MO-PHDMM-1 PROPOSAL AS DIRECTOR OF STUDIES & Rev. 04 del 19/03/2021 RESEARCH PROJECT Page 1 di 2

PROJECT 1

DoS: Enrico Milan

Title: Harnessing production against dyscrasias

Curriculum: Cellular and Molecular Biology

Link to OSR/UniSR personal page: https://www.unisr.it/offerta-formativa/medicina-chirurgia/post-lauream/dottorato-medicina-molecolare/director-of- studies/milan-enrico

Project description (Number of characters, including spaces: 2.000 - 3.000): Systemic light chain (AL) is a plasma cell (PC) dyscrasia caused by the clonal production of an aggregation-prone immunoglobulin (Ig) light chain (LC), which deposits systemically in tissues resulting in organ dysfunction and death. The abundant misfolded byproducts of intense Ig production cause a critical dependence of normal and pathological PCs on pathways that maintain proteostasis. Such non-oncogene addiction has relevant therapeutic implications: indeed, the clinical use of proteasome inhibitors (PI) has showed extraordinary efficacy against AL and the PC (MM). Dissecting how PCs regulate the secretory capacity, in efficient coordination with degradative pathways, may be fundamental to disclose new therapeutic targets against AL. An attractive target is autophagy – a highly conserved lysosomal recycling strategy capable to compensate for proteasome insufficiency. Autophagy engulfs through dedicated receptor intracellular substrates in double-membrane vesicles (autophagosomes) subsequently delivered to the lysosome for content degradation and recycling. Notably, we discovered that intense constitutive autophagy is essential for normal and pathological PC survival and that the receptor for ubiquitinated , SQSTM1/p62 is a critical mediator of the autophagic clearance of ubiquitin-conjugated proteins and affords specific protection against PI (Pengo et al., 2013; Milan et al., 2015; Oliva et al., 2017). Dissecting the specific degradative targets of p62 in PCs, we identified a previously unrecognized interaction with MM-specific tumor suppressor FAM46C/TENT5C (Fucci et al., Cell Reports 2020). We disclosed that FAM46C is a poly(A)polymerase that localizes at the endoplasmic reticulum (ER) membrane interacting with FNDC3 proteins. At the ER, FAM46C stabilizes mRNAs encoding for Igs and ER-targeted proteins acting as a rapid and potent tuner of ER expansion and antibody secretion. A project is available to dissect the role of FAM46C-associated circuits in regulating endoplasmic reticulum (ER) homeostasis and antibody secretion in amyloidogenic and malignant PCs.

Skills to be acquired by the student: The PhD candidate will adopt in vitro and in vivo models of AL amyloidosis to evaluate the effect of p62 and its interactors in order to disclose new therapeutic targets to reduce toxic LC secretion or AL cell survival. Moreover, in collaboration with the Italian Referral Center for Systemic Amyloidoses in Pavia, the Italian leading institution for AL study and treatment, the student will analyze the genomic and proteomic profile of AL primary samples in order to discover novel correlations with patient clinical history and treatment response. Skills to be acquired by the student: -Cell culture - Molecular biology techniques (qRT-PCR, cloning, lentiviral transfection, gene sequencing) - Protein biochemistry techniques (Western Blot, Immunoprecipitation, Immunofluorescence, SILAC-based MO-PHDMM-1 PROPOSAL AS DIRECTOR OF STUDIES & Rev. 04 del 19/03/2021 RESEARCH PROJECT Page 2 di 2 quantitative proteomics, generation and expression of mutant proteins) -Ex vivo purification and analysis of human primary samples -In vivo analysis in AL models.

References (max. 3)

Fucci C, Resnati M, Riva E, Perini T, Ruggieri E, Orfanelli U, Paradiso F, Cremasco F, Raimondi A, Pasqualetto E, Nuvolone M, Rampoldi L, Cenci S, Milan E. The interaction of the tumor suppressor FAM46C with p62 and FNDC3 proteins integrates protein and secretory homeostasis. Cell Reports 2020 Sep 22;32(12):108162. doi: 10.1016/j.celrep.2020.108162. PMID: 32966780

Milan E, Perini T, Resnati M, Orfanelli U, Oliva L, Raimondi A, Cascio P, Bachi A, Marcatti M, Ciceri F, Cenci S. A Plastic SQSTM1/p62-Dependent Autophagic Reserve Maintains Proteostasis and Determines Susceptibility in Multiple Myeloma Cells. Autophagy 2015; 11(7):1161-78

Pengo N, Scolari M, Oliva L, Milan E, Mainoldi F, Raimondi A, Fagioli C, Merlini A, Mariani E, Pasqualetto E, Orfanelli U, Ponzoni M, Sitia R, Casola S, Cenci S. Plasma cells require autophagy for sustainable immunoglobulin production. Nat Immunol. 2013; 14(3):298-305.