Not so lost in translation: RPS15 mutations in CLL

The mutational landscape of CLL is heterogeneous spanning from recurrent mutations in classic tumor suppressor genes (eg, TP53) to mutations in genes involved in the splicing machinery (eg, SF3B1).²  Recently, RPS15 mutations were discovered at low frequencies in newly diagnosed and untreated cohorts,³  but they affected up to 20% of CLL patients who relapsed after chemoimmunotherapy (ie, fludarabine, cyclophosphamide, and rituximab [FCR]).^4,5  In addition, it was noted that approximately one-third of RPS15-mutated patients also carried a TP53 aberration (ie, 17p deletion and/or TP53 mutations).^5,6 

The translation of RNA into proteins is a complex and fundamental cellular process, and the recent discoveries of somatic RPS15 mutations in CLL as well as mutations in RPL10, RPL5, and RPL11 in T-cell acute lymphoblastic leukemia⁷  have sparked interest in how these mutations may tie in to oncogenesis and disease progression in hematologic malignancies. Several ribosomal proteins, including RPS15, have a suggested alternative function besides protein translation in interaction with the MDM2-p53 axis in conditions with nucleolar stress, subsequently leading to cell cycle arrest.⁸  Although preliminary functional analysis of mutant RPS15 pointed to increased ubiquitin-mediated p53 degradation, this could not fully explain abrogation of this function as the specific oncogenic mechanism.⁵ RPS15 mutations in CLL predominantly represent heterozygous missense single nucleotide variants occurring almost exclusively in the evolutionary conserved C-terminal region between amino acids 129 and 145, indicating strong functional constraints for mutations occurring in the decoding center of the ribosome. This mutational pattern indicates an oncogenic rather than a tumor suppressor function of the mutant protein; however, the exact effects of RPS15 mutations on ribosome function have not previously been described.

Bretones et al investigated the cellular impact of RPS15 mutations. They started by investigating the half-life of mutant RPS15 compared with wild-type protein by stable expression of transcripts with 8 different RPS15 mutations, and they detected a decreased half-life of mutant RPS15, most likely by increased ubiquitination. An important question concerning the effect of RPS15 mutations is whether the mutant protein is incorporated in the ribosome or not. The authors show that mutant RPS15 is indeed incorporated in the ribosome and could therefore have an effect on the translational properties of RPS15-mutated cells. Next, they assessed the effects of mutant RPS15 on ribosome function and detected increased cap-independent translation for 5 of the mutants, a decreased translation fidelity for 2 of the mutants, and a significantly decreased ability in the detection of stop-codons for 5 mutants (see figure). Taken together, these features strongly indicate that RPS15 mutations lead to altered ribosomal function regarding both protein synthesis and translational fidelity.

Finally, the authors applied a high-throughput proteome analysis to delineate global protein alterations for 2 of the most frequently occurring RPS15 mutations (RPS15^P131S and RPS15^S138F) compared with wild-type RPS15 in a nonhematologic cell line (HEK293T). The proteome profiles of replicates with the same mutations clustered together, and detailed investigation of the altered protein classes showed that the RPS15^P131S mutant led to increased amount of proteins associated with replication and DNA elongation. In turn, RPS15^S138F led to enrichment of proteins associated with messenger RNA (mRNA) and peptide processing. CLL cell lines often prove challenging in functional studies, and a replicate study in the CLL cell line MEC-1 did not reveal mutant-specific proteome profiles. However, comparison of all mutants to the wild-type profile revealed patterns similar to those in the HEK293T experiments. These profiles also involve downregulation of pathways that could lead to metabolic reprograming toward aerobic glycolysis, which is in line with the previously described Warburg effect.

All in all, this study demonstrates that RPS15 mutations, mainly occurring in the decoding center of the ribosome, lead to reduced RPS15 levels and an altered ribosomal efficiency that in turn causes global changes of the proteome. On the basis of data from different ribosomopathies, the authors speculate that ribosomal mutations may result in a switch from a hypo- to a hyperproliferative phenotype. Initially, ribosomal stress may select mutations that bypass the ribosome quality control, hence increasing the amount of defective ribosomes. Conversely, mutated ribosomes may cause altered protein expression patterns, which may contribute to transformation and leukemia development. Although data presented here are mainly based on experiments performed in cell lines, it will be important to further investigate whether RPS15 mutations cause similar differences in ribosome efficiency and protein synthesis in primary CLL cells as well, particularly exploring effects in vivo (ie, in the context of the tumor microenvironment). To this end, detailed analysis of key pathways and processes affected by impaired mRNA translation and their downstream effects will be necessary to pinpoint the causative effect of ribosomal mutations on CLL pathobiology. As shown in the article by Bretones et al and by others,^3,-5 RPS15 mutations are particularly enriched in patients with aggressive disease who have a higher propensity of disease relapse after treatment with chemo-immunotherapy. Notably, the authors observed an increased resistance to fludarabine, and to a lesser extent B-cell receptor signaling inhibitors, at least for cells carrying selected RPS15 mutations. Hence, it will be important to study additional potential treatment targets or combinations of therapy for this poor-prognosis group of CLL patients.