Ywhae/14-3-3ε Expression Promotes Translation Initiation and Protein Synthesis in Multiple Myeloma Regulating Proteasome Load and Contributing to Proteasome Inhibitor Sensitivity/Resistance

Dysregulated translational control and high protein turnover is a feature of multiple myeloma (MM) cells; as a result, proteasome inhibitors (PI), triggering a misfolded protein stress and death response, have been extremely successfully in MM therapy. But despite success of PIs and other novel therapeutic approaches, majority of patients progress; thus, investigating the mechanisms driving the drug sensitivity and/or resistance is important. We here report a novel chaperone protein 14-3-3ε with significant impact on MM cell pathobiology and patient outcome.

14-3-3 proteins are a family of master regulators of intracellular signaling that influence several cellular processes through binding to specific serine/threonine-phosphorylated residues on a diverse array of cellular proteins. With known interaction of 14-3-3 proteins with several functionally diverse molecules and well recognized chaperon function, we sought to evaluate their role in impacting proteasome activity. We compared the sensitivity of 14 MM cell lines to two proteasome inhibitors, Bortezomib (BTZ) and Carfilzomib (CFZ) with expression of all 14-3-3 proteins at both mRNA and protein levels. We compared the measured drug activity and individual 14-3-3 gene expression across all cell lines by Pearson correlation coefficients and observed that the expression level of only YWHAE (coding gene for the isoform 14-3-3ε) showed a significant negative correlation with both BTZ and CFZ response. We therefore evaluated its expression in primary MM cells and found its lower expression to be associated with poor outcome in MM patients receiving a bortezomib (Btz)-based therapy.

To elucidate the underlying molecular mechanism responsible for 14-3-3ε contribution to PIs sensitivity, we performed integrated analysis of protein interactome and transcriptomic changes following 14-3-3ε gene modulation. These studies showed that 14-3-3ε impacts mTORC1 signaling in MM cells by binding to serine-phosphorylated residues on mTOR and its upstream negative regulator TSC2, resulting in mTORC1 activation. Conversely, depletion of 14-3-3ε inhibits TSC2 phosphorylation, causing a subsequent inhibition of mTORC1 signaling. One major conserved function of mTORC1 is to promote mRNA translation and therefore protein synthesis via activation of S6 kinase 1 (S6K1) as well as inhibition of the eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP). We have therefore assessed the impact of 14-3-3ε on translational efficiency in MM cells. In the viable MM cell population, 14-3-3ε depletion caused up to 50% reduction of protein synthesis in three MM cell lines, whereas 14-3-3ε overexpression in the KMS20 cell line or addback in YWHAE-KO cells (H929 and KMS11) resulted in a marked upregulation of protein synthesis. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of 14-3-3ε perturbation on intracellular and secretion light chains production. We observed a significant decrease in the intracellular abundance and secretion of the light chains with 14-3-3ε KD in all MM cell lines tested. These observations were corroborated by gain-of-function studies where ectopic overexpression of 14-3-3ε in MM cells was associated with increased protein load (and an enhanced sensitivity to PIs) in vitro as well as in vivo.

Importantly, we confirmed a significant correlation between 14-3-3ε expression, PIs sensitivity and protein load (evaluated as M protein production) in primary MM cells from two independent datasets. Moreover, MM patients with del17p, where 14-3-3ε is located, have lower 14-3-3ε expression and decreased monoclonal protein level, providing an explanation for inability of BTZ to overcome high-risk feature associated with del17p.

In conclusion, we here report for the first time a unique function for 14-3-3ε in modulating the sensitivity to PIs through regulation of protein synthesis and M protein load in MM cells. Altogether these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PIs sensitivity in MM.