The E3 Ubiquitin Ligase HUWE1 Is a Potential Therapeutic Target for Multiple Myeloma

Background

The use of proteasome inhibitors to disrupt the ubiquitin proteasome system (UPS) has provided a significant therapeutic advance and highlights the importance of this pathway in Multiple Myeloma (MM). Selective inhibition of other enzymes that regulate the UPS may enable more specific targeting of aberrant signalling pathways with reduced side-effects. Using UPS-specific microarrays we found higher expression of the E3 ligase HUWE1 in MM cell lines compared to normal counterparts. HUWE1 is involved in the regulation of key proteins such as ARF, p53 and MYC. It has previously been reported to be deregulated in tumorigenesis and small molecule inhibitors of HUWE1 have recently shown efficacy in in vitro models of colorectal cancer. The aim of this study was to elucidate the role and therapeutic potential of HUWE1 in MM.

Methods

To validate our UPS microarray studies, HUWE1 expression levels were analysed in publically available datasets (GSE6477, GSE6691, GSE2113), CD138+ cells from healthy bone marrow (n=3) and MM patients (n=5) and MM cell lines [U266 (p53 mutant), OPM-2 (p53 mutant), KMS-18 (p53 WT/-), JJN3(p53 WT/-), MM.1S (p53 WT)]. shRNA-mediated gene knockdown (Source Biosciences) was used to investigate the therapeutic potential of targeting HUWE1. HUWE1 knockdown cells were analysed for viability and cell cycle status; effects on known downstream substrates were analysed by Western blotting. Snapshot Proteomics (AVM Biomed) was performed on HUWE1 knockdown cells to identify novel ubiquitinated substrates of HUWE1.

Results

Elevated HUWE1 expression (≥ 2-fold) was confirmed in MM cell lines and primary CD138+ MM cells compared to healthy CD138+ cells at both the gene and protein level. Analysis of publically available datasets (GSE6477, GSE6691) also demonstrated that HUWE1 was significantly increased in MM patients compared with healthy donors. Moreover, further analysis of datasets GSE6477 and GSE2113 revealed that HUWE1 expression increased significantly (p≤0.05) through a spectrum of plasma cell diseases (MGUS, smoldering MM, newly diagnosed MM, plasma cell leukemia). shRNA knockdown of HUWE1 resulted in a significant increase of cells in G2/M, an accumulation of p53 and MIZ1 and a decrease in MYC protein levels (p≤0.05). HUWE1 depletion significantly decreased (p≤0.01) the proliferation of all MM cell lines, cultured alone or in co-culture with MM patient-derived bone marrow stromal cells, regardless of p53 status. Conversely, we show that MM cell lines that carry mutant p53 were largely resistant to the MDM2 inhibitor Nutlin-3. Finally, proteomic profiling identified more than 80 putative substrates of HUWE1, confirming its known roles as an RNA binding protein and in core histone regulation. Furthermore, this analysis revealed previously unidentified involvement of HUWE1 in regulation of cell cycle (3 proteins) and MAPK signalling (4 proteins).

Conclusions

The proteasome is an established target in MM and there are opportunities for more specific targeting of enzymes within the UPS with reduced side-effects. Here we demonstrate that the E3 ligase HUWE1 is elevated in MM and inhibition of HUWE1 with shRNA results in growth arrest of MM cell lines, decreased expression of MYC and increased levels of p53. These effects were independent of p53 status, suggesting that inhibition of HUWE1 offers a more valuable approach than MDM2 inhibitors which show efficacy only in p53 wild type myeloma. Future studies will validate novel substrates of HUWE1 identified in this study and investigate recently identified small molecule inhibitors of HUWE1 in MM. This study demonstrates that HUWE1 is important in the growth and survival of MM cells and may provide a novel therapeutic target in MM.