Supplementary Fig. S2. Comparison of Phosphotyrosine Enrichment from Fresh and Frozen PDX

Supplementary Figure Legends

Supplementary Fig. S1. Phosphotyrosine sites identified by immunoaffinity profiling in ALL PDXs. (A) Total number of sites and biological function of phosphoproteins.A total of 540 Class I phosphotyrosine (pY) sites along with 61 phosphoserine (pS) and 49 phosphothreonine (pT) sites were identified in 2 T-ALL (ALL-8 and ALL-31) and 2 BCP-ALL (ALL-17 and ALL-19) PDXs. These sites correlated to 424 phosphoproteins of which 17% were kinases. The functional annotation of all the identified phosphoproteins was conducted using Blast2GO software and represented as a pie chart. (B) Pie charts showing the relative intensities of TK phosphotyrosine sites within each T-ALL and BCP-ALL PDX. LCK was the predominant TK enriched in the T-ALL PDXs (ALL-8 and ALL-31). The predominant TKs in the BCP-ALL PDXs (ALL-17 and ALL-19) were BTK, SYK and LYN. ABL1 was markedly over expressed in ALL-19.

Supplementary Fig. S2. Comparison of phosphotyrosine enrichment from fresh and frozen PDX.

(A). Average number of phosphotyrosinepY sites enriched from four replicates of freshly prepared ALL-2 PDX samples and two frozen samples (ALL-2 F). (B). Intensity of enriched Flt3 phosphotyrosine sites (Y842 and Y969) from fresh (ALL-2) and frozen (ALL-2F) PDX samples.

Supplementary Fig. S3. Optimization of quantitative phosphotyrosine profiling of pediatric ALL PDXs.Each panel depicts the log intensities of enriched phosphopeptides on the y axis versus individual peptides on the x axis. Blue bars, phosphopeptides identified from the test PDX (PALLSD); red bars, phosphopeptides with a heavy SILAC label from the cell line mixture. While 590 phosphopeptides were identified qualitatively in PALLSD alone (A), only approximately 10% of the identified phosphopeptides were ‘light’ when tested in a 1:1 (w/w) ratio with the ‘heavy’ SILAC ‘spike in’ cell line mixture (B). The proportion of PALLSD (light) phosphopeptides identified increased to approximately 45% when the ratio of PALLSD : SILAC mixture was increased to 14:1 (C). Further, the total number of PDX phosphopeptides increased by another 1.6-fold when the ratio was increased to 20:1 (D).

Supplementary Fig. S4. Number of identified Class I phosphosites in each of the PDXs. Asterisk represents number of sites that had a light (PDX) and heavy (SILAC) intensity and could be quantified.

Supplementary Fig. S5. Unsupervised hierarchical clustering of Class I phosphosites in ALL PDXs segregated based on molecularly-defined subgroups. Hierarchical Clustering of (A) all the quantified tyrosine phosphorylated sites and (B) all the quantified TK tyrosine phosphorylation sites. The ETP-ALL (Green), Ph-like (Yellow) and BCR-ABL1 (Red) PDXs were separated from the B (Red) - and T (Blue) -lineage PDXs, and each subjected to cluster analysis based on the average normalized ratios (PDX/SILAC) of tyrosine phosphorylated sites enriched by immunoaffinity profiling. The colours in the heatmaps represent the relative expression per phosphosite across all samples. Red indicates relative high (PDX/SILAC) ratio and blue indicates relative low ratio.

Supplementary Fig. S6. Comparison of ETP-ALL, Ph-like-ALL, BCP-ALL and T-ALL PDXs by gene expression profiling. A total of 14 PDXs were profiled on Illumina Human HT-12 Beadchip arrays. (A) Representative heat map (35 genes) and un-supervised hierarchical clustering of all the genes expressed in ETP-ALL (Green), Ph-like- ALL (Yellow), BCP-ALL (Red) and T-ALL(Blue) subtypes. (B) Heat map and un-supervised hierarchical clustering of the TK genes expressed in the 14 PDXs. The colors in the heatmaps represent the relative expression per gene across all samples. Red indicates relative high expression and blue indicates relative low expression.

Supplementary Fig S7.Comparison of phosphorylation levels of pTyr sites with gene expression profiling.(A) Comparison of pTyr site phosphorylation levels of TKs ABL1, FLT3 and JAK1 with their gene expression profiles in PDXs. (B) Comparison of pTyr site phosphorylation levels of key TK signalling substrates with their gene expression profiles.

Supplementary Fig. S8. In vitro sensitivity of ALL PDXs to Sunitinib.

Supplementary Fig. S9. Venn diagram showing the number of Light PDX phosphopeptides and Heavy SILAC labelled phosphopeptides identified in each of the 16 PDXs along with the percentage of overlap between PDX and SILAC phosphopeptides.