FAM46C and FNDC3A Are Multiple Myeloma Tumor Suppressors That Act in Concert to Impair Clearing of Protein Aggregates and Autophagy

Author Manuscript Published OnlineFirst on September 22, 2020; DOI: 10.1158/0008-5472.CAN-20-1357 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

FAM46C and FNDC3A are multiple myeloma tumor suppressors that act in concert to impair clearing of protein aggregates and autophagy

Nicola Manfrini1,2,*, Marilena Mancino1,3,*, Annarita Miluzio1, Stefania Oliveto1,2, Matteo Balestra1, Piera Calamita1,2, Roberta Alfieri1,#, Riccardo L. Rossi1, Marco Sassoè-Pognetto4, Chiara Salio5, Alessandro Cuomo6, Tiziana Bonaldi6, Marcello Manfredi7,8,9 , Emilio Marengo7,8,10, Elia Ranzato10, Simona Martinotti10, Davide Cittaro11, Giovanni Tonon11,12 and Stefano Biffo1,2. 1 INGM, National Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi”, Milan, Italy. 2 Dept. of Biological Sciences, University of Milan, Milan, Italy. 3 Dept. of Clinical Sciences and Community, University of Milan, Milan, Italy 4 Dept. of Neuroscience “Rita Levi Montalcini”, University of Turin, C.so Massimo d’Azeglio 52, 10126 Torino, Italy 5 Dept. of Veterinary Sciences, University of Turin, Largo Paolo Braccini 2, 10095 Grugliasco (To), Italy 6 Dept. of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy 7 Center for Translational Research on Autoimmune and Allergic Diseases, University of Piemonte Orientale, Corso Trieste 15, 28100 Novara, Italy; 8 ISALIT, Via Canobio 4/6, 28100 Novara, Italy; 9 Dept. of Translation Medicine, University of Piemonte Orientale, 28100 Novara, Italy; 10 Dept. of Sciences and Technological Innovation, University of Piemonte Orientale, Viale T. Michel 11, 15121 Alessandria, Italy; 11 Center for Omics Sciences, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy. 12 Functional Genomics of Cancer Unit, Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.

# Current address: IGM- Institute of Molecular Genetics – CNR, Pavia, Italy. * The two authors contributed equally to the work. Running title: Role of the FAM46C/FNDC3A complex in multiple myeloma.

Keywords: UPR, proteasome, FNDC3A, lysosome, secretion. Additional information: This paper was supported by grant AIRC IG 19973 to SB, by grant AIRC 9965 5 ‰ to GT and by unrestricted grant from “Fondazione Romeo ed Enrica Invernizzi”.

Correspondence: Stefano Biffo, INGM National Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi”, Via Francesco Sforza 35, 20122 Milano, Italy. Tel: +390200660304; Email: [email protected]; [email protected]

Competing interests: The authors declare no competing interests. Number of words in main text: 7607, figures: 7.

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 22, 2020; DOI: 10.1158/0008-5472.CAN-20-1357 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Multiple myeloma (MM) is a plasma cell neoplasm characterized by the production of unfolded immunoglobulins which cause endoplasmic reticulum (ER) stress and sensitivity to proteasome inhibition. The genomic landscape of MM is characterized by the loss of several genes rarely mutated in other cancers that may underline specific weaknesses of MM cells. One of these is

FAM46C that is lost in more than 10% of MM patients. 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. Reconstitution of

FAM46C in MM 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 translation of IgG mRNA but rather to impairment of autophagy. Biochemical analysis showed that FAM46C requires interaction with ER-bound protein FNDC3A in order to reside in the cytoplasmic side of the ER. FNDC3A was lost in some MM 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 MM 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 (MM), the second most common hematologic malignancy, is caused by the accumulation of abnormal plasma cells. MM cells retain the plasma cell capability to synthesize and secrete immunoglobulins (Ig)s (1). Ig mRNAs are translated by endoplasmic reticulum (ER) resident ribosomes and undergo conventional secretion. Nascent Ig chains translocate to the ER lumen where they are folded. During this process, fractions of immunoglobulins remain unfolded. The accumulation of unfolded proteins triggers the unfolded protein response (UPR), a three-branch mechanisms which 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 MM treatment (4,5), before clinically resistance develops (6).

Accumulation of cytoplasmic proteins that escape proteasome digestion can trigger the formation of intracellular aggregates, aka aggresomes. Aggresomes can be degraded by autophagy, an intricated pathway of cellular events that results in the clearing of doubled- 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 cooperate to clear ubiquitinylated targets. In addition, autophagy-relevant proteins possess activities that intervene on cellular functions linked to membrane biology, such as endocytosis, intracellular vesicular trafficking and conventional and non-conventional secretion (8).

Genetic analysis has shown that MM 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 of MM (9-11). FAM46C induces apoptosis in MM cell lines (12).

FAM46C is a member of a gene family composed of four highly similar proteins, FAM46A,

FAM46B, FAM46C, and FAM46D. With the exception of FAM46D, which is lost in 3% of gastric

cancer patients (13), no mutations of other FAM46 family members have been observed in cancer. Understanding the reason that underlies the specific loss of FAM46C in MM, may open the avenue for specific therapies.

Unbiased high throughput screening picked up FAM46C as an interferon-regulated modulator of viral production. In some cases, FAM46C overexpression mildly increased viral production, as for yellow fever virus, or had no effect, as with hepatic HCV (14). In other cases, FAM46C strongly inhibited viral propagation, as in the case of the influenza virus H1N1 (15). These observations suggest that the proviral or antiviral effect of FAM46C may depend from specific differences in the way viral particles are replicated and egressed, rather than from a common process. In this context, autophagy modulation plays important roles in viral intracellular amplification (16).

An early in silico analysis predicted that FAM46 proteins constituted a non-canonical terminal transferase (NT) family containing PAP/OAS1 SBD domains (17). Structural resolution of

FAM46B, a FAM46C paralog, did not confirm the existence of PAP/OAS1 domains (18) and suggested structural homology to bacterial nucleotidyl transferases. Interestingly, motif analysis of FAM46B scores the presence of VHS (19), GAT (20) and GAE domains. These domains regulate trafficking pathways for cargo retrieval and degradation (21). In short, structural studies suggest that FAM46 family members may also interact with the trafficking machinery.

The NT activity of FAM46C was reported to add short A-tails to the 3’UTR of ER-bound mRNAs encoding for immunoglobulins (22,23). These data favored the model that FAM46C increases mRNA stability and translation of immunoglobulin(s) mRNA at the ER, increasing IgG secretion

(22-24). These studies, largely based on the comparison of wt FAM46C to mock controls, did not show whether all ER-bound mRNAs increased their translational efficiency, and left the

unresolved question whether FAM46C directly binds mRNA. In addition, an unbiased high throughput screening for secretion modifiers, revealed that FAM46A, a close paralog of

FAM46C, represses conventional secretion rather than increasing it (25). Finally, the recent observations that FAM46C may repress oncogenic Akt signaling (26,27) seems consistent both with its tumor suppressor role and with repression of protein synthesis, given the well-known stimulatory role of the PI3K pathway on translation (28). Thus, alternative models for FAM46C function may explain how the tumor suppressor function of FAM46C is specifically linked to the environment of MM.

To answer why FAM46C is lost in MM, we focused on the differences between the phenotypes induced by the wt and mutant forms of the protein and on the molecular partners of FAM46C.

We conclude that FAM46C functions at the crossroads between secretion and autophagy. Our data indicate that FAM46C requires the presence of FNDC3A for its tumor suppressor function and its association with the endoplasmic reticulum. The FAM46C/FNDC3A complex is cytotoxic in MM cells, where it impairs autophagy, causes accumulation of aggregates and apoptosis.

Materials and Methods

Cell lines and culturing

All the cell lines used in this study were obtained from the American Type Culture Collection

(ATCC). Cell line authentication was performed through RT-qPCR on specific target genes and testing for micoplasma contamination was performed monthly through PCR.

Cell lines at passages greater than 10 were not used for the experiments described in this study.

Detailed description of cell lines in Supplementary Methods.

Manipulation of FAM46C and FNDC3A expression

Cell lines expressing inducible constructs of wt and mutant FAM46C alleles were induced by administration of 2g/ml Doxycycline hydrochloride (Sigma cat no. D3447) for up to 8 days.

Lentiviral vectors expressing shRNAs for FAM46C and FNDC3A were obtained by calcium phosphate transient transfection of HEK293T cells. FAM46C and FNDC3A downmodulation was obtained by infection of multiple myeloma cell lines with the respective shRNA lentiviral vectors. Plasmids, lentiviral vectors, transfection and infection procedures are all detailed in

Supplementary methods.

Cell Biology assays

Immunofluorescence stainings were performed on multiple myeloma LP1 cells and HEK cells, using anti-calnexin, anti-FLAG, anti-HA and anti-LC3-B antibodies. FACS analysis was perfomed on multiple myeloma and HEK cells following propidium iodide, aggresome, autophagosome or LAMP1 staining.

Electron microscopy was perfomed in LP-1 cells expressing either wt FAM46C or the D90G mutant allele. The Treg in vitro assay was performed using conditioned media from multiple myeloma cell lines expressing either wt FAM46C or the D90G mutant allele and Treg

Suppression Inspector beads as described in Supplementary Methods.

Detailed procedures for each technique can be found in the Supplementary Methods section.

Xenografts

OPM-2 xenografts were obtained by injecting NOD scid gamma male mice. All experimental procedures were performed complying with national regulations and ethical approval by

IACUC688. Methods and immunohistochemistry are detailed in Supplementary Methods.

Biochemical procedures

Samples derived from LP-1 multiple myeloma cells were prepared for gel filtration analysis as detailed in Supplementary Methods. Nucleus, cytoplasm and ER isolation was performed using the NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Pierce, Rockford, IL, USA) or the

Endoplasmic Reticulum Isolation Kit (Sigma, cat.no ER0100), respectively, as described in

Supplementary Methods. Western blotting and the full list of antibodies used are listed in

Supplementary Methods. The poly(A) tail-length assay was performed using the Poly(A) Tail-

Length Assay Kit (Thermofisher scientific cat.no 764551KT), using the oligonucleotides listed in

Supplementary Methods. Polysomal profiles were prepared by ultracentrifugation of purified polyribosomes loaded on 15%-50% sucrose gradients. The subsequent analysis of translation through polysomal profiling is fully described in the Supplementary Methods section.

The double immuno-purification, mass spectrometry, secretomics and SWATH analyses were performed on LP-1 multiple myeloma or HEK cells. Details are described in Supplementary

Methods.

RNAseq

RNA sequencing was performed on total RNA samples or on RNA extracted from ER fractions.

Microarray analysis was perfomed on total RNAs. RNA extraction techniques, RNAseq analysis, datamining procedures and microarray analysis are all detailed in Supplementary Methods.

Quantitation and Statistical Analysis

All quantitation are expressed as means ±SD. Statistical p-values were calculated by t-tests, NS: p-value > 0.05; * : p-value <0.05, ** : p- value < 0.01, *** : p-value <0.001. The exact type of t- test used can be found in the legend corresponding to each figure.

Results

Expression of wt FAM46C triggers apoptosis and induces the UPR

We screened for multiple myeloma (MM) cells lacking functional FAM46C in order to re-express near-physiological levels of the wt allele. For the screening of endogenous levels of FAM46C, we had to rely on mRNA data and genetic analysis, since we could not specifically detect the endogenous protein, using both in-house produced and three commercially available antibodies, including those from published sources. Examples of these observations are in Supplementary

Fig. S1A and S1B that show a) the lack of correlation between FAM46C protein staining and mRNA levels and b) the lack of loss of FAM46C staining upon shRNA treatment.

After the genetic screening, two cell lines were selected: the LP-1 cell line, which harbors a homozygous deletion of FAM46C and the OPM-2 cell line, which expresses a mutated FAM46C allele accompanied by LOH (Supplementary Fig. S1C). For analysis of FAM46C-induced effects we produced a doxycycline-inducible FLAG-tagged version of the gene. We made sure that the expression levels of the mRNA of C-TERM-FLAG-FAM46C were comparable to those of endogenous FAM46C, at least at the mRNA level (example, Supplementary Fig. S1D), and then opted for confronting wt FAM46C with selected mutant alleles of FAM46C, as found in tumors

(Supplementary Fig. S1E). Given the fact that all mutants gave similar effects on apoptosis and cell cycle progression, the point-mutant variant D90G, one of the most frequently found in MM patients (11) (Supplementary Fig. S1E), was consequently used throughout the project. This approach resulted in a set of observations that pinpoint the differences between tumor suppressor FAM46C and a FAM46C allele that has lost tumor suppressor functions.

Expression of the wt form of FAM46C slowed down cell duplication (Fig. 1A), caused a modest reduction in G1/S cell cycle progression (Supplementary Fig. S1F), inhibited the colony-forming capacity of MM cells plated on soft agar up to 65% (Fig. 1B) and robustly induced apoptosis (Fig.

1C). Expression of neither the D90G mutant allele, nor of other alleles frequently found in MM patients (Supplementary Fig. S1E), had apoptotic effects (Fig. 1C, Supplementary Fig. S1G and

S1H). Given the fact that FAM46C is mutated only in MM, we asked whether the pro-apoptotic effect of FAM46C could be extended to other cell lines or not. Expression of FAM46C in

HEK293 cells did not trigger apoptosis (Fig. 1D) suggesting that FAM46C-induced apoptosis is not due to a general effect.

FAM46C was proposed to polyadenylate and stabilize mRNAs (23). We therefore compared the effects of wt FAM46C to those of the D90G mutant and of a mock control, on gene expression and on the capability to add a poly(A) tail to an acceptor substrate (23). First, we checked if specific mRNAs were more abundant in cells expressing wt FAM46C vs those expressing the

D90G mutant vs the mock. Strikingly, re-expression of wt FAM46C in OPM-2 cells induced a minimal modulation of mRNAs at the single gene level, compared to D90G-expressing cells

(Supplementary Table S1). To detect coordinated changes of gene expression that may give a hint on the proapoptotic changes driven by FAM46C, we performed Gene Set Enrichment

Analysis (GSEA). GSEA revealed that wt FAM46C expression triggered, when compared to the expression of the D90G mutant and the mock, an ER stress and UPR signature (Fig. 1E), in agreement with (12). Many of the induced genes were ATF6 targets (Fig. 1E; annotated

Supplementary Table S1, sheets FAM vs MOCK and UPR). Chop (DDIT3) was mildly but always significantly induced (Supplementary Table S1, sheet CHOP increase).

We tested whether FAM46C was able to add poly(A) to (A)20 RNA substrates when compared to the D90G mutant . We found that, in our hands, the polyadenylation activity of wt FAM46C was: a) just above the background level when compared to mock controls, b) identical to the activity of the loss of function D90G mutant. In the same assay, with as little as 1/10 of exposure time, the activity of E. Coli Poly(A) Polymerase was very robust (Supplementary Fig. S1I). Next, we tested whether FAM46C bound mRNAs, by performing iCLIP analysis. We cross-linked

radioactively labelled RNA to the immunoprecipitated FAM46C and then analyzed by autoradiography the presence of RNA, either naïve or after urea washes. Urea washes allow to discriminate whether FAM46C directly and specifically binds mRNAs. FAM46C bound mRNAs, however, urea washes completely abolished the binding, indicating that FAM46C binding is not direct (Supplementary Fig. S1J).

The combination of a) the strong pro-apoptotic role of the wt versus the mutant, with the lack of a robust effect on the expression levels of specific mRNAs, with b) the lack of difference between the transferase activity of the wt versus the mutant, suggests that polyadenylation cannot account for the tumor suppressor phenotypes induced by wt FAM46C.

However, in agreement with (12,23) our results demonstrate that FAM46C is a tumor suppressor in MM cells, where it induces a peculiar ER stress response through unknown mechanisms.

FAM46C triggers accumulation of protein aggregates

We characterized the relationship between FAM46C and ER stress by defining its cellular localization and the UPR. First, we addressed FAM46C cellular localization. By cell fractionation we found that FAM46C was cytoplasmic (Fig. 2A and Supplementary Fig. S2A), and barely detectable in the nucleus. Nuclear FAM46C could be ascribed to contamination since: a) it correlated with residual cytoplasmic markers and b) it is never visible by immunofluorescence

(Fig. 2B and Supplementary Fig. S2B). Immunofluorescence experiments in MM cells and epithelial cells, demonstrated that FAM46C localizes in the cytoplasm, in an external ring, partly overlapping with ER marker calnexin (Fig. 2B, and Supplementary B). The localization of

FAM46C in close apposition with the ER is consistent with the ER stress signature and leads to

two questions, 1) why and 2) how FAM46C is sorted to the ER in the absence of ER-localization signals.

We addressed the first question. Classically, the UPR is described by three connected branches,

PERK-eIF2, XBP1 splicing and ATF6 translocation, that converge on CHOP (DDIT3) transcription. DDIT3 mRNA was increased in cells expressing wt FAM46C vs the D90G mutant

(Supplementary Fig. S2C). We did not detect clear changes in XBP1 splicing (Supplementary

Fig. S2D). As previously described, ATF6 targets were upregulated (Fig. 1E; Supplementary

Tables S1-S2-S5). SESN2 is an important target, downstream of ATF6 activation (29). Indeed, we confirmed in multiple conditions that SESN2 mRNA is highly upregulated by FAM46C expression (Supplementary Fig. S2E). These data suggest that the ER stress signature driven by

FAM46C occurs mainly through ATF6.

Next, we wanted to define the closest cellular event connected to ER stress. Analytically, an increase in ER stress can be due to increased proteasomal load, which results from increased protein synthesis (Supplementary Fig. S2F). Analysis of ER polysomes showed that FAM46C expression did not stimulate initiation of translation (Supplementary Fig. S2G), moreover, expression of FAM46C had no detectable effect on eIF2 phosphorylation (Fig. 2C), a marker of unfolded protein accumulation in the ER. A second possibility is that FAM46C affects proteasomal efficiency (Supplementary Fig. S2F). We found that polyubiquitin accumulation did not increase in wt FAM46C-expressing cells vs D90G-expressing cells (Fig. 2D), moreover, the sensitivity to proteasome inhibitors was not altered in wt FAM46C-expressing cells

(Supplementary Fig. S2H). Taken together, these data rule out increased protein synthesis load in the ER.

Next, we asked whether FAM46C could induce accumulation of protein aggregates that escaped the proteasome system. By using the Proteostat reagent (30), which detects hydrophobic

stretches of aggregated cytoplasmic proteins, we found that wt FAM46C induces a steady-state accumulation of protein aggregates which peaks 4 days after induction (Fig. 2E and 2F). The formation of aggregates induced by FAM46C expression was confirmed in several cell lines and, consistently, FAM46C downregulation reduced aggregate accumulation (Supplementary Fig.

S2I-K).

FAM46C negatively affects autophagy both in vitro and in vivo

Given the role of autophagy in clearing protein aggregates (31), we checked whether FAM46C expression affected autophagy. The analysis was performed on different cell lines, with similar results. It is known that p62, the classical autophagy receptor, accumulates during autophagic inhibition. We found that FAM46C-expressing cells had an increase in p62 protein abundance, as compared to the mock or to the D90G mutant, as shown in the FAM46C reconstituted cell line LP-1 (Fig. 3A). The number of LC3B puncta is a marker of autophagy. So, next, we double- stained non-synchronized MM cells for LC3B and FAM46C, and quantitated LC3B puncta. The expression of wt FAM46C led to a reduction in LC3B puncta when compared to expression of the D90G mutant (Fig. 3B). Next, we measured the rates of lysosomal degradation by monitoring the ratio of LC3B-II/LC3B-I, which is a specific indicator of autophagic flux, in the presence or absence of autophagic inhibitor Bafilomycin A1 (Baf) which blocks autophagosome- lysosome fusion causing accumulation of autophagosomes. We found that LP-1 cells that re- expressed wt FAM46C had less LC3BII accumulation when compared to cells expressing the

D90G mutant allele (Fig. 3C) both in the presence and absence of Baf indicating a reduction in the autophagic flux. We also showed the inverse correlation by performing shRNA experiments on the RPMI cell line, which naturally expresses normal levels of wt FAM46C. We found that

50% FAM46C downregulation (Fig. 3D, right) caused an increase in the LC3B-II/LC3B-I ratio

(Fig. 3D, left). Reconstitution of FAM46C in OPM-2 cells decreased the LC3BII/LC3BI ratio in the presence of Baf (Supplementary Fig. S3A). To further understand the dynamics of

autophagic inhibition driven by wtFAM46C, we performed FACS analysis using a monodansylcadaverine derivate which labels autophagic vesicles. We found that FAM46C- expressing cells had a reduction in the number of autophagosomes (Fig. 3E). Depletion of

FAM46C with two shRNA increased autophagosome formation in the U266 MM cell line

(Supplementary Fig. S3B). Taken together all these data show that the increase of FAM46C expression causes a decrease of autophagic fluxes in all the analyzed cells, and viceversa. Finally, we tested whether FAM46C could physically interact with the autophagic machinery by immunoprecipitation of FAM46C, followed by LC3B detection in the pulldowns. We found that

FAM46C does not interact with LC3B (Supplementary Fig. S3C).

To check whether the in vitro results could be confirmed in vivo, we performed in vivo experiments in NOD scid gamma mice, which are more supportive to xenograft growth due to their complete immunodeficiency. MM xenograft models were established with the human MM

OPM-2 cell line harboring either wt FAM46C or the D90G mutant doxycycline-inducible constructs (Fig. 4A). We evaluated the effects of FAM46C expression over an 8-day period after doxycycline administration. As expected, cells expressing wt FAM46C produced smaller tumours compared to cells expressing the D90G mutant (Fig. 4B), confirming the oncosuppressant role of FAM46C in vivo. Next, we characterized by immunohistochemistry the tumour biopsies derived from our xenograft mice models. As predicted, FAM46C-expressing tumours had reduced proliferation, as shown by Ki67 staining, and increased apoptosis, as shown by detection of cleaved-caspase 3 (Fig. 4C and Supplementary Fig. S3D). Moreover, we found a drastic increase in p62 abundance only in tumours expressing the wt form of the protein

(Fig. 4C and Supplementary Fig. S3D). Taken together our data indicate that the expression of

FAM46C reduces autophagy , and leads to the accumulation of intracellular aggregates in vitro, and disfavors tumor growth with concurrent accumulation of p62 in vivo. The lack of a direct

interaction of FAM46C with the LC3B suggests that FAM46C effects are due to a crosstalk with the autophagic flux through unknown elements.

FAM46C localizes at the ER through interaction with FNDC3A

We reasoned that the analysis of FAM46C proteomic network could help us to develop testable hypotheses regarding its mechanism. We first addressed whether FAM46C was monomeric or part of a complex. By performing gel filtration chromatography followed by quantitative analysis we found that 40% of FAM46C elutes with large molecular weight (MW) particles (Fig. 5A). As a positive control for gel filtration, we tested eIF6 which associates with ribosomes (32) (Fig. 5A) and, as expected, it elutes with both soluble and large MW particles. In order to find partners of

FAM46C which are relevant for MM progression, we combined: a) data-mining of available databases, b) direct mass-spec studies using the inducible FAM46C construct, c) mutational analysis of MM databases. This stringent approach was meant tohelp pin down FAM46C interactors that have a role in MM. We found that FNDC3A, an ER-associated protein, fulfilled all the three criteria.

In short, we demonstrated the interaction of FNC3A and FAM46C in multiple cell lines and through several independent approaches (Fig. 5A-D; Supplementary Fig. S4A-H). Mass spectrometry identifies FNDC3A as a FAM46C interactor (Supplementary Table S3). We show that recombinant FAM46C is able to bind FNDC3A by in vitro pulldown assays (Fig. 5B).

Immunoprecipitation of FAM46C leads to the co-precipitation of FNDC3A (Fig. 5C).

Immunoprecipitation of FNDC3A leads to the co-precipitation of FAM46C (Supplementary Fig.

S4A). The interaction between FAM46C and FNDC3A is not affected by RNase treatment

(Supplementary Fig. S4B). Colocalization of FAM46C and FNDC3A was visible by immunofluorescence (Fig. 5D). The FAM46C-FNDC3A interaction was also seen in epithelial

cells (Supplementary Fig. S4C) and in several other multiple myeloma cell lines (Supplementary

Fig. S4D-F). These data indicate the unequivocal existence of a FAM46C/FNDC3A complex.

The carboxy-terminus of FNDC3A is similar to the one of ER 'tail-anchored' proteins (33).

Indeed, we found, by co-staining of FNDC3A with the ER luminal marker calnexin, that

FNDC3A localized at the ER (Fig. 5E). These data are in agreement with the in silico topology of

FNDC3A, which harbors a C-terminal transmembrane domain (Fig. 5F), and with proteomics, which further confirm its localization at the cytosolic side of the ER. The co-localization between

FNDC3A and FAM46C was also confirmed in microsomal preparations and by fluorescence microscopy (Supplementary Fig. S4G-H). Given FNDC3A localization, we checked if FAM46C subcellular localization depended on its interaction with FNDC3A. We exploited a FAM46C- mutant that does not efficiently bind FNDC3A, the L288insG (Fig. 5G), and is frequently found in MM patients (Supplementary Fig. S1E). FAM46C-L288insG mutant showed a disrupted ER localization (Fig. 5H). In conclusion, FAM46C interacts with FNDC3A and this interaction may be required for its localization at the ER.

FNDC3A is a MM-specific tumour suppressor required for FAM46C function

Datasets of MM patients (34) show that low mRNA levels of either FNDC3A or FAM46C correlate with decreased overall survival (Fig. 6A;Supplementary Fig. S5A). Loss of FNDC3A is found in the MM genomic DNA dataset of (11), encompassing 203 patients. FNDC3A mutations generate loss-of-function proteins at low frequency (4/203), all mutually exclusive with

FAM46C mutations (Fig. 6B), suggesting a genetic interaction between FAM46C and FNDC3A.

Given that the low frequency of FNDC3A mutations was not sufficient to rule out a random association with the maintenance of wt FAM46C, we tested their genetic interaction in vitro.

Mixing LP-1 MM cells expressing wtFAM46C (90%) with cells devoid of FAM46C (10%) leads to the progressive loss of FAM46C expression, possibly due to the reduced fitness of FAM46C- expressing cells. The same experiment, when performed by mixing 90% of D90G FAM46C- expressing cells with 10%of cells devoid of D90G, does not lead to loss of D90G FAM46C expression (Supplementary Fig. S5B). Next, we tested whether depletion of FNDC3A delayed loss of FAM46C. Indeed, depletion of as little as 50% of endogenous FNDC3A, in this context, resulted in the retained expression of wt FAM46C (Fig. 6C-D), indicating that FNDC3A expression mediates the loss of fitness of FAM46C-expressing cells.

To further investigate, whether FNDC3A acted as a modulator of FAM46C function and as a putative MM tumor suppressor, we screened for MM cell lines that had lost both alleles of

FNDC3A (Supplementary Fig. S5C-D). We found that the Delta47 cell line had lost FNDC3A mRNA expression due to bi-allelic mutations. Re-expression of FNDC3A in Delta47 cells

(Supplementary Fig. S5E), impaired cell growth (Supplementary Fig. S5F) and induced apoptosis (Fig. 6E, top). Analysis of apoptosis in various conditions of FAM46C expression, confirmed that a) reinduction of FAM46C in FNDC3A-mutated Delta47 cells, and of b) FNDC3A in FAM46C-mutated LP-1 cells did not significantly increase apoptosis, confirming the mutual necessity for both proteins (Supplementary Fig. S5G-H). Interestingly, FNDC3A expression in

HEK293 cells did not trigger apoptosis (Fig. 6E, bottom), nor did overexpression of both

FNDC3A and FAM46C (Supplementary Fig. S5I) thus confirming that the tumour suppressor function of the FAM46C/FNDC3A complex is limited to the MM scenario.

Next, we analyzed whether the phenotype induced by the re-expression of FNDC3A in cells that had lost it, but retained normal levels of wt FAM46C, triggered similar effects to the one observed by FAM46C expression (Figs. 2-4). We therefore analyzed the gene expression signature, aggregate formation, and autophagy impairment. RNAseq data indicated

(Supplementary Table S2) that FNDC3A expression, triggered a significant enrichment in GO

terms related to ER stress (Fig. 6F). FNDC3A re-expression caused also protein aggregate accumulation (Fig. 6G). To close the circle, we checked for FNDC3A involvement in autophagy.

Basal autophagy in Delta47 cells was higher than in either OPM-2 or LP-1 cells; this said, expression of FNDC3A caused a reduction in the LC3B-II/LC3B-I ratio (Fig. 6H) and reduced the number of autophagosomes (Fig. 6I). Taken together these data strongly suggest that the

FAM46C/FNDC3A complex coordinately induces autophagic impairment, accumulation of intracellular aggregates and apoptosis.

FAM46C and FNDC3A work in concert with a larger network of ER-associated proteins involved in trafficking

We asked then whether FAM46C/FNDC3A is part of a larger network of proteins and how it might reduce autophagy. We designed a multi-step purification assay based on a double- immunoprecipitation (double-IP) strategy, performing first, a FAM46C pulldown on total lysates and then, FNDC3A immunoprecipitation on the FAM46C purified protein

(Supplementary Fig. S6A). The experiment was done both in cells not undergoing apoptosis,

HEK293T, and cells undergoing FAM46-induced apoptosis (Fig. 1D), to discriminate for potential differential partners and common pathways. Data obtained will be briefly described

(Supplementary Table S3). Copurification was achieved for both FAM46C and FNDC3A

(Supplementary Fig. S6B) in both cell lines (Supplementary Table S3). Enrichment analysis confirms that a fraction of FAM46C is not part of the FAM46C/FNDC3A complex

(Supplementary Fig. S6C), in line with gel filtration experiments (Fig. 5A). Mass spectrometry analysis of the purified complex showed that the more prominent proteins were represented by

FAM46C, FNDC3A, ER-resident proteins and trafficking proteins (Supplementary Table S3).

We performed GO analysis on the list of FAM46C and FNDC3A interactors, and found a strong enrichment in terms related to vacuole structure and to extracellular exosomes (Fig. 7A).

Overall, these data suggest that the function of FAM46C/FNDC3A complex is related to the regulation of secretion, and as such it may indirectly interfere with autophagic secretion (8). We therefore analyzed which secretory pathway is affected by FAM46C.

The FAM46C/FNDC3A complex alters secretion independently from mRNA levels

To provide evidence that the tumor suppression capability of FAM46C was linked to secretory fluxes, we analyzed FAM46C-induced secretome, comparing it to the D90G mutant. In order to assess if secretion depended on mRNA levels on ER polysomes, we performed secretomics paired with RNAseq of ER-associated mRNAs (Fig. 7B and Supplementary Tables S4-S5). By our approach, we may distinguish if increased secretion of a protein follows the rate of translation of its mRNA or is due to a trafficking alteration, namely by scoring if the amount of secreted proteins trends with the amount of increased mRNA on the ER or not.

First, RNAseq data of LP-1 cells expressing wt FAM46C, compared to the D90G mutants fully confirms, as with OPM-2 cells, the presence of an ATF6 signature (Supplementary Table S5, sheet ALL DATA and GO). Unbiased analysis of IgG mRNAs does not confirm the existence of consistent variations in IgG mRNA levels (23) driven by FAM46C (Supplementary Table S5, sheet Immunoglobulins). The secretome of FAM46C-expressing cells vs that of D90G- expressing cells contained several differentially-secreted proteins (Supplementary Table S4).

FAM46C expression caused increased secretion of proteins involved in alternative vesicle- mediated secretion pathways (Fig. 7C; Supplementary Table S4). The secretome showed strong association with the lysosome/exosome, containing Cathepsin B, Legumain and DNase2 and with immune modulation (Supplementary Fig. S6D). The comparison between the mRNA levels present on the ER and the secretome indicated that the lysosomal enzymes Cathepsin B and

Legumain were secreted more efficiently in the presence of increased mRNA levels on the ER

(Fig. 7D; Supplementary Fig. S6E; Supplementary Table S4), but proteins from the secretory pathway like SMOC1 were less secreted (Fig. 7D, left) in spite of significantly increased mRNA

levels (Fig. 7D, right; Supplementary Table S4, sheetRNAseqprot). It has been reported that

FAM46C increases IgG secretion through increased mRNA stability (23). Expression of wt

FAM46C, compared to the D90G mutant, did not alter canonical immunoglobulin secretion

(Supplementary Fig. S6F and Supplementary Table S4). On the same line, we tested by RT- qPCR on polysomes and PAT assays whether LGMN, DNase2, and CATB mRNAs had preferential translation and/or polyadenylation. Within the sensitivity limits of our approach, we confirmed transcriptional changes (Supplementary Fig. S6G, top), but we did not detect preferential loading on polysomes (Supplementary Fig. S6G, bottom) or increased polyadenylation (Supplementary Fig. S6H). Summarizing, our data confirm a connection between the FAM46C complex with secretion, and a crosstalk with autophagic fluxes by lysosomal re-routing or trafficking. In addition, the absence of a strict correlation between mRNA levels and the amount of the corresponding secreted protein favors the conclusion that wt FAM46C alters secretory fluxes independently from translational efficiency and mRNA stability. We also rule out a relationship between the tumor suppressor capability of FAM46C and the increased secretion of IgG proteins.

To test if lysosomal exocytosis, as suggested by DNas2 in the medium, was increased by

FAM46C expression, we measured the levels of membrane-bound LAMP1, a lysosomal marker enriched at the plasma membrane during lysosomal exocytosis(35). Plasma membrane-bound

LAMP1 was higher in FAM46C- vs D90G-expressing cells (Fig. 7E). Last, we tested if the immune modulation signature predicted by the bioinformatics analysis was accompanied by changes in the biological activity. Since conditioned media from MM cells is capable of favouring differentiation of Treg cells and immunosuppression, we tested if Treg polarization could be inhibited by the FAM46C-driven secretome. Indeed, co-colturing MM cells expressing wt

FAM46C with differentiating CD4+ Tregs (Supplementary Fig. S6I, left), caused an overall reduction of their differentiation efficiency compared to co-colturing with cells expressing the

D90G mutant allele (Supplementary Fig. S6I, center). The effect did not require cell-to-cell interaction, as it was seen also using conditioned media (Supplementary Fig. S6I, right). We conclude that the FAM46C/FNDC3A complex alters intracellular trafficking, and suggest that it diverts lysosomes from aggresome-autophagosome fusion to exocytosis.

In the attempt to visualize alterations of intracellular structures driven by trafficking or autophagic inhibition, we performed electron microscopy imaging on MM cells expressing either wt FAM46C or the D90G mutant. We found that MM cells expressing wt FAM46C had intracellular peculiarities, including: dilated ER cisternae, vacuole structures and small-sized vesicles, such as endosomes (Fig. 7F), which are compatible with altered secretion and/or vesicular transport and simultaneous accumulation of protein aggregates.

The FAM46C/FNDC3A complex alters sensitivity to sphingosine kinase inhibitors

The previous experiments underlining changes in autophagic and secretory pathways, suggest that reconstitution of the FAM46C/FNDC3A complex may also alter the pharmacological sensitivity of MM cells. To unveil potential pharmacological weaknesses of MM cells expressing a functional FAM46C/FNDC3A complex, we performed a connectivity map analysis [35] that predicted sensitivity to inhibitors of the sphingosine kinase (SK) pathway, DL-PMP and SA-

792728 (Supplementary Fig. S7A). Hypothesizing that the complex could affect the sensitivity to

SK inhibitors, we tested the effects of SK inhibition on cells reconstituted or depleted for the

FAM46C/FNDC3A complex. Treatment with SK1-I, which inhibits both SK1 and SK2, inhibited cell growth of FAM46C-expressing LP-1 cells (Supplementary Fig. S7B) and OPM-2 cells

(Supplementary Fig. S7C) compared to D90G-expressing cells. In agreement with these data, the down-modulation of FAM46C in U266 MM cells, which express physiological levels of wt

FAM46C, caused a decrease in the sensitivity to SK inhibitors (Supplementary Fig. S7D). Re- expression of FNDC3A in Delta47 cells also increased sensitivity to SK1-I (Supplementary Fig.

S7E) treatment, thus suggesting that FAM46C/FNDC3A complex alters sensitivity to specific inhibitors.

Discussion In this work we have collected data that indicate the existence of the novel FAM46C/FNDC3A complex, on the cytoplasmic side of the endoplasmic reticulum. The FAM46C/FNDC3A complex alters trafficking and secretion and, in MM cells, it interferes with the autophagic flux, leading to aggregate formation and apoptosis. Indeed, it is well known that autophagy-relevant genes broadly interact with the trafficking machinery (36). The FAM46C/FNDC3A complex, in MM cells, may steer lysosomes from fusion with autophagosomes to the plasmamembrane, thus impairing autophagy.

FAM46C is lost or mutated in more than 10% of MM patients, but not in other cancers (9-11), raising the intriguing question of which specific process makes MM cells sensitive to FAM46C expression. To answer this question, we characterized the suppressor function of wt FAM46C in comparison with loss of function alleles, such as the D90G mutant. We then searched for

FAM46C potential role by combining proteomics, genetics and cell biology data. Our conclusion is that the proapoptotic role of FAM46C in MM cells is accompanied by an accumulation of protein aggregates due to impaired autophagy. Our data sustain the hypothesis that FAM46C is part of a multi-protein network that regulates secretion, and its interaction with FNDC3A is necessary for its tumor suppressor role. Three results confirm the physiological relevance of the

FNDC3A/FAM46C complex: a) re-expression of FNDC3A in MM cells that had naturally lost the gene, recapitulates the effects of FAM46C re-expression, b) depletion of FNDC3A in cells with functional FAM46C leads to increased survival, and c) loss of interaction between FAM46C and

FNDC3A results in defective FAM46C localization. Finally, despite the low frequency of

FNDC3A mutations in MM patients, high FNDC3A levels positively correlate with OS. In summary, FAM46C/FNDC3A is a new MM-specific tumour suppressor complex.

The proapototic role of FAM46C in MM is well established (12,23,24), as well as the high incidence of FAM46C mutations (9-11). All the studies published so far, revealed that expression of FAM46C in MM cells, causes an ER-stress signature (12,26). One possible cause of this signature was an increased proteasome burden due to the increased translation of IgG mRNAs.

In general, others and us have confirmed that the sensitivity of MM cells to proteasome inhibitors is not affected by FAM46C expression (12,26). The lack of increased sensitivity to proteasome inhibitors is in line with the observations that wt FAM46C does not induce a general increase of immunoglobulins mRNA translation on ER-resident ribosomes. It should be noted that this finding does not challenge the general conclusion that, through other mechanisms, the expression of FAM46C modulates the transition from immature B cells to antibody- producing plasmacells (22). The transition from a B cell to an antibody-producing plasmacell requires a striking remodelling of the trafficking apparatus, with an increase in the endoplasmic reticulum and of the endocytic pathway, which is required for membrane homeostasis. FAM46C increases lysosomal secretion, reduces the autophagic flux, causes accumulation of protein aggregates and triggers programmed cell death. All these phenomena suggest that, by diverting lysosomal trafficking toward secretion, rather than to autophagosome-lysosome fusion, FAM46C may inhibit a pro-survival autophagy route. Finally, autophagic inhibition driven by FAM46C was proapoptotic only in MM cells. This observation indicates that the FAM46C/FNDC3A complex alters secretory pathways that interfere with lysosomal activity, but also that this event becomes rate limiting for survival only in the setup of a cell that is subject to proteotoxic stress.

The physiological role of the FAM46C/FNDC3A complex is intriguing. Several data point to the existence of multiple FAM46/FNDC3 complexes which regulate trafficking and secretion.

FAM46C is part of a family of proteins which includes FAM46A, FAM46B and FAM46D.

FAM46C knockout leads to the disassembly of the manchette, a structure essential for trafficking (37) and FAM46A depletion affects BMP signaling (38). Analysis on the protein sequence of FAM46B homolog F7E7M3 (http://www.rcsb.org/pdb/protein/F7E7M3) shows the presence of VHS (19), GAT (20) and GAE domains (39), which are typical of proteins necessary for transport and lysosomal targeting. Concerning FAM46 family partners, high throughput proteomic studies have shown that FAM46A interacts with both FNDC3A and a closely related gene, FNDC3B (40). Secretion of surfactant-associated proteins that are associated with lamellar bodies, lysosome-related organelles, is decreased in the lung of FNDC3B knockouts

(41). FNDC3A depletion alters the deposition of the extracellular matrix (42), a phenomenon requiring remodeling by secreted proteases. Last, genetic studies suggest the existence of specific complexes in specific organs. Deletion of either FNDC3A (33) or FAM46C causes male sterility and abnormal sperm maturation (37), whereas a bone defect is observed with either

FAM46A or FNDC3B knockouts (43,44). In the context of B cells, lysosomal exocytosis has been suggested to regulate antigen extraction at the immunological synapse (45). In conclusion, each cell may have a unique combination of FAM46 and FNDC3 isoforms that may become rate limiting for secretion-related phenotypes.

An unbiased search for potential sensitivities of FAM46C-expressing cells, by connectivity map analysis (46), predicted also a potential sensitivity to sphingosine kinase inhibitors.

Experimental data confirmed the prediction, since we provide evidence that the expression of physiological levels of the FAM46C/FNDC3A complex increases sensitivity to SK1-I. The connection between MM, FAM46C, lysosome biology and sphingosine kinases is new, but not totally surprising. Sensitivity of MM cells to sphingosine kinase inhibitors has been recently proposed (47,48). ATF6, a major mammalian UPR sensor, is activated by specific sphingolipids

(49), and is also robustly induced by re-expression of either wt FAM46C or FNDC3A. Gaucher

disease, which affects lysosomal processing of sphingolipids has an increased and unexplained risk of development of MM (50). We hypothesize that clinical studies of their efficacy may take in account genetic data regarding the components of the FAM46C/FNDC3A network.

The final exciting question relates to the enzymatic activity of FAM46C. Our work shows a lack of correlation between FAM46C nucleotidyl transferase activity, and its tumor suppressor capability when compared to the D90G loss-of-function mutant. Is there a possibility that the enzymatic activity of FAM46C is different? Structural and biochemical studies have shown that

FAM46B, a FAM46C paralog, has a much higher nucleotidyl transferase activity, compared to

FAM46C, and similarities to bacterial nucleotidyl transferases (18). One possibility is that the

D90G mutation alters the specificity of FAM46C to unknown substrates. In this context, the known superfamily of transferases capable to bind ATP, the substrate of FAM46B (18), is composed of more than 40 different sub-families including: protein kinases, lipid kinases and others. Up to now, in spite of the lack of homology of FAM46 members to eukaryotic nucleotidyl transferases, we have only tested the possibility that FAM46C acts as a nucleotidyl transferase.

The weak activity of FAM46C (18) may be therefore due to the fact that its correct substrate still needs to be identified.

Author Contributions

Conception and design: Nicola Manfrini, Stefano Biffo, Marilena Mancino

Acquisition of data: Nicola Manfrini, Marilena Mancino, Annarita Miluzio, Stefania Oliveto,

Matteo Balestra, Piera Calamita, Marco Sassoè-Pognetto, Chiara Salio, Alessandro Cuomo,

Marcello Manfredi, Elia Ranzato, Simona Martinotti

Analysis and interpretation of data: Nicola Manfrini, Marilena Mancino, Roberta Alfieri,

Riccardo L. Rossi, Tiziana Bonaldi, Emilio Marengo, Marco Sassoè-Pognetto, Chiara Salio,

Davide Cittaro, Giovanni Tonon, Stefano Biffo.

Writing, review and/or revision of the manuscript: all authors

Study supervision: Stefano Biffo

Acknowledgments

We would like to thank Ilaria Mariani and Deborah Salvi Mesa for their initial help with

FNDC3A downmodulation experiments and fluorescence microscopy experiments; Alessia

Tommasini and Eugenio Graceffo for technical support in mass spectrometry analysis of

FAM46C and FAM46C/FNDC3A interactomics; Mariacristina Crosti for her help with FACS experiments and Chiara Cordiglieri for supervision with fluorescence microscopy; Simone Cenci and Enrico Milan for help with early autophagic analysis. This paper was supported by grant

AIRC IG 19973 to SB, by AIRC 9965 5 ‰ to GT, and MIUR project “Dipartimenti di Eccellenza

2018 – 2022” to Dept. of Neuroscience “Rita Levi Montalcini” to M S-P.

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Figure legends

Figure 1. FAM46C is a MM specific tumour suppressor whose expression triggers apoptosis and a UPR signature. A) Population doublings and B) colony forming capacity of

FAM46C- and D90G-expressing MM cells. C) Apoptotic rates of MM cells expressing either wt

FAM46C or different FAM46C mutant alleles. D) Apoptosis induction in LP-1 (left) and HEK cells (right) expressing either wt FAM46C or the D90G mutant allele. Representative FACS histograms (above) and bar graphs (below) are shown. F AM46C induces apoptosis only in MM cells E) Gene set enrichment analysis (GSEA) on data derived from OPM-2 multiple myeloma cell lines expressing either wt FAM46C or the D90G mutant allele. Data are means ± SD of at least three independent experiments. Statistical p-values were calculated using double-tailed unpaired t tests. (ns: p-value >0.05; *: p-value <0.05; **: p-value < 0.01; ***: p-value < 0.001).

Figure 2. FAM46C localizes in a ring external to the ER and its expression causes accumulation of protein aggregates. A-B) FAM46C is cytoplasmic in a ring external to calnexin. A) Analysis of FAM46C in nuclear and cytoplasmic fractions of LP-1 cells expressing wt FAM46C. FAM46C was detected using anti-FLAG antibodies. Lamin B, nuclear marker; tubulin, cytoplasmic marker. B) Fluorescence microscopy of LP-1 cells stained for FAM46C and

ER marker calnexin. Nuclei were stained with DAPI. Enlargement inset is shown. Scale-bar:

7m, inset scale bar: 3.5m C-F) FAM46C induces aggregates without affecting eIF2a phosphorylation C) Representative western blot of P-eIF2 levels in LP-1 cells. D)

Representative western blot of poly-ub protein levels in LP-1 cells. Histograms (E) and fluorescence (F) of protein aggregates in LP-1 cells expressing wt FAM46C or the D90G mutant

allele. Data are means ± SD of at least three independent experiments. Scale-bar: 7µm.

Statistical p-values were calculated using double-tailed unpaired t tests. (ns: p-value >0.005; *: p-value <0.05; **: p-value < 0.01; ***: p-value < 0.001).

Figure 3. FAM46C inhibits autophagy in vitro. A) (Left) Representative western blot showing the increase of p62 levels in LP-1 cells expressing wt FAM46C or the D90G mutant allele and relative quantitation (Right). B) (Left) Fluorescence microscopy images of LC3B in

LP-1 cells expressing wt FAM46C or the D90G mutant, showing the reduction of LC3B puncta

(Right). Scale bars: 12m. C-D) Assays showing the reduction of autophagic LC3BII/LC3BI ratio driven by FAM46C. Representative western blot of LC3B-I and LC3B-II levels in LP-1 cells expressing wt FAM46C or the D90G mutant. LC3B-II/LC3B-I ratios are shown. D) (Left)

Western blot of LC3B-I and LC3B-II levels in RPMI multiple myeloma cells depleted of FAM46C

(sh1 and sh2) or a scramble control. LC3B-II/LC3B-I ratios are shown. (Right) RT-qPCR for

FAM46C mRNA levels. E) MM cells expressing either wt FAM46C or the D90G mutant allele were treated with 50M chloroquine or 0,1% DMSO (solvent control, -), stained with a monodansylcadaverine analogue and analyzed by flow cytometry. FAM46C reduces autophagic vesicle formation induced by chloroquine. Data are means ± SD of at least three independent experiments. Statistical p-values were calculated using double-tailed unpaired t tests. (ns: p- value >0.005; *: p-value <0.05; **: p-value < 0.01; ***: p-value < 0.001).

Figure 4. FAM46C expression in vivo is associated with increased apoptosis and p62 accumulation. A) In vivo model of xenografts. Experimental scheme. B) Effects of

FAM46C expression on tumour growth in vivo. N=10. C) Immunohistochemistry images of tumour biopsies derived from mice models described in Fig. 4A, analyzed for Ki67 (proliferation marker), cleaved caspase-3 (apoptosis marker), p62. (Right) Quantitation of p62. Scale bars

50m. Statistical p-values were calculated using double-tailed unpaired t tests. (ns: p-value

>0.005; *: p-value <0.05; **: p-value < 0.01; ***: p-value < 0.001)

Figure 5. FAM46C localizes at the ER through interaction with FNDC3A. A) (Top)

Gel filtration chromatography shows that part of FAM46C elutes in a non-monomeric form. MW of fractions is indicated. (Bottom) Histograms represent quantification of FAM46C and eIF6 protein levels. B) Pulldown experiment. Protein lysates derived from LP-1 cells expressing

FNDC3A were loaded on a FAM46C resin. Proteins interacting with FAM46C were eluted and analyzed. Controls are indicated. C) Representative blot of co-immunoprecipitation experiments performed in LP-1 cells. IP for FLAG-FAM46C, western blotting for HA-FNDC3A. D) Double fluorescence images of LP-1 cells expressing FAM46C-FLAG and FNDC3A-HA. Inset is shown.

E) Double fluorescence images of cells expressing FNDC3A-HA and stained with anti-calnexin antibodies. Inset is shown. F) Scheme of FNDC3A ORF. G-H) FAM46CL288insG poorly binds

FNDC3A and loses ER localization. G) Representative blot of co-immunoprecipitation experiments performed in HEK cells expressing a FAM46CL288insG. H) Fluorescence microscopy images of cells transfected with either wt FAM46C-FLAG (top) or the FAM46C-

L288insG-FLAG mutant allele (bottom).Nuclei were stained with DAPI and cytoplasms with phalloidin. Insets show enlargement. Scale-bar: 7m; inset scale bars: 3.5 m. At least three independent experiments were performed for all data. Statistical p-values were calculated using double-tailed unpaired t tests. (ns: p-value >0.005; *: p-value <0.05; **: p-value < 0.01; ***: p- value < 0.001).

Figure 6. FNDC3A is a novel tumour suppressor which is required for FAM46C function and which induces apoptosis, reduces autophagy and increases protein aggregate formation.

A) Kaplan-Meier survival curves of MM patients clustered for high or low levels of FNDC3A.

Data from (34). B) FNDC3A mutations found in 204 MM patients, retrieved from (11). The panel on the left shows the type of mutation found in FNDC3A and in other genes frequently mutated in MM. The panel on the right shows the mutational status of FNDC3A in patients harbouring deletions of FAM46C. C-D) Genetic interaction between FNDC3A and FAM46C, experimental scheme (C) and results (D). FAM46C expressing cells were mixed 9:1 with normal cells, with or without FNDC3A depletion. FAM46C expression is retained in the absence of

FNDC3A indicating the improved fitness of FAM46C expressing cells in the absence of FNDC3A.

Downmodulation of FNDC3A was 50% as detected by RT-qPCR (left). E) Apoptosis induction in

Delta47 (left) and HEK cells (right) expressing either FNDC3A or a mock control. FNDC3A is pro apoptotic only in MM cells F) FNDC3A Gene ontology (GO) term enrichment analysis. G)

Bar graphs representing the level of protein aggregates in Delta47 cells expressing either

FNDC3A or a mock control. H-I) Reduced autophagic flux in MM Delta47 cells re-expressing

FNDC3A, shown by western blot of LC3B-I and LC3B-II levels (H) or by monodansylcadaverine staining (I). Statistical p-values were calculated using two-tailed t-tests (ns: p-value > 0.05; *: p- value < 0.05; **: p-value < 0.01; ***: p-value < 0.001).

Figure 7. FAM46C and FNDC3A are part of a network of ER-associated proteins regulating secretion and trafficking. A) Gene ontology (GO) term enrichment analysis of proteins detected by mass spectrometry in the sequential purification of FAM46C and FNDC3A.

B) Experimental setup of ER RNAseq/secretomics approach. C-E) FAM46C increases unconventional and lysosomal secretion C) GO of differentially secreted proteins of LP-1 MM cells expressing wt FAM46C vs the D90G mutant. D) Levels of differentially secreted proteins

between cells expressing wt FAM46C and the D90G mutant and relative mRNA levels. Blue bars represent the levels of proteins which are more abundant in the secretome of FAM46C- expressing cells (left) and their relative mRNAs (right), black bars represent levels of proteins which are more abundant in the secretome of D90G-expressing cells (left) and their relative mRNAs (right). Refer to Supplementary Tables S4-S5. E) Plasma membrane-bound LAMP1 levels in LP-1 cells expressing either FAM46C or the D90G mutant allele. F) Representative electron microscopic images of LP-1 cells expressing either wt FAM46C or the D90G mutant allele. Structures with altered morphology in FAM46C-expressing cells are shown with higher magnification. Scale bars: 0.5 and 1m. Statistical p-values were calculated using two-tailed t- tests (ns: p-value >0.05; *: p-value < 0.05; **: p-value <0.01; ***: p-value <0.001). A minimum of three independent samples for each experiment was done.

FAM46C and FNDC3A are multiple myeloma tumor suppressors that act in concert to impair clearing of protein aggregates and autophagy

Nicola Manfrini, Marilena Mancino, Annarita Miluzio, et al.

Cancer Res Published OnlineFirst September 22, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-20-1357

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/09/22/0008-5472.CAN-20-1357.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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