Cellular Commitment Research Articles

The Impact of Low Protein Diet on the Molecular and Cellular Development of the Fetal Kidney.

Low nephron number has a direct impact on the development of hypertension and chronic kidney disease later in life. While intrauterine growth restriction caused by maternal low protein diet (LPD) is thought to be a significant cause of reduced nephron endowment in impoverished communities, its influence on the cellular and molecular processes which drive nephron formation are poorly understood. We conducted a comprehensive characterization of the impact of LPD on kidney development using tomographic and confocal imaging to quantify changes in branching morphogenesis and the cellular and morphological features of nephrogenic niches across development. These analyses were paired with single-cell RNA sequencing to dissect the transcriptional changes that LPD imposes during renal development to affect nephron number. Single cell analysis at E14.5 and P0 revealed differences in the expression of genes and pathways involved in metabolism, cell cycle, epigenetic regulators and reciprocal inductive signals in most cell types analyzed, yielding imbalances and shifts in cellular energy production and cellular trajectories. In the nephron progenitor cells, LPD impeded cellular commitment and differentiation towards pre-tubular and renal vesicle structures. Confocal microscopy revealed a reduction in the number of pre-tubular aggregates and proliferation in nephron progenitor cells. We also found changes in branching morphogenesis, with a reduction in cell proliferation in the ureteric tips as well as reduced tip and tip parent lengths by optical projection tomography which causes patterning defects. This unique profiling demonstrates how a fetal programming defect leads to low nephron endowment which is intricately linked to changes in both branching morphogenesis and the commitment of nephron progenitor cells. The commitment of progenitor cells is pivotal for nephron formation and is significantly influenced by nutritional factors, with a low protein diet driving alterations in this program which directly results in a reduced nephron endowment. While a mother's diet can negatively impact the number of nephrons in the kidneys of her offspring, the root cellular and molecular drivers of these deficits have not been rigorously explored. In this study we use advanced imaging and gene expression analysis in mouse models to define how a maternal low protein diet, analogous to that of impoverished communities, results in reduced nephron endowment. We find that low protein diet has pleiotropic effects on metabolism and the normal developmental programs of gene expression. These profoundly impact the process of branching morphogenesis necessary to establish niches for nephron generation and change cell behaviors which regulate how and when nephron progenitor cells commit to differentiation.

Apoptosis-dependent head development during metamorphosis of the cnidarian Hydractinia symbiolongicarpus

Apoptosis is a regulated cell death that depends on caspases. It has mainly been studied as a mechanism for the removal of unwanted cells. However, apoptotic cells can induce fate or behavior changes of their neighbors and thereby participate in development. Here, we address the functions of apoptosis during metamorphosis of the cnidarian Hydractinia symbiolongicarpus. We describe the apoptotic profile during metamorphosis of the larva and identify Caspase3/7a, but no other executioner caspases, as essential for apoptosis in this context. Using pharmacological and genetic approaches, we find that apoptosis is required for normal head development. Inhibition of apoptosis resulted in defects in head morphogenesis. Neurogenesis was compromised in the body column of apoptosis-inhibited animals but there was no effect on the survival or proliferation of stem cells, suggesting that apoptosis is required for cellular commitment rather than for the maintenance of their progenitors. Differential transcriptomic analysis identifies TRAF genes as downregulated in apoptosis-inhibited larvae and functional experiments provide evidence that they are essential for head development. Finally, we find no major role for apoptosis in head regeneration in this animal, in contrast to the significance of apoptosis in Hydra head regeneration.

ATRT-07. PRC1/BMI1 ACTIVITY ALTERS CHROMATIN ACCESSIBILITY TO REGULATE DIFFERENTIATION IN ATYPICAL TERATOID RHABDOID TUMORS

Abstract BACKGROUND SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. Atypical Teratoid Rhabdoid Tumor (ATRT) is a highly aggressive pediatric brain tumor and characterized by SMARCB1 deletion. METHODS Genome wide RNAi screen, RNA-seq and ChIP-Seq analysis, colony formation assay, cell cycle, western blotting, immunoprecipitation, mass spectrometry analysis, mouse xenograft model, IVIS and MRI imaging. RESULTS We performed an RNAi-based functional genomic screen to identify key epigenetic regulators that co-operate with SMARCB1 loss and contribute to tumorigenesis in ATRT. 406 genes associated with epigenetic regulation in patient-derived ATRT cell lines were targeted. This screen identified BMI1, a component of the Polycomb Repressive Complex 1 (PRC1), as one of the top targets essential for ATRT cell growth. Using RNA-seq and ChIP-Seq analysis we demonstrated that BMI1 co-operates with SMARCB1 deletion to suppress transcription of pro-differentiation pathways and promote self-renewal of tumor stem cells. In SMARCB1 deficient cells BMI1 forms a partial PRC1 complex devoid of DNA binding components. We found that genetic inactivation of BMI1 suppresses ATRT cell clonogenicity and neurosphere formation, leads to S-phase arrest and induces ATRT cell differentiation in vitro. In vivo BMI1 inhibition induces apoptosis, leading to significantly increasing survival of treated mice. By Doxycycline-inducible SMARCB1 re-expression system we revealed activation of proapoptotic proteins p16 and p21. With immunoprecipitation for BMI1, followed by mass spectrometry analysis we demonstrated that re-expression of SMARCB1 activates two PRC1 chromatin localizing components CBX4 and CBX8. Moreover, SMARCB1 re-expression significantly prolongs mice survival and decreases tumor size as evaluated by IVIS imaging system and T2-MRI. CONCLUSION Thus, we demonstrated that SMARCB1 deletion results in reprograming of BMI1 chromatin occupancy away from lineage specification by altering the components of the PRC1 complex and that PRC1/BMI1 inhibition is a novel therapeutic approach in ATRT.

B4galt1 Regulates the WNT-β-Catenin Axis to Control Hematopoietic Stem and Progenitor Cells (HSPCs) Fitness

In achieving the optimal numbers of fully functional blood cells across different lineages, intrinsic developmental programs within the cells and external guidance mechanisms are crucial. Glycans on the cell surface and extracellular environment are pivotal in regulating cell maintenance, differentiation, and function. We recently discovered that the glycosyltransferase β-1,4-galactosyltransferase 1 (B4galt1) leads to dysplastic megakaryocytes, impaired thrombopoiesis, and increased hematopoietic stem cells (HSC). To investigate the relationship between B4galt-dependent glycosylation and HSC function, we analyzed the glycan composition in control and B4galt1 null (B4 -/-) femurs by spatial mass spectrometry using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI). In control femurs, we observed a distinct gradient of complex N-glycans, with higher expression at the distal ends and decreasing complexity towards the shaft, showcasing diverse glycan expression in the hematopoietic environment. However, this gradient was absent in B4 -/-femurs, where we detected an increase of immature N-glycans and a concurrent decrease in complex N-glycan structures (p<0.05). Additionally, we observed aberrant O-glycosylation in B4 -/- LT-HSCs, as determined using lectin arrays. The glycan composition in the absence of B4galt1-dependent glycosylation mirrors cancer-associated glycan patterns. To further understand the role of B4galt1 in steady-state hematopoiesis, we next examined the bone marrow (BM) immunophenotypic composition of B4 -/- mice. Flow cytometry analysis revealed a significant expansion of immunophenotypically defined long term (LT)-HSCs (p=0.0061) and multipotent progenitors (MPPs) (p<0.05) in B4 -/-BM. Additionally, B4 -/- LT-HSCs showed higher expression of the platelet marker CD41 (p=0.0171), suggesting the presence of an expanded megakaryocyte (MK) biased LT-HSCs population (LT-HSC CD41+ cells). This expansion occurred without pro-inflammatory cues, as determined through cytokine arrays. To investigate if the global transcriptional landscape in B4 -/- cells could explain the observed expansion phenotype, we conducted droplet-based single-cell RNA-seq. Transfer learning classification allowed us to identify an expansion of the transcriptional output associated with LT-HSC and MPP2 signatures in B4 -/- cells (p<0.05). Further transcriptional analysis revealed an increased number of B4 -/- cells expressing MKs specific markers (CD41, Pf4, GP9, Vwf, GP1ba), providing additional support for the expansion of B4 -/- MK-biased LT-HSCs. Gene Set Enrichment Analysis (GSEA) of B4 -/- LT-HSCs demonstrated enrichment in metabolism-associated pathways and cell cycle regulation (FDR<0.05). Conversely, critical differentiation processes at the stem cell level, such as cellular adhesion and lineage commitment, were downregulated (FDR<0.05). To investigate the role of B4galt1 in HSC expansion, we transcriptionally classified LT-HSCs based on their cell cycle status (G1, G2M, and S). Analysis of LT-HSC CD41+ cell cycle distribution revealed an increased in S phase in B4 -/- LT-HSC CD41+ cells. Additionally, B4 -/- LT-HSC CD41+ cells exhibited enrichment in cell cycle regulation pathways, including DNA replication, DNA strand elongation, and G1 to S cell cycle control (FDR<0.05). Conversely, critical pathways associated with HSC homeostasis and quiescence were depleted in the absence of B4galt1 (FDR<0.05). GSEA analysis unveiled upregulation of Wnt and Myc pathways in B4 -/- LT-HSC CD41+ cells. Immunofluorescence and immunoblot analysis demonstrated elevated total β-catenin levels in B4 -/- LSK cells and enhanced nuclear translocation of non-phosphorylated Ser33/37/Thr41 β-catenin. To confirm the functional significance, we treated B4 -/- LSK cells with the Wnt inhibitor XAV939 which normalized cell numbers to control levels and cause a reduction in the observed expansion at the stem and progenitor compartments. These findings suggest that B4galt1 is vital in regulating HSC expansion, likely through the Wnt/β-catenin signaling pathway. Our data provide compelling evidence that B4galt1 regulates steady-state hematopoiesis, through its influence on glycosylation in the bone marrow niche, highlighting the critical role of the B4galt1-Wnt-β-catenin axis in regulating LT-HSC fitness and expansion.

Chromatin-modifying enzymes as modulators of nuclear size during lineage differentiation

The mechanism of nuclear size determination and alteration during normal lineage development and cancer pathologies which is not fully understood. As recently reported, chromatin modification can change nuclear morphology. Therefore, we screened a range of pharmacological chemical compounds that impact the activity of chromatin-modifying enzymes, in order to get a clue of the specific types of chromatin-modifying enzymes that remarkably effect nuclear size and shape. We found that interrupted activity of chromatin-modifying enzymes is associated with nuclear shape abnormalities. Furthermore, the activity of chromatin-modifying enzymes perturbs cell fate determination in cellular maintenance and lineage commitment. Our results indicated that chromatin-modifying enzyme regulates cell fate decision during lineage differentiation and is associate with nuclear size alteration.

Analyzes In Silico Indicate the lncRNAs MIR31HG and LINC00939 as Possible Epigenetic Inhibitors of the Osteogenic Differentiation in PDLCs.

Chromatin conformation, DNA methylation pattern, transcriptional profile, and non-coding RNAs (ncRNAs) interactions constitute an epigenetic pattern that influences the cellular phenotypic commitment and impacts the clinical outcomes in regenerative therapies. Here, we investigated the epigenetic landscape of the SP7 transcriptor factor (SP7) and Distal-Less Homeobox 4 (DLX4) osteoblastic transcription factors (TFs), in human periodontal ligament mesenchymal cells (PDLCs) with low (l-PDLCs) and high (h-PDLCs) osteogenic potential. Chromatin accessibility (ATAC-seq), genome DNA methylation (Methylome), and RNA sequencing (RNA-seq) assays were performed in l- and h-PDLCs, cultured at 10 days in non-induced (DMEM) and osteogenic (OM) medium in vitro. Data were processed in HOMER, Genome Studio, and edgeR programs, and metadata was analyzed by online bioinformatics tools and in R and Python environments. ATAC-seq analyses showed the TFs genomic regions are more accessible in l-PDLCs than in h-PDLCs. In Methylome analyses, the TFs presented similar average methylation intensities (AMIs), without differently methylated probes (DMPs) between l- and h-PDLCs; in addition, there were no differences in the expression profiles of TFs signaling pathways. Interestingly, we identified the long non-coding RNAs (lncRNAs), MIR31HG and LINC00939, as upregulated in l-PDLCs, in both DMEM and OM. In the following analysis, the web-based prediction tool LncRRIsearch predicted RNA:RNA base-pairing interactions between SP7, DLX4, MIR31HG, and LINC00939 transcripts. The machine learning program TriplexFPP predicted DNA:RNA triplex-forming potential for the SP7 DNA site and for one of the LINC00939 transcripts (ENST00000502479). PCR data confirmed the upregulation of MIR31HG and LINC00939 transcripts in l-PDLCs (× h-PDLCs) in both DMEM and OM (p < 0.05); conversely, SP7 and DLX4 were downregulated, confirming those results observed in the RNA-Seq analysis. Together, these results indicate the lncRNAs MIR31HG and LINC00939 as possible epigenetic inhibitors of the osteogenic differentiation in PDLCs by (post)transcriptional and translational repression of the SP7 and DLX4 TFs.

Chitosan/Hyaluronan and Alginate-Nanohydroxyapatite Biphasic Scaffold as a Promising Matrix for Osteoarthritis Disorders.

Regenerative medicine offers new techniques for osteoarthritis (OA) disorders, especially while considering simultaneous chondral and subchondral regenerations. Chitosan and hyaluronan were chemically bound as the chondral phase and the osteogenic layer was prepared with alginate and nano-hydroxyapatite (nHAP). These scaffolds were fixed by fibrin glue as a biphasic scaffold and then examined. Scanning electron microscopy (SEM) confirmed the porosity of 61.45±4.51 and 44.145±2.81 % for the subchondral and chondral layers, respectively. The composition analysis by energy dispersive X-ray (EDAX) indicated the various elements of both hydrogels. Also, their mechanical properties indicated that the highest modulus and resistance values corresponded to the biphasic hydrogel as 108.33±5.56 and 721.135±8.21 kPa, despite the same strain value as other groups. Their individual examinations demonstrated the proteoglycan synthesis of the chondral layer and also, the alkaline phosphatase (ALP) activity of the subchondral layer as 13.3±2.2 ng. After 21 days, the cells showed a mineralized surface and a polygonal phenotype, confirming their commitment to bone and cartilage tissues, respectively. Immunostaining of collagen I and II represented greater extracellular matrix (ECM) secretion in the biphasic composite group due to the paracrine effect of the two cell types on each other. For the first time, the ability of this biphasic scaffold to regenerate both tissue types was evaluated and the results showed satisfactory cellular commitment to bone and cartilage tissues. Thus, this scaffold can be considered a new strategy for the preparation of implants for OA.

Structural and biological investigation of alginate-nano-hydroxyapatite with chitosan-hyaluronic acid for potential osteochondral regeneration

Cartilage joint lesions have created a significant challenge in the field of durable reconstruction and recovery due to their limited long-term repair and reconstruction. In this study, a biphasic scaffold was obtained using chitosan (CS)-hyaluronic acid (HA) and alginate-nanohydroxyapatite (nHAP) for osteoarthritis diseases. The scaffolds revealed porous structures and hydrophilic chemical groups by scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) methods, respectively. The resulting three-dimensional hydrogels were assembled by using fibrin glue and seeded with human chondrocytes cells (hCHCs) in the chondral layer and human adipose-derived mesenchymal stem cells (hAdMSCs) in the subchondral one. Then, the assessments including 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), SEM, compressive assay, weight loss, alizarin red staining, alkaline phosphatase (ALP) activity, alcian blue staining and Real-Time PCR were performed. The associated cell viability confirmed that there were no interventions between the cell types and SEM illustrated cell attachment and spreading. The subchondral and chondral layers were investigated separately to detect their ability for cellular commitment. Finally, the Real-time PCR was carried out on the bilayer scaffold and the related observations approved the osteogenic/chondrogenic differentiation of both cell types. Taken together, the Alg-nHAP/CS-HA scaffold provided an appropriate environment for cartilage and bone regenerations and could be recommended for all aspects of osteochondral tissue engineering.

Nicotinamide N-methyltransferase (NNMT) regulates the glucocorticoid signaling pathway during the early phase of adipogenesis

Obesity is associated with adipose tissue dysfunction through the differentiation and expansion of pre-adipocytes to adipocytes (hyperplasia) and/or increases in size of pre-existing adipocytes (hypertrophy). A cascade of transcriptional events coordinates the differentiation of pre-adipocytes into fully differentiated adipocytes; the process of adipogenesis. Although nicotinamide N-methyltransferase (NNMT) has been associated with obesity, how NNMT is regulated during adipogenesis, and the underlying regulatory mechanisms, remain undefined. In present study we used genetic and pharmacological approaches to elucidate the molecular signals driving NNMT activation and its role during adipogenesis. Firstly, we demonstrated that during the early phase of adipocyte differentiation NNMT is transactivated by CCAAT/Enhancer Binding Protein beta (CEBPB) in response to glucocorticoid (GC) induction. We found that Nnmt knockout, using CRISPR/Cas9 approach, impaired terminal adipogenesis by influencing the timing of cellular commitment and cell cycle exit during mitotic clonal expansion, as demonstrated by cell cycle analysis and RNA sequencing experiments. Biochemical and computational methods showed that a novel small molecule, called CC-410, stably binds to and highly specifically inhibits NNMT. CC-410 was, therefore, used to modulate protein activity during pre-adipocyte differentiation stages, demonstrating that, in line with the genetic approach, chemical inhibition of NNMT at the early stages of adipogenesis impairs terminal differentiation by deregulating the GC network. These congruent results conclusively demonstrate that NNMT is a key component of the GC-CEBP axis during the early stages of adipogenesis and could be a potential therapeutic target for both early-onset obesity and glucocorticoid-induced obesity.

Recovering hidden information from live-cell trajectories to predict the biological pathway activity underlying cell state change decisions

Extracellular signals induce changes in cellular phenotype, activating diverse cellular programs and decisions at the single-cell level-- proliferation or cell-cycle arrest, multicellular clustering or contact avoidance, and enhanced collective or individual motility. High-throughput, extended duration, and single-cell resolved trajectories can be obtained via live-cell imaging assays. Trajectory analysis indicates that the single-cell responses to perturbation from extracellular signals can be heterogenous dynamically in the transition pathways and transition times between cell states, as well as in the extent of overall response at the final timepoint. It is unknown the extent to which the dynamics of cell state transitions are intrinsically stochastic and random, or deterministic and thus predictable. We present a live-cell morphodynamical trajectory-based analysis which extracts “hidden” information present in extended single-cell trajectory windows, and indicates much of the observed stochasticity in cell state transition dynamics can be attributed to incomplete single-timepoint cell characterization, and not intrinsically random cell state change dynamics. Our analysis enables the extraction of information rich single-cell trajectories of cell state changes, enabling data-driven modeling and translation from live-cell imaging morphodynamical cell states to gene transcript levels. We map the biological pathway activity along transition intermediates as the cellular commitment to extracellular signal-induced cell state change occurs, investigating specific pathway activity gating the development of multicellular clusters, and the switch from individualized to collective motility.

Nutrient metabolism of the nucleus pulposus: A literature review

Cells take in, consume, and synthesize nutrients for numerous physiological functions. This includes not only energy production but also macromolecule biosynthesis, which will further influence cellular signaling, redox homeostasis, and cell fate commitment. Therefore, alteration in cellular nutrient metabolism is associated with pathological conditions.Intervertebral discs, particularly the nucleus pulposus (NP), are avascular and exhibit unique metabolic preferences. Clinical and preclinical studies have indicated a correlation between intervertebral degeneration (IDD) and systemic metabolic diseases such as diabetes, obesity, and dyslipidemia. However, a lack of understanding of the nutrient metabolism of NP cells is masking the underlying mechanism. Indeed, although previous studies indicated that glucose metabolism is essential for NP cells, the downstream metabolic pathways remain unknown, and the potential role of other nutrients, like amino acids and lipids, is understudied.In this literature review, we summarize the current understanding of nutrient metabolism in NP cells and discuss other potential metabolic pathways by referring to a human NP transcriptomic dataset deposited to the Gene Expression Omnibus, which can provide us hints for future studies of nutrient metabolism in NP cells and novel therapies for IDD.

Stimulatory Effect of Tofacitinib on Bone Marrow Adipocytes Differentiation.

BackgroundSystemic inflammation is the main factor underlying secondary osteoporosis in patients with rheumatoid arthritis (RA). Janus kinase inhibitors (JAKi), such as tofacitinib (Tofa), can control systemic inflammation and may have beneficial effects on bone in various models. This might be due to direct effects on the bone microenvironment and not exclusively based on their anti-inflammatory function. Bone marrow adipocytes (BMAds) are abundant in the bone microenvironment. The effect of JAKi on BMAds is unknown, but evidence suggests that there is competition between human bone marrow-derived stromal cell (hBMSC) differentiation routes towards BMAds and osteoblasts (Ob) in osteoporosis.ObjectivesThe aims of the study are to determine whether Tofa influences BMAds and Ob derived from hBMSCs and to investigate the potential effects of Tofa on bone marrow adiposity in RA patients.MethodsTo determine the effect of Tofa on cellular commitment, hBMSCs were differentiated to BMAds or OBs for 3 days together with Tofa at 200, 400, or 800 nM and TNFα. This study was also conducted using differentiated BMAds. The impact of Tofa was determined by gene and protein expression analysis and cell density monitoring. In parallel, in a pilot study of 9 RA patients treated with Tofa 5 mg twice a day (NCT04175886), the proton density fat fraction (PDFF) was measured using MRI at the lumbar spine at baseline and at 6 months.ResultsIn non-inflammatory conditions, the gene expression of Runx2 and Dlx5 decreased in Ob treated with Tofa (p <0.05). The gene expression of PPARγ2, C/EBPα, and Perilipin 1 were increased compared to controls (p <0.05) in BMAds treated with Tofa. Under inflammatory conditions, Tofa did not change the expression profiles of Ob compared to TNFα controls. In contrast, Tofa limited the negative effect of TNFα on BMAd differentiation (p <0.05). An increase in the density of differentiated BMAds treated with Tofa under TNFα was noted (p <0.001). These findings were consolidated by an increase in PDFF at 6 months of treatment with Tofa in RA patients (46.3 ± 7.0% versus 53.2 ± 9.2% p <0.01).ConclusionTogether, these results suggest a stimulatory effect of Tofa on BMAd commitment and differentiation, which does not support a positive effect of Tofa on bone.

P1399: EXTRAMEDULLARY HEMATOPOIESIS IN THE ADULT ADRENAL GLAND AS A MODEL OF THE MINIMAL HEMATOPOIETIC NICHE

Background: Hematopoietic Stem and Progenitor Cells (HSPCs) reside in the hematopoietic niche, a structure that regulates the balance of cellular quiescence, self-renewal and commitment towards progenitor cells in a demand-adapted manner. The bone marrow (BM) hematopoietic niche is formed by several cellular players, mainly endothelial cells, osteoblasts, adipocytes, and a variety of stromal cells. While the BM niche contains a complex stromal population, evidence exists for simpler, albeit functional, extramedullary hematopoietic (EMH) niches. EMH can be found associated to adipose tissue. An example of adipose-associated EMH is myelolipoma, an adrenal tumor composed of myeloid and adipose tissue. Myelolipoma is frequent in patients with congenital adrenal hyperplasia, an endocrine disorder that associates an increase in ACTH. Aims: We aimed to produce an EMH animal model that would recapitulate myelolipoma to study a minimalistic, boneless hematopoietic niche. Methods: We designed a strategy to induce EMH in the adrenal gland. For this, we injected hematopoietic cytokines (Granulocyte colony-stimulating factor (G-CSF)) and pituitary axis hormones (ACTH) as well as androgens (testosterone) in splenectomized C57BL/6J mice and studied the hematopoietic-supportive capacity of the induced adrenal glands. Results: Here, we show that the adult adrenal gland can be transformed into a hematopoietic supportive environment. Upon hormonal stimulation, the adult adrenal gland can be induced to recruit and host hematopoietic cells, including serially transplantable HSCPs. The adrenal stroma supports HSPC proliferation and function; furthermore, the adrenal stroma contains a CXCL12+ population, reminiscent of BM CXCL12-Abundant Reticular (CAR). These findings are mirrored by the presence of CXCL12+ cells in human myelolipoma samples, a benign adrenal tumor composed of adipose and hematopoietic tissue. Summary/Conclusion: We present our model as a novel tool to increase our understanding of the physiology of hematopoietic support and to facilitate the development of a boneless niche model.

Abstract 5268: The spectrum of FGFR mutations in pediatric and young adult solid tumor

Abstract Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases expressed on the cell membrane that play crucial roles in cellular lineage commitment, differentiation, proliferation, and apoptosis. Deregulated FGFR signaling is observed in a subset of tumors across various histologies, making FGFRs ideal therapeutic targets. We sought to determine the genetic landscape of FGFR-family variations in a cohort of pediatric and young adult patients with solid tumors. The CHOP Comprehensive Solid Tumor Panel was performed on 1,420 patients. The panel covers 238 cancer genes and screens for single nucleotide variants (SNVs), indels, copy number alterations, and 117 fusion gene partners interrogating over 700 exons for known and novel fusions. Identified variants were categorized and reported according to the AMP/ASCO/CAP guidelines. Fifty-six patients (4.1%), including 47 children and 9 young adults, were found to carry at least one FGFR alteration in their tumors. CNS tumors accounted for most of the cases (51 total, 87.9%), with pilomyxoid astrocytoma/pilocytic astrocytoma and dysembryoplastic neuroepithelial tumor the most common (13 and 12 patients, respectively). Non-CNS solid tumors included rhabdomyosarcoma (4 patients), neuroblastoma/ganglioneuroblastoma (2), and follicular thyroid carcinoma (1). FGFR somatic alterations were found in 56 tumors including 41 SNVs and small indels, 6 internal tandem duplications (ITDs), and 15 fusions genes. The most common SNVs observed were hotspot mutations p.K656E and p.N546K of FGFR1. Sequence alterations in FGFR1 contained 35 SNVs and small indels, mostly gain of function mutations located in the kinase domain, and 6 kinase domain ITDs. One SNV was identified in FGFR2 in the immunoglobulin domain. Two SNVs were reported in FGFR3, both of which were in the fibroblast growth factor receptor family domain, and 3 SNVs were identified in FGFR4, all occurring at the p.V550 codon located on the kinase domain. Companion mutations in non-FGFR genes were detected in 27 tumors, predominantly involving RAS signaling pathway genes including NF1 (14 variants), PIK3CA (8), PTPN11 (6) and PIK3R1 (4). Among fusion variants, FGFR1-TACC1 fusions were found in 5 patients, mostly in pediatric patients. One FGFR3-TACC3 fusion was identified in one young adult patient. Seven pediatric patients tested positive for FGFR2 fusions; all with different 3’ partners. The detection of an FGFR alteration defined or changed the histologic diagnosis for 22 patients. Our results reveal that FGFR alterations account for 4.1% (56/1420) of the patients with solid tumors tested in our laboratory. The majority of the FGFR-positive tumors are low-grade CNS tumors. Further, the identification of FGFR alterations can significantly improve the tumor diagnosis and provide genomic evidence for potential targeted treatment with FGFR inhibitors. Citation Format: Jinhua Wu, Jeffrey Schubert, Feng Xu, Ariel Long, Maha Patel, Netta Golenberg, Weixuan Fu, Kajia Cao, Jiani Chen, Elizabeth H. Denenberg, Elizabeth A. Fanning, Rochelle Bagatell, Theodore W. Laetsch, Adam Resnick, Mariarita Santi, Phillip Jay B. Storm, Minjie Luo, Lea F. Surrey, Yiming Zhong, Marilyn M. Li. The spectrum of FGFR mutations in pediatric and young adult solid tumor [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 5268.

ATRT-23. SIRT2 cooperates with SMARCB1 to induce a differentiation block in ATRT

Atypical Teratoid Rhabdoid Tumor is a highly aggressive pediatric brain tumor with poor prognosis driven by loss of the chromatin remodeling factor SMARCB1 that is responsible for determining cellular pluripotency and lineage commitment. The mechanisms by which SMARCB1 deletion results in tumorigenesis remain unclear. We investigated the effect of SIRT2 inhibition in ATRT which was identified as a primary dependency in ATRT. SIRT2 inhibition with shRNA or Thiomyristoyl (TM) decreased ATRT cell growth, inhibited clonogenic potential and leaded to the cell cycle arrest. SIRT2 inhibition effectively suppresses pluripotency-associated genomic programs, significantly changed stem cell frequency, decreased tumor-sphere formation of ATRT cells and attenuated tumor cell self-renewal. In vivo SIRT2 inhibition decreased oncogenic markers and increased accumulation neuronal differentiation markers. Furthermore, SIRT2 induced apoptosis, decreased tumor growth and prolonged survival in orthotopic xenograft models. Single-cell RNA transcriptome analysis of xenoftaft tumors reveals elimination of tumor cells expressing stem cell genes and expansion of tumor cells expressing differentiated genes following TM treatment in ATRT. We demonstrated that SIRT2 inhibition is a molecular vulnerability in SMARCB1-deleted ATRT.

3073 – HUMAN HAEMATOPOIETIC STEM AND PROGENITOR CELLS FORM KINSHIPS THAT ARE SIMILAR IN FATE AND SYNCHRONOUS IN DIVISION

While the heterogeneity of human haematopoietic stem and progenitor cells has become apparent, linking this heterogeneity to functional properties at the single-cell level remains a challenge. Here, we have developed a combination of flow cytometry, live-cell imaging, and mathematical modelling to track the cell division properties and cell commitment in human haematopoietic stem and progenitor cells. We identified two key properties: synchronicity in division and similarity in fate among progenies of individual stem and progenitor cells. Each human stem and progenitor cell give rise to a cellular kinship that share a strong resemblance in fate and divide synchronously, suggesting an intrinsic cellular memory transmitted from mother to daughter cells. Interestingly, we observe that the time to divide shared by two sibling cells is not correlated with the division time of its mother cell. These behaviours are observed and shared across the entire lifespan, from cord blood to aged bone marrow cells. Moreover, we collected evidence that these behaviours are altered in haematological malignancies such as acute myeloid leukemia. Using a multi-agent mathematical model, we were able to recapitulate the cellular early commitment, where two sister cells descending from the same ancestor share a strong correlation in fate. In conclusion, we propose a model that describe the human haematopoiesis as a heterogeneous system formed by homogeneous cell families. While the heterogeneity of human haematopoietic stem and progenitor cells has become apparent, linking this heterogeneity to functional properties at the single-cell level remains a challenge. Here, we have developed a combination of flow cytometry, live-cell imaging, and mathematical modelling to track the cell division properties and cell commitment in human haematopoietic stem and progenitor cells. We identified two key properties: synchronicity in division and similarity in fate among progenies of individual stem and progenitor cells. Each human stem and progenitor cell give rise to a cellular kinship that share a strong resemblance in fate and divide synchronously, suggesting an intrinsic cellular memory transmitted from mother to daughter cells. Interestingly, we observe that the time to divide shared by two sibling cells is not correlated with the division time of its mother cell. These behaviours are observed and shared across the entire lifespan, from cord blood to aged bone marrow cells. Moreover, we collected evidence that these behaviours are altered in haematological malignancies such as acute myeloid leukemia. Using a multi-agent mathematical model, we were able to recapitulate the cellular early commitment, where two sister cells descending from the same ancestor share a strong correlation in fate. In conclusion, we propose a model that describe the human haematopoiesis as a heterogeneous system formed by homogeneous cell families.

Protein abundance of the cytokine receptor γc controls the thymic generation of innate-like T cells.

Innate-like T (iT) cells comprise a population of immunoregulatory T cells whose effector function is imposed during their development in the thymus to provide protective immunity prior to antigen encounter. The molecular mechanism that drives the generation of iT cells remains unclear. Here, we report that the cytokine receptor γc plays a previously unappreciated role for thymic iT cells by controlling their cellular abundance, lineage commitment, and subset differentiation. As such, γc overexpression on thymocytes dramatically altered iT cell generation in the thymus, as it skewed the subset composition of invariant NKT (iNKT) cells and promoted the generation of IFNγ-producing innate CD8 T cells. Mechanistically, we found that the γc-STAT6 axis drives the differentiation of IL-4-producing iNKT cells, which in turn induced the generation of innate CD8 T cells. Collectively, these results reveal a cytokine-driven circuity of thymic iT cell differentiation that is controlled by the abundance of γc proteins.

Expression and Role of Ubiquitin-Specific Peptidases in Osteoblasts.

The ubiquitin-proteasome system regulates biological processes in normal and diseased states. Recent investigations have focused on ubiquitin-dependent modifications and their impacts on cellular function, commitment, and differentiation. Ubiquitination is reversed by deubiquitinases, including ubiquitin-specific peptidases (USPs), whose roles have been widely investigated. In this review, we explore recent findings highlighting the regulatory functions of USPs in osteoblasts and providing insight into the molecular mechanisms governing their actions during bone formation. We also give a brief overview of our work on USP53, a target of PTH in osteoblasts and a regulator of mesenchymal cell lineage fate decisions. Emerging evidence addresses questions pertaining to the complex layers of regulation exerted by USPs on osteoblast signaling. We provide a short overview of our and others’ understanding of how USPs modulate osteoblastogenesis. However, further studies using knockout mouse models are needed to fully understand the mechanisms underpinning USPs actions.

The Mechanical Interplay Between Differentiating Mesenchymal Stem Cells and Gelatin-Based Substrates Measured by Atomic Force Microscopy.

Traditional methods to assess hMSCs differentiation typically require long-term culture until cells show marked expression of histological markers such as lipid accumulation inside the cytoplasm or mineral deposition onto the surrounding matrix. In parallel, stem cell differentiation has been shown to involve the reorganization of the cell’s cytoskeleton shortly after differentiation induced by soluble factors. Given the cytoskeleton’s role in determining the mechanical properties of adherent cells, the mechanical characterization of stem cells could thus be a potential tool to assess cellular commitment at much earlier time points. In this study, we measured the mechanical properties of hMSCs cultured on soft gelatin-based hydrogels at multiple time points after differentiation induction toward adipogenic or osteogenic lineages. Our results show that the mechanical properties of cells (stiffness and viscosity) and the organization of the actin cytoskeleton are highly correlated with lineage commitment. Most importantly, we also found that the mechanical properties and the topography of the gelatin substrate in the vicinity of the cells are also altered as differentiation progresses toward the osteogenic lineage, but not on the adipogenic case. Together, these results confirm the biophysical changes associated with stem cell differentiation and suggest a mechanical interplay between the differentiating stem cells and their surrounding extracellular matrix.

Hydrostatic Pressure Modulates Intervertebral Disc Cell Survival and Extracellular Matrix Homeostasis via Regulating Hippo-YAP/TAZ Pathway.

Established studies proved that hydrostatic pressure had multiple effects on the biological behavior of the intervertebral disc (IVD). However, the conclusions of the previous studies were inconsistent, due to the difference in hydrostatic loading devices and observing methods used in these studies. The current study is aimed at investigating the role of dynamic hydrostatic pressure in regulating biological behavior of the notochordal nucleus pulposus (NP) and fibrocartilaginous inner annulus fibrosus (AF) and its possible mechanism using our novel self-developed hydrostatic pressure bioreactor. The differences in the biological behavior of the rabbit IVD tissues under different degree of hydrostatic pressure were evaluated via histological analysis. Results revealed that low-loading dynamic hydrostatic pressure was beneficial for cell survival and extracellular matrix (ECM) homeostasis in notochordal NP and fibrocartilaginous inner AF via upregulating N-cadherin (N-CDH) and integrin β1. In comparison, high-magnitude dynamic hydrostatic pressure aggravated the breakdown of ECM homeostasis in NP and inner AF via enhancing the Hippo-YAP/TAZ pathway-mediated cell apoptosis. Moreover, inner AF exhibited greater tolerance to physiological medium-loading degree of hydrostatic pressure than notochordal NP. The potential mechanism was related to the differential expression of mechanosensing factors in notochordal NP and fibrocartilaginous inner AF, which affects the fate of the cells under hydrostatic pressure. Our findings may provide a better understanding of the regulatory role of hydrostatic pressure on the cellular fate commitment and matrix metabolism of the IVD and more substantial evidence for using hydrostatic pressure bioreactor in exploring the IVD degeneration mechanism as well as regeneration strategies.