Impaired Response to Oxidative DNA Damage Underlies Genomic Instability in B-Cell Leukemia Driven By RPS15 Mutation

The ribosomal protein RPS15 is recurrently mutated in chronic lymphocytic leukemia (CLL) and confers adverse prognosis. While translational rewiring is a recognized consequence of RPS15 mutation (RPS15-mut), its impact on genome stability and the precise mechanisms governing RPS15-mut driven B-cell leukemogenesis remain unclear. To delineate the sequence of cellular events that underlie malignant transformation and progression in the context of RPS15 mutation, we functionally interrogated a conditional knock-in mouse model of the RPS15-S138F mutation established using the Cd19-Cre/loxP system (Gutierrez et al, ASH 2020). Despite enrichment for Myc target genes in pre-leukemic Rps15-mut B splenocytes, these cells displayed a hypoproliferative phenotype characterized by increased G1 cell cycle checkpoint activation and reduced ex vivo proliferative capacity accompanied by increased apoptosis, compared to Rps15-WT splenocytes.

We hypothesized that decreased proliferation and increased apoptosis could result from a p53-mediated response to cellular stress arising from Rps15 mutation. Informed by the genomic landscape of CLL developing in older Rps15-mut mice that displayed an enrichment of A•T>C•G and C•G>T•A transversions indicative of peroxide-induced DNA damage, we interrogated the transcriptomes of Rps15-mut pre-leukemic B splenocytes, which showed upregulation of reactive oxygen species (ROS) genes (NES 1.5, p<0.05). We experimentally quantified mitochondrial ROS which confirmed increased oxidative stress in Rps15-mut compared to Rps15-WT splenocytes (p<0.001). We reasoned that unresolved oxidative stress could lead to DNA damage accumulation. Accordingly, we demonstrated increased γH2AX accumulation, a marker of DNA damage, in Rps15-mut B splenocytes compared to their Rps15-WT counterparts (p=0.01), as well as hypersensitivity of Rps15-mut splenocytes to oxidative stress overload with parthenolide (p=0.0009). RPS15 is known to stabilize p53 via MDM2 inhibition, hence Rps15-mut cells exhibited reduced p53 expression. Nevertheless, in response to DNA damage, p53 and p21 were significantly induced, the latter a p53-dependent mediator of G1 checkpoint activation, highlighting the functional importance of residual p53 activity in Rps15-mut B cells.

Given their heightened level of DNA damage, we further hypothesized that Rps15-mut pre-leukemic B splenocytes are hyper-dependent on DNA damage response (DDR). Conversely, defective DDR could precipitate genomic instability and facilitate acquisition of additional genomic alterations, such as TP53 deletion or loss-of-function mutation, that might promote transition toward a hyperproliferative or malignant state. To determine whether Rps15-mut B splenocytes could effectively respond to further genomic insults, we assessed cellular response to hydroxyurea (HU) and ionizing radiation (IR) that induce replication stress and DNA double-strand breaks (DSBs) respectively. While the ATR-Chk1 response to HU was preserved, the ATM-Chk2 response to IR was defective with diminished phosphorylation of downstream ATM targets and impaired induction of G2/M arrest. Indeed, Rps15-mut B cells subsequently acquired a spectrum of additional mutations and chromosomal alterations (e.g. Myc/Mapk amplification, Trp53 loss) that reflect genomic instability secondary to defective DDR.

Finally, we postulated that translational defects directly contribute to oxidative DNA damage accumulation and genomic instability in Rps15-mut B cells. We therefore performed ribosome profiling that showed differential translation efficiency (TE) in 342 genes between Rps15-mut and Rps15-WT B splenocytes, with significant enrichment of DNA replication, DDR and cell cycle genes by GSEA that was recapitulated in RPS15-mut vs WT isogenic HG3 CLL cell lines. In particular, Gpx1, a glutathione peroxidase that functions as a key cellular antioxidant, was confirmed by western blot to be downregulated in Rps15-mut B cells secondary to reduced TE (0.6-fold change vs WT, p=0.018), alongside other proteins important for the amelioration of oxidative DNA damage and DSBs (e.g. Cyb5r4, Otud4, Fance, Tlk1). Our analyses thus link RPS15-induced translational alterations with a cascade of cellular defects that compromise genome stability and highlight the critical nature of these defects in B-cell transformation and CLL progression.