Title: Establishing ER-associated degradation (ERAD) mediated by the Hrd1 ubiquitin ligase complex as a therapeutic target in cancer
Funder: Cancer Research UK

Abstract: BACKGROUND: Cellular stress is a cancer hallmark and the restorative homeostatic response mechanisms often become constitutively engaged to adapt populations to hyperproliferative and metabolically dysregulated states, stabilise malignancy, and elevate resistance to therapeutic agents. Multiple myeloma (MM) is B-cell derived malignancy with elevated immunoglobulin production and an increased dependency on protein homeostasis mechanisms in the endoplasmic reticulum (ER). ER-associated degradation (ERAD) is an adaptive, ubiquitin-proteasome dependent process eliminating misfolded proteins from the ER necessary to maintain organelle homeostasis by restoring secretory flux. Robust and dynamically responsive, ERAD unwittingly supports cancer onset and progression by resisting cell death under atypical conditions. The multi-subunit Hrd1 ubiquitin ligase complex is the principal moderator of ERAD. Proteasome inhibitor-based therapies currently treating MM block the final proteolytic step in Hrd1-mediated ERAD and while initially efficacious, eventually become ineffective. Direct targeting of Hrd1 activity to block ERAD offers a potential strategy to avoid pleiotropic consequences of general proteolytic impairment and thus, represents an “Achilles heel” for stress-adapted cancers. AIMS: This programme explores ERAD as a therapeutic target in cancer, using MM as a model system. It seeks to biochemically and structurally define Hrd1 complexes responsible for ERAD and ER stress resolution to understand how dynamic adaptability supports MM proliferation. It aims to identify small molecules selectively impairing Hrd1 complexes locally, thereby avoiding pleiotropic effects associated with global proteolytic impairment. METHODS: The programme leverages existing insight into Hrd1 complexes to facilitate biochemical and structural determinations of its composition, assembly, and mechanism. Reporter cell lines and in vitro assays will screen for small molecules that impair Hrd1-mediated ERAD and are synthetically lethal with ER stress. Quantitative mass spectrometry of native Hrd1 complexes will establish changes to composition and stoichiometry resulting from ER stress. This will inform CRISPR/Cas9 target choices among cofactors to produce knockout cell lines that directly test contributions to ERAD activity and proteotoxic ER stress resolution. Recombinantly producing Hrd1 complex cytoplasmic domains will facilitate; direct interaction and relative affinity measurements, reconstitution of ubiquitination in vitro, and structural determination of Hrd1 assemblies. Scaling native Hrd1 complex immunoprecipitation for Cryo-EM will be explored for added structural insight. USE OF RESEARCH RESULTS: This programme will ascertain whether ERAD and specifically the Hrd1 complex is a druggable target in MM. If successful, it will open new avenues for therapeutic target identification among ER ubiquitin ligases. Moreover, it may prove suitable for other cancers “addicted” to homeostatic stress resolving pathways.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS