Biology subjects

Vagnarelli, P.

Publications and source records attributed to Vagnarelli, P..

7 recordsLinked to original sources

bioRxiv2025

A CDCA2 - MYC positive feedback loop controls cancer cells survival

MYC transcription factors are encoded by a small family of genes which include, in addition to the prototype member MYC, MYCN and MYCL. The majority of human cancers display expression alterations of MYC genes and their products, making this group of oncoproteins among the most relevant therapeutic targets in oncology. MYC is regulated at multiple levels, and protein phosphorylation is a critical mechanism used by the cell to modulate MYC stability and activity. Although there is a reasonable knowledge of the kinases required for MYC modifications, the counteracting phosphatases have been relatively understudied. Here we have investigated the role of the chromatin remodelling factor and protein phosphatase 1 (PP1) regulatory subunit CDCA2, also known as RepoMan, in the regulation of MYC proteins in cancer cells. Using RNA interference and regulatable degron-mediated degradation of CDCA2, we demonstrate that the PP1 subunit is required for MYC and MYCN stabilisation and viability of triple negative breast cancer, neuroblastoma and colon cancer cells. Proximity ligation assays indicate that both MYC and MYCN are in close proximity of CDCA2 in vivo. Furthermore, chromatin immunoprecipitation data and promoter mutation studies establish that CDC2A is a bone-fide MYC target gene in cancer cells, revealing a reciprocal regulatory loop that could be exploited for therapeutic purposes.

cell biology↗

bioRxiv2025

Illuminating oncogenic KRAS signaling by multi-dimensional chemical proteomics

Mutated KRAS is among the most frequent activating genetic alterations in cancer and drug discovery efforts have led to inhibitors that block its activity. To better understand oncogenic KRAS signaling and the cytostatic effects of drugs, we performed comprehensive dose-dependent proteome-wide target deconvolution, pathway engagement and protein expression characterization of KRAS, MEK, ERK, SHP2 and SOS1 inhibitors in pancreatic (KRAS G12C, G12D) and lung cancer (KRAS G12C) cells. Analysis of the resulting 687,954 dose-response curves available online revealed both common and cell line-specific signaling networks dominated by oncogenic KRAS activity. Time-dose experiments separated early KRAS-MEK-ERK from CDK-mediated signaling that cause cells to exit from the cell cycle. This transition to a quiescent state occurred without substantial proteome re-modelling but extensive changes of protein phosphorylation and ubiquitylation. The collective data highlights the complexity of KRAS signaling in cancer and places a large number of new proteins into this functional context.

cell biology↗

bioRxiv2024

Multi-omic analyses reveal a differential contribution of chromatin-associated PP1 holoenzymes to mitotic exit and G1 re-establishment.

Mitotic exit is an important part of the cell cycle that requires coordination of many chromatin and cytoskeleton remodelling events to successfully complete cell division and maintain cell identity. Protein de-phosphorylation is a key step in directing mitotic exit and Protein phosphatase (PP1) is essential to this process, however the specific contribution of its numerous targeting subunits is still unknown. Here we have investigated the function of three chromatin-associated PP1 targeting subunits in exiting mitosis Repo-Man, Ki-67 and PNUTS. We have generated endogenously tagged, auxin-degradable alleles for each subunit and used a multi-omic approach to address their specific contribution towards transcription resumption, chromatin accessibility and protein phosphorylation at the transition from mitosis to G1. This approach has identified their distinct role in mitotic exit, provided unique datasets for the cell cycle community, and highlighted novel functions for Ki-67 and Repo-Man in genome stability and organisation.

cell biology↗

bioRxiv2023

Ki-67 is necessary during DNA replication for forks protection and genome stability

BackgroundThe proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years but investigations on its biological functions have lagged. Recently, Ki-67 was shown to regulate both the composition of the chromosome periphery and chromosome behaviour in mitosis as well as to play a role in heterochromatin organisation and gene transcription. However, a role for Ki-67 in regulating cell cycle progression has never been reported. The progress towards understanding Ki-67 function have been limited by the tools available to deplete the protein coupled to its abundance and fluctuation during the cell cycle. ResultsHere we have used an auxin-inducible degron tag (AID) to achieve a rapid and homogeneous degradation of Ki-67 in HCT116 cells. This system, coupled with APEX2 proteomics and phospho-proteomics approaches, allowed us to show for the first time that Ki-67 plays a role during DNA replication. In its absence, DNA replication is severely delayed, the replication machinery is unloaded, causing DNA damage that is not sensed by the canonical pathways and dependant on HUWE1 ligase. This leads to replication and sister chromatids cohesion defects, but it also triggers an interferon response mediated by the cGAS/STING pathway in all the cell lines tested. ConclusionsWe have unveiled a new function of Ki-67 in DNA replication and genome maintenance that is independent of its previously known role in mitosis and gene regulation.

cell biology↗

bioRxiv2022

CCDC86/Cyclon is a novel Ki-67 interacting protein important for cell division

The chromosome periphery is a network of proteins and RNAs that coats the outer surface of mitotic chromosomes. Despite the identification of new components, the functions of this complex compartment are poorly characterised. In this study we identified a novel chromosome periphery-associated protein CCDC86/cyclon. Using a combination of RNAi (RNA interference), microscopy and biochemistry, we studied the functions of CCDC86/cyclon in mitosis. CCDC86/cyclon depletion resulted in partial disorganisation of the chromosome periphery with partial alterations in the localization of Ki-67 and Nucleolin and the formation of abnormal cytoplasmic aggregates. Furthermore, CCDC86/cyclon-depleted cells displayed errors in chromosome alignment, altered spindle length and increased apoptosis. These results suggest that, within the chromosome periphery, different subcomplexes that include CCDC86/cyclon, Nucleolin and B23 are required for mitotic spindle regulation and correct kinetochore-microtubule attachments, thus contributing to chromosome segregation in mitosis. Moreover, we have identified CCDC86/cyclon as a MYC-N regulated gene whose expression levels represent a powerful marker for prognostic outcomes in neuroblastoma. Summary statementHere we report the identification of CCDC86/cyclon as novel component of the perichromosomal layer. CCDC86 is important for chromosome segregation and represents a strong prognostic marker for neuroblastoma patients.

cell biology↗

bioRxiv2020

Non-redundant functions of H2A.Z.1 and H2A.Z.2 in chromosome segregation and cell cycle progression.

H2A.Z is a H2A-type histone variant essential for many aspects of cell biology ranging from gene expression to genome stability. From deuterostomes, H2A.Z evolved into two paralogues H2A.Z.1 and H2A.Z.2 that differ by only three amino acids and are encoded by different genes (H2AFZ and H2AFV respectively). Despite the importance of this histone variant in development and cellular homeostasis, very little is known about the individual functions of each paralogue in mammals. Here we investigated the distinct roles of two paralogues in cell cycle regulation. Using a specific siRNA approach for each paralogue in human cells, we unveiled non-redundant functions for H2A.Z.1 and H2A.Z.2 in cell division: H2A.Z.1 regulates the expression of important cell cycle genes (including Myc and Ki-67) and its depletion leads to a G1 arrest, whereas H2A.Z.2 is essential for centromere integrity and function, thus playing a key role in chromosome segregation.

cell biology↗

bioRxiv2017

CDK1 and PLK1 co-ordinate the disassembly and re-assembly of the Nuclear Envelope in vertebrate mitosis.

Micronuclei (MN) arise from chromosomes or fragments that fail to be incorporated into the primary nucleus after cell division. These structures are a major source of genetic instability caused by DNA repair and replication defects coupled to aberrant Nuclear Envelope (NE). These problems ultimately lead to a spectrum of chromosome rearrangements called chromothripsis, a phenomenon that is a hallmark of several cancers. Despite its importance, the molecular mechanism at the origin of this instability is still not understood. Here we show that lagging chromatin, although it can efficiently assemble Lamin A/C, always fails to recruit Nuclear Pore Complexes (NPCs) proteins and that Polo-Like Kinase (PLK1) negatively regulates the NPC assembly. We also provide evidence for the requirement of PLK1 activity for the disassembly of NPCs, but not Lamina (A/C), at mitotic entry. Altogether this study reveals the existence of independent regulatory pathways for Lamin A/C and NPC reorganization during mitosis where Lamin A targeting to the chromatin is controlled by CDK1 activity (a clock-based model) while the NPC loading is also spatially monitored by PLK1.

cell biology↗