Abstract 2908: Mesothelioma phylogeny reveal MTAP as a solitary clonal deletion, exposing vulnerability to the PRMT5 perturbagen, quinacrine
Authors: Busacca Sara , Brannan Lee , Nakas Apostolos , Sharkey Annabel , Riganti Chiara , Waller David , Richards Cathy , Salaroglio Iris , Milosevic Vladan , Wells-Jordan Peter , Dawson Alan , Sheaff Michael , LeQuesne John , Gaba Aarti , Hastings Robert , Martinson Luke , Lo Jin-Li , Bajaj Amrita , Boutros Paul , John Tom , Thapa Bibhusal , Wilson Gareth , Shaw Jacqui , Swanton Charles , Dudbridge Frank , Hollox Edward , Fennell Dean A. 
Published: August 2019

Abstract
Background: Malignant Pleural Mesothelioma (MPM) remains an incurable cancer that is caused by asbestos, and for which there is a paucity of effective therapy. Stratified medicine for MPM is in its infancy. We hypothesized that deciphering the phylogenetic architecture of mesothelioma would yield a census of recurrent clonal homozygous copy number losses as potential therapeutic vulnerabilities.
Methods and Results: We prospectively enrolled 125 patients with MPM undergoing radical pleurectomy decortication, into the MEDUSA (Mesothelioma Evolution: DrUgging Somatic Alterations) study. Multi-region whole exome sequencing was conducted on 106 tumours from 20 patients (Medusa20 cohort). Up to 5 consistent regions were sampled: apex, pericardium, anterior/ posterior costophrenic angles, and the oblique fissure. For each patient, matching whole blood DNA was also whole exome sequenced to allow identification of tumour- specific somatic variations. Somatic copy number alterations (SCNAs) in each tumour region were called using SEQUENZA. We inferred phylogeny for each patient’s tumour using the SCNA calls by maximum parsimony (TUMULT), which revealed branched evolution in all MPMs. The total number of SCNAs ranged from 78 to 380 across the cohort with biphasic MPMs exhibiting a significantly larger total and clonal SCNA burden compared to epithelioid MPMs (p=0.024) . Only 9p21 which harbours CDKNA and methylthioadenosine phosphorylase (MTAP), exhibited clonal homozygous loss in 3 patients (15%). Clonal heterozygous loss was seen in 2 patients (10%). A further 5 patients showed with evidence of parallel evolution involving MTAP loss in distant MPM regions (25%), with one patient's MPM having late homozygous deletion in a single branch (5%). MTAP loss was validated by array based SCNA analysis and was found to be negatively prognostic in an independent cohort. Protein arginine methyltransferase 5 (PRMT5) has been recently identified as a vulnerability in MTAP deleted cancer. We found that siRNA silencing of PRMT5, caused MTAP selective loss of clonogenicity with proliferative arrest. Utilizing the connectivity map, quinacrine was validated as a PRMT5 perturbagen, which suppressed c-jun-dependent PRMT5 expression without inhibiting its methyltransferase activity. Quinacrine phenocopied PRMT5 siRNA, reducing global symmetrical arginine dimethylation of histone H4 (H4R3me2S). Finally, exogenous wild-type PRMT5 rescued quinacrine-mediated cell arrest in MTAP-negative cells, an effect not seen using the PMRT5 E444Q methyltransferase dead mutant.
Conclusion: MTAP deletion is a clonal homozygous event in mesothelioma, with potential as a therapeutically tractable Achilles heel, via PRMT5 silencing using a repurposed small molecule, quinacrine.
Citation Format: Sara Busacca, Lee Brannan, Apostolos Nakas, Annabel Sharkey, Chiara Riganti, David Waller, Cathy Richards, Iris Salaroglio, Vladan Milosevic, Peter Wells-Jordan, Alan Dawson, Michael Sheaff, John LeQuesne, Aarti Gaba, Robert Hastings, Luke Martinson, Jin-Li Lo, Amrita Bajaj, Paul Boutros, Tom John, Bibhusal Thapa, Gareth Wilson, Jacqui Shaw, Charles Swanton, Frank Dudbridge, Edward Hollox, Dean A. Fennell. Mesothelioma phylogeny reveal MTAP as a solitary clonal deletion, exposing vulnerability to the PRMT5 perturbagen, quinacrine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2908.