The T-cell leukemia-associated ribosomal RPL10 R98S mutation enhances JAK-STAT signaling

Abstract

Several somatic ribosome defects have recently been discovered in cancer, yet their oncogenic mechanisms remain poorly understood. Here we investigated the pathogenic role of the recurrent R98S mutation in ribosomal protein L10 (RPL10 R98S) found in T-cell acute lymphoblastic leukemia (T-ALL). The JAK-STAT signaling pathway is a critical controller of cellular proliferation and survival. A proteome screen revealed overexpression of several Jak-Stat signaling proteins in engineered RPL10 R98S mouse lymphoid cells, which we confirmed in hematopoietic cells from transgenic Rpl10 R98S mice and T-ALL xenograft samples. RPL10 R98S expressing cells displayed JAK-STAT pathway hyper-activation upon cytokine stimulation, as well as increased sensitivity to clinically used JAK-STAT inhibitors like pimozide. A mutually exclusive mutation pattern between RPL10 R98S and JAK-STAT mutations in T-ALL patients further suggests that RPL10 R98S functionally mimics JAK-STAT activation. Mechanistically, besides transcriptional changes, RPL10 R98S caused reduction of apparent programmed ribosomal frameshifting at several ribosomal frameshift signals in mouse and human Jak-Stat genes, as well as decreased Jak1 degradation. Of further medical interest, RPL10 R98S cells showed reduced proteasome activity and enhanced sensitivity to clinical proteasome inhibitors. Collectively, we describe modulation of the JAK-STAT cascade as a novel cancer-promoting activity of a ribosomal mutation, and expand the relevance of this cascade in leukemia.

Figure 1. Mass spectrometry screen for proteins and pathways showing differential expression between RPL10 WT and R98S cells.

A) Volcano plot of the quantitative proteomics data comparing RPL10 WT and R98S samples. The dashed line illustrates the cut-off for significance (p<0.01; T-test). Red dots represent the 178 proteins that are significantly upregulated in the R98S samples, green dots correspond to the 68 proteins that are significantly downregulated. B) Pathways/processes that are significantly up- (left) or down- (right) regulated in R98S samples. The numbers in the pie chart represent the number of significant pathways corresponding to each process that are up or down in the cells. The individual pathways are listed in Tables S6-S7. C) Pie charts reporting mutation frequencies in JAK and STAT genes (left) or in IL7RA (right) detected in RPL10 WT versus R98S mutant pediatric T-ALL patients. The P-value was calculated using a 2-tailed Fisher’s test.

Figure 2. RPL10 R98S Ba/F3 cells express altered levels of Jak-Stat signaling components, are hyper-reactive towards cytokine stimulation and are sensitized to JAK-STAT inhibitors.

A) Left: immunoblot validation of differential expression of Jak-Stat pathway genes in three independent clones of RPL10 R98S versus WT expressing cells. The figure shows a representative blot of 3 independent experiments. Right: quantification of the immunoblot validations. Signal of the JAK-STAT pathway proteins was normalized for loading. The quantification represents the average +/- standard deviation of a representative experiment comparing 3 independent RPL10 WT versus 3 independent R98S cell clones. B) Left: immunoblot analysis of protein phosphorylation of Jak-Stat pathway genes in RPL10 R98S versus WT expressing cells at different time points after addition of 1 ng/ml of IL3. The figure shows a representative blot of 6 independent experiments. Right: phospho-levels were quantified by dividing the phospho-signal through the signal of the loading control on the blot. The quantification represents the average +/- standard deviation of a representative experiment comparing 3 biologically independent RPL10 WT versus 3 independent R98S cell clones. P-values were calculated using a T-test. C) Relative proliferation of RPL10 WT and R98S cells treated with the JAK1/JAK2 inhibitor ruxolitnib and the STAT5 inhibitor pimozide measured using the ATPlite luminescence assay (Perking Elmer). The panel shows the average +/- standard deviation of a representative experiment comparing 4 biologically independent RPL10 WT versus 4 independent R98S cell clones. P-values were calculated using a T-test *p<0.05, **p<0.01.

Figure 3. RPL10 R98S mouse knock-in hematopoietic cells display increased expression and phosphorylation of Jak-Stat signaling components.

A) Representative immunoblots of expression and phosphorylation of Jak-Stat proteins in hematopoietic cells derived from Rpl10^{cKI R98S} (labeled as WT in the figure) and Mx1-Cre Rpl10^{cKI R98S} (labeled as R98S in the figure) mice. The blots show data from cells derived from 3 biologically independent RPL10 WT versus 3 independent R98S mice. Cells were cultured in cytokine-rich Methocult GF M3534 media containing stem cell factor (SCF), IL3 and IL6. B) Quantification representing the average +/- standard deviation of the experiment shown in A). P-values were calculated using a T-test.

Figure 4. RPL10 R98S T-ALL patient samples have elevated expression and activation of the JAK-STAT cascade and are sensitized to JAK-STAT inhibitors.

A) Left: immunoblot analysis of JAK-STAT pathway protein expression in 3 RPL10 WT and 3 RPL10 R98S mutant human T-ALL xenograft samples. Only those components of the JAK-STAT pathway for which significant changes were observed are represented. Right: quantification of the immunoblots. The bars indicate the average +/- standard deviation of 3 independent RPL10 WT T-ALL patient samples versus 3 RPL10 R98S positive patient samples. P-values were calculated using a T-test. B) Left: Immunoblot analysis of STAT5 and ERK phosphorylation in RPL10 WT (XD83, X10, X12) versus R98S mutant (X13, X15, XB41, X14) T-ALL xenograft samples in unstimulated conditions or after 30 mins of stimulation with 10 ng/ml IL7. Right: quantification of the immunoblots. The bars indicate the average +/- standard deviation of 3 independent RPL10 WT T-ALL patient samples versus 4 RPL10 R98S positive patient samples that were analyzed. P-values were calculated using a T-test. C) Left: Immunoblot analysis of STAT5 and ERK phosphorylation in RPL10 WT versus R98S mutant T-ALL xenograft samples treated for 30 minutes with DMSO, ruxolitinib (500 nM) or pimozide (5 µM) followed by addition of 10 ng/ml IL7 for 30 mins. Right: quantification of the immunoblots. The bars represent the fold reduction in phospho-protein in the drug treated versus DMSO condition and show the average +/- standard deviation of all analyzed RPL10 WT T-ALL patient samples versus R98S positive patient samples. P-values were calculated using a T-test. D) Relative proliferation of 3 RPL10 WT (X09, X10 and X12) and 3 RPL10 R98S mutant (X13, X14 and X15) in vitro cultured T-ALL xenograft samples treated with the STAT5 inhibitor pimozide. Read out was done using the ATPlite assay (Perkin Elmer). The panel shows the average +/- standard deviation of three technical replicates per xenograft sample. P-values were calculated using a T-test. Due to limited sample availability, all experiment in this figure could only be performed once.*p<0.05, **p<0.01, ***p<0.001.

Figure 5. Jak-Stat pathway genes contain functional apparent -1 PRF signals, the frameshifting levels on some of which are influenced by the RPL10 R98S mutation.

A) Results of enrichment analysis within all human genes containing predicted -1 PRF signals and extracted from the PRF database. Analysis was performed using the G:profiler software and KEGG databases, and statistical significance was calculated using Fisher’s one-tailed test. B) Results from dual luciferase reporter assays (explained in Figure S3A) testing apparent -1 PRF levels on computationally predicted -1 PRF signals in the indicated mouse genes. The out-of-frame (OOF) is a negative control. Assays were performed in Ba/F3 cells expressing RPL10 WT or R98S. The bars indicate the average +/- standard error of at least 5 biologically independent measurements. C) Percentages of -1 PRF on human IL7RA and JAK1 mRNAs as determined by dual luciferase reporter assays performed in WT versus R98S Ba/F3 cells. Plots show the average +/- standard error of at least 5 biologically independent measurements.

Figure 6. RPL10 R98S expressing cells show altered proteasome expression and activity and enhanced stability of Jak1.

A) Chymotrypsin-like, caspase-like and trypsin-like proteasomal activity of Ba/F3 cells expressing either WT or R98S RPL10. Plots show the average +/- standard deviation of three independent experiments comparing 3 biologically independent RPL10 WT versus 3 independent R98S cell clones. B) Relative proliferation of RPL10 WT and R98S cells treated with the indicated proteasome inhibitors measured using the ATPlite luminescence assay (Perking Elmer). The panel shows the average +/- standard deviation of a representative experiment comparing 3 biologically independent RPL10 WT versus 3 independent R98S cell clones. C) Left: immunoblots illustrating stability of Jak1 and Csf2rb/2 proteins as assessed by cycloheximide chase assays. Right: quantification of the immunoblots representing the average +/- standard deviation of three independent experiments comparing 3 biologically independent RPL10 WT versus 3 independent R98S cell clones. P-values were calculated using a T-test *p<0.05, **p<0.01, ***p<0.001. CHX: cycloheximide

Figure 7. Model of RPL10 R98S promoted JAK-STAT upregulation and T-ALL pathogenesis.

Expression of RPL10 R98S alters protein stability and -1 PRF rates. These and potentially other mechanisms such as altered transcription drive a cancer promoting cellular protein profile, including overexpression of the JAK-STAT cascade. Particular conditions, such as cytokine stimulation, provide mutant cells with a competitive advantage, causing clonal selection and T-ALL development. Attractive targets for which clinically used drugs are available, some of which RPL10-R98S cells are more sensitive to, are indicated.