Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition

Targeted therapeutics that block signal transduction through the RAS–RAF–MEK and PI3K–AKT–mTOR pathways offer significant promise for the treatment of human malignancies. Dual inhibition of MAP/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) with the potent and selective small-molecule inhibitors GDC-0973 and GDC-0941 has been shown to trigger tumor cell death in preclinical models. Here we have used phosphomotif antibodies and mass spectrometry (MS) to investigate the effects of MEK/PI3K dual inhibition during the period immediately preceding cell death. Upon treatment, melanoma cell lines responded by dramatically increasing phosphorylation on proteins containing a canonical DNA damage-response (DDR) motif, as defined by a phosphorylated serine or threonine residue adjacent to glutamine, [s/t]Q. In total, >2,000 [s/t]Q phosphorylation sites on >850 proteins were identified by LC-MS/MS, including an extensive network of DDR proteins. Linear mixed-effects modeling revealed 101 proteins in which [s/t]Q phosphorylation was altered significantly in response to GDC-0973/GDC-0941. Among the most dramatic changes, we observed rapid and sustained phosphorylation of sites within the ABCDE cluster of DNA-dependent protein kinase. Preincubation of cells with the inhibitors of the DDR kinases DNA-dependent protein kinase or ataxia-telangiectasia mutated enhanced GDC-0973/GDC-0941–mediated cell death. Network analysis revealed specific enrichment of proteins involved in RNA metabolism along with canonical DDR proteins and suggested a prominent role for this pathway in the response to MEK/PI3K dual inhibition.Dysregulation of the RAS–RAF–MAP/MEK and PI3K–AKT–mTOR pathways represents a common theme in human cancer. The importance of these interconnected pathways is highlighted by the frequency of mutational activation of pathway members including RAS, RAF, and PI3K, as well as inactivation of the inhibitory phosphatase and tensin homolog (PTEN) (1, 2). Targeted therapies that block signaling through the RAS–RAF–MEK pathway, including specific inhibitors of oncogenic forms of BRAF (e.g., BRAF-V600E, which is observed in ∼50% of melanomas) and of the downstream effector MEK have shown clinical efficacy in melanoma and other tumor types (3, 4). Likewise, suppression of cell-survival signaling through inhibition of PI3K has been shown to kill cancer cells (5, 6). Although inhibition of either pathway individually can elicit measureable responses, feedback through signal-transduction networks often limits the effectiveness of single-agent therapies. Mounting evidence suggests that dual inhibition of the RAS–RAF–MEK and PI3K–AKT–mTOR pathways will demonstrate improved efficacy over single-agent therapies.At the heart of the RAS–RAF–MEK and PI3K–AKT–mTOR pathways is a web of phosphorelay networks in which individual phosphorylation sites serve as nodes. Many key nodes reside on protein kinases, where phosphorylation individually and in aggregate modulates the amplitude and specificity of downstream signaling. Our understanding of these networks has been shaped by studies using phosphospecific antibodies against these individual, site-specific phosphorylation events including ERK1/2 at Thr202/Tyr204 (7, 8) and AKT at Thr308 (9, 10). Although this strategy has proven successful, the generation of sensitive phosphospecific reagents capable of reading out signal unambiguously remains a challenge. Likewise, multiply phosphorylated sequences or those occurring adjacent to other posttranslational modifications can confound data interpretation. A key limitation is that phosphospecific antibodies are intended to interrogate only a single node in a signal-transduction network, so that even when multiplexed they provide only a narrow portal through which to view the dynamic system.Mass spectrometry (MS) proteomics provides a platform to dissect signaling networks in breadth and depth. Although theoretically the phosphorylated peptides can be profiled directly from digested cell lysates, interrogating signal-transduction networks requires enrichment of modified peptides from the cellular milieu. One approach involves immunoaffinity enrichment (IAE) with antibodies recognizing classes of phosphopeptides, such as phosphotyrosine (11). IAE methods also have been reported for assaying phosphorylation in the AKT (12) and DNA damage-response (DDR) signaling networks (13, 14), using antibodies that recognize phosphorylation within a local sequence context. For substrates of AKT family kinases, phosphorylation occurs within an RXRXX[s/t] sequence, where X represents any amino acid and [s/t] refers to the phosphorylated serine or threonine (12). For substrates of ataxia-telangiectasia mutated (ATM) and other DDR kinases, phosphorylation occurs on serine or threonine adjacent to glutamine, [s/t]Q (15). In contrast to phosphospecific antibodies directed against a single sequence, motif-specific antibodies recognize a degenerate motif and permit enrichment of an array of peptides. Likewise, these reagents provide a readout for screening conditions by immunoblot before phosphopeptide enrichment and LC-MS/MS.In the current study, we noticed that MEK/PI3K dual inhibition in melanoma lines resulted in a marked DDR. Using motif-directed IAE and MS proteomics, we investigated the signaling elicited by small-molecule inhibitors of MEK and PI3K currently in clinical development to establish a molecular understanding of this response.