Translatome analysis reveals altered serine and glycine metabolism in T-cell acute lymphoblastic leukemia cells

Somatic ribosomal protein mutations have recently been described in cancer, yet their impact on cellular transcription and translation remains poorly understood. Here, we integrate mRNA sequencing, ribosome footprinting, polysomal RNA sequencing and mass spectrometry datasets from a mouse lymphoid cell model to characterize the T-cell acute lymphoblastic leukemia (T-ALL) associated ribosomal RPL10 R98S mutation. Surprisingly, RPL10 R98S induces changes in protein levels primarily through transcriptional rather than translation efficiency changes. Phosphoserine phosphatase (PSPH), encoding a key serine biosynthesis enzyme, was the only gene with elevated transcription and translation leading to protein overexpression. PSPH upregulation is a general phenomenon in T-ALL patient samples, associated with elevated serine and glycine levels in xenograft mice. Reduction of PSPH expression suppresses proliferation of T-ALL cell lines and their capacity to expand in mice. We identify ribosomal mutation driven induction of serine biosynthesis and provide evidence supporting dependence of T-ALL cells on PSPH.

A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Data analysis
All statistics were performed using the R software (and the following R packages: Babel, DESeQ2) or IBM SPSS 23 (IBM Analytics) software. The following open source softwares were also used for analysis: seqtk, fastq-mcf, Bowtie, TopHat2, iRegulon, WebGestalt. All used softwares are referenced in the text. Custom code was used for metagene analyses around start and stop codons and ribosome footprint density profiles as described in the methods section.

April 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability All RNA sequencing and proteomics datasets were generated from isogenic mouse lymphoid Ba/F3 cells engineered to express the WT or R98S mutant form of human RPL10, with three independent WT and three R98S cell clones analyzed in each experiment. The quantitative mass spectrometry dataset has been described previously (

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
No sample-size calculations were performed. Sample sizes were chosen to have enough replicates to do statistics and were determined by availability of biological samples and/or based on experience with our biological models.
Data exclusions total RNA seq sample 1-WT36 matched with the polysome profiling was excluded because it clustered with the R98S mutants in PCA analysis.
Besides this, no data were excluded.

Replication
All figure legends of experimental data contain clear descriptions of the biological and technical replicates.
Randomization Allocation of mice to experimental or control groups was random.

Blinding
Blinding was not performed.
Reporting for specific materials, systems and methods

Validation
Western blotting: All antibodies used showed a specific band at the expected MW according to the Precision Plus Protein Dual Color (Biorad) protein marker. For flow cytometry: The anti-human CD45 antibody specificity was validated using counter staining with anti-mouse CD45. For BRDU flow cytometry, non-cycling cells were used as a negative control for aspecific BRDU incorporation and antibody staining.

Eukaryotic cell lines Policy information about cell lines
Cell line source(s) The Ba/F3 cell model and hematopoietic cell cultures derived from RPL10 R98S conditional knock-in mice were previously described (Girardi et al., Leukemia, 2018) . Jurkat, DND41, RPMI8402 and KE37 T-ALL cell lines were obtained from Leibniz-Institute DSMZ.
Authentication RPMI8402 and DND41 were authenticated by confirming unique NOTCH1 mutational status. Other used cell lines were not authenticated.

Mycoplasma contamination
Cell lines were tested for mycoplasma on a regular basis and always tested negative.

Commonly misidentified lines (See ICLAC register)
Name any commonly misidentified cell lines used in the study and provide a rationale for their use.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
Xenografting of human T-ALL samples was performed in 6-8 weeks old male or female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice.

Wild animals
Not applicable Field-collected samples Not applicable

Human research participants
Policy information about studies involving human research participants

Population characteristics
For the experiments using human T-ALL xenograft material, approval was obtained by the ethics committees of UZ/ KU Leuven and Universiteit Gent (S54608 and S59975). After getting written informed consent, the mononuclear cell fraction of bone marrow from pediatric T-ALL patients was obtained and the cells were xenografted into NSG mice.

Recruitment
Human T-ALL samples from patients that gave approval have been systematically collected by our collaborator Jan Cools and have been injected into NSG mice for expansion.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Sample preparation
All measurements were performed on suspension hematopietic cell lines. Proliferation was measured by counting viable cells