HUWE1 Inhibition As a Therapeutic Strategy to Target MYC in MM

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). There is increasing interest in developing therapeutic strategies to target enzymes that regulate the UPS, with the aim of gaining more specificity and reducing side-effects. We previously identified elevated expression of the E3 ligase HUWE1 in MM compared to normal cells and demonstrated that inhibition of HUWE1 with shRNA results in growth arrest of MM cell lines (Blood 2016, 128:240). HUWE1 is involved in the regulation of MYC and its expression is deregulated in a number of tumor types. The aim of this study was to elucidate the role and therapeutic potential of inhibiting HUWE1 in MM using small molecule inhibitors.

Methods

Inhibitors of HUWE1 (BI8622, BI8626) as described in Peter et al. (EMBO Mol Med 2014, 6:1525-1541) were purchased from Syngene. The effect of the inhibitors on MM cell lines (OPM-2, KMS-18, JJN3, MM.1S, MM.1R, ANBL6.WT, ANBL6.BR), patient cells and normal bone marrow (NBM) from healthy donors was assessed using CellTitre-glo and WST1 assays. Cells were analysed for cell cycle status (propidium iodide and EdU staining), apoptosis and β-galactosidase activity. Snapshot Proteomics (AVM Biomed) was performed on HUWE1 knockdown cells to identify novel ubiquitinated substrates of HUWE1. Signalling pathways and substrates of HUWE1 were analysed using UbiQapture-Q (Enzo Life Sciences), Western blotting and RQ-PCR. Calcusyn software was used to calculate a combination index (CI) to assess synergistic anti-MM activity.

Results

UbiQapture assays demonstrated that BI8622 and BI8626 blocked auto-ubiquitination of HUWE1 in a concentration-dependent manner, with an associated decrease in the ubiquitination of MIZ1 (a validated HUWE1 substrate). BI8622 and BI8626 significantly reduced the growth of MM cell lines, including those resistant to dexamethasone (MM.1R) and bortezomib (ANBL6.BR), and patient-derived MM cells (p ≤ 0.01; IC50 range 9-20 µM) without significantly affecting NBM, suggesting a favourable therapeutic index. Furthermore, both compounds inhibit the proliferation of MM cell lines when co-cultured with MM patient-derived bone marrow stromal cells. HUWE1 inhibition induced cell cycle arrest, associated with a reduction of cells undergoing DNA synthesis (p = 0.005), cellular senescence demonstrated by β-galatosidase staining and increased p16 protein expression. HUWE1 inhibition in MM cell lines resulted in a significant decrease in MYC mRNA and protein expression (p < 0.001), accompanied by downregulation of multiple MYC-dependent target genes (CCND2, HSPE1, ODC1, CCNA2, CDC25A; p ≤ 0.02). Proteomic profiling along with ingenuity pathway analysis revealed novel putative substrates of HUWE1 involved in the regulation of MYC. Finally, the combination of HUWE1 inhibitors with proteasome inhibitors (carfilzomib and bortezomib) and dexamethasone results in synergistic anti-MM activity (CI range 0.57 - 1).

Conclusions

MYC is frequently dysregulated in MM and represents an attractive therapeutic target. Here we demonstrate that inhibitors of the E3 ligase HUWE1 lead to growth arrest of MM cell lines, associated with decreased expression of MYC and MYC-dependent target genes. While BI8622 and BI8626 have previously been reported to repress MYC activity in models of colorectal cancer, we demonstrate for the first time that in MM this is accompanied with a decrease in MYC expression. Studies are ongoing to elucidate the molecular mechanism of MYC downregulation following HUWE1 inhibition both in vitro and in vivo. In summary, this study demonstrates that HUWE1 is important in the growth and survival of MM cells and presents a novel therapeutic strategy for the inhibition of MYC function in MM.