Elucidating the role of the CTLH complex in NSCLC

In this project, I investigated the roles of RANBP9 and RANBP10, two paralogous scaffolds of the CTLH E3 ligase complex, in non-small cell lung cancer (NSCLC). Through CRISPR-engineered knockouts, inducible expression systems, proteomics, and ubiquitylome profiling, I demonstrated that both proteins can independently stabilize the CTLH core but also dynamically cross-regulate each other. Analyses of patient tumors showed consistent overexpression of RANBP9 and its partner GID8, alongside downregulation of RANBP10. Functionally, a high RANBP9/RANBP10 ratio correlated with increased tumor proliferation, whereas elevated RANBP10 slowed growth by reducing proliferation-associated proteins and reshaping ubiquitylation networks. These findings establish RANBP9 and RANBP10 as a rheostat that fine-tunes oncogenic signaling in NSCLC, positioning them as potential therapeutic targets. Collectively, this work advanced understanding of RANBP9 and RANBP10 and suggest their role as targets for the treatment of NSCLC.

RANBP9 presence affects levels of Tip60 and activated p53 in lung cancer cells in response to DNA damage

Shimaa Soliman, Arturo Orlacchio, Anna Tessari, Marina Capece, Rosa Visone, Carlo Croce, Dario Palmieri, Vincenzo Coppola

AACR Annual Meeting 2019

RANBP9 and RANBP10 cooperate in regulating non-small cell lung cancer proliferation

Arturo Orlacchio, Yasuko Kajimura, Lara Rizzotto, Anna Tessari, Shimaa H. A. Soliman, Rosa Visone, Liwen Zhang, Brian Fries, Lino Tessarollo, Joseph Amann, David P. Carbone, Alessia Lodi, Amer Ahmed, Ruggiero Gorgoglione, Giuseppe Fiermonte, Mike Freitas, Dario Palmieri, Jacob Kaufman & Vincenzo Coppola

Abstract

Background: RANBP9 and RANBP10, also called Scorpins, are essential components of the C-terminal to LisH (CTLH) complex, an evolutionarily conserved poorly investigated multisubunit E3 ligase. Their role in non-small cell lung cancer (NSCLC) is unknown.

Methods: In this study, first we used stable loss-of function and overexpression inducible cell lines to investigate the ability of either RANBP9 or RANBP10 to form their own functional CTLH complex. Then, we probed lysates from patient tumors and analyzed data from publicly available repositories to investigate the expression of RANBP9 and RANBP10. Finally, we used inducible cell lines in vitro to recapitulate the expression observed in patients and investigate the changes of the proteome and the ubiquitylome associated with either RANBP9 or RANBP10 in NSCLC.

Results: Here, we show that the two Scorpins are both expressed in NSCLC cells and that either of them can independently support the formation of the CTLH complex. Short-term experiments revealed that the RANBP9 and RANBP10 proteins balance each other in terms of expression, and the acute overexpression of one or the other results in significant reshaping of the NSCLC cell proteome and ubiquitylome. A higher RANBP9/RANBP10 ratio is associated with greater proliferation in both NSCLC cell lines and patients. Acute increased expression of RANBP10 slows NSCLC cell proliferation and decreases the level of proliferation-associated proteins, including key players in DNA replication.

Conclusions: We present evidence that the Scorpins act as partial antagonists and work together as one sophisticated rheostat to modulate the CTLH complex ubiquitylation output, which regulates cell proliferation and other key biological processes in NSCLC. These results suggest that the two Scorpins can be considered as targets for the treatment of NSCLC.

Cancer Research 2017

An in vivo "turning model" reveals new RanBP9 interactions in lung macrophages

Yasuko Kajimura, Shuxin Dong, Anna Tessari, Arturo Orlacchio, Alexandra Thoms, Maria Concetta Cufaro, Federica Di Marco, Foued Amari, Min Chen, Shimaa H A Soliman, Lara Rizzotto, Liwen Zhang, Damu Sunilkumar, Joseph M Amann, David P Carbone, Amer Ahmed, Giuseppe Fiermonte, Mike A Freitas, Alessia Lodi, Piero Del Boccio, Lino Tessarollo, Dario Palmieri, Vincenzo Coppola

Abstract

The biological functions of the scaffold protein Ran Binding Protein 9 (RanBP9) remain elusive in macrophages or any other cell type where this protein is expressed together with its CTLH (C-terminal to LisH) complex partners. We have engineered a new mouse model, named RanBP9-TurnX, where RanBP9 fused to three copies of the HA tag (RanBP9-3xHA) can be turned into RanBP9-V5 tagged upon Cre-mediated recombination. We created this model to enable stringent biochemical studies at cell type specific level throughout the entire organism. Here, we have used this tool crossed with LysM-Cre transgenic mice to identify RanBP9 interactions in lung macrophages. We show that RanBP9-V5 and RanBP9-3xHA can be both co-immunoprecipitated with the known members of the CTLH complex from the same whole lung lysates. However, more than ninety percent of the proteins pulled down by RanBP9-V5 differ from those pulled-down by RanBP9-HA. The lung RanBP9-V5 associated proteome includes previously unknown interactions with macrophage-specific proteins as well as with players of the innate immune response, DNA damage response, metabolism, and mitochondrial function. This work provides the first lung specific RanBP9-associated interactome in physiological conditions and reveals that RanBP9 and the CTLH complex could be key regulators of macrophage bioenergetics and immune functions.

Cell Death Discovery 2025

Tagging enhances histochemical and biochemical detection of Ran Binding Protein 9 in vivo and reveals its interaction with Nucleolin

Shimaa H A Soliman, Aaron E Stark, Miranda L Gardner, Sean W Harshman, Chelssie C Breece, Foued Amari, Arturo Orlacchio, Min Chen, Anna Tessari, Jennifer A Martin, Rosa Visone, Michael A Freitas, Krista M D La Perle, Dario Palmieri, Vincenzo Coppola

Abstract

The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin.

Scientific Reports 2020

RANBP9 as potential therapeutic target in non-small cell lung cancer

Anna Tessari, Shimaa H A Soliman, Arturo Orlacchio, Marina Capece, Joseph M Amann, Rosa Visone, David P Carbone, Dario Palmieri, Vincenzo Coppola

Abstract

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths in the Western world. Despite progress made with targeted therapies and immune checkpoint inhibitors, the vast majority of patients have to undergo chemotherapy with platinum-based drugs. To increase efficacy and reduce potential side effects, a more comprehensive understanding of the mechanisms of the DNA damage response (DDR) is required. We have shown that overexpressby live cell imaging (Incuyion of the scaffold protein RAN binding protein 9 (RANBP9) is pervasive in NSCLC. More importantly, patients with higher levels of RANBP9 exhibit a worse outcome from treatment with platinum-based drugs. Mechanistically, RANBP9 exists as a target and an enabler of the ataxia telangiectasia mutated (ATM) kinase signaling. Indeed, the depletion of RANBP9 in NSCLC cells abates ATM activation and its downstream targets such as pby live cell imaging (Incuy53 signaling. RANBP9 knockout cells are more sensitive than controls to the inhibition of the ataxia and telangiectasia-related (ATR) kinase but not to ATM inhibition. The absence of RANBP9 renders cells more sensitive to drugs inhibiting the Poly(ADP-ribose)-Polymerase (PARP) resulting in a “BRCAness-like” phenotype. In summary, as a result of increased sensitivity to DNA damaging drugs conferred by its ablation in vitro and in vivo, RANBP9 may be considered as a potential target for the treatment of NSCLC. This article aims to report the results from past and ongoing investigations focused on the role of RANBP9 in the response to DNA damage, particularly in the context of NSCLC. This review concludes with future directions and speculative remarks which will need to be addressed in the coming years.

Journal of Cancer Metastasis and Treatment 2021