Protein Tyrosine Phosphatase Non-Receptor 11 (PTPN11/Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC)

Abstract

<i>PTPN11</i> encodes the SHP2 protein tyrosine phosphatase that activates the mitogen-activated protein kinase (MAPK) pathway upstream of <i>KRAS</i> and MEK. <i>PTPN11</i>/Shp2 somatic mutations occur frequently in Juvenile myelomonocytic leukaemia (JMML); however, the role of mutated <i>PTPN11</i> in lung cancer tumourigenesis and its utility as a therapeutic target has not been fully addressed. We applied mass-spectrometry-based genotyping to DNA extracted from the tumour and matched the normal tissue of 356 NSCLC patients (98 adenocarcinomas (LUAD) and 258 squamous cell carcinomas (LUSC)). Further, <i>PTPN11</i> mutation cases were identified in additional cohorts, including TCGA, Broad, and MD Anderson datasets and the COSMIC database. <i>PTPN11</i> constructs harbouring <i>PTPN11</i> E76A, A72D and C459S mutations were stably expressed in IL-3 dependent BaF3 cells and NSCLC cell lines (NCI-H1703, NCI-H157, NCI-H1299). The MAPK and PI3K pathway activation was evaluated using Western blotting. <i>PTPN11</i>/Shp2 phosphatase activity was measured in whole-cell protein lysates using an Shp2 assay kit. The Shp2 inhibitor (SHPi) was assessed both in vitro and in vivo in a <i>PTPN11</i>-mutated cell line for improved responses to MAPK and PI3K targeting therapies. Somatic <i>PTPN11</i> hotspot mutations occurred in 4/98 (4.1%) adenocarcinomas and 7/258 (2.7%) squamous cells of 356 NSCLC patients. Additional 26 <i>PTPN11</i> hotspot mutations occurred in 23 and 3 adenocarcinomas and squamous cell carcinoma, respectively, across the additional cohorts. Mutant <i>PTPN11</i> significantly increased the IL-3 independent survival of Ba/F3 cells compared to wildtype <i>PTPN11</i> (<i>p</i> < 0.0001). Ba/F3, NCI-H1703, and NCI-H157 cells expressing mutant <i>PTPN11</i> exhibited increased <i>PTPN11</i>/Shp2 phosphatase activity and phospho-ERK1/2 levels compared to cells expressing wildtype <i>PTPN11</i>. The transduction of the <i>PTPN11</i> inactivating mutation C459S into NSCLC cell lines led to decreased phospho-ERK, as well as decreased phospho-AKT in the <i>PTPN11</i>-mutated NCI-H661 cell line. NCI-H661 cells (<i>PTPN11</i>-mutated, <i>KRAS</i>-wild type) were significantly more sensitive to growth inhibition by the PI3K inhibitor copanlisib (IC50: 13.9 ± 4.7 nM) compared to NCI-H1703 (<i>PTPN11</i>/<i>KRAS</i>-wild type) cells (IC50: >10,000 nM). The SHP2 inhibitor, in combination with the PI3K targeting therapy copanlisib, showed no significant difference in tumour development in vivo; however, this significantly prevented MAPK pathway induction in vitro (<i>p</i> < 0.0001). <i>PTPN11</i>/Shp2 demonstrated the in vitro features of a driver oncogene and could potentially sensitize NSCLC cells to PI3K inhibition and inhibit MAPK pathway activation following PI3K pathway targeting.