The IMiD target CRBN determines HSP90 activity toward transmembrane proteins essential in multiple myeloma

•CRBN functions as a transmembrane protein-specific co-chaperone of HSP90•Disruption CRBN-HSP90 interaction determines the anti-tumor activity IMiDs•The CD98hc/LAT1 complex is central target IMiDs in multiple myeloma•CD98hc-Anticalin theranostic tool myeloma The architecture proteins requires quality control (QC) folding, membrane positioning, and trafficking prerequisites for cellular homeostasis intercellular communication. However, it has remained unclear whether QC hubs exist. Here we identify cereblon (CRBN), immunomodulatory drugs (IMiDs), that specifically chaperone HSP90 toward by means counteracting AHA1. This function abrogated IMiDs, which disrupt CRBN with HSP90. Among protein clients CRBN-AHA1-HSP90 revealed cell surface proteomics, amino acid transporter LAT1/CD98hc determinant IMiD (MM) present an Anticalin-based CD98hc radiopharmaceutical MM radio-theranostics. These data establish axis biogenesis proteins, link to tumor metabolism, nominate LAT1 attractive diagnostic therapeutic targets MM. Faithful (TPs) vital homeostasis, adhesion, migration, communication (Guna Hegde, 2018Guna A. Hegde R.S. Transmembrane domain recognition during control.Curr. Biol. 2018; 28: R498-R511Abstract Full Text PDF PubMed Scopus (41) Google Scholar). Aberrant TPs have been established cancer hallmarks are successfully targeted immunotherapeutic molecular treatments (Lin et al., 2019Lin C.Y. Lee C.H. Chuang Y.H. J.Y. Chiu Y.Y. Wu Jong Y.J. Hwang J.K. Huang S.H. Chen L.C. al.Membrane protein-regulated networks across human cancers.Nat. Commun. 2019; 10: 3131Crossref (22) Central tasks TP include folding extra-membrane domains well insertion hydrophobic domains, thereby avoiding aggregation erroneous interactions (Cymer 2015Cymer F. von Heijne G. White Mechanisms integral folding.J. 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Langlais Luo Jedlowski al.Identification cereblon-binding relationship response survival after myeloma.Blood. 124: 536-545Crossref (130) led development IMiD-based proteolysis-targeting chimeras (PROTACs), between CRL4CRBN typically undruggable neo-targets induce (Winter 2015Winter G.E. Buckley D.L. Paulk Roberts Souza Dhe-Paganon development. Phthalimide conjugation strategy vivo degradation.Science. 348: 1376-1381Crossref (773) pleiotropic effects adverse teratogenicity neurotoxicity, important striking synergy proteasome inhibitors (PIs) suggest additional mechanisms action. Along line, structure CRBN, comprises large LON shared Clp family chaperones Lon proteases (Smith 1999Smith Baker T.A. Sauer R.T. share homologous substrate-recognition domains.Proc. Natl. Acad. Sci. U S 1999; 96: 6678-6682Crossref (159) Scholar), next pocket binding (TBD), suggests beyond activity. Indeed, previously found exist ligase-independent complexes, where stabilizes two TPs, CD147 (also known basigin) monocarboxylate (MCT1) (Eichner 2016Eichner Heider Fernández-Sáiz van Bebber Garz Lemeer Rudelius Targosz Jacobs Knorn A.M. cereblon-CD147-MCT1 exert antitumor teratogenicity.Nat. Med. 22: 735-743Crossref (93) We further demonstrated interfere competitive manner teratotoxic confers other whose functional loss may contribute efficacy. set out study Starting approach combined unbiased proteomics interactome screens, identified global TP-specific network comprising Herein, regulates antagonizing AHA1, allows correct maturation TPs. various novel CRBN/HSP90/AHA1 candidates, specify (SLC7A5)/CD98hc (SLC3A2) new vulnerability MM, destabilized inactivated upon treatment. thus metabolism characterize radio-theranostic approaches. To start investigating impact performed quantitative proteomic screens proteome using biotinylation, streptavidin affinity purification, mass spectrometry (Figures 1A, S1A, S1B). Strikingly, observed broad alterations abundance (Figure 1A). cross-validated our previous dataset Scholar) order distinguish clients. yielded (CD98 heavy chain) significant hit 1A; Figure S1C). forms heterodimeric LAT1, preferentially transports neutral branched or aromatic acids (Yan 2019Yan Zhao Lei Zhou Q. Structure LAT1-4F2hc heteromeric complex.Nature. 568: 127-130Crossref (97) levels progression disorders associated high proliferation poor prognosis patients (Bolzoni 2016Bolzoni Accardi Vescovini Airoldi Storti Todoerti Agnelli Missale Andreoli al.Dependence glutamine uptake addiction cells: target.Blood. 128: 667-679Crossref (73) Isoda 2014Isoda Kaira Iwashina Oriuchi N. Tominaga Nagamori Kanai Oyama Asao Matsumoto Sawamura Expression L-type (LAT1) prognostic indicator myeloma.Cancer 105: 1496-1502Crossref (36) validate screen, treated thalidomide, lenalidomide, substantial destabilization 1B). therefore went investigate potential CRBN-dependent proteins. CRISPR-Cas9-mediated depletion reduced 1C). Unchanged elevated mRNA confirmed post-transcriptional independent neo-substrate transcription factors S1D). subcellular mis-localization cytoplasmic accumulation at ER/Golgi, indicative 1D, S1E, S6F, S6G). 1E, 1F, S1F) mapped site intracellular regions S1F). binding-defective CRBNYW/AA (mutation Tyr384 Trp386 Ala) failed interact compete CRBNs TBD 1G S1G). Together, cross-validated, biochemical experiments interactor elucidated mediates Further interrogation hypothesized involved HSP90-AHA1 complex. HSP90α endogenous 2A S2A). Importantly, ruled possibility client. Although glucocorticoid receptor concentration- time-dependent induced HSP70 (Sittler 2001Sittler Lurz Lueder Priller Lehrach Hayer-Hartl M.K. Hartl F.U. Wanker E.E. Geldanamycin inhibits huntingtin culture model Huntington’s disease.Hum. Genet. 2001; 1307-1315Crossref (351) stable S2B S2C) did not accumulate insoluble fraction S2D). Using bidirectional mapping studies, C-terminal S3A S3B) bind DDB1-binding region S3C–S3F). Notably, also co-precipitated S3C). relation cycle, made use mutants keep locked (HSP90E47A) open (HSP90D93A) (Panaretou 2002Panaretou Siligardi Meyer Maloney Sullivan Singh Millson Clarke P.A. Naaby-Hansen Stein al.Activation stress-regulated 2002; 1307-1318Abstract (407) conformation, pattern p23 2B) (Ali 2006Ali Crystal Hsp90-nucleotide-p23/Sba1 440: 1013-1017Crossref (656) A point (HSP90V410E) (Retzlaff interaction, supporting 2B). Molybdate nucleotide-bound conformation-specific co-chaperones (Sullivan 1997Sullivan Stensgard Caucutt Bartha McMahon Alnemri Litwack Toft Nucleotides states hsp90.J. Chem. 1997; 272: 8007-8012Abstract (222) 2015Sun Hartson S.D. Matts R.L. HeLa proteomics.Biochim. Biophys. Acta. 1854: 365-380Crossref (10) addition molybdate extracts markedly stabilized confirming 2C S3G). contrast, proposed changed S3H S3I). if both just protomer within dimer, chose based overexpressing either HA-tagged FLAG-tagged HSP90WT protomers HA-HSP90WT FLAG-HSP90V410E (abrogating HSP90) 2017Woodford Sager Marris Dunn Blanden A.R. Murphy Rensing Shapiro O. al.Tumor suppressor Tsc1 kinase non-kinase clients.EMBO 36: 3650-3665Crossref (32) sequential FLAG HA immunoprecipitation (IP), isolated dimers consisting wild-type FLAG-HSP90WT 2D, lane 2) mutated 3). As opposed asymmetrically require only protomer, 2D). Next, investigated HSP90-CRBN interaction. almost completely while unaffected 2E, 2F, S4A, S4B). inhibitor geldanamycin (GA) mimics ADP-bound state, inhibiting locking cycle early (Neckers, 2003Neckers Development small inhibitors: utilizing forward reverse chemical genomics identification.Curr. 2003; 733-739Crossref (84) Zhang 2008Zhang Hamza Cao Yu Zhan Hsp90/Cdc37 against pancreatic cells.Mol. Ther. 2008; 7: 162-170Crossref (312) GA analog 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) suppressed association CRBNWT CRBNYW/AA, 2E 2F). findings interacts nucleotide bound, domain. At same YW motif containing abrogates CRBN. Because implicated reasoned stabilizing axis. phosphorylation-dependent phospho-mimic form (AHA1Y223E) (Wolfgeher 2015Wolfgeher Bourboulia Bratslavsky Mollapour Kron S.J. dynamic Y223 phosphorylation.Data Brief. 5: 752-755Abstract (5) Phosphorylation enhances leading increased compromised test affects interplay, first AHA1Y223E 3A). Surprisingly, contrast affect CRBN-AHA1 additionally 3B S4C). introduction AHA1E67K mutation, disrupts alter AHA1-CRBN 3C S4D), change S4E). excluded indirectly interacting performing studies 3D S4F). independently tested effect stability CFTR. forced reversed negative whereas 3E 3F). Both (CRBNYW/AA) DDB1 (CRBNΔmid) had no AHA1-induced destabilization, excluding any unspecific 3E). showed S4G), line directly vitro, determined HSP90-AHA1, applying assay detects ATP hydrolysis