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Phosphoproteomic Characterization of DNA Damage Response in Melanoma Cells Following MEK/PI3K Dual Inhibition

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Phosphoproteomic Characterization of DNA Damage Response in Melanoma Cells Following MEK/PI3K Dual Inhibition Corrections CELL BIOLOGY Correction for “Phosphoproteomic characterization of DNA dam- which appeared in issue 48, November 26, 2013, of Proc Natl age response in melanoma cells following MEK/PI3K dual in- Acad Sci USA (110:19426–19431; first published November 11, hibition,” by Donald S. Kirkpatrick, Daisy J. Bustos, Taner Dogan, 2013; 10.1073/pnas.1309473110). Jocelyn Chan, Lilian Phu, Amy Young, Lori S. Friedman, Marcia The authors note that Figure 1 appeared incorrectly. The cor- Belvin, Qinghua Song, Corey E. Bakalarski, and Klaus P. Hoeflich, rected figure and its legend appear below. A A2058: DMSO (-) vs MEKi/PI3Ki combo (+) -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ pRSK pAKT pERK pS6 s/tQ s/tQG CDK CK PXsP PXtP tP PKA PDK tXR RXXs/tRXRXXs/t 888MEL: DMSO (-) vs MEKi/PI3Ki combo (+) -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ pRSK pAKT pERK pS6 s/tQ s/tQG CDK CK PXsP PXtP tP PKA PDK tXR RXXs/tRXRXXs/t B MEKi -+ -+ -+ -+ PI3Ki --++ --++ 191 191 97 97 64 64 51 51 39 39 28 28 s/tQ RXXs/t C MEKi PI3Ki MEKi + Pi3Ki EC 50 000.25 .5 11124 0.25.5 2 4 .25 .5 24 p-p53 (S15) p53 p-H2AX(S139) H2A.X cl PARP p-AKT(T308) AKT p-ERK1/2 ERK1/2 Actin GAPDH Fig. 1 Dual inhibition of MEK and PI3K induces phosphorylation of DDR substrates. (A) A2058 or 888MEL melanoma cells were treated for 6 h with DMSO or GDC-0973+GDC-0941 (MEKi/PI3Ki combo; 4× EC50) and were subjected to KinomeView Profiling. A2058 and 888MEL cells were treated with 10 μM GDC-0973 + 10 μM GDC-0941 or 0.2 μM GDC-0973 + 10 μM GDC-0941, respectively. Blots were probed using an antibody mixture recognizing pRSK, pAKT, pERK, and pS6 or phosphomotif antibodies (e.g., DDR substrates with the [s/t]Q motif and AKT substrates with the RXX[s/t] and RXRXX[s/t] motifs). (B) A2058 lysates probed with the [s/t]Q and RXX[s/t] antibodies after treatment with DMSO, 10 μM GDC-0973 (MEKi), 10 μM GDC-0941 (PI3Ki), or the combination. (C) Dose response of A2058 cells to increasing concentrations of MEKi and PI3Ki alone or in combination. Blots were performed against DDR (p53 pSer15, histone 2AX pSer139), cell survival/ cell death (AKT pThr308, cleaved PARP), and cell signaling (ERK1/2 pThr202/Tyr204) markers and controls. Actin and GAPDH served as loading controls. www.pnas.org/cgi/doi/10.1073/pnas.1322630111 562–563 | PNAS | January 7, 2014 | vol. 111 | no. 1 www.pnas.org Downloaded by guest on September 27, 2021 ENVIRONMENTAL SCIENCES GENETICS Correction for “Genome of an arbuscular mycorrhizal fungus Correction for “Whole-genome sequencing identifies a recurrent provides insight into the oldest plant symbiosis,” by Emilie functional synonymous mutation in melanoma,” by Jared J. Tisserant, Mathilde Malbreil, Alan Kuo, Annegret Kohler, Gartner, Stephen C. J. Parker, Todd D. Prickett, Ken Dutton- Aikaterini Symeonidi, Raffaella Balestrini, Philippe Charron, Regester, Michael L. Stitzel, Jimmy C. Lin, Sean Davis, Vijaya L. Nina Duensing, Nicolas Frei dit Frey, Vivienne Gianinazzi-Pearson, Simhadri, Sujata Jha, Nobuko Katagiri, Valer Gotea, Jamie K. Luz B. Gilbert, Yoshihiro Handa, Joshua R. Herr, Mohamed Teer, Xiaomu Wei, Mario A. Morken, Umesh K. Bhanot, NISC Hijri, Raman Koul, Masayoshi Kawaguchi, Franziska Krajinski, Comparative Sequencing Program, Guo Chen, Laura L. Elnitski, Peter J. Lammers, Frederic G. Masclaux, Claude Murat, Michael A. Davies, Jeffrey E. Gershenwald, Hannah Carter, Emmanuelle Morin, Steve Ndikumana, Marco Pagni, Denis Rachel Karchin, William Robinson, Steven Robinson, Steven A. Petitpierre, Natalia Requena, Pawel Rosikiewicz, Rohan Riley, Rosenberg, Francis S. Collins, Giovanni Parmigiani, Anton A. Katsuharu Saito, Hélène San Clemente, Harris Shapiro, Diederik Komar, Chava Kimchi-Sarfaty, Nicholas K. Hayward, Elliott H. van Tuinen, Guillaume Bécard, Paola Bonfante, Uta Paszkowski, Margulies, and Yardena Samuels, which appeared in issue 33, Yair Y. Shachar-Hill, Gerald A. Tuskan, Peter W. Young, Ian R. August 13, 2013, of Proc Natl Acad Sci USA (110:13481–13486; Sanders, Bernard Henrissat, Stefan A. Rensing, Igor V. Grigoriev, first published July 30, 2013; 10.1073/pnas.1304227110). Nicolas Corradi, Christophe Roux, and Francis Martin, which The authors note that the following statement should be appeared in issue 50, December 10, 2013, of Proc Natl Acad Sci added to the Acknowledgments: “Y.S. is supported by the Israel USA (110:20117–20122; first published November 25, 2013; Science Foundation (Grants 1604/13 and 877/13) and the Eu- 10.1073/pnas.1313452110). ropean Research Council (Grant StG-335377).” The authors note that the author name Peter W. Young should instead appear as J. Peter W. Young. The corrected au- www.pnas.org/cgi/doi/10.1073/pnas.1323160111 thor line appears below. The online version has been corrected. Emilie Tisserant, Mathilde Malbreil, Alan Kuo, Annegret Kohler, Aikaterini Symeonidi, Raffaella Balestrini, Philippe Charron, Nina Duensing, Nicolas Frei dit Frey, MEDICAL SCIENCES Vivienne Gianinazzi-Pearson, Luz B. Gilbert, Yoshihiro “ β Correction for Integrin 1-focal adhesion kinase signaling CORRECTIONS Handa, Joshua R. Herr, Mohamed Hijri, Raman Koul, directs the proliferation of metastatic cancer cells dissemi- Masayoshi Kawaguchi, Franziska Krajinski, Peter J. nated in the lungs,” by Tsukasa Shibue and Robert A. Weinberg, Lammers, Frederic G. Masclaux, Claude Murat, which appeared in issue 25, June 23, 2009, of Proc Natl Acad Sci Emmanuelle Morin, Steve Ndikumana, Marco Pagni, USA (106:10290–10295; first published June 5, 2009; 10.1073/ Denis Petitpierre, Natalia Requena, Pawel Rosikiewicz, pnas.0904227106). Rohan Riley, Katsuharu Saito, Hélène San Clemente, The authors note that on page 10295, right column, 2nd full “ ” Harris Shapiro, Diederik van Tuinen, Guillaume Bécard, paragraph, line 8 10 mg/kg xylene should instead appear as “10 mg/kg xylazine.” Paola Bonfante, Uta Paszkowski, Yair Y. Shachar-Hill, Gerald A. Tuskan, J. Peter W. Young, Ian R. Sanders, www.pnas.org/cgi/doi/10.1073/pnas.1322177111 Bernard Henrissat, Stefan A. Rensing, Igor V. Grigoriev, Nicolas Corradi, Christophe Roux, and Francis Martin www.pnas.org/cgi/doi/10.1073/pnas.1322697111 PNAS | January 7, 2014 | vol. 111 | no. 1 | 563 Downloaded by guest on September 27, 2021 Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition Donald S. Kirkpatricka,1, Daisy J. Bustosa,2, Taner Doganb,2, Jocelyn Chanb,2, Lilian Phua, Amy Youngb, Lori S. Friedmanb, Marcia Belvinb, Qinghua Songc, Corey E. Bakalarskid, and Klaus P. Hoeflichb Departments of aProtein Chemistry, bTranslational Oncology, cNonclinical Biostatistics, and dBioinformatics and Computational Biology, Genentech, Inc., South San Francisco, CA 94080 Edited by James E. Cleaver, University of California, San Francisco, CA, and approved October 15, 2013 (received for review May 22, 2013) Targeted therapeutics that block signal transduction through the specificity of downstream signaling. Our understanding of these RAS–RAF–MEK and PI3K–AKT–mTOR pathways offer significant networks has been shaped by studies using phosphospecific promise for the treatment of human malignancies. Dual inhibition antibodies against these individual, site-specific phosphorylation of MAP/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) events including ERK1/2 at Thr202/Tyr204 (7, 8) and AKT at with the potent and selective small-molecule inhibitors GDC-0973 Thr308 (9, 10). Although this strategy has proven successful, and GDC-0941 has been shown to trigger tumor cell death in pre- the generation of sensitive phosphospecific reagents capable of clinical models. Here we have used phosphomotif antibodies and reading out signal unambiguously remains a challenge. Like- mass spectrometry (MS) to investigate the effects of MEK/PI3K dual wise, multiply phosphorylated sequences or those occurring inhibition during the period immediately preceding cell death. Upon adjacent to other posttranslational modifications can confound treatment, melanoma cell lines responded by dramatically increasing data interpretation. A key limitation is that phosphospecific phosphorylation on proteins containing a canonical DNA damage- antibodies are intended to interrogate only a single node in response (DDR) motif, as defined by a phosphorylated serine or a signal-transduction network, so that even when multiplexed threonine residue adjacent to glutamine, [s/t]Q. In total, >2,000 they provide only a narrow portal through which to view the [s/t]Q phosphorylation sites on >850 proteins were identified by dynamic system. LC-MS/MS, including an extensive network of DDR proteins. Linear Mass spectrometry (MS) proteomics provides a platform to mixed-effects modeling revealed 101 proteins in which [s/t]Q phos- dissect signaling networks in breadth and depth. Although the- phorylation was altered significantly in response to GDC-0973/GDC- oretically the phosphorylated peptides can be profiled directly 0941. Among the most dramatic changes, we observed rapid and from digested cell lysates, interrogating signal-transduction net- sustained phosphorylation of sites within the ABCDE cluster of DNA- works requires enrichment of modified peptides from the cellular dependent protein kinase. Preincubation of cells with the inhibitors milieu. One approach involves immunoaffinity enrichment (IAE) of the DDR kinases DNA-dependent protein kinase or ataxia-tel- with antibodies recognizing classes of phosphopeptides, such angiectasia mutated enhanced GDC-0973/GDC-0941–mediated as phosphotyrosine (11). IAE methods also have been reported cell death. Network
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