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Kinome Profiling of Primary Endometrial Tumors Using Multiplexed Inhibitor Beads and Mass Spectrometry Identifies SRPK1 as Candidate Therapeutic Target

Open AccessPublished:September 29, 2020DOI:https://doi.org/10.1074/mcp.RA120.002012
      Endometrial carcinoma (EC) is the most common gynecologic malignancy in the United States, with limited effective targeted therapies. Endometrial tumors exhibit frequent alterations in protein kinases, yet only a small fraction of the kinome has been therapeutically explored. To identify kinase therapeutic avenues for EC, we profiled the kinome of endometrial tumors and normal endometrial tissues using Multiplexed Inhibitor Beads and Mass Spectrometry (MIB-MS). Our proteomics analysis identified a network of kinases overexpressed in tumors, including Serine/Arginine-Rich Splicing Factor Kinase 1 (SRPK1). Immunohistochemical (IHC) analysis of endometrial tumors confirmed MIB-MS findings and showed SRPK1 protein levels were highly expressed in endometrioid and uterine serous cancer (USC) histological subtypes. Moreover, querying large-scale genomics studies of EC tumors revealed high expression of SRPK1 correlated with poor survival. Loss-of-function studies targeting SRPK1 in an established USC cell line demonstrated SRPK1 was integral for RNA splicing, as well as cell cycle progression and survival under nutrient deficient conditions. Profiling of USC cells identified a compensatory response to SRPK1 inhibition that involved EGFR and the up-regulation of IGF1R and downstream AKT signaling. Co-targeting SRPK1 and EGFR or IGF1R synergistically enhanced growth inhibition in serous and endometrioid cell lines, representing a promising combination therapy for EC.

      Graphical Abstract

      Endometrial carcinoma (EC) is the most common gynecologic malignancy in the United States with 60,050 new cases and 10,470 deaths expected in 2020 (
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      ). There are two major histological types of EC, Type I and Type II, each displaying distinctive overall prognosis and survival outcomes (
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      ). Type 1 ECs are composed of low-grade endometrioid tumors representing the majority of EC (80%) with early stage detection and favorable prognosis. In contrast, Type II ECs are high-grade, display poor prognosis, and consist of 3 distinct histologies: serous adenocarcinomas, clear cell adenocarcinomas, and carcinosarcomas (
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      Targeted Therapies in Type II Endometrial Cancers: Too Little, but Not Too Late.
      ). Uterine serous carcinoma (USC) is the most lethal form of Type II EC because of late stage detection and high recurrence rates with current treatments only modestly impacting survival (
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      ). Moreover, there has been a steady increase in the mortality rate for EC that has been attributed to higher proportions of patients presenting with USC (
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      ). EC tumors are characterized by alterations in TP53, PPP2R1A, FBXW7, CDKN2A, PTEN, EGFR, ERBB2, PIK3CA, and KRAS (
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      ). Importantly, these proteomics approaches can measure the levels of numerous understudied kinases, providing a strategy to interrogate the unexplored cancer kinome. Several methods for quantitating kinases enriched by MIBs or kinobeads have been explored, including isobaric tagging (i.e. iTRAQ or TMT), label-free quantitation (LFQ) approaches, and labeled methods such as stable isotope labeling with amino acids in cell culture (SILAC) (
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      ). In our prior work, we utilized a s-SILAC reference mixed with tumor tissues to measure kinase abundance in ovarian tumors (
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      ).
      Here, we employed MIB-MS kinome profiling using LFQ and s-SILAC quantitation to measure the abundance of kinases in patient endometrial carcinoma (EC) tumors and normal endometrial (NE) tissues. MIB-MS profiling identified several kinases overexpressed in endometrial tumors including Serine/Arginine-Rich Splicing Factor Kinase 1 (SRPK1). Inhibition of SRPK1 in USC cells using SPHINX31 altered RNA splicing, blocked cell growth, and induced apoptosis under nutrient-deficient conditions. Moreover, SRPK1 inhibitors had minimal impact on EC cell growth in the presence of serum because of compensatory EGFR, IGF1R, and AKT signaling. Together, our findings nominate SRPK1 as a putative target in combination with EGFR, IGF1R, or AKT inhibitors for the treatment of EC.

      DISCUSSION

      Endometrial cancer is the fourth most common cancer in women, and incidence of this cancer is on the rise (
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      ). The protein kinome represents an attractive target for the treatment of EC as these tumors harbor frequent alterations in kinase pathways (
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      ). However, therapies targeting well-established kinases such as EGFR, IGF1R, or AKT have shown limited therapeutic benefit, prompting the search for novel kinase therapeutic targets (
      • Remmerie M.
      • Janssens V.
      Targeted Therapies in Type II Endometrial Cancers: Too Little, but Not Too Late.
      ). Here, we performed kinome profiling of patient endometrial tumors and normal tissues using MIB-MS and identified several kinases overexpressed in tumors, including SRPK1. Loss-of-function studies targeting SRPK1 in endometrial cancer cell lines revealed an integral role for SRPK1 in RNA splicing, as well as cell proliferation and survival under nutrient-deficient conditions. Moreover, we discovered a resistance mechanism to SRPK1 inhibitors that involved activation of the EGFR-IGF1R-AKT signaling axis, which could be overcome by inhibition of EGFR, IGF1R, or AKT.
      Several methods for quantitating kinases enriched by MIB-MS have been explored, including both labeled and label-free approaches (
      • Kurimchak A.M.
      • Herrera-Montávez C.
      • Brown J.
      • Johnson K.J.
      • Sodi V.
      • Srivastava N.
      • Kumar V.
      • Deihimi S.
      • O'Brien S.
      • Peri S.
      • Mantia-Smaldone G.M.
      • Jain A.
      • Winters R.M.
      • Cai K.Q.
      • Chernoff J.
      • Connolly D.C.
      • Duncan J.S.
      Functional proteomics interrogation of the kinome identifies MRCKA as a therapeutic target in high-grade serous ovarian carcinoma.
      ,
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      Enhancer remodeling during adaptive bypass to MEK inhibition is attenuated by pharmacologic targeting of the P-TEFb complex.
      ). Here, we compared LFQ versus s-SILAC measurement of MIB-enriched kinases from endometrial tissues, which revealed LFQ measured a greater number of MIB-enriched kinases at higher frequency among tissue samples than s-SILAC quantitation. These findings are consistent with the s-SILAC method being limited by measurement of only those kinases present in the s-SILAC standard. The heterogeneity of the kinome among cancers, and even within tumor tissues, makes the design of an optimal s-SILAC cancer cell line mix capable of quantitating all MIB-enriched kinases at high frequency a nearly impossible task. Additionally, over-or-under represented kinases in the s-SILAC cancer cell line mixture or inefficient SILAC-labeling of kinases because of low turnover rates could potentially skew the measurement of kinases in tissue samples, resulting in technical artifacts. Generation of SILAC-labeled peptides for each kinase within the kinome that could be spiked into tissue samples at a standardized concentration could improve the coverage and frequency of s-SILAC measurements, though would be costly. Comparison of the quantitation of MIB-enriched kinases using LFQ or s-SILAC in endometrial tissues revealed ∼30 kinases commonly up-regulated or down-regulated in tumor versus normal tissues by both LFQ and s-SILAC quantitative strategies. Despite overlap, numerous kinases exhibited discordant kinase measurements among the LFQ and s-SILAC, suggesting the 2 methods were not uniform. Additional studies including global proteome profiling using intensity-based absolute quantitation (iBAQ) (
      • Krey J.F.
      • Wilmarth P.A.
      • Shin J.-B.
      • Klimek J.
      • Sherman N.E.
      • Jeffery E.D.
      • Choi D.
      • David L.L.
      • Barr-Gillespie P.G.
      Accurate label-free protein quantitation with high- and low-resolution mass spectrometers.
      ) and immunoblot analysis of tissues will be required to determine the abundance of these discordant kinases in tumors versus normal endometrial tissues. Based on our findings, we propose LFQ should be the primary quantitative method for measuring MIB-enriched kinases, with s-SILAC used as a complementary validation method.
      Pairing MIB-MS with LFQ measured >280 kinases per sample, providing MIB-enriched abundance values for a significant proportion of the kinome. Kinases are enriched by MIB-resins based on several factors including their affinity, abundance, and in some instances their activation state (
      • Duncan J.S.
      • Whittle M.C.
      • Nakamura K.
      • Abell A.N.
      • Midland A.A.
      • Zawistowski J.S.
      • Johnson N.L.
      • Granger D.A.
      • Jordan N.V.
      • Darr D.B.
      • Usary J.
      • Kuan P.-F.
      • Smalley D.M.
      • Major B.
      • He X.
      • Hoadley K.A.
      • Zhou B.
      • Sharpless N.E.
      • Perou C.M.
      • Kim W.Y.
      • Gomez S.M.
      • Chen X.
      • Jin J.
      • Frye
      • Stephen V.
      • Earp H.S.
      • Graves L.M.
      • Johnson G.L.
      Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer.
      ,
      • Ruprecht B.
      • Zecha J.
      • Heinzlmeir S.
      • Médard G.
      • Lemeer S.
      • Kuster B.
      Evaluation of kinase activity profiling using chemical proteomics.
      ). Notably, these MIB-binding properties could interfere with quantitation of the actual abundance of the kinase within a tissue sample. Recent advances in global proteome analysis and iBAQ quantitation methods (
      • Cox J.
      • Hein M.Y.
      • Luber C.A.
      • Paron I.
      • Nagaraj N.
      • Mann M.
      Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.
      ) capable of measuring numerous kinases without enrichments may represent an alternative or complementary method for quantitating kinase abundance in tissues. Here, we identified SRPK1 to be elevated in endometrial tumors using MIB-MS and validated increased SRPK1 protein abundance by querying a recent global proteome analysis of endometrial tumors by CPTAC. Nevertheless, MIB-MS remains a valuable tool, particularly in determining abundance of those kinases expressed at low levels within samples. In our hands, applying single-run proteome analysis using the LC–MS/MS equipment and methods detailed, we measured ∼190 kinases/sample by single-run proteome analysis in SPEC-2 cells, whereas MIB-MS enrichment quantified ∼240 kinases/sample. Thus, combining MIB-MS and single-run proteome profiling may be the optimal strategy for quantitating kinase abundance in samples increasing the overall kinome coverage measured and providing additional validation.
      SRPK1 has emerged as a promising therapeutic target in cancer because of its roles in a variety of oncogenic process including RNA splicing, cell proliferation and survival, as well as migration and metastasis (
      • Bullock N.
      • Oltean S.
      The many faces of SRPK1.
      ). SRPK1 has been shown to regulate alternative splicing in cancer cells through phosphorylation of SRSF (SR Splicing Factors) by SRPK1 controlling splicing, RNA export, and other processes of RNA metabolism in cells (
      • Aubol B.E.
      • Wu G.
      • Keshwani M.M.
      • Movassat M.
      • Fattet L.
      • Hertel K.J.
      • Fu X.-D.
      • Adams J.A.
      Release of SR proteins from CLK1 by SRPK1: a symbiotic kinase system for phosphorylation control of pre-mRNA splicing.
      ). Consistent with these findings, we observed a substantial increase in alternative splicing following treatment with the SRPK1 inhibitor SPHINX31, including mis-spliced gene targets enriched in VEGF pathways, cell adhesion, and cell cycle. Inhibition of SRPK1 has been previously shown to induce splicing of VEGF-A to VEGF165b reducing angiogenesis in cells and tumor models, represent a promising anti-angiogenic therapy (
      • Charo L.M.
      • Plaxe S.C.
      Recent advances in endometrial cancer: a review of key clinical trials from 2015 to 2019.
      ). SPHINX31 treatment or knockdown of SRPK1 in USC cells altered splicing of VEGF-A and increased VEGF165b protein levels, suggesting SRPK1 inhibitors could impact angiogenesis in endometrial cancer models. However, further studies in USC xenograft tumor models will be required to assess whether SPHINX31 treatment increases VEGF165b and blocks angiogenesis observed with other tumor models such as melanoma and prostate cancers (
      • Bullock N.
      • Potts J.
      • Simpkin A.J.
      • Koupparis A.
      • Harper S.J.
      • Oxley J.
      • Oltean S.
      Serine-arginine protein kinase 1 (SRPK1), a determinant of angiogenesis, is upregulated in prostate cancer and correlates with disease stage and invasion.
      ,
      • Gammons M.V.
      • Lucas R.
      • Dean R.
      • Coupland S.E.
      • Oltean S.
      • Bates D.O.
      Targeting SRPK1 to control VEGF-mediated tumour angiogenesis in metastatic melanoma.
      ).
      In addition to identifying a role of SRPK1 in RNA splicing in endometrial cancer cells, our proteogenomics characterization of SPHINX31 treated USC cells revealed SRPK1 was required for growth and survival of cells under nutrient deprivation. Comparative pathway analysis of gene targets mis-spliced and those mRNA and proteins down-regulated by SPHINX31 treatment showed they were commonly enriched for cell cycle and cell division signaling. SRPK1 has been implicated in cell cycle progression in other cancer models (
      • Xu X.
      • Wei Y.
      • Wang S.
      • Luo M.
      • Zeng H.
      Serine-arginine protein kinase 1 (SRPK1) is elevated in gastric cancer and plays oncogenic functions.
      ). Our findings suggest SRPK1 may be integral for cell cycle progression when nutrients are limiting through regulation of alternative splicing of cell cycle associated proteins. SRPK1 has been shown to shuttle to the nucleus upon growth factor stimulation where it in turn phosphorylates SRSF proteins influencing mRNA splicing (
      • Zhou Z.
      • Qiu J.
      • Liu W.
      • Zhou Y.
      • Plocinik R.M.
      • Li H.
      • Hu Q.
      • Ghosh G.
      • Adams J.A.
      • Rosenfeld M.G.
      • Fu X.-D.
      The Akt-SRPK-SR axis constitutes a major pathway in transducing EGF signaling to regulate alternative splicing in the nucleus.
      ). Further characterization of the cellular localization of SRPK1, as well as defining SRPK1 interacting proteins in serum-competent and nutrient-deprived cells could provide insight into the mechanisms by which SRPK1 promotes growth and survival in the absence of growth factors. Notably, SRPK1 has been shown to directly interact with activated AKT following EGF stimulation, where AKT activates SRPK1 promoting its nuclear localization, facilitating growth factor induced mRNA splicing (
      • Zhou Z.
      • Qiu J.
      • Liu W.
      • Zhou Y.
      • Plocinik R.M.
      • Li H.
      • Hu Q.
      • Ghosh G.
      • Adams J.A.
      • Rosenfeld M.G.
      • Fu X.-D.
      The Akt-SRPK-SR axis constitutes a major pathway in transducing EGF signaling to regulate alternative splicing in the nucleus.
      ). In the absence of EGF-driven AKT-SRPK1 interactions, SRPK1 could have distinct functions in the regulation of cell cycle, potentially through kinase-independent roles when nutrients are limiting. However, further studies exploring the AKT-SRPK1 interactions through co-immunoprecipitations in serum-competent or serum-starved USC cells, as well as in response to SPHINX31 treatment would help address the impact of AKT-binding on SRPK1 function following serum-starvation. Additionally, performing phosphoproteomics in response to SPHINX31 treatment or utilizing ATP-analog sensitive SRPK1 mutants (
      • Hertz N.T.
      • Wang B.T.
      • Allen J.J.
      • Zhang C.
      • Dar A.C.
      • Burlingame A.L.
      • Shokat K.M.
      Chemical genetic approach for kinase-substrate mapping by covalent capture of thiophosphopeptides and analysis by mass spectrometry.
      ) to identify SRPK1 substrates would help define signaling pathways regulated by SRPK1 under serum starvation.
      Drug resistance to kinase inhibitors frequent occurs representing a significant limitation in the treatment of cancer (
      • Lovly C.M.
      • Shaw A.T.
      Molecular pathways: resistance to kinase inhibitors and implications for therapeutic strategies.
      ). One mechanism by which cancer cells bypass kinase inhibition is through adaptive kinome reprogramming, a process involving compensatory activation of alternative kinase survival pathways (
      • Duncan J.S.
      • Whittle M.C.
      • Nakamura K.
      • Abell A.N.
      • Midland A.A.
      • Zawistowski J.S.
      • Johnson N.L.
      • Granger D.A.
      • Jordan N.V.
      • Darr D.B.
      • Usary J.
      • Kuan P.-F.
      • Smalley D.M.
      • Major B.
      • He X.
      • Hoadley K.A.
      • Zhou B.
      • Sharpless N.E.
      • Perou C.M.
      • Kim W.Y.
      • Gomez S.M.
      • Chen X.
      • Jin J.
      • Frye
      • Stephen V.
      • Earp H.S.
      • Graves L.M.
      • Johnson G.L.
      Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer.
      ). Combination therapies that block these compensatory kinases often provide more durable therapeutic responses (
      • Knight Z.A.
      • Lin H.
      • Shokat K.M.
      Targeting the cancer kinome through polypharmacology.
      ). Here, we identified EGF and IGF-1 receptors and their downstream AKT signaling to promote resistance to SRPK1 inhibitors in endometrioid and serous endometrial cancer cell lines. SPHINX31 treatment or SRPK1 knockdown in SPEC-2 cells resulted in up-regulation of IGF1R protein and activating phosphorylation that coincided with increased AKT phosphorylation. Notably, although we did not observe an increase in EGFR total or activating phosphorylation, inhibition of EGFR or IGF1R blocked SPHINX31-mediated AKT activation, suggesting both EGFR and IGF1R contributed to AKT activation. Our findings are consistent with SRPK1 knockout studies, which showed genetic loss of SRPK1 resulted robust activation of AKT and MTOR signaling (
      • Wang P.
      • Zhou Z.
      • Hu A.
      • Ponte de Albuquerque C.
      • Zhou Y.
      • Hong L.
      • Sierecki E.
      • Ajiro M.
      • Kruhlak M.
      • Harris C.
      • Guan K.-L.
      • Zheng Z.-M.
      • Newton A.C.
      • Sun P.
      • Zhou H.
      • Fu X.-D.
      Both decreased and increased SRPK1 levels promote cancer by interfering with PHLPP-mediated dephosphorylation of Akt.
      ). These studies showed SRPK1 was essential for maintaining a protein-complex consisting of SRPK1, activated AKT and the AKT phosphatase PHLPP2, where knockout of SRPK1 destabilizes the interaction between AKT and PHLPP2, leading to increased phosphorylation of AKT. Defining the impact of SRPK1 inhibitors on the SRPK1-AKT-PHLPP2 complex stability in endometrial cancer cells could help decipher whether disruption of AKT-PHLPP2 interactions promotes the activated AKT observed with SPHINX31 treatment. Notably, our findings suggest that EGFR and IGF1R were integral to the SPHINX31-mediated activation of AKT, as inhibition of either growth factor receptor blocked SPHINX31-induced AKT activation. Cross-talk among EGFR and IGF1R has been shown to occur at many levels in cancer cells, including direct interactions, altering availability of ligands, or indirectly through downstream effectors (
      • Veeken J.V D.
      • Oliveira S.
      • Schiffelers R.M.
      • Storm G.
      • van Bergen En Henegouwen P.M.
      • Roovers R.C.
      Crosstalk between epidermal growth factor receptor- and insulin-like growth factor-1 receptor signaling: implications for cancer therapy.
      ). Determining the mechanism by which IGF1R is up-regulated in response to SRPK1 inhibition and how this impacts EGFR activity will be of particular interest, as both these receptors are frequently altered in EC. Several compensatory feedback mechanisms have been reported to regulate IGF1R signaling in cancer cells, including epigenetic and/or negative feedback phosphorylation events (
      • Simpson A.
      • Petnga W.
      • Macaulay V.M.
      • Weyer-Czernilofsky U.
      • Bogenrieder T.
      Insulin-like growth factor (IGF) pathway targeting in cancer: role of the IGF axis and opportunities for future combination studies.
      ). Molecular characterization of the impact of SRPK1 inhibitors on these established IGF1R feedback responses should provide insight into the link between SRPK1 and EGF/IGF-1 signaling in EC cells.
      Overexpression of EGFR, IGF1R and activation of AKT signaling frequently occurs in endometrial tumors and is associated with poor overall survival (
      • Konecny G.E.
      • Venkatesan N.
      • Yang G.
      • Dering J.
      • Ginther C.
      • Finn R.
      • Rahmeh M.
      • Fejzo M.S.
      • Toft D.
      • Jiang S.W.
      • Slamon D.J.
      • Podratz K.C.
      Activity of lapatinib a novel HER2 and EGFR dual kinase inhibitor in human endometrial cancer cells.
      ,
      • Bruchim I.
      • Sarfstein R.
      • Werner H.
      The IGF hormonal network in endometrial cancer: functions, regulation, and targeting approaches.
      ,
      • Roncolato F.
      • Lindemann K.
      • Willson M.L.
      • Martyn J.
      • Mileshkin L.
      PI3K/AKT/mTOR inhibitors for advanced or recurrent endometrial cancer.
      ). However, single agent therapies targeting EGFR, IGF1R, or AKT have not shown therapeutic benefit in EC (
      • Remmerie M.
      • Janssens V.
      Targeted Therapies in Type II Endometrial Cancers: Too Little, but Not Too Late.
      ). Here, we observed drug synergy combining SRPK1 and EGFR, IGF1R, or AKT inhibitors in serous or endometrioid endometrial cancer cell lines. Our findings suggest blocking SRPK1 activity using the selective inhibitor SPHINX31 may provide more durable therapeutic responses than single agent EGFR, IGF1R, or AKT inhibitors in EC. Furthermore, our studies demonstrated SRPK1 was required to maintain growth and survival under nutrient-deprived conditions, suggesting SRPK1 may contribute to EGFR or IGF1R inhibitor resistance observed in EC cells. Additional studies investigating the impact of overexpression of SRPK1 WT or kinase-dead mutants in EC cells on EGFR, IGF1R, or AKT inhibitor sensitivity could provide insight into the role of SRPK1 in resistance these inhibitors. Furthermore, determining the in vivo efficacy of SRPK1 inhibitors alone or in combination with EGFR, IGF1R, or AKT inhibitors in EC tumor models, as well as the potential cytotoxic effects of these combinations will be essential for therapeutic proof-of-concept.
      Together, profiling endometrial tumors using MIB-MS in combination with loss-of-function studies led to the discovery of SRPK1 as an integral kinase regulating mRNA splicing in EC cells, as well as cell proliferation and survival under nutrient deprivation. Moreover, dual blockade of SRPK1 and EGFR, IGF1R, or AKT synergized to block growth and survival providing more durable therapeutic response than single agent therapies. As several inhibitors targeting EGFR, IGF1R, or AKT are avidly being explored in clinical trials, our findings support the addition of SRPK1 inhibitors to improve anti-tumor responses for the treatment of EC.

      DATA AVAILABILITY

      All relevant proteomics files are available through the PRIDE partner repository (http://www.ebi.ac.uk) with the data set identifier PXD020357. Annotated spectra for each proteomics data set have been deposited in MS-Viewer (http://msviewer.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer), the search keys are provided in Data File S13. The BioProject ID number for the mRNA sequencing data reported in this paper is PRJNA608466 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA608466). All other data needed to evaluate the conclusion in the paper are present in the paper or the supplemental material.

      Acknowledgments

      We thank Chunxiao Zhou (University of North Carolina) for providing the SPEC-2 cell line.
      Competing interests—J.S.D. is an inventor on International patent application PCT/US2019/048053 for using multiplexed inhibitor beads for measuring kinase levels in patient tumors. The other authors declare that they have no competing interests.

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