Abstract SY01-01: Adaptive protein and phosphoprotein networks which promote therapeutic sensitivity or acquired resistance
Authors: Haley John D. , Thomson Stuart , Bean James , Xie Ruixi , Epstein David , Miglarese Mark , Buck Liz 
Published: May 2019

&lt;abstract&gt; Multiple signal transduction pathways can be concurrently active within a single cell, and extensive crosstalk can occur between RTKs. Additionally, tumor tissues can be comprised of a heterogeneous collection of cell states utilizing distinct RTKs for maintenance of tumor cell growth and survival. As a consequence of this complexity, many tumors may be only partially sensitive to single agent therapies and would require the interdiction of multiple RTKs and other protein signaling targets for optimal anti-cancer therapy. Understanding pathway crosstalk is vital to guide the rational combination of approved and experimental anti-cancer agents.

Receptor tyrosine kinases (RTKs) are key mediators of tumor cell survival, proliferation and migratory pathways, and inhibitors of RTKs have demonstrated anti-tumor efficacy in both the preclinical and clinical settings. Multiple sensitivity and resistance mechanisms have been described for EGF receptor inhibitors for the treatment of NSCLC. In carcinomas with an epithelial phenotype, onco-addiction and tumor progression have been associated with autocrine ligand-dependent activation of EGFR (1, 2). Notably, EGFR dependence can also occur through genetic mutations in exons 19-21 of the EGFR gene (3-5) which renders the encoded protein less able to bind ATP. Resistance can occur, primarily in these patients with these primary activating mutations, from acquisition of a secondary mutation of EGFR at the T790M gatekeeper site (6, 7) which increases ATP affinity and thereby decreases binding of ATP competitive inhibitors (8). Importantly, resistance can also occur through up regulation of alternate receptor tyrosine kinases (RTKs), and therefore understanding crosstalk between RTKs is critical for optimizing the use of RTK inhibitors in the clinic.

Tumor tissues progress from in situ to metastatic states through the reacquisition of developmental programs allowing invasion and metastasis. The acquisition of an invasive phenotype can occur by epithelial-mesenchymal transition (EMT). The molecular characteristics of epithelial and mesenchymal cell phenotypes were extensively characterized by intersection of proteomic, phosphoproteomic and gene expression profiling approaches. Tumor cells that have undergone EMT show a marked reduction in sensitivity to EGFR TKIs and anti-EGFR MAbs (9). In several instances, the EMT-derived mesenchymal-like tumor cells have gained sensitivity to PDGFR and/or FGFR1 inhibitors. Significant switching of receptor tyrosine kinases, from EGFR, Met/Ron and IGF-1R to cells utilizing PDGFR and FGFR was observed. The acquisition of autocrine fibronectin - integrin was also observed in several tumor lines and inducible models. Therefore, EMT can promote use of alternative signaling pathways.

Resistance can also derive from over activation of partially redundant pathways concurrently active in a cell. For example, activation of the IGF-1 receptor (IGF-1R) or the HGF receptor (Met) has been shown to obviate the need for EGFR signaling in epithelial-derived lung tumors (10). Crosstalk has been well documented with EGF, HGF and IGF1 receptors. These receptors, when activated by ligand binding, can create network redundancies (for example by IGF stimulation of EGFR onco-addicted cells; (11)). Reciprocal activation of one receptor following the inhibition of a distinct receptor has also been observed. For example PDGFRα was observed to be activated and substrates phosphorylated when EGFR was inhibited in the NSCLC line H1703(12). These studies involved a quantitative anti-phosphotyrosine profiling (13) coupled to an LC-MS/MS shotgun approach. In a second example in the Ewings sarcoma line A673, IGF-1R inhibition, by kinase inhibition resulted in the reciprocal activation of the insulin receptor and cell survival (14). We have previously shown reciprocal activation of EGFR by IGF1R inhibition and activation of IGF1R through EGFR inhibition (15). The mechanisms by which reciprocity is achieved are under investigation. The ability of tumor cells to reciprocally induce alternative RTKs following the inhibition of a given receptor (in this case EGFR or IGF1R) highlights the need for rational combination anti-cancer therapy.

One RTK may also positively affect the activity of another in a process that can be termed receptor co-option. Here, one dominant ligand-stimulated RTK, possibly amplified or mutated, directly or indirectly tyrosine phosphorylates additional RTKs to create functional signaling scaffolds and to engage signaling networks beyond the normal capabilities of the original dominant kinase. Where the co-option of RTK signaling networks is observed, markers of RTK activation (e.g. the extent of receptor tyrosine phosphorylation) are not necessarily predictive of onco-addiction. Direct or indirect crosstalk between EGFR, ErbB2, Met, Ron and other RTKs has been observed in a bidirectional manner. Crosstalk can occur on the RON Y1238/Y1239 autophosphorylation site where MET and/or EGFR are the active kinases. Such phosphorylation in trans (either direct or indirect) may promote RON kinase activity in the absence of ligand. Alternatively or in addition, cross phosphorylation of RTKs in trans can allow signaling scaffolds to be established, mimicking kinase activation in the absence of ligand. Three NSCLC models illustrate co-option of RTKs as described below:

The NSCLC line H1650 harbors a gain-of-function exon 19 deletion that is strongly onco-addicting in adenocarcinoma of the lung and is often associated with exquisite sensitivity to EGFR blockade. Pharmacological inhibition of EGFR in H1650 by the EGFR-ErbB2 inhibitor erlotinib results in a rapid dephosphorylation of ErbB2, Src family kinases and the direct and downstream substrates Cbl-B and Erk2 respectively. Interestingly attenuation of Met tyrosine phosphorylation is also observed suggesting that hyperactive mutant EGFR (del19) can also co-opt additional RTKs.

In the NSCLC line H292, ~50% of the tyrosine phosphate on Met and on Ron is inhibitable by EGFR blockade. Crosstalk between EGFR, Met and Ron kinases was measured by combined anti-phosphotyrosine immunoaffinity selection, stable isotope peptide labeling (iTRAQ) and LC-electrospray tandem MS. The phosphotyrosine content on Met and Ron was decreased following exposure to erlotinib relative to mock control cells. The inhibition of phospho-Met and phospho-Ron followed kinetics for the erlotinib-dependent decrease in the SH2 adapter proteins phospho-SHC, -Erk2 and Grb2. The data suggest either direct phosphorylation of Met and Ron by EGFR or rapid recruitment and activation of intermediary non-transmembrane tyrosine kinases. These non-transmembrane tyrosine kinases could include Src/Yes/Fyn family kinases or Brk family kinases, possibly in a cell-specific manner, but this remains to be tested.

A more extreme example of co-option of diverse kinase signaling networks by a single RTK is observed in the NSCLC H1993 cell line. Here exposure to small molecule Met tyrosine kinase inhibitors attenuated both Met and Met-associated SH2/PTB domain adapter proteins as well as more distantly related RTK signaling networks. Exposure of H1993 epithelial carcinoma cells to Met inhibitors markedly inhibited ~250 Met substrates, both known and unknown, including the cell surface signaling proteins Met, Ron, EGFR, ErbB2, DDR1, CSFR2, ITG4, ITG6, EphA2, EphB4. This in turn results in a comparatively complete dephosphorylation of a wide array of signaling adaptors, cell-cell junction proteins, cytoskeletal reorganizing elements and folding chaperones. While marked attenuation of EGFR was observed in response to Met inhibition alone, the combination of both Met and EGFR inhibitors was required for full dephosphorylation of both Erk1/2 and STAT5A. These findings suggest that while Met can co-opt EGFR, phosphorylate EGFR, and establish initial SH2/PTB domain dependent complexes on EGFR via active Met kinase, the extent of Met-dependent EGFR tyrosine phosphorylation is insufficient for full Erk and STAT5 activation. Several conclusions can be drawn. First, Met is the principle source of phosphotyrosine in H1993, and the cell line is likely onco-addicted solely to Met. Second, Met essentially ‘highjacks’ other RTKs as signaling adapters. Third, effective blockade of Erk activation was only observed with dual Met and EGFR inhibition suggesting synergy relies on erlotinib directed Erk inhibition in the H1993 model.

The quantitative measurement of cancer cell signaling under dynamic conditions of pharmacological or siRNA mediated inhibition of specific signaling nodes gives insight into the requirements for effective cellular blockade of survival and invasive networks associated with cancer progression. The use of quantitative shotgun LC-MS/MS methods incorporating stable isotopes provides a rapid means to identify proteins and phosphoproteins perturbed in particular cancer cell states. These methods have allowed interrogation of RTK crosstalk in tumor cell lines and xenografts so as to generate specific hypotheses relating to targeted drug combinations. In addition such data have better defined the role of EMT in survival signaling switching and anticancer drug resistance, again suggesting combination therapy options for further investigation.

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Citation Format: John D. Haley, Stuart Thomson, James Bean, Ruixi Xie, David Epstein, Mark Miglarese, Liz Buck. Adaptive protein and phosphoprotein networks which promote therapeutic sensitivity or acquired resistance [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY01-01