XBP1 variant 1 (Xv1) is the most abundant XBP1 variant and is highly enriched across cancer types but nearly none in normal tissues. Its expression is associated with poor patients’ survival and is specifically required for survival of malignant cells, but the underlying mechanism is not known. Here we report that Xv1 upregulates the polyglutamylase tubulin tyrosine ligase-like 6 (TTLL6) and promotes mitosis of cancer cells. Like the canonical XBP1, Xv1 mRNA undergoes unconventional splicing by IRE1α under endoplasmic reticulum stress, but it is also constitutively spliced by IRE1β. The spliced Xv1 mRNA encodes the active form of Xv1 protein (Xv1s). RNA sequencing in HeLa cells revealed that Xv1s overexpression regulates expression of genes that are not involved in the canonical unfolded protein response, including TTLL6 as a highly upregulated gene. Gel shift assay and chromatin immunoprecipitation revealed that Xv1s bind to the TTLL6 promoter region. Knockdown of TTLL6 caused death of cancer cells but not benign and normal cells, similar to the effects of knocking down Xv1. Moreover, overexpression of TTLL6 partially rescued BT474 cells from apoptosis induced by either TTLL6 or Xv1 knockdown, supporting TTLL6 as an essential downstream effector of Xv1 in regulating cancer cell survival. TTLL6 is localized in the mitotic spindle of cancer cells. Xv1 or TTLL6 knockdown resulted in decreased spindle polyglutamylation and interpolar spindle, as well as congression failure, mitotic arrest and cell death. These findings suggest that Xv1 is essential for cancer cell mitosis, which is mediated, at least in part, by increasing TTLL6 expression.

The endoplasmic reticulum (ER) maintains protein homeostasis through chaperone-mediated folding and ER-associated degradation (ERAD). Disruption of this quality control, particularly involving the ER chaperone GRP94, contributes to diseases such as hypercholesterolemia, cancer, and immune disorders, where defective GRP94-dependent folding and the trafficking of client proteins like PCSK9, integrins, and Toll-like receptors drive pathology. Here, we characterize NSC637153 (cp153), a small molecule identified in a drGFP-based ERAD dislocation screen, as a selective probe of GRP94-dependent processes. cp153 inhibits the dislocation of ERAD substrates, preferentially affecting luminal clients, increases PCSK9 secretion, and promotes LDLR degradation. Unlike ATP-competitive HSP90 inhibitors, cp153 does not induce HSP70 or destabilize AKT, suggesting that it perturbs GRP94 function by interfering with client interaction or folding. The identification of cp153 provides a useful tool to for probing GRP94’s role in protein folding, trafficking, ER quality control, and disease-relevant signaling pathways, and supports the development of client-selective GRP94-targeted therapies.

Endoplasmic reticulum-associated degradation (ERAD) must dynamically adapt to cellular protein-folding demands, yet mechanisms regulating ERAD-associated adaptors remain incompletely defined. Here, we identify the E3 ubiquitin ligase RNF5 as a regulator of the ERAD adaptors HERPUD1 (HERP1) and OS9. RNF5 depletion increased HERP1 and OS9 protein abundance, while semiquantitative endpoint RT-PCR did not reveal obvious changes in their transcript signals. Available cycloheximide-chase experiments were consistent with increased adaptor persistence and more rapid loss of the ERAD-L substrate null Hong Kong α1-antitrypsin (NHK) after RNF5 depletion. In contrast, expression of the catalytically inactive RNF5 C42S mutant was associated with slower NHK loss, indicating that RNF5 depletion and catalytic inactivation produce distinct functional outcomes. Proteomic screening and co-immunoprecipitation supported association of RNF5 with HERP1- and OS9-containing complexes. RNF5 depletion reduced compound 225-induced HERP1 ubiquitination and adaptor loss, while wild-type RNF5 produced stronger HERP1 ubiquitination than C42S, supporting a contribution of RNF5 catalytic activity. RNF5 depletion also attenuated selected tunicamycin-induced UPR markers and 225-associated cytotoxic/apoptosis-related readouts while increasing SA-β-galactosidase- and γH2AX-associated phenotypes. Together, these findings identify RNF5 as a regulator of ERAD-adaptor abundance and NHK processing, while leaving the causal relationship between adaptor regulation and the RNF5-depletion phenotype to be resolved by formal rescue and epistasis studies.