Project description:Abstract: RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine, and the mechanisms of how they contribute to tumorigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine. We identify non-canonical roles of RAL GTPases not as RAS effectors, but rather by acting upstream of RAS activation via induction of EGFR internalisation . Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to impacting stem cell proliferation and damage-induced intestinal regeneration, this function of RAL GTPases drives EGFR-dependent tumorigenic growth in the intestine and in human mammary epithelium. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of EGFR-driven tissue homeostasis and malignant transformation. Results: RalA is required within ISCs to induce midgut adult midgut regeneration following damage by oral infection with Erwinia carotovora carotovora 15 (Ecc15) (Johansson et al., 2019). To achieve a global view of intestinal pathways affected by RalA, we performed a transcriptomic analysis by RNAseq of whole midguts from vehicle treated (Mock) or damaged (Ecc15 fed) control animals or following RalA knockdown in intestinal stem and progenitor cells using the escargot-gal4 driver (ISC/EB>) (Micchelli and Perrimon, 2006). Consistent with its effect on ISC proliferation (Johansson et al., 2019), RalA knockdown significantly impaired damage-induced upregulation of cell cycle genes in the midgut. Additionally, levels of multiple transcriptional targets of the EGFR/MAPK pathway (Golembo et al., 1996; Hsu et al., 2001; Jin et al., 2015; Meng and Biteau, 2015), such as argos (aos), rhomboid (rho), Sox21a and string (stg) were increased following Ecc15 infection in control midguts. The upregulation of these target genes was significantly impaired upon RalA knockdown.
Project description:The neural behavior of glioblastoma, including the formation of tumor microtubes and synaptic circuitry, is increasingly understood to be pivotal for disease manifestation (Osswald et al. 2015; Venkatesh et al. 2015; Weil et al. 2017; Venkataramani et al. 2019; Venkatesh et al. 2019; Alcantara Llaguno et al. 2019; Venkataramani et al. 2022). Nonetheless, the few approved treatments for glioblastoma target its oncological nature, while its neural vulnerabilities remain incompletely mapped and clinically unexploited. Here, we systematically survey the neural molecular dependencies and cellular heterogeneity across 27 glioblastoma patients and diverse model systems. In patient tumor samples taken directly after surgery, we identify a spectrum of neural stem cell morphologies indicative of poor prognosis, and discover a set of repurposable neuroactive drugs with unexpected and consistent anti-glioma efficacy. Glioblastoma cells exhibit functional dependencies on highly expressed drug targets including neurological ion channels and receptors, while interpretable molecular machine learning reveals downstream convergence on secondary drug targets (COSTAR) involving AP-1-driven tumor suppression. COSTAR enables in silico drug screening on >1 million compounds that are validated with high accuracy. Multi-omic profiling of drug-treated glioblastoma cells confirms rapid Ca2+-driven AP-1 pathway induction to represent a tumor-intrinsic vulnerability at the intersection of oncogenesis and neural activity-dependent signaling. Finally, the consistent anti-glioma activity across patients and model systems is epitomized by the antidepressant Vortioxetine, which synergizes in vivo with approved glioblastoma chemotherapies. In all, our global analysis reveals that the neural vulnerabilities of glioblastoma converge on an AP-1 mediated gene regulatory network with direct translatable potential.
Project description:Το investigate the role of Rac1 and Rac3 GTPases in the development of MGE-derived cortical interneurons we generated a transgenic mice where both Rac1 and 3 were depleted from the medial ganglionic eminence. First we generated a conditional knockout for Rac1 (Vidaki et al. 2012) by crossing animals carrying a floxed allele of Rac1 (Rac1fl/fl) (the fourth and fifth exon of the Rac1 gene are flanked with loxP sites, Walmsley et al. 2003) to the Nkx2.1Tg(Cre) mice (Nkx2.1 transgenic Cre, Fogarty et al. 2007). The ROSA26fl-STOP-fl-YFP allele was also inserted as an independent marker (Srinivas et al. 2001). Next, we crossed the Rac1 conditional knockout with the Rac3 KO line (Corbetta et al. 2005) and obtained a mouse line where Rac1 and Rac3 were both depleted from the MGE-derived interneurons.
Project description:In recent years, several small molecule cytotoxic drugs have been identified as potential inhibitors of ribosome biogenesis (Drygin et al., 2011; Peltonen et al., 2014a; Peltonen et al., 2014b). CX-5461 is one such drug that has also demonstrated anticancer potential for a wide range of malignancies (Bywater et al., 2012; Cornelison et al., 2017; Devlin et al., 2015; Drygin et al., 2011; Hald et al., 2019; Hein et al., 2017; Ismael et al., 2019; Lawrence et al., 2018; Lee et al., 2017; Negi and Brown, 2015; Taylor et al., 2019; Xu et al., 2017; Yan et al., 2017) (Haddach et al., 2012), and is presently under phase I trials for the treatment of both hematological cancers and solid tumours (Group, 2016; Khot et al., 2019). CX-5461 was initially characterized as an inhibitor of RNA Polymerase I (RPI/PolR1/PolI) that is responsible for the synthesis of the major ribosomal RNAs and the initial step in ribosome biogenesis (Drygin et al., 2011). Since RPI and its corresponding core transcription factors are dedicated to this task alone, they present ideal molecular targets by which to modulate ribosome biogenesis. However, the specificity of CX-5461 has been questioned and it has been suggested that this drug may also act by stabilizing DNA G-quadruplexes or by “poisoning” topoisomerase II (Topo II). Thus, the primary target of this drug and its mode of action are still in doubt. Here we used Deconvolution-ChIP-Seq in NIH3T3 and HEK293T cells treated for different times with CX-5461. The data show that the primary target of CX5461 is the initiation of ribosomal RNA gene (rDNA) transcription. CX-5461 blocks transcription initiation in vitro and in vivo by arresting RNA polymerase I (RPI/Pol1) within the preinitiation complex. In contrast to previous suggestions, CX-5461 does not effect recruitment of the TBP-TAF complex SL1 to the rDNA promoter, the recruitment of the initiation competent RPI-Rrn3 complex or ongoing transcription elongation, arguing against a role for G-quadruplex stabilization or topoisomerase II poisoning. Inhibition of transcription by CX-5461 is not reversible, the RPI-Rrn3 complex remains arrested in the preinitiation complex even after drug removal. This leads to nucleolar stress, extensive DNA damage and cell senescence. Our data show that the cytotoxicity of CX-5461 is the downstream result of the highly specific inhibition of rDNA transcription. The observation that this inhibition is irreversible will be important for the future design of chemotherapeutic strategies and the avoidance of drug resistance.
Project description:Precise deposition of CpG methylation is critical for mammalian development and tissue homeostasis and is often dysregulated in human diseases. The localization of de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) is facilitated by PWWP domain recognition of histone H3 lysine 36 (H3K36) methylation (Baubec et al. 2015, Weinberg et al. 2019) and is normally excluded from CpG islands (CGIs) (Wu et al. 2010). However, CpG methylation of CGIs that are regulated by Polycomb repressive complexes (PRCs) has been observed during embryogenesis (Chen et al. 2019), cellular differentiation (Mohn et al. 2008), and cancer progression (Ohm et al. 2007, Schlesinger et al. 2007, Widschwendter et al. 2007), suggesting that an uncharacterized mechanism exists to compete for de novo DNMT recruitment. Here we report that DNMT3A PWWP domain mutations recently identified in paragangliomas (Remacha et al. 2018) and microcephalic dwarfism (Heyn et al. 2019) promote localization of DNMT3A to CGIs in a PRC1-dependent manner. Genome-wide analysis shows that DNMT3A PWWP mutants redistribute to regions containing ubiquitylation of histone H2A at lysine 119 (H2AK119Ub) deposited by PRC1, irrespective of the levels of PRC2-catalyzed tri-methylation of histone H3 at lysine 27 (H3K27me3). DNMT3A, but not DNMT3B, is capable of directly interacting with H2AK119Ub-modified nucleosomes through a putative amino-terminal ubiquitin-dependent recruitment (UDR) region, which serves as an alternative form of genomic targeting in cells upon loss of PWWP reader function. Ablation of PRC1 abrogates localization of DNMT3A PWWP mutants to CGIs and prevents aberrant hypermethylation at these sites. Our study implies that a balance between DNMT3A recruitment by distinct reader domains guides de novo CpG methylation and may underlie the abnormal DNA methylation landscapes observed in human cancers and developmental disorders.