UBE2G1 governs the destruction of cereblon neomorphic substrates

Abstract

The cereblon modulating agents (CMs) including lenalidomide, pomalidomide and CC-220 repurpose the Cul4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic substrates via polyubiquitination in conjunction with E2 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the K48-linked polyubiquitination of CRL4CRBN neomorphic substrates via a sequential ubiquitination mechanism. Blockade of UBE2G1 diminishes the ubiquitination and degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent IKZF1/3 degrader CC-220. Collectively, it will be of fundamental interest to explore if loss of UBE2G1 activity is linked to clinical resistance to drugs that hijack the CRL4CRBN to eliminate disease-driving proteins.

Genome-wide CRISPR Screens Reveal Genetic Mediators of Cereblon Modulator Toxicity in Primary Effusion Lymphoma

“…Overall, our data suggest a preference for CRL4 CRBN for UBE2G1, similar to that seen in multiple myeloma 37,38,43 .…”

“…We confirmed that inactivation of UBE2G1 in PEL cells confers resistance to LEN and to POM, but not CC-122. LEN and POM resistance of UBE2G1inactivated cells was recently also reported in multiple myeloma by Sievers et al 37 and Lu et al 38 . These groups proposed a "prime-extend" mechanism, where the E2 conjugating enzyme UBE2D3 mediates neosubstrate mono-ubiquitination, while In summary, we propose a model where overall expression levels of CRBN, CUL4A, CUL4B, UBE2G1, and SENP8 dictate sensitivity of CMs in PEL (Fig 7A-C).…”

“…Sievers et al 37 and Lu et al 38 confirmed a role for UBE2G1 in CM-induced toxicity in MM and proposed a "prime-extend" mechanism, whereby the E2 enzyme UBE2D3 mediates substrate mono-ubiquitination, while UBE2G1 extends the Ub chain via addition of further K48-linked Ub moieties. Inactivation of UBE2D3 alone, which did not score in our screens, did not confer significant CM resistance in PEL, suggesting the existence of redundant E2 enzymes that can fulfill this function in our system (Fig.…”

“…These results parallel two recent reports 37,43 , which similarly identified UBE2G1 as hits in genome-wide CRISPR/Cas9 screens for LEN or POM resistance in multiple myeloma. UBE2G1 was also identified as the preferred E2 in CM-induced CRL4 CRBN activity in an E2 enzyme focused CRISPR screen in multiple myeloma 38 .…”

“…An enrichment for guides against CRBN was expected, because its inactivation has previously been shown to confer CM resistance in PEL 6,7 and MM cell lines 21,22 . Other genes with strong sgRNA enrichments in at least a subset of settings include: the CRL4 cullin subunit CUL4B; GLMN, a previously identified binding partner of RBX1 36 ; and the E2 Ub conjugating enzyme UBE2G1, which has very recently been implicated as the preferred E2 enzyme for ubiquitin chain elongation by CRL4 37,38 . sgRNAs targeting four other genes (ILF2, ILF3, YPEL5, SENP8) were highly and specifically enriched in LENresistant cells (Table S4, Fig.…”