Summary p97 is a AAA-ATPase with multiple cellular functions, one of which is critical regulation of protein homeostasis pathways. We describe the characterization of CB-5083, a potent, selective and orally bioavailable inhibitor of p97. Treatment of tumor cells with CB-5083 leads to accumulation of poly-ubiquitinated proteins, retention of endoplasmic reticulum associated degradation (ERAD) substrates and generation of irresolvable proteotoxic stress leading to activation of the apoptotic arm of the unfolded protein response (UPR). In xenograft models, CB-5083 causes modulation of key p97-related pathways, induces apoptosis and has antitumor activity in a broad range of both hematological and solid tumor models. Molecular determinants of CB-5083 activity include expression of genes in the ERAD pathway providing a potential strategy for patient selection.

SUMMARY Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors. Leading inhibitors in clinical development act by binding to a common site within Smoothened, a critical pathway component. Acquired Smoothened mutations, including SMOD477G, confer resistance to these inhibitors. We report here that itraconazole and arsenic trioxide, two agents in clinical use that inhibit Hedgehog signaling by mechanisms distinct from that of current Smoothened antagonists, retain inhibitory activity in vitro in the context of all reported resistance-conferring Smoothened mutants and GLI2 overexpression. Itraconazole and arsenic trioxide, alone or in combination, inhibit the growth of medulloblastoma and basal cell carcinoma in vivo, and prolong survival of mice with intracranial drug-resistant SMOD477G medulloblastoma.

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system (CNS). B lymphocytes in the cerebrospinal uid (CSF) of MS patients contribute to in ammation and secrete oligoclonal immunoglobulins. Epstein-Barr virus (EBV) infection has been linked to MS epidemiologically, but its pathological role remains unclear. Here we demonstrate high-a nity molecular mimicry between the EBV transcription factor EBNA1 and the CNS protein GlialCAM, and provide structural and in-vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identi ed by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSFderived antibodies against MS-associated viruses. Sequence analysis, a nity measurements, and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment allowed for tracking the development of the naïve EBNA1-restricted antibody to a mature EBNA1/GlialCAM crossreactive antibody. Molecular mimicry is facilitated by a post-translational modi cation of GlialCAM. EBNA1 immunization exacerbates the mouse model of MS and anti-EBNA1/GlialCAM antibodies are prevalent in MS patients. Our results provide a mechanistic link for the association between MS and EBV, and could guide the development of novel MS therapies. Main TextThe presence of oligoclonal bands (OCB) in cerebrospinal uid (CSF) and the e cacy of B cell depleting therapies emphasize the importance of B cells in the pathobiology of multiple sclerosis (MS) 2 . Anti-viral antibodies against mumps, measles, varicella-zoster, and Epstein-Barr Virus (EBV) are often present in MS 4,5 , but their relevance is unclear. Anti-EBV antibody titers in over 99% of MS patients provide evidence for an epidemiological link between MS and EBV 6 . Symptomatic infectious mononucleosis during EBV infection increases risk for MS 7 . Molecular mimicry between virus and self-antigens is a potential mechanism that might explain this association 8 . Antibodies against certain EBV nuclear antigen 1 (EBNA1) regions have been found in MS patients, including the region AA365-426 5,9-12 , which we describe here in our identi cation of molecular mimicry between EBNA1 and the glial cellular adhesion molecule GlialCAM. The potential signi cance of this mimicry in the pathophysiology of MS is described in detail.The B cell repertoire in MS CSF plasmablasts is highly clonal CSF and blood samples were obtained from MS patients during the onset of disease (clinically isolated syndrome, n=5) or an acute episode of relapsing-remitting MS (n=4). Patients with a CSF pleocytosis of >10 cells/µl were selected (Extended Data Table 1, Supplementary Discussion). Single B cells were sorted by ow cytometry (Extended Data Fig. 1a,b). Characteristic phenotypic differences of B cells in blood and CSF were observed 13,14 , including (i) high plasmablast (PB) counts in CS...