibiting Pol II elongation. Further experiments demonstrated that the other NELF complex subunits (Nelfa and Nelfe) played similar roles in controlling granulocytic development. Together, our studies suggested that NELF is critical in controlling the proper granulocytic development in vivo, and that promoter proximal pausing might help maintain the undifferentiated state of hematopoietic progenitor cells.

Preclinical studies of Flonoltinib Maleate, a novel JAK2/FLT3 inhibitor, in treatment of JAK2V617F-induced myeloproliferative neoplasms

Janus kinase 2 (JAK2) hyperactivation by JAK2V617F mutation leads to myeloproliferative neoplasms (MPNs) and targeting JAK2 could serve as a promising therapeutic strategy for MPNs. Here, we report that Flonoltinib Maleate (FM), a selective JAK2/FLT3 inhibitor, shows high selectivity for JAK2 over the JAK family. Surface plasmon resonance assays verified that FM had a stronger affinity for the pseudokinase domain JH2 than JH1 of JAK2 and had an inhibitory effect on JAK2 JH2V617F. The cocrystal structure confirmed that FM could stably bind to JAK2 JH2, and FM suppressed endogenous colony formation of primary erythroid progenitor cells from patients with MPNs. In several JAK2V617F-induced MPN murine models, FM could dose-dependently reduce hepatosplenomegaly and prolong survival. Similar results were observed in JAK2V617F bone marrow transplantation mice. FM exhibited strong inhibitory effects on fibrosis of the spleen and bone marrow. Long-term FM treatment showed good pharmacokinetic/pharmacodynamic characteristics with high drug exposure in tumor-bearing tissues and low toxicity. Currently, FM has been approved by the National Medical Products Administration of China (CXHL2000628), and this study will guide clinical trials for patients with MPNs.

MyD88-dependent TLR signaling oppositely regulates hematopoietic progenitor and stem cell formation in the embryo.

Hemogenic endothelial (HE) cells in the dorsal aorta undergo an endothelial-to-hematopoietic transition (EHT) to form multipotent progenitors, lympho-myeloid biased progenitors (LMPs), pre-hematopoietic stem cells (pre-HSCs) and adult-repopulating HSCs. These briefly accumulate in intra-arterial hematopoietic clusters (IAHCs) before being released into the circulation. It is generally assumed that the number of IAHC cells correlates with the number of HSCs. Here, we show that changes in the number of IAHC cells, LMPs and HSCs can be uncoupled. Mutations impairing MyD88-dependent toll-like receptor (TLR) signaling decreased the number of IAHC cells and LMPs, but increased the number of HSCs in the aorta-gonad-mesonephros region of mouse embryos. TLR4-deficient embryos generated normal numbers of HE cells, but IAHC cell proliferation decreased. Loss of MyD88-dependent TLR signaling in innate immune myeloid cells had no effect on IAHC cell numbers. Instead, TLR4 deletion in endothelial cells (ECs) recapitulated the phenotype observed with germline deletion, demonstrating that MyD88-dependent TLR signaling in ECs and/or in IAHCs regulates the numbers of LMPs and HSCs.

Using thrombocytopenia modeling to investigate the mechanisms underlying platelet depletion induced by pan‐proteasome inhibitors

Pan‐proteasome inhibitors (pPIs) significantly improve outcomes in patients with multiple myeloma; however, their indiscriminate inhibition of multiple proteasome and immunoproteasome subunits causes diverse toxicities, including thrombocytopenia. We investigated the mechanisms underlying the platelet depletion induced by the pPIs bortezomib, carfilzomib, and ixazomib. An established thrombocytopenia model was adapted for each compound (bortezomib, ixazomib, and carfilzomib) to compare the following two pharmacodynamic mechanisms: a reversible inhibition of new progenitor cell formation (the myelosuppression model) and a reversible effect on the function of megakaryocytes to bud new platelets (platelet formation model). Bortezomib, ixazomib, and carfilzomib plasma concentration profiles and platelet counts were extracted from the literature. Pharmacokinetic (PK) and thrombocytopenia models were developed to predict the PK of these drugs and to describe their effects on proliferating cells and platelet budding. The PK models reproduced the exposure of the three compounds at steady state well compared with those reported in the literature. Both the platelet formation and myelosuppression models seemed able to describe the platelet depletion caused by bortezomib, ixazomib, and carfilzomib. Estimated structural parameters in the myelosuppression model were in the range of the values reported in the literature, whereas the mean transit time estimated with the platelet formation model was 3‐fold to 10‐fold higher than the highest reported value. The model of drug‐induced myelosuppression yielded estimates of structural parameters in the range of those previously reported. The platelet formation model captured the temporal variation reported in clinical studies.