Karunaraj, P. | bioRxiv Science

bioRxiv2025

A Hotspot Phosphorylation Site on SHP2 Drives Oncoprotein Activation and Drug Resistance

SHP2 is a phosphatase and a critical mediator of receptor tyrosine kinase (RTK)-driven RAS/mitogen-activated protein kinase (MAPK) signaling. Despite promising preclinical data, SHP2 inhibitors have shown minimal clinical efficacy, with no defined clinical mechanisms of primary resistance. Here, we elucidate phosphorylation of SHP2 at tyrosine 62 (pY62) as a hotspot phosphorylation site in the proteome and RTK-driven tumor types in patients. We demonstrate that SRC family kinases directly phosphorylate SHP2 at Y62, downstream of but not directly phosphorylated by RTKs. Using biochemical and biophysical analyses, we show that SHP2 Y62D enforces an open, active conformation, resulting in constitutive phosphatase activation that is sufficient to activate MAPK signaling and confer resistance to allosteric SHP2 inhibitors. These findings establish that SHP2 pY62 is a phosphorylation hotspot phenocopying mutational activation, a mechanism of primary resistance to SHP2 inhibitors, and a cancer drug target distinct from wildtype SHP2. Statement of significanceThis study identifies phosphorylation of SHP2 at tyrosine 62 (pY62) as a conserved mechanism of resistance to allosteric SHP2 inhibitors. By stabilizing an open, active SHP2 conformation, pY62 phenocopies oncogenic PTPN11 mutations and sustains MAPK signaling across cancer types. These findings redefine SHP2 inhibitor resistance as a phosphorylation-driven, target-intrinsic process, nominate pY62 as a potential biomarker for therapeutic response, and propose phosphorylated SHP2 as a distinct drug target.

cancer biology↗

bioRxiv2025

A Drosophila model for Costello Syndrome caused by Ras mutation K117R

Germline mutations that increase signaling through the Ras pathway can cause developmental disorders called RASopathies. The RASopathy Costello syndrome has been described to present with hallmarks that include short stature, intellectual disability, cardiac issues, and characteristic facial abnormalities and has been associated with gain-of-function mutations in HRas. The most common HRas mutations in Costello Syndrome occur at G12 and G13, but there are also other rare mutation sites such as K117 including HRasK117R. RasK117R mutations are also found in colorectal cancer. Drosophila studies modeling gain-of-function in Ras primarily utilize the common cancer-associated mutation G12V, and previous Drosophila RASopathy models assessing Ras gain-of-function mutations have used human sequences for KRas G12D and HRas G12S. To augment these studies, we characterized the phenotype of engineering the rare gain-of-function mutation K117R in the Drosophila Ras sequence. We report here that constitutive low-level expression of RasK117R increased lethality and reduced body size while also causing rough eye and ectopic wing vein phenotypes in those flies that survived to adulthood. Ras pathway inhibitors Trametinib and Rigosertib suppressed the lethality but not the reduced size phenotypes. Trametinib strongly suppressed the K117R wing vein phenotype whereas Rigosertib had only subtle effects. Trametinib is a direct MEK inhibitor. Rigosertib has been reported to have strong effects on PI3K signaling and to indirectly inhibit the Raf-ERK branch. Therefore, this data is consistent with an interpretation that some lethality in the fly RasK117R model depends on elevated signaling through the Raf-ERK branch and potentially some lethality depends on the PI3K branch. In contrast, the lack of effects on the reduced size phenotypes would be consistent with small stature resulting from Raf- and PI3K-independent processes. We propose that this model can be useful for future mechanistic analysis and pharmacological screening and evaluation.

developmental biology↗