Patients with multiple myeloma (MM) carrying 17p deletion (del17p) have poor outcome. Around a third of MM patients with del(17p) are also reported to have a TP53 mutation. The acquisition of TP53 mutations seems to occur after allelic loss of 17p, and confers worse clinical outcomes. However, a recent publication has raised possibility that other additional genes resident at 17p13 may play a role in the clinical outcome of this subgroup of MM patients.
The YWHAE gene is located at 17p13.3 and encodes the 14-3-3ε protein, one of the mammalian 14-3-3 protein family members that are highly conserved in eukaryotes. Altered 14-3-3ε expression is associated with development and progression of cancer. It has a context dependent pleotropic effect both as oncogene or tumor suppressor gene. However, its precise role in tumorigenesis has yet to be fully discovered.
Using a large cohort of MM patients (n=369) RNA sequencing and SNP array data with complete clinical annotation, we observed a significant positive correlation (r=0.7) between YWHAE and TP53 at gene and copy number levels. In MM patients with del17p, YWHAE expression was significantly lower compared to normal plasma cells. Importantly, low expression of YWHAE correlated with poor clinical outcome (both EFS and OS), suggesting that YWHAE may be important in this specific myeloma clinical setting. However, in 2 independent MM datasets, we observed that overall YWHAE expression is higher in CD138+ MM cells compared to normal plasma cells. Moreover, we confirmed high expression of 14-3-3ε at the protein level in a large panel of MM cell lines and primary MM cells.
To clarify this apparent paradox and shed light on the role of 14-3-3ε in MM, we used gene-specific CRISPR screen as well as shRNA knock down (KD) to study the functional impact of perturbation of YWHAE in MM. For the loss-of-function studies, we selected a large panel of MM cell lines based on their p53 status (WT, HD, Mu). We found a significant inhibitory effect of YWHAE KD on MM cell growth and survival over time, and induction of apoptosis via PARP cleavage and BAX increase. The antiproliferative effects were even more pronounced in p53 WT myeloma cell lines, such as MM1S and H929.
Gene expression profiling (GEP) analysis showed a significant number of differentially expressed genes in YWHAE KD cells compared to control cells. Gene enrichment analysis revealed a major impact of YWHAE KD on a large set of genes involved in the mTORC1 signaling pathway. WB analysis confirmed a reduction of p-mTOR along with a decreased of p-p70 S6 kinase and p-4E BP1 after YWHAE KD. Considering the crucial role of mTOR pathway in the regulation of autophagy, we are now in the process of analyzing the effect of YWHAE on the autophagic response. Moreover, we found that YWHAE KD exerted a significant suppression of known E2F1 gene targets. Notably, a significant decrease in the expression of genes involved in the unfolded protein response was observed upon silencing/knock-out of YWHAE. As a consequence, myeloma cell sensitivity to bortezomib was increased after 14-3-3ε KD.
In conclusion, YWHAE and its coding protein 14-3-3ε are constitutively expressed in MM cells. Perturbation of 14-3-3ε expression impacts MM cell growth via induction of apoptosis and inhibition of mTOR pathway. 14-3-3ε KD increase cell sensitivity to bortezomib. Ongoing efforts will further elucidate the potential molecular mechanism of YWHAE/14-3-3ε action in MM.
Anderson: Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.
Asterisk with author names denotes non-ASH members.