BAP1 Malignant Pleural Mesothelioma Mutations in Caenorhabditis elegans Reveal Synthetic Lethality between ubh-4/BAP1 and the Proteasome Subunit rpn-9/PSMD13

Abstract

The deubiquitinase BAP1 (BRCA1-associated protein 1) is associated with BAP1 tumor predisposition syndrome (TPDS). BAP1 is a tumor suppressor gene whose alterations in cancer are commonly caused by gene mutations leading to protein loss of function. By CRISPR-Cas, we have generated mutations in ubh-4, the BAP1 ortholog in Caenorhabditis elegans, to model the functional impact of BAP1 mutations. We have found that a mimicked BAP1 cancer missense mutation (UBH-4 A87D; BAP1 A95D) resembles the phenotypes of ubh-4 deletion mutants. Despite ubh-4 being ubiquitously expressed, the gene is not essential for viability and its deletion causes only mild phenotypes without affecting 20S proteasome levels. Such viability facilitated an RNAi screen for ubh-4 genetic interactors that identified rpn-9, the ortholog of human PSMD13, a gene encoding subunit of the regulatory particle of the 26S proteasome. ubh-4[A87D], similarly to ubh-4 deletion, cause a synthetic genetic interaction with rpn-9 inactivation affecting body size, lifespan, and the development of germ cells. Finally, we show how ubh-4 inactivation sensitizes animals to the chemotherapeutic agent Bortezomib, which is a proteasome inhibitor. Thus, we have established a model to study BAP1 cancer-related mutations in C. elegans, and our data points toward vulnerabilities that should be studied to explore therapeutic opportunities within the complexity of BAP1 tumors.

AKIR-1 regulates proteasome subcellular function in Caenorhabditis elegans

“…UBH-4, and its human homolog UCHL5, have previously been shown to interact with the 19S subunit RPN-13 34 , 39 , 40 , 41 and to be broadly expressed in C. elegans tissues, including the intestine. 34 , 42 Here, we used a CRISPR-engineered UBH-4:GFP strain 42 , 43 and observed that these animals displayed strong nuclear fluorescence in intestinal nuclei similar to the pattern detected with the anti-20S antibody ( Figures 3 C and 3A). In accordance with the 20S immunofluorescence and GFP::RPT-5 results, UBH-4:GFP fluorescence decreased in the intestinal nuclei of animals exposed to akir-1 RNAi when compared to control animals ( Figure 3 ).…”

“…This study utilizes our new and previously generated fluorescent reporter systems 34 , 36 , 37 , 38 , 42 to uncover cell- and tissue-specific aspects of akir-1 depletion on the in vivo function of the UPS. Unfortunately, the expression of our current fluorescent polyubiquitin-binding reporter and photoconvertible UPS activity reporter is yet restricted to a few cell types, excluding, e.g., germ cells, which limits a more comprehensive comparative in vivo analysis of proteasome function.…”

“…; Vicencio et al.) 42 , 43 N/A C. elegans: Strain YD116[ rrf-3(pk1426);xzIs2[unc-54p::UIM2::ZsProSensor] ] (Jha & Holmberg) 35 N/A C. elegans: Strain YD114[ xzIs2[unc-54p::UIM2::ZsProSensor] ] (Pispa et al.) 36 WBStrain00049246 C. elegans: Strain YD25 [xzEx25[vha-6p::Dendra2]] (Li et al.)…”

“…The akir-1 mutant allele gk528 was backcrossed three times with wild-type N2 animals. Following strains were also used: YD3[ xzEx3[unc-54p::UbG76V::Dendra2] ], 37 YD27[ xzEx27[vha-6p::UbG76V::Dendra2] ], 38 YD90[ xzIs1[vha-6p::UIM2::ZsProSensor] ], 34 GR1702[ Is1[sur-5p::NLS-GFP] ], 33 CER395 [ubh-4(cer68[ubh-4::egfp])II] , 42 , 43 YD116[ rrf-3(pk1426);xzIs2[unc-54p::UIM2::ZsProSensor] ], 35 and YD114[ xzIs2[unc-54p::UIM2::ZsProSensor] ]. 36 To generate YD117[ xzEx113[rpt-5p::GFP::RPT-5] ] reporter strain, GFP::RPT-5 translational fusion construct was created by substituting the let-858 promoter of the GFP vector pPD118.25 (L3786 was a gift from Andrew Fire; Addgene plasmid #1593; http://n2t.net/addgene:1593; RRID:Addgene_1593; Watertown, MA, USA) with a 2010-bp putative rpt-5 promoter sequence, mutating the GFP stop codon, and replacing the let858 3′ UTR with a 1747-bp sequence covering the 1504-bp rpt-5 coding region with introns and the following 183-bp 3′ UTR region.…”