The Effect of the Proteasome Inhibitor Ixazomib (MLN9708) and UPR Mediated Therapy in Multiple Myeloma Cells and Bone Marrow Niche

Background: The concept of disrupting proteostasis in order to weaken cancer cells and challenge their microenvironment has shown striking clinical success, especially by applying proteasome inhibitors (PIs) in multiple myeloma (MM). The proteasome inhibitor MLN9708 (ixazomib) demonstrated significant preclinical and clinical anti-myeloma activity combined with a favorable toxicity profile. Proteostasis is also maintained by different adaptive pathways, including heat shock response (HSR) and the unfolded protein response (UPR). MM cells are characterized by mis- and overproduction and thus accumulation of immunoglobulin (Ig) within the bone marrow, relying on the UPR system to overcome stress due to this accumulation of unfolded proteins. The UPR mediates its function through three pathways, namely IRE-1α, PERK and ATF6. The most conserved UPR pathway is mediated by IRE-1α which reduces the ER stress load by splicing the XBP1 mRNA through removal of a 26-nucleotide intron.

Aim: The UPR represents a vulnerability of MM cells which we aimed to harness with a novel combination therapy of UPR inhibition and proteasome inhibition.

Methods: We used two MM cell lines (KMS11 and RPMI-8226) to validate the cytotoxic effects of ixazomib together with the IRE-1α inhibitors A106 and STF-083010 by measuring cell viability and proliferation, assessing synergistic or additive effects with the Bliss formula and quantifying the apoptotic cell fraction. For gene expression assays, total RNA was isolated and relative quantification of target gene expression was calculated by the comparative threshold cycle method.

Results: Our in vitro results demonstrate that the combination of ixazomib and IRE-1α inhibitors has a synergistic effect on reducing of viability and proliferation of multiple myeloma cells and induces apoptosis as a single agent and in combination with IRE-1α inhibitors (Viability of KMS11 with ixazomib treatment 13.5% ± 0.35 vs 7.3% ± 0.23 combination with STF-083010, RPMI-8226 with ixazomib treatment 33.4% ± 0.56 vs 15.7% ± 0.07 combination with STF-083010; apoptotic fraction of KMS11 with ixazomib treatment 67.1% vs 76.9% combination with STF-083010, RPMI-8226 with ixazomib treatment 31.7% vs 36.3% combination with STF-083010). Ixazomib in combination with STF-083010 increased proteolytic cleavage of PARP, a signature of apoptosis and downstream target of Caspase-3, increased G1 phase regulators (p21^cip1, p27^kip1) and up-regulated pro-apoptotic regulator protein, BAX. In addition, we found that combination treatment increased the expression of pro-apoptotic BH3-only family members, PUMA and NOXA and decreased the expression of Bcl2 pro-survival family members (Bcl2, Bcl-xl).

Mesenchymal stromal cells (MSCs) mimic the positive effects of bone marrow niche on MM cells in vitro . Co-culture of MM cells with Telomerase-immortalized Human Mesenchymal Stem Cell (hMSC-TERT) resulted in support of survival and cell growth of the two MM cell lines, (KMS11-hMSC-TERT with ixazomib treatment 55.4% ± 2.3 vs 24.2% ± 1.0 combination with STF-083010, RPMI-8226 with ixazomib treatment 68.6% ± 0.14 vs 17.8% ± 0.42 combination with STF-083010). However, the combination therapy of ixazomib with IRE-1α inhibitors significantly reduced the viability of multiple myeloma cells even when protected by the bone marrow niche microenvironment, confirming the notion that this combination therapy abrogates microenvironmental support of myeloma cells.

Quantitative PCR (qRT-PCR) revealed a relevant up-regulation of XBP1s mRNA levels by ixazomib monotherapy in the two MM cell lines whereas the combination of proteasome inhibition by ixazomib with IRE-1α inhibition significantly reduced the expression level of XBP1s (KMS11 with ixazomib 77.3% ± 3.4 vs 34.8% ± 2.7 combination with STF-083010, RPMI-8226 with ixazomib 22.2.0% ± 1.7 vs 11.9% ± 1.7 combination with STF-083010), again demonstrating the IRE-1α pathway of the UPR as an important target in the therapy of multiple myeloma. A second experimental set with the IRE-1α inhibitor A106 showed similar results, for which data are not explicitly mentioned here.

Conclusion: Taken together, our findings demonstrate that targeting the UPR specifically via inhibition of IRE-1α in combination with ixazomib is a promising therapeutic strategy in multiple myeloma.