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.
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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
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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 analysis revealed specific enrichment of pro- for assaying phosphorylation in the AKT (12) and DNA teins involved in RNA metabolism along with canonical DDR pro- teins and suggested a prominent role for this pathway in the Significance response to MEK/PI3K dual inhibition. Growing evidence suggests that successful intervention in phosphoproteomics | PI3 kinase | PRKDC many human cancers will require combinations of therapeutic agents. Critical to this effort will be a detailed understanding ysregulation of the RAS–RAF–MAP/MEK and PI3K–AKT– of the crosstalk between signaling networks that modulate DmTOR pathways represents a common theme in human can- proliferation, cell death, drug sensitivity, and acquired re- cer. The importance of these interconnected pathways is highlighted sistance. Here we investigated DNA-damage signaling elicited by the frequency of mutational activation of pathway members by small-molecule inhibitors against MAP/ERK kinase (MEK) including RAS, RAF, and PI3K, as well as inactivation of the and PI3K in melanoma cells. This work, performed using cut- inhibitory phosphatase and tensin homolog (PTEN) (1, 2). ting-edge mass spectrometry proteomics, uncovered a burst Targeted therapies that block signaling through the RAS–RAF– of signaling among proteins in the DNA-damage pathway MEK pathway, including specific inhibitors of oncogenic forms upon initiation of the cell-death program by agents targeting the – – – – of BRAF (e.g., BRAF-V600E, which is observed in ∼50% of RAS RAF MEK and PI3K AKT mTOR pathways. These sig- melanomas) and of the downstream effector MEK have shown nals may prove important to the short- and long-term sensi- clinical efficacy in melanoma and other tumor types (3, 4). tivity of tumor cells to MEK- and PI3K-targeted therapies. Likewise, suppression of cell-survival signaling through inhibition Author contributions: D.S.K. and K.P.H. designed research; D.S.K., D.J.B., T.D., J.C., L.P., of PI3K has been shown to kill cancer cells (5, 6). Although in- and A.Y. performed research; L.S.F., M.B., Q.S., and C.E.B. contributed new reagents/ hibition of either pathway individually can elicit measureable analytic tools; D.S.K., D.J.B., T.D., J.C., L.P., A.Y., Q.S., C.E.B., and K.P.H. analyzed responses, feedback through signal-transduction networks often data; and D.S.K., D.J.B., T.D., J.C., C.E.B., and K.P.H. wrote the paper. limits the effectiveness of single-agent therapies. Mounting evi- Conflict of interest statement: All authors are employees and shareholders of Genentech dence suggests that dual inhibition of the RAS–RAF–MEK and Inc., a member of the Roche group. PI3K–AKT–mTOR pathways will demonstrate improved effi- This article is a PNAS Direct Submission. cacy over single-agent therapies. Data deposition: The large data tables provided as Supporting Information also are available At the heart of the RAS–RAF–MEK and PI3K–AKT– at http://research-pub.gene.com/proteomics_data. mTOR pathways is a web of phosphorelay networks in which 1To whom correspondence should be addressed. E-mail: [email protected]. individual phosphorylation sites serve as nodes. Many key 2D.J.B., T.D., and J.C. contributed equally to this work. nodes reside on protein kinases, where phosphorylation in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dividually and in aggregate modulates the amplitude and 1073/pnas.1309473110/-/DCSupplemental.
19426–19431 | PNAS | November 26, 2013 | vol. 110 | no. 48 www.pnas.org/cgi/doi/10.1073/pnas.1309473110 damage-response (DDR) signaling networks (13, 14), using anti- A A2058: DMSO (-) vs MEKi/PI3Ki combo (+) bodies 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 pRSK amino acid and [s/t] refers to the phosphorylated serine or threo- pAKT nine (12). For substrates of ataxia-telangiectasia mutated (ATM) pERK and other DDR kinases, phosphorylation occurs on serine or pS6 threonine adjacent to glutamine, [s/t]Q (15). In contrast to phosphospecific antibodies directed against a single sequence, s/tQ s/tQG CDK CK PXsP PXtP tP PKA PDK tXR RXXs/tRXRXXs/t motif-specific antibodies recognize a degenerate motif and permit 888MEL: DMSO (-) vs MEKi/PI3Ki combo (+) enrichment of an array of peptides. Likewise, these reagents -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ provide a readout for screening conditions by immunoblot be- fore phosphopeptide enrichment and LC-MS/MS. In the current study, we noticed that MEK/PI3K dual in- pRSK pAKT hibition in melanoma lines resulted in a marked DDR. Using pERK motif-directed IAE and MS proteomics, we investigated the sig- pS6 naling elicited by small-molecule inhibitors of MEK and PI3K currently in clinical development to establish a molecular un- s/tQ s/tQG CDK CK PXsP PXtP tP PKA PDK tXR RXXs/tRXRXXs/t derstanding of this response. Results B MEKi -+ -+ -+ -+ Published studies have shown that the compounds GDC-0973 PI3Ki --++ --++ (MEKi) and GDC-0941 (PI3Ki) potently and selectively inhibit 191 191 97 kinase activities of MEK and PI3K, respectively (16, 17). Recent 97 – – 64 work indicates that concurrent inhibition of the RAS RAF 64 MEK and PI3K–AKT–mTOR pathways is more potent at killing 51 cells than individual agents (17, 18). In 4-d viability assays, A2058 51 fi μ 39 (BRAF-V600E, PTEN-de cient; EC50: 2.5 M GDC-0973, 2.5 39 μM GDC-0941) and 888MEL (BRAF-V600E; EC50: 0.05 μM 28 GDC-0973, 2.5 μM GDC-0941) cells displayed synergistic de- 28 creases in cell number after combined pathway inhibition (17). To investigate the mechanism of cell death, A2058 and 888MEL s/tQ RXXs/t cells were treated with 4× EC50 concentrations of MEKi and PI3Ki (hereafter referred to as “combo” treatment). After 6-h C MEKi PI3Ki MEKi + Pi3Ki EC treatment, cell lysates were subjected to multiplexed Western 50 000.25.5111224 0.25 .52 4 .25 .5 4 blot profiling. Consistent with PI3K inhibition, permissive (RXX p-p53 (S15) [s/t]) and rigid (RXRXX[s/t]) AKT-substrate motif antibodies p53 showed robust decreases for the majority of observable bands p-H2AX(S139) (Fig. 1A). Diminished signal also was observed for the PXtP, H2A.X PXsP, and MAPK substrate motifs, consistent with MEK in- cl PARP hibition blocking canonical MAPK signaling. Also striking p-AKT(T308) were elevated levels of DDR phosphorylation reported by two AKT ATM-substrate motif antibodies, [s/t]Q and [s/t]QG (Fig. 1A). p-ERK1/2 To understand the individual contributions of MEK and PI3K inhibition, A2058 cells were left untreated or were treated with ERK1/2 × Actin MEKi,PI3Ki,orthecombination(4 EC50). AKT-substrate GAPDH motif phosphosignals were decreased by PI3Ki alone or in combination with MEKi. ATM-substrate phosphomotif blots Fig. 1. Dual inhibition of MEK and PI3K induces phosphorylation of DDR showed a single, low molecular weight feature which similarly substrates. (A) A2058 or 888MEL melanoma cells were treated for 6 h with + × decreased in cells when PI3K was inhibited (i.e., in cells DMSO or GDC-0973 GDC-0941 (MEKi/PI3Ki combo; 4 EC50) and were sub- treated with PI3Ki alone or combo). With this exception, jected to KinomeView Profiling. A2058 and 888MEL cells were treated with μ + μ μ + μ immunoblots showed increased DDR signaling in cells treated 10 M GDC-0973 10 M GDC-0941 or 0.2 M GDC-0973 10 M GDC-0941, with combo but not in cells treatedwitheithersingleagent respectively. Blots were probed using an antibody mixture recognizing pRSK, alone (Fig. 1B). 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). Given that neither GDC-0941 nor GDC-0973 causes geno- (B) A2058 lysates probed with the [s/t]Q and RXX[s/t] antibodies after toxicity or directly damages DNA in mutagenicity, clastogenicity, treatment with DMSO, 10 μM GDC-0973 (MEKi), 10 μM GDC-0941 (PI3Ki), or micronucleus formation assays, we speculated that increased or the combination. (C) Dose response of A2058 cells to increasing concen- [s/t]Q motif phosphorylation was a consequence of cell death. In trations of MEKi and PI3Ki alone or in combination. Blots were performed a dose–response experiment, we first confirmed that pThr202/ against DDR (p53 pSer15, histone 2AX pSer139), cell survival/cell death (AKT pTyr204 ERK1/2 and pThr308 AKT were reduced by MEKi or pThr308, cleaved PARP), and cell signaling (ERK1/2 pThr202/Tyr204) markers PI3Ki as single agents, respectively (Fig. 1C). Immunoblots and controls. Actin and GAPDH served as loading controls. against pSer15 p53 and pSer139 of the histone variant H2AX (γH2AX) revealed dose-dependent activation of DDR signaling, most notably in combo-treated cells. H2AX and p53 phosphor- and Table S1). To confirm that induction of the DDR was not ylation correlated with increased poly-ADP ribose polymerase unique to these particular inhibitors, experiments were carried out (PARP) cleavage in both dose–response (Fig. 1C) and time- using combinations of either GDC-0973 and the PI3K/mTOR course (Fig. S1A) experiments. Increased signals for cleaved inhibitor GDC-0980 (19) or GDC-0941 and the BRAF inhibitor PARP, pSer15 p53, pSer139 H2AX, and [s/t]Q phosphomotif PLX-4720 (20). As expected, inhibition of various signaling nodes blots were also notable across five of six additional melanoma resulted in a similar extent of PARP cleavage, DDR signaling, and cell lines (C32, 888MEL, Colo829, Hs695T, and G361, but not p53/H2AX phosphorylation (Fig. S1C). Additionally, caspase in- CELL BIOLOGY LOX-IMVI), all carrying the BRAF-V600E mutation (Fig. S1B hibition by z-Val-Ala-Asp-fluoromethylketone partially protected
Kirkpatrick et al. PNAS | November 26, 2013 | vol. 110 | no. 48 | 19427 cells from the effects of dual inhibition, decreasing PARP cleavage singly and multiply modified forms (Fig. 2B). In line with spec- and H2AX phosphorylation (Fig. S1D). Taken together, activation tral-count data, extracted ion chromatograms for the doubly of DDR signaling was dependent on efficient and sustained in- phosphorylated pThr2609/pSer2612 peptide from PRKDC in- hibition of the MEK/PI3K pathway and was coincident with the creased >10-fold in combo-treated cells compared with either induction of apoptosis across multiple treatments, time points, and single agent (Fig. 2C). Enhanced phosphorylation of pThr2609 cell lines. and pSer2612 on PRKDC following MEKi + PI3Ki was ob- We next used IAE and MS proteomics to profile DDR sig- served in six additional melanoma lines (Fig. S2B). In contrast, naling at the level of individual substrates (Fig. 2A). In total, many proteins (e.g., G3P/GAPDH; >90phosphoPSMs per sample) 1,066 unique phosphopeptides were identified via 4,314 phos- displayed little or no difference in the frequency of [s/t]Q phos- phopeptide spectral matches (phosphoPSMs) with false discov- phoPSMs between untreated and treated groups. ery rates (FDRs) of 0.74% and 2.07% at the peptide (pepFDR) To interrogate phosphopeptide results systematically, we as- A and protein (protFDR) levels, respectively (Fig. S2 ). Using the sembled a filtered list of phosphoPSMs from confidently identi- Ascore algorithm (AScore) to optimize phosphorylation-site lo- fi fi ed spectra containing an [S/T]Q motif, regardless of whether calization (21), we identi ed 690 unique phosphopeptides (3,072 phosphorylation was formally assigned to the motif. Areas under PSMs) in which the phosphorylation was assigned to a serine or the curve (AUCs) were determined using the VistaQuant algo- threonine residing immediately adjacent to a glutamine [Ascore > fi rithm (25) in either direct or cross-quantitation (XQuant) mode. algorithm values (AScore) 13; 95% con dence score for locali- To identify proteins most significantly affected by the stimulus zation]. Filtering the data to contain only the 1,004 phosphopep- and to address the challenges posed by missing values, we used tides (4,219 PSMs) bearing either a [s/t]Q motif phosphorylation linear mixed-effects modeling (LiME). LiME, an improvement event or an available [S/T]Q motif (unphosphorylated) was over ad hoc cutoffs or simple feature averaging, takes advantage sufficient to eliminate the remaining decoy peptides from the dataset. Conversely, identified phosphopeptides lacking an of inherent replicate structure of the data and leverages in- > formation from a series of biological conditions to identify the [S/T]Q motif (62 unique, 95 PSMs) displayed an FDR of 33%. fi This result suggests that the majority of 314 unique phospho- signi cantly affected proteins (26, 27). For PRKDC, LiME peptides which carry a [S/T]Q motif but in which the modifi- analysis revealed a systematic increase in peak area for the [s/t]Q containing phosphoPSMs, with >100-fold increase (P < 0.001) cation is not explicitly localized within the motif represent D high-confidence hits in which reassignment of the phosphory- between combo and control treatments (Fig. 2 ). This change lation event may be appropriate even when localization scores and other protein-level fold changes were calculated for each remain ambiguous. treatment group relative to the control using a mixed-effect For a subset of identified proteins, the number of phos- model, with P values determined based on the fit between in- phoPSMs increased dramatically after combo treatment relative dividual phosphoPSMs and the model. Volcano plots show that to no treatment (control) or treatment with either single agent. PRKDC, NUCL, HMGA1, and K1967 each displayed >30-fold Among these proteins were known DDR substrates including the change and a P value < 0.001 between combo and control DNA-dependent protein kinase (PRKDC), Nucleolin (NUCL), treatments (Fig. 2E). Although less pronounced, similar trends high-mobility group AT-hook 1 (HMGA1), and K1967 (Dataset were observed for PRKDC and NUCL in lysates from cells S1). For each, no phosphoPSMs were observed in untreated treated with GDC-0973 (MEKi) or GDC-0941 (PI3Ki) as single cells. A total of five phosphorylation sites were assigned to the agents (Fig. 2E). These LiME plots, their associated fold changes, ABCDE cluster of PRKDC (22–24); many were based on both and P values are provided in Dataset S2.
DMSO MEKiPI3Ki combo A B PRKDC 2609/2612/2620/2638/2647 tQ/sQ/tQ/tQ/tQ 1 4128 Lyse, Digest, Desalt HEAT1 HEAT2Leu- TPR1 KIP- FAT PI3K/ FATC Zipper binding TPR2/3 PI4K IAE: ATM motif s/tQ C PRKDC pThr2609/pSer2612 MS Analysis (2x) STVLTPMFVEtQAsQGTLQTR
DMSO D fold chg 2.7 14.3 138.1 9 Pvalue 0.03 <0.001 <0.001