Biogenesis Factors Research Articles

Arabidopsis AN3 and OLIGOCELLULA genes link telomere maintenance mechanisms with cell division and expansion control.

Telomeres are conserved chromosomal structures necessary for continued cell division and proliferation. In addition to the classical telomerase pathway, multiple other genes including those involved in ribosome metabolism and chromatin modification contribute to telomere length maintenance. We previously reported that Arabidopsis thaliana ribosome biogenesis genes OLI2/NOP2A, OLI5/RPL5A and OLI7/RPL5B have critical roles in telomere length regulation. These three OLIGOCELLULA genes were also shown to function in cell proliferation and expansion control and to genetically interact with the transcriptional co-activator ANGUSTIFOLIA3 (AN3). Here we show that AN3-deficient plants progressively lose telomeric DNA in early homozygous mutant generations, but ultimately establish a new shorter telomere length setpoint by the fifth mutant generation with a telomere length similar to oli2/nop2a -deficient plants. Analysis of double an3 oli2 mutants indicates that the two genes are epistatic for telomere length control. Telomere shortening in an3 and oli mutants is not caused by telomerase inhibition; wild type levels of telomerase activity are detected in all analyzed mutants in vitro. Late generations of an3 and oli mutants are prone to stem cell damage in the root apical meristem, implying that genes regulating telomere length may have conserved functional roles in stem cell maintenance mechanisms. Multiple instances of anaphase fusions in late generations of oli5 and oli7 mutants were observed, highlighting an unexpected effect of ribosome biogenesis factors on chromosome integrity. Overall, our data implicate AN3 transcription coactivator and OLIGOCELLULA proteins in the establishment of telomere length set point in plants and further suggest that multiple regulators with pleiotropic functions can connect telomere biology with cell proliferation and cell expansion pathways.

Peroxisome biogenesis factor PEX14 is crucial for survival and fecundity of female brown planthopper, Nilaparvata lugens (Stål)

Peroxisomes are ubiquitous cellular organelles participating in a variety of critical metabolic reactions. PEX14 is an essential peroxin responsible for peroxisome biogenesis. In this study, we identified the human PEX14 homolog in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). N. lugens PEX14 (NlPEX14) showed significant topological similarity to its human counterpart. It is expressed throughout all developmental stages, with the highest expression observed in adult insects. Down-regulation of NlPEX14 through injection of NlPEX14-specific double-strand RNA impaired nymphal development. Moreover, females subjected to dsNlPEX14 treatment exhibited a significantly reduced lifespan. Additionally, we found abnormal ovarian development and a significant decrease in the number of eggs laid in NlPEX14-downregulated females. Further experiments support that the shortening of lifespan and the decrease in female fecundity can be attributed, at least partially, to the accumulation of fatty acids and reduced expression of vitellogenin. Together, our study reveals an indispensable function of NlPEX14 for insect reproduction and establishes a causal connection between the phenotypes and peroxisome biogenesis, shedding light on the importance of peroxisomes in female fecundity.

Endocytosis blocks the vesicular secretion of exosome marker proteins.

Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.

The uS10c-BPG2 module mediates ribosomal RNA processing in chloroplast nucleoids.

In plant chloroplasts, certain ribosomal proteins (RPs) and ribosome biogenesis factors (RBFs) are present in nucleoids, implying an association between nucleoids and ribosome biogenesis. In Arabidopsis, the YqeH-type GTPase Brassinazole-Insensitive Pale Green2 (BPG2) is a chloroplast nucleoid-associated RBF. Here, we investigated the relationship between nucleoids and BPG2-involved ribosome biogenesis steps by exploring how BPG2 targets ribosomes. Our findings demonstrate that BPG2 interacts with an essential plastid RP, uS10c, in chloroplast nucleoids in a ribosomal RNA (rRNA)-independent manner. We also discovered that uS10c is a haploinsufficient gene, as theheterozygous deletion of this gene leads to variegated shoots and chlorophyll aggregation. uS10c is integrated into 30S ribosomal particles when rRNA is relatively exposed and also exists in polysome fractions. In contrast, BPG2 exclusively associates with 30S ribosomal particles. Notably, the interaction between BPG2 and 30S particles is influenced by the absence of uS10c, resulting in BPG2 diffusing in chloroplasts instead of targeting nucleoids. Further, our results reveal that the loss of BPG2 function and the heterozygous deletion of uS10c impair the processing of 16S and 23S-4.5S rRNAs, reduce plastid protein accumulation, and trigger the plastid signaling response. Together, these findings indicate that the uS10c-BPG2 module mediates ribosome biogenesis in chloroplast nucleoids.

The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis

DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.

RIO-kinase 2 is essential for hematopoiesis.

Regulation of protein synthesis is a key factor in hematopoietic stem cell maintenance and differentiation. Rio-kinase 2 (RIOK2) is a ribosome biogenesis factor that has recently been described an important regulator of human blood cell development. Additionally, we have previously identified RIOK2 as a regulator of protein synthesis and a potential target for the treatment of acute myeloid leukemia (AML). However, its functional relevance in several organ systems, including normal hematopoiesis, is not well understood. Here, we investigate the consequences of RIOK2 loss on normal hematopoiesis using two different conditional knockout mouse models. Using competitive and non-competitive bone marrow transplantations, we demonstrate that RIOK2 is essential for the differentiation of hematopoietic stem and progenitor cells (HSPCs) as well as for the maintenance of fully differentiated blood cells in vivo as well as in vitro. Loss of RIOK2 leads to rapid death in full-body knockout mice as well as mice with RIOK2 loss specific to the hematopoietic system. Taken together, our results indicate that regulation of protein synthesis and ribosome biogenesis by RIOK2 is essential for the function of the hematopoietic system.

The transcription of the main gene associated with Treacher–Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP)

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

Abstract 3160: PufA promotes proliferation, migration and tumorigenesis through NF-kB pathway in cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) exhibits a poor prognosis despite the recent advances in targeted therapy and immunotherapy. Therefore, exploring the novel mechanism of CCA tumorigenesis and identifying the effective targeted therapy is critical for advanced CCA to improve future treatment. A novel biogenesis factor of the 90S pre-ribosome named PufA, is higher expressed in tumors than normal tissue based on the TCGA databank implying the role of CCA tumorigenesis. Our results showed that PufA expression was significantly associated with larger tumor size, more lymph node metastasis, more tumor relapse, death, and unfavorable survivals in 205 patients with resectable CCA in Chang Gung Memorial Hospital. We demonstrated the inhibited effect of cell survival, migration ability, and the sphere-formation potential by silencing of PufA in CCA cell lines. Tumor growth is also suppressed in PufA knockdown xenograft mice. Mechanistic investigation showed that PufA was expression associated with nuclear factor kappa B p65 (RELA) activity and facilitated activity of the NF-κB transactivation. RELA, a transcription factor, is the principal regulator of the inflammatory response and is crucial for the homeostasis of the immune system and the expression of the immune checkpoint. Furthermore, decreased PufA levels also downregulated PD-L1 expression in CCA. Together, these findings suggested that the PufA-RELA axis could be a potential target for further application of targeted therapy in combination with immunotherapy. Citation Format: Chiao-Ping Chen, Chun-Nan Yeh, Yi-Ru Pan, Yu-Tien Hsiao, Chih-Hong Lo, Chia-Hsien Lin, Chiao-En Wu. PufA promotes proliferation, migration and tumorigenesis through NF-kB pathway in cholangiocarcinoma [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 3160.

Uncovering the photosystem I assembly pathway in land plants.

Photosystem I (PSI) is one of two large pigment-protein complexes responsible for converting solar energy into chemical energy in all oxygenic photosynthetic organisms. The PSI supercomplex consists of the PSI core complex and peripheral light-harvesting complex I (LHCI) in eukaryotic photosynthetic organisms. However, how the PSI complex assembles in land plants is unknown. Here we describe PHOTOSYSTEM I BIOGENESIS FACTOR 8 (PBF8), a thylakoid-anchored protein in Arabidopsis thaliana that is required for PSI assembly. PBF8 regulates two key consecutive steps in this process, the building of two assembly intermediates comprising eight or nine subunits, by interacting with PSI core subunits. We identified putative PBF8 orthologues in charophytic algae and land plants but not in Cyanobacteria or Chlorophyta. Our data reveal the major PSI assembly pathway in land plants. Our findings suggest that novel assembly mechanisms evolved during plant terrestrialization to regulate PSI assembly, perhaps as a means to cope with terrestrial environments.

FacZ is a GpsB-interacting protein that prevents aberrant division-site placement in Staphylococcus aureus

Staphylococcus aureus is a Gram-positive pathogen responsible for antibiotic-resistant infections. To identify vulnerabilities in cell envelope biogenesis that may overcome resistance, we enriched for S. aureus transposon mutants with defects in cell surface integrity or cell division by sorting for cells that stain with propidium iodide or have increased light-scattering properties, respectively. Transposon sequencing of the sorted populations identified more than 20 previously uncharacterized factors impacting these processes. Cells inactivated for one of these proteins, factor preventing extra Z-rings (FacZ, SAOUHSC_01855), showed aberrant membrane invaginations and multiple FtsZ cytokinetic rings. These phenotypes were suppressed in mutants lacking the conserved cell-division protein GpsB, which forms an interaction hub bridging envelope biogenesis factors with the cytokinetic ring in S. aureus. FacZ was found to interact directly with GpsB in vitro and in vivo. We therefore propose that FacZ is an envelope biogenesis factor that antagonizes GpsB function to prevent aberrant division events in S. aureus.

Apical papilla stem cell-derived exosomes regulate lipid metabolism and alleviate inflammation in the MCD-induced mouse NASH model

Stem cells from the apical papilla(SCAPs) exhibit remarkable tissue repair capabilities, demonstrate anti-inflammatory and pro-angiogenic effects, positioning them as promising assets in the realm of regenerative medicine. Recently, the focus has shifted towards exosomes derived from stem cells, perceived as safer alternatives while retaining comparable physiological functions. This study delves into the therapeutic implications of exosomes derived from SCAPs in the methionine-choline-deficient (MCD) diet-induced mice non-alcoholic steatohepatitis (NASH) model. We extracted exosomes from SCAPs. During the last two weeks of the MCD diet, mice were intravenously administered SCAPs-derived exosomes at two distinct concentrations (50 μg/mouse and 100 μg/mouse) biweekly. Thorough examinations of physiological and biochemical indicators were performed to meticulously evaluate the impact of exosomes derived from SCAPs on the advancement of NASH in mice induced by MCD diet. This findings revealed significant reductions in body weight loss and liver damage induced by the MCD diet following exosomes treatment. Moreover, hepatic fat accumulation was notably alleviated. Mechanistically, the treatment with exosomes led to an upregulation of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) levels in the liver, enhancing hepatic fatty acid oxidation and transporter gene expression while inhibiting genes associated with fatty acid synthesis. Additionally, exosomes treatment increased the transcription levels of key liver mitochondrial marker proteins and the essential mitochondrial biogenesis factor. Furthermore, the levels of serum inflammatory factors and hepatic tissue inflammatory factor mRNA expression were significantly reduced, likely due to the anti-inflammatory phenotype induced by exosomes in macrophages. The above conclusion suggests that SCAPs-exosomes can improve NASH.

VAV2 orchestrates the interplay between regenerative proliferation and ribogenesis in both keratinocytes and oral squamous cell carcinoma

VAV2 is an activator of RHO GTPases that promotes and maintains regenerative proliferation-like states in normal keratinocytes and oral squamous cell carcinoma (OSCC) cells. Here, we demonstrate that VAV2 also regulates ribosome biogenesis in those cells, a program associated with poor prognosis of human papilloma virus-negative (HPV−) OSCC patients. Mechanistically, VAV2 regulates this process in a catalysis-dependent manner using a conserved pathway comprising the RAC1 and RHOA GTPases, the PAK and ROCK family kinases, and the c-MYC and YAP/TAZ transcription factors. This pathway directly promotes RNA polymerase I activity and synthesis of 47S pre-rRNA precursors. This process is further consolidated by the upregulation of ribosome biogenesis factors and the acquisition of the YAP/TAZ-dependent undifferentiated cell state. Finally, we show that RNA polymerase I is a therapeutic Achilles’ heel for both keratinocytes and OSCC patient-derived cells endowed with high VAV2 catalytic activity. Collectively, these findings highlight the therapeutic potential of modulating VAV2 and the ribosome biogenesis pathways in both preneoplastic and late progression stages of OSCC.

Human RPF1 and ESF1 in Pre-rRNA Processing and the Assembly of Pre-Ribosomal Particles: A Functional Study.

Ribosome biogenesis is essential for the functioning of living cells. In higher eukaryotes, this multistep process is tightly controlled and involves a variety of specialized proteins and RNAs. This pool of so-called ribosome biogenesis factors includes diverse proteins with enzymatic and structural functions. Some of them have homologs in yeast S. cerevisiae, and their function can be inferred from the structural and biochemical data obtained for the yeast counterparts. The functions of human proteins RPF1 and ESF1 remain largely unclear, although RPF1 has been recently shown to participate in 60S biogenesis. Both proteins have drawn our attention since they contribute to the early stages of ribosome biogenesis, which are far less studied than the later stages. In this study, we employed the loss-of-function shRNA/siRNA-based approach to the human cell line HEK293 to determine the role of RPF1 and ESF1 in ribosome biogenesis. Downregulating RPF1 and ESF1 significantly changed the pattern of RNA products derived from 47S pre-rRNA. Our findings demonstrate that RPF1 and ESF1 are associated with different pre-ribosomal particles, pre-60S, and pre-40S particles, respectively. Our results allow for speculation about the particular steps of pre-rRNA processing, which highly rely on the RPF1 and ESF1 functions. We suggest that both factors are not directly involved in pre-rRNA cleavage but rather help pre-rRNA to acquire the conformation favoring its cleavage.

METTL17 is an Fe-S cluster checkpoint for mitochondrial translation

Friedreich's ataxia (FA) is a debilitating, multisystemic disease caused by the depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry, and cryoelectron microscopy, we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint, promoting translation of Fe-S cluster-rich oxidative phosphorylation (OXPHOS) proteins only when Fe-S cofactors are replete.

GENES FROM PEROXISOME AND INTEGRATED STRESS RESPONSE IN REGULATION OF HUMAN LONGEVITY

Abstract Dietary/caloric restriction shares pathways with peroxisome, a cellular organelle that regulates the synthesis and turnover of complex lipids. PEX3, a peroxisomal biogenesis factor involved in selective type of autophagy. EIF2AK4, a sensor of amino acid deprivation in the integrated stress response (ISR) involved in lifespan regulation. We hypothesize that interplay between EIF2AK4 and PEX3 is associated with human survival. We applied logistic regression model with the interaction term to the analysis of Health and Retirement Study (HRS) and UK Biobank (UKB) parents data to test whether interactions of SNPs from the EIF2AK4 and SNPs from the PEX3 are associated with the dichotomous survival trait (ST) with case group defined as: lifespan ≥ 85; and control as: 75 ≤ (lifespan or age at last follow-up) < 85. Analyses of HRS data showed that interactions between SNPs rs4924405, rs2250402, rs8031319, rs16970049, rs3816899, rs16970112, rs2412459 from the EIF2AK4 gene and SNP rs161070 from the PEX3 gene are significantly associated with ST. In the UKB data, interactions of rs161070 from the PEX3 gene and rs7176881 and rs117778159 SNPs from the EIF2AK4 gene showed nominally significant associations with ST. Analyses of linkage disequilibrium (LD) between the EIF2AK4 SNPs detected in HRS and UKB data showed that their LD are close to the highest values for SNPs with allele frequencies specific for detected variants. Our results confirm association of interplay between EIF2AK4 and PEX3 with human longevity. They also show how information from experimental studies could be transformed to the form useful for human applications.

Defective kernel 58 encodes an Rrp15p domain-containing protein essential to ribosome biogenesis and seed development in maize.

Ribosome biogenesis is a highly dynamic and orchestrated process facilitated by hundreds of ribosomal biogenesis factors and small nucleolar RNAs. While many of the advances are derived from studies in yeast, ribosome biogenesis remains largely unknown in plants despite its importance to plant growth and development. Through characterizing the maize (Zea mays) defective kernel and embryo-lethal mutant dek58, we show that DEK58 encodes an Rrp15p domain-containing protein with 15.3% identity to yeast Rrp15. Over-expression of DEK58 rescues the mutant phenotype. DEK58 is localized in the nucleolus. Ribosome profiling and RNA gel blot analyses show that the absence of DEK58 reduces ribosome assembly and impedes pre-rRNA processing, accompanied by the accumulation of nearly all the pre-rRNA processing intermediates and the production of an aberrant processing product P-25S*. DEK58 interacts with ZmSSF1, a maize homolog of the yeast Ssf1 in the 60S processome. DEK58 and ZmSSF1 interact with ZmCK2α, a putative component of the yeast UTP-C complex involved in the small ribosomal subunit processome. These results demonstrate that DEK58 is essential to seed development in maize. It functions in the early stage of pre-rRNA processing in ribosome biogenesis, possibly through interacting with ZmSSF1 and ZmCK2α in maize.

Structural insights into the role of GTPBP10 in the RNA maturation of the mitoribosome

Mitochondria contain their own genetic information and a dedicated translation system to express it. The mitochondrial ribosome is assembled from mitochondrial-encoded RNA and nuclear-encoded ribosomal proteins. Assembly is coordinated in the mitochondrial matrix by biogenesis factors that transiently associate with the maturing particle. Here, we present a structural snapshot of a large mitoribosomal subunit assembly intermediate containing 7 biogenesis factors including the GTPases GTPBP7 and GTPBP10. Our structure illustrates how GTPBP10 aids the folding of the ribosomal RNA during the biogenesis process, how this process is related to bacterial ribosome biogenesis, and why mitochondria require two biogenesis factors in contrast to only one in bacteria.

The subset of peroxisomal tail-anchored proteins do not reach peroxisomes via ER, instead mitochondria can be involved.

Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, bile acid synthesis and biosynthesis of sterols and ether lipids. Defects in peroxisomes result in severe genetic diseases, such as Zellweger syndrome and neonatal adrenoleukodystrophy. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter the endoplasmic reticulum (ER) in both wild type (WT) and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. Finally, to investigate peroxisomal TA protein targeting in cells with fully functional peroxisomes we used a proximity biotinylation approach. We showed that while ER-targeted TA construct was exclusively inserted into the ER, peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that some peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes, instead, mitochondria can be involved.

NPM1c Mutations Are Associated with Reduced Levels of Multiple Ribosome Biogenesis Factors Leading to Therapeutic Vulnerabilities

Objectives: Mutations in the NPM1 gene are found in more than 30% of acute myeloid leukaemia (AML) cases. The mutations disrupt a nucleolar localization signal (NoLS) and create a novel nuclear export signal (NES), leading to cytoplasmic displacement of the protein (NPM1c). NPM1cmutations prime haematopoietic progenitors to leukaemic transformation, but their precise molecular consequences remain elusive. Here, we first examine the effects of isolated NPM1c mutations on mouse haematopoietic cells and explore their impact on their transcriptome, including at single-cell resolution, and their proteome. Then, using several murine and human AML models and datasets we investigate the role of such NPM1c-associated changes in fully transformed AML cells and explore their potential as therapeutic vulnerabilities. Methods: We performed RNA-seq, CITE-seq and TMT global quantitative proteomics using lineage-depleted bone marrow progenitor cells from NPM1 flox-cA/+;Mx1-cre mice ( Vassiliou et al, Nat Genet, 2011). Proteomic changes were contrasted to those identified in a recently published dataset of 44 human AMLs, including 8 NPM1-mutant AMLs. Candidate vulnerabilities were identified using datasets from CRISPR-Cas9 drop-out screens and were validated in NPM1 cA/+-harbouring cell lines and primary mouse NPM1 cA/+;FLT3 ITD/+ AMLs. Results: We found a global increase in the protein abundance of several members of the KPN family in pre-leukaemic Npm1 cA/+ cells, an effect also observed in human NPM1c+ AMLs. Moreover, we observed a decrease in the relative abundance of several ribosome biogenesis-related proteins in pre-leukaemic Npm1 cA/+ cells and confirmed that this effect persists in human NPM1c+ AML patient samples. We go on to show that, when compared to NPM1 +/+ cells, pre-leukaemic NPM1 cA/+cells show high sensitivity to Actinomycin D and other RNA pol I inhibitors, but not to the translation inhibitor cycloheximide. Combination treatment with Actinomycin D and Venetoclax inhibited the growth and colony forming ability of both pre-leukaemic NPM1 cA/+cells and primary mouse Npm1 cA/+;Flt3 ITD/+AMLs. By contrasting our findings with data from CRISPR drop-out screens, including a genome-wide screen in primary mouse Npm1 cA/+;Flt3 ITD/+/Cas9AMLcells, we identified and validated ribosome biogenesis factors, such as Tsr3, whose knock-out selectively affected cell growth, survival and clonogenicity of NPM1 +/+AML cells, therefore representing novel potential therapeutic targets that could be exploited in the clinic. Conclusions: We show that NPM1c mutations are associated with reduced levels in several ribosome biogenesis factors resulting in therapeutic vulnerabilities of NPM1-mutant pre-leukaemic and leukaemic (AML) cells. The development of novel therapeutics to target these factors may help improve the survival of patients with NPM1-mutant AML. Figure Legend: (A) Volcano plot showing protein abundance in mouse NPM1 cA/+vs NPM1 +/+ pre-leukaemic haematopoetic progenitors. (B) Relative abundance of ribosome-related proteins in human NPM1 cA/+vs NPM1 +/+AML samples (dataset from Kramer et al., 2022).

Inhibiting NR5A2 targets stemness in pancreatic cancer by disrupting SOX2/MYC signaling and restoring chemosensitivity

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a profoundly aggressive and fatal cancer. One of the key factors defining its aggressiveness and resilience against chemotherapy is the existence of cancer stem cells (CSCs). The important task of discovering upstream regulators of stemness that are amenable for targeting in PDAC is essential for the advancement of more potent therapeutic approaches. In this study, we sought to elucidate the function of the nuclear receptor subfamily 5, group A, member 2 (NR5A2) in the context of pancreatic CSCs.MethodsWe modeled human PDAC using primary PDAC cells and CSC-enriched sphere cultures. NR5A2 was genetically silenced or inhibited with Cpd3. Assays included RNA-seq, sphere/colony formation, cell viability/toxicity, real-time PCR, western blot, immunofluorescence, ChIP, CUT&Tag, XF Analysis, lactate production, and in vivo tumorigenicity assays. PDAC models from 18 patients were treated with Cpd3-loaded nanocarriers.ResultsOur findings demonstrate that NR5A2 plays a dual role in PDAC. In differentiated cancer cells, NR5A2 promotes cell proliferation by inhibiting CDKN1A. On the other hand, in the CSC population, NR5A2 enhances stemness by upregulating SOX2 through direct binding to its promotor/enhancer region. Additionally, NR5A2 suppresses MYC, leading to the activation of the mitochondrial biogenesis factor PPARGC1A and a shift in metabolism towards oxidative phosphorylation, which is a crucial feature of stemness in PDAC. Importantly, our study shows that the specific NR5A2 inhibitor, Cpd3, sensitizes a significant fraction of PDAC models derived from 18 patients to standard chemotherapy. This treatment approach results in durable remissions and long-term survival. Furthermore, we demonstrate that the expression levels of NR5A2/SOX2 can predict the response to treatment.ConclusionsThe findings of our study highlight the cell context-dependent effects of NR5A2 in PDAC. We have identified a novel pharmacological strategy to modulate SOX2 and MYC levels, which disrupts stemness and prevents relapse in this deadly disease. These insights provide valuable information for the development of targeted therapies for PDAC, offering new hope for improved patient outcomes.Graphical A Schematic illustration of the role of NR5A2 in cancer stem cells versus differentiated cancer cells, along with the action of the NR5A2 inhibitor Cpd3. B Overall survival of tumor-bearing mice following allocated treatment. A total of 18 PDX models were treated using a 2 x 1 x 1 approach (two animals per model per treatment); n=36 per group (illustration created with biorender.com).