Fig 2: PKC? and PKM? expression and PKC? pseudosubstrate efficacy in ALL cell lines. (a) PRKMZ and PRKCZ mRNA expression in 12 ALL cell lines. One embryonic kidney cell line (HEK293T) and one acute myeloid leukaemia cell line (THP-1) were included as positive controls. RPS20 was used as a loading control. Asterisks denote precursor B-ALL cell lines carrying a TCF3 translocation. (b) PKC/M? protein expression in ALL cell lines and HEK293T as determined by Reverse-phase protein arrays (RPPA). TCF3-rearranged cell lines are represented by hatched bars. Protein levels are expressed in units of fluorescence, corrected for background and total protein signals. (c) Survival curve of a PKM?-high (KASUMI-2), PKM?-low (697), PKC?-high (THP-1) and PKC/M?-negative (REH) leukaemic cell line after 72 h of exposure to PKC? pseudosubstrate. Cell viability was determined with an MTS assay and expressed as a percentage of control cells exposed for 72 h to solvent only.

Fig 3: PKC? and PKM? expression and phosphorylation levels in ALL patients. (a) PRKCZ and PRKMZ mRNA expression in paediatric ALL patient samples (n=20). RPS20 was used as a loading control. Asterisks indicate samples with PKC/M? protein levels below the median as determined in Figure 2d. (b) Western blot analysis of PKC? and PKM? expression using a C-terminus- (left panel) or N-terminus (right panel)-binding antibody detected with a red or green fluorescent secondary antibody, respectively, enabling simultaneous hybridization. ß-actin was used as a protein loading control. Reverse-phase protein arrays (RPPA) were used to determine PKC/M? protein levels (c) and T410/403 phosphorylation levels of PKC?/? (d) in genetic subtypes of precursor B-ALL (n=237), T-ALL (n=40) and normal bone marrow (nBM; n=14). Y axes present protein expression in units of fluorescence, corrected for background and total protein signals. Statistical significance is indicated for nBM vs ALL, T-ALL vs precursor B-ALL, and for each genetic subtype of precursor B-ALL vs all other precursor B-ALL subtypes.

Fig 4: Schematic comparison of PKC? and PKM? protein and transcript domains. (a) The protein sequences of PKC? and PKM? contain an identical C-terminus including the complete catalytic domain. PKC? activation requires phosphorylation at a threonine residue in the activation loop (T410) and subsequent autophosphorylation at the turn motif site (T560). A flexible hinge region connects the catalytic domain with the N-terminal regulatory region, which consists of a PB1 (Phox and Bem 1) domain not present in the conventional and novel PKCs and responsible for protein–protein interaction, an autoinhibitory pseudosubstrate domain (ps) and adjacent atypical C1 domain which both interact with the catalytic domain to keep PKC? in a closed and inactive conformation.26 PKM?, however, lacks this N-terminal regulatory region and is therefore constitutively active. (b) PRKCZ and PRKMZ transcripts contain a unique 5'-end but share the final 1864 bp, including part of the coding sequence (CDS) and the complete 3'-untranslated region (UTR). The PRKCZ sequence was taken from the NCBI database (accession nr. NM_002744.4). The sequence of PRKMZ was derived from the NCBI database (PRKCZ isoform 2, accession nr. NM_001033581) and Hernandez et al.19

Fig 5: High PKC/M? protein expression is associated with increased sensitivity to thiopurines. Primary leukaemic cells of precursor B-ALL patients (n=13) were exposed to 1.6 µg/ml 6-TG or 15.6 µg/ml 6-MP. Leukaemic cell viability was determined with an MTS assay after 96 h and expressed as a percentage of cells treated with solvent only. Cases were categorized into those with low and high PKC/M? protein levels with a cut-off of 13 513 fluorescence units (that is, the 90th percentile of 277 ranked patient samples). Open circles represent TCF3-rearranged cases. (a) Comparison of cell viability after exposure of low-PKC/M? and high-PKC/M? cases to 6-TG (left) and 6-MP (right). Horizontal lines indicate group medians. (b) Correlation plots of PKC/M? protein expression and leukaemic cell viability after exposure to 6-TG (left) or 6-MP (right). The vertical dotted line indicates the cut-off for low–high expression. Graphs present the best fit and Spearman correlation coefficient (rs). (c) PRKCZ and PRKMZ mRNA expression in thiopurine-resistant low-PKC/M? and sensitive high-PKC/M? cases.