Relationship between HUWE1 Mutation and Functionality in Multiple Myeloma

Background

Proteasome inhibitors have provided significant therapeutic advance in the treatment of Multiple Myeloma (MM), however resistance and dose limiting side effects remain a clinical challenge. Recent research has focused on developing strategies to target other enzymes within the ubiquitin proteasome system, with the aim of overcoming resistance and toxicity. We have previously reported deregulated expression of the E3 ligase HUWE1 in MM and demonstrated that knockdown or inhibition of HUWE1 leads to a decrease in MYC activity (Blood 2016, 128:240; 2017, 130:3077). HUWE1 is a large HECT domain E3 ligase that is involved in the regulation of key proteins such as p53, MYC and MCL-1. Mutations in HUWE1 have recently been identified in MM patients, significantly associated with the t(11;14) subgroup (blood-2018-03-840132). A recurrently mutated splice site mutation in the DUF913 (domain of unknown function 913) of HUWE1 was identified in 18% of patients, the remainder are predominantly missense mutations distributed across the coding sequence (CDS). Other studies have demonstrated that point mutations in the dimerization interface of HUWE1, which acts to regulate its activity, result in hyper-activation of HUWE1 (eLife 2017;6:e21036; Sci Rep 2017;7:15050). While 7% of the mutations reported in MM patients are found in this region, the effect of the majority of mutations on the functional significance of HUWE1 has yet to be determined. The aim of this study was to analyse HUWE1 expression and activity in HUWE1 mutant MM cell lines.

Methods

HUWE1 mutational status was analysed in a publically available dataset of MM cell lines (www.keatslab.org). Inhibitors of HUWE1 (BI8622, BI8626; described in EMBO Mol Med 2014, 6:1525-1541) were purchased from Syngene. The effect of the inhibitors on HUWE1 mutant MM cell lines was assessed using CellTitre-glo. HUWE1 auto-ubiquitination activity was analysed using UbiQapture-Q and Western blotting.

Results

HUWE1 mutations were identified in 6 out of 69 MM cell lines. HUWE1 mutational status was confirmed in 5 cell lines (U266, XG-1, XG-2, KMS-27, H1112) by Sanger sequencing. In agreement with MM patient data, HUWE1 mutations were predominantly found in cell lines expressing the t(11;14) translocation (4/6 cell lines) and are distributed in a similar manner. H1112 cells harbor the recurrent splice site mutation observed in patients, whereas the other cell lines contain missense mutations across the CDS. HUWE1 protein expression in mutant cell lines was compared with expression in 5 MM cell lines expressing wild type (WT) HUWE1 (JJN3, MM.1S, ANBL-6, KMS-18, OPM-2). No significant difference in expression was observed in the majority of HUWE1 mutant cell lines, however, HUWE1 expression was significantly lower in the H1112 cell line (p=0.002) compared to HUWE1 WT cell lines. Accordingly, HUWE1 auto-ubiquitination activity was reduced only in H1112 cells. XG-2 and U266 displayed similar sensitivity to HUWE1 inhibitors as HUWE1 WT cell lines (IC50 12-18 µM vs 9-20 µM), while XG-1, H1112 and KMS-27 were less sensitive (IC50 20-33 µM). The effect of HUWE1 on substrate proteins (e.g. MYC) varies depending on tumor type. In HUWE1 WT MM cell lines, inhibition of HUWE1 leads to significantly decreased expression of MYC and MCL-1 in (p<0.01), through increased proteasomal degradation. A similar decrease in MYC and MCL-1 expression is observed in XG-2, and in MCL-1 in U266 cells (which lack expression of c-MYC). Conversely, no significant effect on MYC or MCL-1 expression was seen in XG-1 cells, while barely detectable levels of MYC and MCL-1 were observed in H1112 and KMS-27 cells, suggesting altered or absent HUWE1 activity in these cell lines.

Conclusion

HUWE1 has recently been identified as a mutational driver in t(11;14) MM, however, little is known about the functional consequence of HUWE1 mutations. Here we show that the H1112 cell line, representative of the most commonly occurring HUWE1 mutation in MM, leads to reduced expression and activity of HUWE1 and is associated with low expression of HUWE1 substrates MYC and MCL-1. Conversely, expression of HUWE1 and activity against selected substrates remains unchanged in XG-2 and U266 cells. Moreover, recent studies demonstrate that certain HUWE1 mutations lead to enhanced catalytic activity. In summary, the pathogenicity of HUWE1 mutations in MM is likely to depend on the type and location of the mutation.