CANCER RESEARCH | MOLECULAR CELL BIOLOGY FAM46C and FNDC3A Are Multiple Myeloma Tumor Suppressors That Act in Concert to Impair Clearing of Protein Aggregates and Autophagy Nicola Manfrini 1,2 , Marilena Mancino 1,3 , Annarita Miluzio 1 , Stefania Oliveto 1,2 , Matteo Balestra 1 , Piera Calamita 1,2 , Roberta Alfieri 1 , Riccardo L. Rossi 1 , Marco Sasso e-Pognetto 4 , Chiara Salio 5 , Alessandro Cuomo 6 , Tiziana Bonaldi 6 , Marcello Manfredi 7,8,9 , Emilio Marengo 7,8,10 , Elia Ranzato 10 , Simona Martinotti 10 , Davide Cittaro 11 , Giovanni Tonon 11,12 , and Stefano Biffo 1,2 ABSTRACT ◥ Multiple myeloma is a plasma cell neoplasm characterized by the production of unfolded immunoglobulins, which cause endo- plasmic reticulum (ER) stress and sensitivity to proteasome inhibition. The genomic landscape of multiple myeloma is char- acterized by the loss of several genes rarely mutated in other cancers that may underline specific weaknesses of multiple mye- loma cells. One of these is FAM46C that is lost in more than 10% of patients with multiple myeloma. We show here that FAM46C is part of a new complex containing the ER-associated protein FNDC3A, which regulates trafficking and secretion and, by impairing autophagy, exacerbates proteostatic stress. Reconstitu- tion of FAM46C in multiple myeloma cells that had lost it induced apoptosis and ER stress. Apoptosis was preceded by an increase of intracellular aggregates, which was not linked to increased trans- lation of IgG mRNA, but rather to impairment of autophagy. Biochemical analysis showed that FAM46C requires interaction with ER bound protein FNDC3A to reside in the cytoplasmic side of the ER. FNDC3A was lost in some multiple myeloma cell lines. Importantly, depletion of FNDC3A increased the fitness of FAM46C-expressing cells and expression of FNDC3A in cells that had lost it recapitulated the effects of FAM46C, inducing aggregates and apoptosis. FAM46C and FNDC3A formed a complex that modulates secretion routes, increasing lysosome exocytosis. The cellular landscape generated by FAM46C/ FNDC3A expression predicted sensitivity to sphingosine kinase inhibition. These results suggest that multiple myeloma cells remodel their trafficking machinery to cope with ER stress. Significance: This study identifies a new multiple myeloma– specific tumor suppressor complex that regulates autophagy and unconventional secretion, highlighting the sensitivity of multiple myeloma cells to the accumulation of protein aggregates. Introduction Multiple myeloma, the second most common hematologic malig- nancy, is caused by the accumulation of abnormal plasma cells. Multiple myeloma cells retain the plasma cell capability to synthesize and secrete immunoglobulins (Ig; ref. 1). Ig mRNAs are translated by endoplasmic reticulum (ER) resident ribosomes and undergo con- ventional secretion. Nascent Ig chains translocate to the ER lumen where they are folded. During this process, fractions of Igs remain unfolded. The accumulation of unfolded proteins triggers the unfolded protein response (UPR), a three-branch mechanism that maintains ER homeostasis (2). As part of the UPR process, unfolded proteins are retro-translocated from the ER to the cytoplasm and degraded by the proteasome (3). Indeed, proteasome inhibitors are highly effective for multiple myeloma treatment (4, 5), before clinical resistance develops (6). Accumulation of cytoplasmic proteins that escape proteasome digestion can trigger the formation of intracellular aggregates, also known as aggresomes. Aggresomes can be degraded by autophagy, an intricated pathway of cellular events that results in the clearing of double membrane vesicles by the lysosomal degradative pathway. Nowadays, most studies converge on the concept that autophagy and the ubiquitin-proteasome system are integrated (7) and coop- erate to clear ubiquitinylated targets. In addition, autophagy rele- vant proteins possess activities that intervene with cellular functions linked to membrane biology, such as endocytosis, intracellular vesicular trafficking, and conventional and nonconventional secretion (8). Genetic analysis has shown that multiple myeloma cells have frequent loss-of-function mutations in genes that are rarely mutated in other cancers (9–11). One of these genes is FAM46C that is mutated in more than 10% of patients with multiple myeloma (9–11). FAM46C induces apoptosis in multiple myeloma cell lines (12). FAM46C is a 1 INGM, National Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi,” Milan, Italy. 2 Department of Biological Sciences, University of Milan, Milan, Italy. 3 Department of Clinical Sciences and Community, University of Milan, Milan, Italy. 4 Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Torino, Italy. 5 Department of Veterinary Sciences, University of Turin, Grugliasco, Torino, Italy. 6 Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy. 7 Center for Translational Research on Autoimmune and Allergic Diseases, University of Piemonte Orien- tale, Novara, Italy. 8 ISALIT, Novara, Italy. 9 Department of Translation Medicine, University of Piemonte Orientale, Novara, Italy. 10 Department of Sciences and Technological Innovation, University of Piemonte Orientale, Alessandria, Italy. 11 Center for Omics Sciences, IRCCS San Raffaele Scientific Institute, Milan, Italy. 12 Functional Genomics of Cancer Unit, Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). N. Manfrini and M. Mancino contributed equally to this article. Current address for R. Alfieri: IGM- Institute of Molecular Genetics – CNR, Pavia, Italy. Corresponding Author: Stefano Biffo, University of Milan, Milano 20133, Italy. Phone: 3902-0066-0304; E-mail: stefano.biffo@unimi.it Cancer Res 2020;80:4693–706 doi: 10.1158/0008-5472.CAN-20-1357 Ó2020 American Association for Cancer Research. AACRJournals.org | 4693 Downloaded from http://aacrjournals.org/cancerres/article-pdf/80/21/4693/2796831/4693.pdf by guest on 21 June 2022