Define the role of mRNA translation reprogramming in multiple myeloma progression and response to therapy

Multiple myeloma (MM) is a rare cancer type of white blood cell called plasma cells within the bone marrow. The exceptionally high rate of protein synthesis and the increased processing load, render MM cells particularly susceptible to perturbations in protein homeostasis. The ubiquitin-proteasome system is a key mechanism by which MM cells maintain a balance between protein synthesis and disposing of damaged proteins. The induction of proteotoxic stress by targeting protein degradation with proteasome inhibitors is an effective therapeutic approach. However, resistance to this treatment modality is inevitable. Protein homeostasis also relies on exquisite regulation of mRNA translation and protein synthesis. Many MM genetic drivers have direct impact on mRNA translational activity. Therefore, investigating altered mRNA translational activity in MM cells will provide new understanding of MM oncogenic transformation and disease progression and identify novel therapeutic approaches for treating MM.

Heterozygous deletion of RPL5, a component of the 60S ribosome subunit, has been found in 20-40% of MM patients. Patients with low RPL5 expression level have a worse survival outcome but respond better to the proteasome inhibitor than patients with high RPL5 expression. We hypothesize that translational adaptation mediated by RPL5 deficiency promotes MM progression and affects therapeutic response. This project aims to interrogate the functional impact of RPL5 deficiency in MM cells.

We have established RPL5 deficient MM cell lines by either CRISPR-Cas9 knock-out or inducible shRNA knockdown. Cell proliferation and drug sensitivity will be assessed. As deficiency in RPL5 can alter ribosome composition leading to ribosome heterogeneity, we will isolate 40S and 60S ribosomal subunits and 80S monosomes by sucrose gradient separation and fractionation and the abundance of ribosomal proteins will be quantified by LC-MS/MS. To determine the effect of RPL5 deficiency on mRNA translation and protein synthesis, we will also perform polysome profiling and proteomics analysis. In addition, the effect of RPL5 deficiency on p53-mediated ribosome biogenesis checkpoint activation will be determined. The findings will provide new insight into mRNA translation reprogramming in MM pathogenesis and provide evidence for RPL5 as a potential biomarker of cancer therapy in MM.