Fig. 1: ERBB2 point mutations found in leukemia samples are oncogenic, enable cytokine-independent proliferation, and downstream activation.

a ERBB2 gene schematic with location of point mutations depicted. The location of the following domains is included: receptor L domain (RLD, domains I and III), furin-like cysteine rich region (FLCRR, domain II), growth factor receptor domain (GRRD4, domain IV), transmembrane domain (TM), and tyrosine kinase domain. b Sanger sequencing chromatograms of patient genomic DNA confirm the presence of ERBB2^R188C, ERBB2^P489L, and ERBB2^L1157R mutations. Peaks correspond to the following nucleotides: A (green), T (red), C (blue), and G (black). Arrows indicate direction of sequencing. c ERBB2^R188C, ERBB2^P489L, and ERBB2^L1157R mutations transform the murine Ba/F3 pro-B cell line to IL-3-independent growth. No growth was observed in parental Ba/F3 cells or cells harboring an empty vector or wild-type (WT) ERBB2. All engineered Ba/F3 cell lines were flow sorted for low, equivalent GFP expression prior to the IL-3 withdrawal assay. Total viable cells are plotted over time after the withdrawal of IL-3. This experiment was repeated at least twice with consistent results. d Stable expression of ERBB2 mutants in NIH 3T3 fibroblasts exhibited spontaneous foci formation in monolayers independent of cell plating density. 3T3 cells transduced with ERBB2^WT lack this phenotype but exhibit an increased rate of cell growth. This experiment was repeated at least twice with consistent results. e Immunoblot analysis of ERBB2-transformed Ba/F3 cells shows increased phosphorylation of ErbB2 with all three mutants compared with ERBB2^WT. GAPDH served as a loading control. Prior to lysis, WT cells were grown in IL-3 supplemented media and all lines were starved overnight in 0.1% BSA RPMI. f In comparison with Ba/F3 cells expressing ERBB2^WT, changes in gene expression were observed in mutant-transformed Ba/F3 cells. Most notably, all mutants resulted in an increase in expression of MAPK signaling. Prior to RNA isolation, WT cells were grown in IL-3 supplemented media and all lines were starved overnight in 0.1% BSA RPMI. Expression analysis was performed in triplicate. g Expression of phosphorylated AKT is increased in mutant-transformed Ba/F3 cells relative to WT cells. ERK phosphorylation was evident only in Ba/F3 cells expressing ERBB2^R188C and ERBB2^L1157R. GAPDH served as a loading control. As noted above, all cell lines were starved overnight in 0.1% BSA RPMI.