Inhibition of p62-ZZ Domain-Mediated Signaling Overcomes Bortezomib Resistance in Multiple Myeloma Cells Independent of Their p53 Status

Multiple myeloma (MM) cells depend on autophagy as well as proteasome activity for their survival. In MM cells, autophagy is essential to reduce the extraordinary ubiquitinated protein system stress derived from high levels of immunoglobulin synthesis. Thus, proteasome inhibitors (PIs) decrease tumor burden in the majority of MM patients but eventually a PI resistant MM clone(s) arises. Bortezomib (BTZ) resistance has been reported to correlate with p53 status of MM cells. Previous studies showed that MM patients harboring p53 genetic abnormalities are commonly resistant to standard therapies, increasing the need for new druggable targets.

A possible mechanism of BTZ resistance involves the cargo receptor for ubiquitin-mediated autophagy SQSTM1/p62, that enhances MM cells survival by increasing their autophagic flux. We found that MM cells with wild type p53, are more sensitive to PI treatment, while MM cells harbouring mutant p53 or lacking p53 are more resistant to BTZ-mediated cytotoxicity. BTZ treatment induced a modest level of apoptosis in MM1.S cells (p53 wild type) at high doses (>5nM) by triggering the cleavage of caspase 3 and PARP-1. As expected BTZ treatment induced a significant compensatory increase in SQSTM1/p62 at the mRNA and protein levels in these cells. Conversely, BTZ treatment of p53 mutant MM cells failed to induce apoptosis, exhibiting only a mild effect on viability. This occurred in spite of a similar increase in SQSTM1/p62 expression as seen in MM.1S cells.

To determine the role of p62 in the BTZ resistance of p53 mutant MM cells, we tested a novel small-molecule p62-ZZ domain inhibitor, XRK3F2 we recently identified. XRK3F2 treatment of mice with established MM, dramatically increased bone formation in MM-bearing bones, although it did not affect tumour burden in vivo . In vitro studies showed that high concentrations (10μM) of XRK3F2 were required to decrease the viability of primary and MM cell lines and induce a time-dependent activation of caspases 3, 7 and 9. XRK3F2 treatment dose-dependently increased levels of the lipidated form of LC3, LC3II, suggesting activation of pro-survival autophagy. Surprisingly, the expression levels of SQSTM1/p62 were either unchanged or increased following XRK3F2 treatment despite the increase of autophagosome formation, indicating that XRK3F2 inhibited the autophagic flux in MM cells. Defective autophagy occurs in tumor cells and leads to accumulation of p62, further promoting tumorigenesis by altering NFκB regulation and gene expression. We found that XRK3F2 treatment also significantly inhibited TNFα-induced NFκB activation, by inhibiting p65 nuclear translocation. These data suggested that XRK3F2 may sensitize MM cells to BTZ-induced cytotoxicity and possibly overcome BTZ resistance.

To test this hypothesis, human MM cell lines MM1.S (p53 wild type), U266 (p53 mutant) and KMS-11 (p53 null) were exposed to different doses of a BTZ-XRK3F2 combination comprised of varying concentrations of BTZ combined with XRK3F2 at 5μM. We found treatment with the BTZ-XRK3F2 combination significantly reduced cell viability compared with either drug alone at the same doses, and increased MM cells sensitivity to BTZ, independent of their p53 status (IC50 BTZ vs BTZ-XRK3F2 for MM1.S 3 vs 1.5nM of BTZ; for U266 10 vs 1.7nM of BTZ; for KMS-11 7 vs 1nM BTZ).

Mechanistic studies revealed that in p53 wild type MM cells, the combination (Bortezomib 3nM - XRK3F2 5 μM) significantly increased annexin V positive cells after 6h of treatment. This was followed by a reduction in expression of the anti-apoptotic Bcl-2 protein (mRNA and protein levels) and extensive activation of caspase 3 and cleaved PARP1 after 24h, demonstrating apoptosis-mediated cell death. Importantly, p62 expression was significantly reduced by the BTZ-XRK3F2 combination. The decrease in p62 level may result as a consequence of its caspase 3-dependent degradation. In p53 mutant cells, the combination significantly induced PARP1 cleavage, although only mildly increased caspase 3 activation. These results imply that the BTZ-XRK3F2 combination activates multiple parallel death pathways to enhance both apoptosis and necrosis.

Taken together, these data suggest that p62-ZZ antagonists, alone or in combination with PIs, may have both anti-tumor and bone anabolic effects, and may overcome PI resistance in MM cells, making p62 an attractive target for the treatment of MM.