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Synergistic targeting of DNA-PK and KIT signaling pathways in KIT mutant acute myeloid leukemia

  • Heather C. Murray
    Affiliations
    School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, and Hunter Cancer Research Alliance, Hunter Medical Research Institute, Callaghan, NSW, 2308, Australia
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  • Kasey Miller
    Affiliations
    School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, and Hunter Cancer Research Alliance, Hunter Medical Research Institute, Callaghan, NSW, 2308, Australia
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  • Joshua S. Brzozowski
    Affiliations
    School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, and Hunter Cancer Research Alliance, Hunter Medical Research Institute, Callaghan, NSW, 2308, Australia
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  • Richard G.S. Kahl
    Affiliations
    School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, and Hunter Cancer Research Alliance, Hunter Medical Research Institute, Callaghan, NSW, 2308, Australia
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  • Nathan D. Smith
    Affiliations
    Analytical and Biomolecular Research Facility, Advanced Mass Spectrometry Unit, University of Newcastle, Callaghan, NSW, 2308, Australia
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  • Sean J. Humphrey
    Affiliations
    School of Life and Environmental Sciences, and The Charles Perkins Centre, The University of Sydney, Sydney, New South Wales, Australia
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  • Matthew D. Dun
    Affiliations
    School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, and Hunter Cancer Research Alliance, Hunter Medical Research Institute, Callaghan, NSW, 2308, Australia
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  • Nicole M. Verrills
    Correspondence
    Corresponding author: Name: Nicole M. Verrills, Address: Level 3, Life Sciences Bldg, The University of Newcastle, Callaghan, NSW, 2308, Australia. . Phone: +61 2 4921 5619. Fax: +61 2 4921 6903
    Affiliations
    School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, and Hunter Cancer Research Alliance, Hunter Medical Research Institute, Callaghan, NSW, 2308, Australia
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Open AccessPublished:January 19, 2023DOI:https://doi.org/10.1016/j.mcpro.2023.100503

      Highlights

      • DNA-PK is phosphorylated (activated) in mutant-KIT cells.
      • Phosphoproteomic characterisation of oncogenic mutant D816V-KIT cells
      • Mutant-KIT cells are sensitive to DNA-PK inhibitors
      • DNA-PK inhibitors synergise with KIT signalling inhibitors in mutant-KIT cells
      • Combined DNA-PK and KIT inhibition reduced ERK, MTOR, MYC and MYB phosphorylation

      Abstract

      Acute Myeloid Leukemia (AML) is the most common and aggressive form of acute leukemia, with a 5-year survival rate of just 24%. Over a third of all AML patients harbor activating mutations in kinases, such as the receptor tyrosine kinases FLT3 and KIT. FLT3 and KIT mutations are associated with poor clinical outcomes and lower remission rates in response to standard-of-care chemotherapy. We have recently identified that the core kinase of the non-homologous end joining DNA repair pathway, DNA-PK, is activated downstream of FLT3; and targeting DNA-PK sensitized FLT3-mutant AML cells to standard-of-care therapies. Herein, we investigated DNA-PK as a possible therapeutic vulnerability in KIT mutant AML, using isogenic FDC-P1 mouse myeloid progenitor cell lines transduced with oncogenic mutant KIT (V560G, D816V), or vector control. Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK in the T2599/T2605/S2608/S2610 cluster in KIT mutant cells, indicative of DNA-PK activation. Accordingly, proliferation assays revealed that KIT mutant FDC-P1 cells were more sensitive to the DNA-PK inhibitors M3814 or NU7441, compared to empty vector controls. DNA-PK inhibition combined with inhibition of KIT signaling using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Global phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib and M3814 single-agent treatments inhibited ERK and AKT/MTOR activity, with greater inhibition of both pathways when used in combination. Combined dasatinib and M3814 treatment also synergistically inhibited phosphorylation of the transcriptional regulators MYC and MYB. This study provides insight into the oncogenic pathways regulated by DNA-PK beyond its canonical role in DNA repair, and demonstrates that DNA-PK is a promising therapeutic target for KIT mutant cancers.

      Graphical abstract

      Abbreviations:

      DNA-PK (DNA dependent protein kinase), KIT (Mast/stem cell growth factor receptor Kit), CD117 (cluster of differentiation 117), FLT3 (Receptor-type tyrosine-protein kinase FLT3), TKI (Tyrosine kinase inhibitor), ERK (Extracellular-signal-regulated kinase), AKT (RAC-alpha serine/threonine-protein kinase), MTOR (Serine/threonine-protein kinase mTOR), PDGFR (Platelet-derived growth factor receptor), FYN (Tyrosine-protein kinase Fyn), LYN (Tyrosine-protein kinase Lyn), RPS6 (Ribosomal protein S6), STAT (Signal transducer and activator of transcription), GRB2 (Growth factor receptor-bound protein 2), PI3K (Phosphoinositide 3-kinase), MAPK (Mitogen activated protein kinase), JAK (Tyrosine-protein kinase JAK1), P70S6K (p70S6 kinase), P90RSK (p90 ribosomal S6 kinase), GSK3A (Glycogen Synthase Kinase 3 Alpha), BAD (BCL2 associated agonist of cell death), ROCK1 (Rho Associated Coiled-Coil Containing Protein Kinase 1), BTK (Bruton’s tyrosine kinase), IFNGR1 (Interferon gamma receptor 1), ATM (Ataxia Telangiectasia Mutated), NUCKS1 (Nuclear casein kinase and cyclin-dependent kinase substrate 1), HNRNPD (Heterogeneous nuclear ribonucleoprotein D), TRIM28 (Transcriptional intermediary factor 1β), RCSD1 (CapZ-interacting protein), TJP2 (Tight junction protein 2)

      Introduction

      Acute Myeloid Leukemia (AML) arises from deregulated proliferation of myeloid progenitor cells, culminating in an overpopulation of leukemic “blast” cells in the bone marrow and other tissues. AML is the most lethal form of leukemia, accounting for less than one third of all diagnoses but nearly two-thirds of leukemia deaths [

      Cancer in Australia 2017. In. Canberra Cat. no CAN100: Australian Institute of Health and Welfare (AIHW); 2017.

      ]. Since 1973, the mainstay of therapy for AML has been the cytotoxic chemotherapy “7+3” regimen; 7 days infusion of the antimetabolite cytarabine, combined with an intravenous anthracycline for the first 3 days [
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      ]. KIT (CD117) is a class III receptor tyrosine kinase (RTK), sharing high sequence similarity with the other class III RTK family members, FLT3 and PDGFR [
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      ,
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      ]. KIT contains 5 immunoglobulin-like extracellular domains, a transmembrane domain, and intracellular juxtamembranous and split kinase domains. Wildtype KIT receptor activity is regulated by its endogenous ligand, stem cell factor (SCF). SCF binding leads to KIT receptor dimerization [
      • Lemmon M.A.
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      • Lax I.
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      Kit receptor dimerization is driven by bivalent binding of stem cell factor.
      ], stimulating trans-autophosphorylation [
      • Mol C.D.
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      ]. Docking and subsequent phosphorylation of signaling mediators such as FYN [
      • Timokhina I.
      • Kissel H.
      • Stella G.
      • Besmer P.
      Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation.
      ], LYN [
      • Linnekin D.
      • DeBerry C.S.
      • Mou S.
      Lyn associates with the juxtamembrane region of c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal progenitor cells.
      ], STAT1 [
      • Deberry C.
      • Mou S.
      • Linnekin D.
      Stat1 associates with c-kit and is activated in response to stem cell factor.
      ], and GRB2 [
      • Thommes K.
      • Lennartsson J.
      • Carlberg M.
      • Ronnstrand L.
      Identification of Tyr-703 and Tyr-936 as the primary association sites for Grb2 and Grb7 in the c-Kit/stem cell factor receptor.
      ] activates downstream networks including PI3K/AKT [
      • Timokhina I.
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      • Stella G.
      • Besmer P.
      Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation.
      ,
      • Wandzioch E.
      • Edling C.E.
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      Activation of the MAP kinase pathway by c-Kit is PI-3 kinase dependent in hematopoietic progenitor/stem cell lines.
      ], MAPK [
      • Wandzioch E.
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      Activation of the MAP kinase pathway by c-Kit is PI-3 kinase dependent in hematopoietic progenitor/stem cell lines.
      ], and JAK/STAT [
      • Brizzi M.F.
      • Zini M.G.
      • Aronica M.G.
      • Blechman J.M.
      • Yarden Y.
      • Pegoraro L.
      Convergence of signaling by interleukin-3, granulocyte-macrophage colony-stimulating factor, and mast cell growth factor on JAK2 tyrosine kinase.
      ].
      Approximately 80% of acute myeloid leukemias express KIT [
      • Ikeda H.
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      • Tamaki T.
      • Kuriu A.
      • Kitayama H.
      • Ishikawa J.
      • Kanayama Y.
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      Expression and functional role of the proto-oncogene c-kit in acute myeloblastic leukemia cells.
      ], and high expression is associated with poor prognosis [
      • Gao X.
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      ,
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      Tripp B et al: Increased C-kit intensity is a poor prognostic factor for progression-free and overall survival in patients with newly diagnosed AML.
      ]. KIT activating mutations occur in a range of neoplasms, including AML [
      • Boissel N.
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      • de Botton S.
      • Auvrignon A.
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      ], systemic mastocytosis [
      • Longley Jr., B.J.
      • Metcalfe D.D.
      • Tharp M.
      • Wang X.
      • Tyrrell L.
      • Lu S.Z.
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      Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis.
      ], melanoma [
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      ]. KIT mutations in AML are most common in the core-binding factor subtype (CBF), occurring in 25-40% of CBF-AML patients [
      • Shen Y.
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      • Fan X.
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      Monitoring of clonal evolution of double C-KIT exon 17 mutations by Droplet Digital PCR in patients with core-binding factor acute myeloid leukemia.
      ]. The most frequent KIT mutations are point mutations in codon 816 within the second tyrosine kinase domain [
      • Cairoli R.
      • Beghini A.
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      • Nadali G.
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      ], most often leading to a valine substitution for aspartic acid (D816V) [
      • Jang W.
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      Significance of KIT exon 17 mutation depends on mutant level rather than positivity in core-binding factor acute myeloid leukemia.
      ,
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      KIT D816 mutation associates with adverse outcomes in core binding factor acute myeloid leukemia, especially in the subgroup with RUNX1/RUNX1T1 rearrangement.
      ]. Although CBF-AML has a relatively favorable prognosis [
      • Dohner H.
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      ], the presence of mutant KIT is associated with a lower complete remission rate [
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      ] and reduced survival [
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      ,
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      ].
      Similarly to activating mutations of FLT3 [
      • Sillar J.R.
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      • DeIuliis G.N.
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      ], KIT activating mutations in the tyrosine kinase domain (D816V) or juxtamembrane domain (V560G) lead to elevated intracellular reactive oxygen species (ROS) [
      • Kim D.-K.
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      • Sillar J.R.
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      ,
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      ]. Elevated DNA double strand break repair activity could enable cell survival in the presence of increased ROS, presenting a potential therapeutic vulnerability. Indeed, oncogenic FLT3-mutations induce activation of the non-homologous end joining DNA repair pathway [
      • Fan J.
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      • Small D.
      • Rassool F.
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      ]. We have recently shown that this is associated with increased phosphorylation of the non-homologous end joining core kinase, DNA-PK; and importantly, that FLT3-mutant cells are sensitive to pharmacological DNA-PK inhibition [
      • Murray H.C.
      • Enjeti A.K.
      • Kahl R.G.S.
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      • Sillar J.
      • Skerrett-Byrne D.A.
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      ].
      Herein, we show that expression of D816V- and V560G-KIT mutations is associated with increased DNA-PK phosphorylation, consistent with DNA-PK activation. Accordingly, mutant D816V and V560G-KIT cell lines are sensitive to DNA-PK inhibitors, and DNA-PK inhibition combined with KIT signaling inhibition induces synergistic cell death. Mechanistically, discovery phosphoproteomic analysis of combined DNA-PK and tyrosine kinase inhibitor (TKI) treatment in D816V-KIT cells revealed co-operative inhibition of AKT/mTOR, P70S6K, and ERK/MAPK signaling, and synergistic inhibition of phosphorylation of the transcription factors MYC and MYB.

      Experimental Procedures

      Cell lines

      Mouse myeloid progenitor FDC-P1 cells transduced with mutant KIT (D816V or V560G), or an empty vector (EV) were generated previously [
      • Frost M.J.
      • Ferrao P.T.
      • Hughes T.P.
      • Ashman L.K.
      Juxtamembrane Mutant V560GKit Is More Sensitive to Imatinib (STI571) Compared with Wild-Type c-Kit Whereas the Kinase Domain Mutant D816VKit Is Resistant.
      ]. All FDC-P1 cell lines were maintained in standard culture conditions (37oC, 5% CO2), in DMEM containing 10% FCS, 20mM HEPES, and 2mM L-Glutamine. EV cell lines were expanded with 0.5ng/mL mouse granulocyte macrophage-colony stimulating factor (mGM-CSF) (BioLegend; San Diego, CA, USA). All lines were routinely confirmed to be free of mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza; Basel, Switzerland), as per manufacturer’s instructions. KIT expression was monitored using flow cytometry (Supplementary Methods).

      Drugs

      Cell lines were treated with the following agents, either alone or in combination as indicated in text. DNA-PK inhibitors: NU7441 (Selleckchem; Houston, TX, USA), M3814 (Merck; Kenilworth, NJ, USA); KIT signaling inhibitors: dasatinib (Cayman Chemical; Ann Arbor, MI, USA), ibrutinib (Selleckchem; Houston, TX, USA), FTY720 (Cayman Chemical), and AAL(S) (synthesized by A/Prof Jonathan Morris, School of Chemistry, UNSW as described [
      • Toop H.D.
      • Dun M.D.
      • Ross B.K.
      • Flanagan H.M.
      • Verrills N.M.
      • Morris J.C.
      Development of novel PP2A activators for use in the treatment of acute myeloid leukaemia.
      ]). DMSO was used as the solvent for all compounds. Final vehicle concentration was below 0.1% for all experiments.

      Cell Viability and Apoptosis Analysis

      Cell viability in response to 72 hour drug treatments was assessed using the metabolic resazurin assay, as previously described [
      • Murray H.C.
      • Enjeti A.K.
      • Kahl R.G.S.
      • Flanagan H.M.
      • Sillar J.
      • Skerrett-Byrne D.A.
      • Al Mazi J.G.
      • Au G.G.
      • de Bock C.E.
      • Evans K.
      • et al.
      Quantitative phosphoproteomics uncovers synergy between DNA-PK and FLT3 inhibitors in acute myeloid leukaemia.
      ]. Apoptotic cells were measured using Annexin V-FITC or Annexin V-APC flow cytometry assays (BD Biosciences), as per manufacturer’s instructions. For combination drug treatments, synergy was assessed using the fractional product method of Webb [

      Webb J: Effect of more than one inhibitor In: Hochster ER, Quastel J (eds). Enzymes and metabolic inhibitors.: Academic Press: New York; 1963.

      ]. Graphs were produced using Graphpad Prism 9 software (La Jolla, CA, USA).

      Synergy analysis

      For combination drug treatments, synergy was evaluated using the method of Webb (also termed the “fractional product method”) [

      Webb J: Effect of more than one inhibitor In: Hochster ER, Quastel J (eds). Enzymes and metabolic inhibitors.: Academic Press: New York; 1963.

      ]. The method of Webb estimates the expected additive effect of two drugs using the fractional product of the effect of each drug alone, ie:
      1Fa(Drug1+Drug2)=(1FaDrug1)*(1FaDrug2)


      Where Fa = the fraction of cells affected, expressed as a decimal
      An observed Fa(Drug1+Drug2) value greater than the expected Fa(Drug1+Drug2) value indicates synergy, whereas an observed Fa(Drug1+Drug2) lower than the expected value indicates antagonism. The Webb result was calculated by subtracting the observed Fa(Drug1+Drug2) from the expected Fa(Drug1+Drug2). A result less than -0.1 was defined as synergistic.
      Where dose-response curves were obtained, synergy was also assessed using the model of Bliss [
      • BLISS C.I.
      THE TOXICITY OF POISONS APPLIED JOINTLY1
      ], evaluated using SynergyFinder [
      • Ianevski A.
      • Giri A.K.
      • Aittokallio T.
      SynergyFinder 3.0: an interactive analysis and consensus interpretation of multi-drug synergies across multiple samples.
      ]. A Bliss result greater than 10 was defined as synergistic.

      Phosphoproteomic profiling

      Label-free global phosphoproteomic profiling was performed using the EasyPhos method [
      • Humphrey S.J.
      • Karayel O.
      • James D.E.
      • Mann M.
      High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform.
      ]. In brief, 200μg of lysate was reduced and alkylated using Tris(2-carboxyethyl)phosphine hydrochloride and 2-chloroacetamide. Lysates were digested using trypsin-LysC (V5072, Promega), overnight at 37oC with shaking (1500rpm). Phosphopeptides were subsequently enriched using TiO2 beads and desalted using SDB-RPS StageTips. The non-modified proteome flow through was desalted using ethylacetate phase separation and desalted using SDB-RPS StageTips using a custom 3D-printed adapter [
      • Harney D.J.
      • Hutchison A.T.
      • Hatchwell L.
      • Humphrey S.J.
      • James D.E.
      • Hocking S.
      • Heilbronn L.K.
      • Larance M.
      Proteomic Analysis of Human Plasma during Intermittent Fasting.
      ].
      Peptides were further purified with an Acclaim PepMap 100 C18 75μM ✕ 20 mm trap column (Thermo Fisher, Bremen, DE) prior to separation on a 75μM ✕ 25cm EASY-Spray PepMap C18 column (Thermo Fisher) using a 5%-35% ACN gradient on an Exploris 480 mass spectrometer (Thermo Fisher; Carlsbad, CA, USA). A FAIMSpro compensation voltage of -60 was used. For DDA analysis, peptides were separated using a 90 minute gradient. Full MS scans of 350-1200m/z were acquired at a resolution of 120,000, with an automatic gain control of 3e6 and maximum injection time 50ms. MS/MS scans were acquired using a resolution of 45,000, automatic gain control of 2.5e6, a normalized collision energy of 36, and maximum injection time of 100ms. For targeted analyses using parallel reaction monitoring [
      • Degryse S.
      • de Bock C.E.
      • Demeyer S.
      • Govaerts I.
      • Bornschein S.
      • Verbeke D.
      • Jacobs K.
      • Binos S.
      • Skerrett-Byrne D.A.
      • Murray H.C.
      • et al.
      Mutant JAK3 phosphoproteomic profiling predicts synergism between JAK3 inhibitors and MEK/BCL2 inhibitors for the treatment of T-cell acute lymphoblastic leukemia.
      ], an Exploris 480 or Oribtrap Eclipse mass spectrometer was used, with a FAIMS compensation voltage of -60. For the Exploris 480, peptides were separated using a 60 minute gradient. Full MS scans of 370-1500m/z were acquired with resolution 60,000, an automatic gain control of 1e6 and maximum injection time 50ms. MS/MS scans were acquired using resolution 15,000, an automatic gain control of 1e6 and maximum injection time of 120ms. For the Orbitrap Eclipse, peptides were purified with a PepMap Neo trap column (Thermo Fisher) prior to separation on a 75μM ✕ 15cm EASY-Spray PepMap Neo column (Thermo Fisher) using a 70 minute gradient. Full MS scans of 370-1500m/z were acquired with resolution 60,000, an automatic gain control of 100%, and maximum injection time 50ms. MS/MS scans were acquired using resolution 15,000, an automatic gain control of 200% and maximum injection time of 120ms.
      DDA data was analyzed using Proteome Discoverer 2.5 (Thermo Fisher) as described [
      • Skerrett-Byrne D.A.
      • Trigg N.A.
      • Bromfield E.G.
      • Dun M.D.
      • Bernstein I.R.
      • Anderson A.L.
      • Stanger S.J.
      • MacDougall L.A.
      • Lord T.
      • Aitken R.J.
      • et al.
      Proteomic Dissection of the Impact of Environmental Exposures on Mouse Seminal Vesicle Function.
      ]. Spectrum files were recalibrated using the PD node “Spectrum files RC.” SEQUEST HT was used to search against the UniProt Mus Musculus proteome (25,342 sequences, downloaded 27.09.2021). Search parameters included a precursor mass tolerance of 10ppm, a fragment mass tolerance of 0.02Da, and trypsin digestion with up to 2 missed cleavages allowed. Cysteine carbamidomethylation was set as a fixed modification while dynamic modifications were phosphorylation (S/T/Y), acetylation (N-terminus, K), oxidation (M), deamidation (N/Q), and N-terminal methionine loss. A site probability threshold of 75/100 was used. Percolator [
      • Käll L.
      • Canterbury J.D.
      • Weston J.
      • Noble W.S.
      • MacCoss M.J.
      Semi-supervised learning for peptide identification from shotgun proteomics datasets.
      ] was used to filter the results to a 1% false discovery rate at the peptide level, using the target-decoy strategy. Label-free quantification was performed using “Minora Feature Detector”, “Feature Mapper”, and “Precursor Ions Quantifier” nodes as described [
      • Skerrett-Byrne D.A.
      • Trigg N.A.
      • Bromfield E.G.
      • Dun M.D.
      • Bernstein I.R.
      • Anderson A.L.
      • Stanger S.J.
      • MacDougall L.A.
      • Lord T.
      • Aitken R.J.
      • et al.
      Proteomic Dissection of the Impact of Environmental Exposures on Mouse Seminal Vesicle Function.
      ]. Minora Feature Detector detects chromatographic peaks and maps them to peptide spectral matches (PSMs). Feature Mapper performs retention time alignment and links features across all raw files. The Precursor Ions Quantifier performs label free precursor ion quantification. T-tests were performed using the background-based method in Proteome Discoverer 2.5 [
      • Skerrett-Byrne D.A.
      • Trigg N.A.
      • Bromfield E.G.
      • Dun M.D.
      • Bernstein I.R.
      • Anderson A.L.
      • Stanger S.J.
      • MacDougall L.A.
      • Lord T.
      • Aitken R.J.
      • et al.
      Proteomic Dissection of the Impact of Environmental Exposures on Mouse Seminal Vesicle Function.
      ]. The peptide list was refined to include only entries with quantitative values in all replicates of at least one treatment group. Where duplicate peptides with the same phosphosite(s) were identified, the peptide with the highest fold-change was used.
      Significant (p value < 0.05) phosphosite changes with a fold-change greater than ±1.5 were subject to pathway enrichment analysis using Ingenuity Pathway Analysis (IPA, QIAGEN; Hilden, Germany). Enriched kinases were identified by mapping the upstream kinase(s) for each identified phosphosite (Table S2) using both mouse and human annotated kinase-substrate relationships in the Phosphosite Plus database [
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      • PhosphoSitePlus
      2014: mutations, PTMs and recalibrations.
      ]. Kinases with more than 5 substrates with significantly increased phosphorylation, or more than 5 substrates with significantly decreased phosphorylation, were classified as enriched. This analysis was complemented with kinase substrate enrichment analysis using RoKAI [
      • Yılmaz S.
      • Ayati M.
      • Schlatzer D.
      • Çiçek A.E.
      • Chance M.R.
      • Koyutürk M.
      Robust inference of kinase activity using functional networks.
      ], wherein data was searched against the PhosphositePlus [
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      • PhosphoSitePlus
      2014: mutations, PTMs and recalibrations.
      ] and DEPOD databases [

      Damle NP, Köhn M: The human DEPhOsphorylation Database DEPOD: 2019 update. Database (Oxford) 2019, 2019.

      ]. Results were filtered for significantly enriched kinases and phosphatases with 5 or more mapped substrates.

      Development and Analytical Validation Targeted MS Assays/Measurements

      We performed Tier 3 PRM analysis. To identify the optimal precursor m/z, the targets for PRM analysis were selected from public and in-house DDA data. PRM data files were analyzed using Skyline 20.2 (MacCoss Lab). The digestion enzyme was set to trypsin, precursor charges 2-4 were allowed, and ion charges 1-3 were allowed. Transitions were selected based on co-elution, and precursor and fragment ion mass error. Peptide abundance was assessed by summing the peak area of the 4-6 highest intensity fragment ions. Results were normalized to the TIC (total ion current) total area. For DNA-PKcs total protein measurements, the quantification values of the 3 peptides (Table S1) were averaged. Statistical analysis was performed using GraphPad Prism version 9 for Windows, using one-way ANOVA with Tukey’s or Dunnett’s adjustment for multiple comparisons. An adjusted p value < 0.05 was considered significant.

      Western blot

      Whole cell lysates were sonicated in ice-cold RIPA buffer freshly supplemented with protease inhibitors (0.05M HEPES pH 7.4, 1% Triton X-100, 0.1% SDS, 50mM Sodium Fluoride, 0.05M EDTA, 5% Sodium Deoxycholate, 1mM Sodium Orthovanadate, Protease Inhibitor Cocktail (Sigma)). Total protein was quantified by Bicinchoninic Acid assay, as per manufacturer’s instructions (Thermo-Scientific).
      Lysates were resolved in reducing conditions on 4-12% gradient Bis-Tris NuPAGE precast gels (Thermo-Scientific) before wet transfer to nitrocellulose. Blots were probed with primary antibodies MTOR (4517S), pMTOR S2448 (5536S), BTK (8547S), pBTK Y223 (5082S), ERK1/2 (9107S) or pERK1/2 T202/Y204 (Mouse T203/Y205, 9101S) (Cell Signaling Technology, Danvers, MA, USA) followed by HRP-conjugated mouse or rabbit secondary antibody. Blots were developed using Immobilon Classico ECL substrate (Millipore; Billerica, MA, USA) before visualization on a Chemidoc imager (BioRad, CA, USA)). Densitometry was performed using Image Lab (BioRad).

      Experimental Design and Statistical Rationale

      Proteomic analysis was performed with n=3 biological replicates per group. For DDA data, the peptide list was refined to include only entries with quantitative values in all replicates of at least one treatment group. Differentially expressed proteins were defined by a fold-change ≥ 1.5 or ≤ −1.5 and p value ≤ 0.05. Statistical analysis for DDA data was performed using Proteome Discoverer 2.5. For all other experiments, the number of biological replicates used in each experiment are presented in the figure legends. Graphs were prepared using GraphPad Prism version 9 for Windows and are presented as mean values ± SEM.
      Figures were produced using Microsoft Excel, and Graphpad Prism 9.

      Results

      Mutational activation of KIT increases DNA-PK phosphorylation

      To investigate if expression or phosphorylation of DNA-PK is associated with KIT activity, we used an established FDC-P1 isogenic myeloid cell line model of oncogenic mutant constitutively active KIT signaling [
      • Roberts K.G.
      • Smith A.M.
      • McDougall F.
      • Carpenter H.
      • Horan M.
      • Neviani P.
      • Powell J.A.
      • Thomas D.
      • Guthridge M.A.
      • Perrotti D.
      • et al.
      Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers.
      ]. Parallel reaction monitoring (PRM) revealed that FDC-P1 cells transduced with V560G-KIT, D816V-KIT, and EV controls expressed an equal level of the catalytic subunit of DNA-PK, DNA-PKcs (Figure 1A). In contrast, the phosphorylation of DNA-PKcs in the T2599/T2605/S2608/S2610 cluster was significantly higher in cell lines expressing mutant KIT (V560G, D816V) compared to the EV control (Figure 1B), suggesting DNA-PK activity is elevated in cells with constitutively active mutant KIT. As expected, acute (1h) treatment of D816V-KIT cells with DNA-PK inhibitors M3814 or NU7441, significantly decreased DNA-PKcs phosphorylation (Figure 1C). Treatment with the TKI, dasatinib, reduced DNA-PKcs phosphorylation at 30nM (Figure 1C). Combining M3814 or NU7441 with dasatinib led to a further reduction in DNA-PKcs phosphorylation (Figure 1C). No significant changes in total DNA-PKcs expression were observed (Figure S1).
      Figure thumbnail gr1
      Figure 1Activated KIT signaling increases DNA-PKcs phosphorylation. A) Protein expression and B) Phosphorylation of DNA-PKcs, assessed by parallel reaction monitoring (PRM) mass spectrometry, in FDC-P1 cells transduced with an empty vector (EV), or factor-independent mutant forms of KIT (V560G, D816V). N = 3 per experiment. *p<0.05, **p<0.01 (ANOVA + Tukey’s test). C) Phosphorylation of DNA-PKcs, assessed by PRM mass spectrometry, in FDC-P1/D816V-KIT cells untreated or treated for 1 h with 4μM M3814, 2μM NU7441, 4μM NU7441, 15nM dasatinib, 30nM dasatinib, or their combinations. N≥2. *p<0.05, **p<0.01 (ANOVA + Dunnett’s test). A singly phosphorylated DNA-PKcs peptide from the T2599/T2605/S2608/S2610 cluster was identified, however the phosphorylation could not be localized to a specific site.

      KIT V560G (juxtamembrane) and D816V (tyrosine kinase domain) mutations are associated with increased sensitivity to DNA-PK inhibitors

      To investigate the therapeutic potential of inhibiting DNA-PK in KIT-mutant cells, we assessed cell survival in response to DNA-PK inhibition. M3814 is a potent, specific inhibitor of DNA-PK (DNA-PKIC50 = 0.0006uM) currently in clinical trial in combination with chemotherapy for relapsed/refractory AML (NCT03983824). NU7441 is an ATP-competitive inhibitor of DNA-PK (DNA-PKIC50 = 0.014 μM, mTORIC50 = 1.7 μM); however the low solubility profile of NU7441 restricts its use to in vitro applications [
      • Zhao Y.
      • Thomas H.D.
      • Batey M.A.
      • Cowell I.G.
      • Richardson C.J.
      • Griffin R.J.
      • Calvert A.H.
      • Newell D.R.
      • Smith G.C.M.
      • Curtin N.J.
      Preclinical Evaluation of a Potent Novel DNA-Dependent Protein Kinase Inhibitor NU7441.
      ]. The FDC-P1 cell lines signaling through activated KIT (D816V, V560G) were more sensitive to M3814 and NU7441, compared to the EV control cells ( Figure S2).

      Inhibition of KIT signaling is synergistic with DNA-PK inhibition in KIT mutant cells

      Subsequently, we assessed whether targeting DNA-PK in combination with inhibition of KIT signaling would lead to an enhanced anti-proliferative effect in KIT-mutant cells. Dasatanib is an ATP-competitive tyrosine kinase inhibitor with activity against KIT, BCR-ABL, and Src family kinases [
      • Shah N.P.
      • Lee F.Y.
      • Luo R.
      • Jiang Y.
      • Donker M.
      • Akin C.
      Dasatinib (BMS-354825) inhibits KITD816V, an imatinib-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis.
      ,
      • O'Hare T.
      • Walters D.K.
      • Stoffregen E.P.
      • Jia T.
      • Manley P.W.
      • Mestan J.
      • Cowan-Jacob S.W.
      • Lee F.Y.
      • Heinrich M.C.
      • Deininger M.W.
      • et al.
      In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants.
      ]. Dasatinib is currently in clinical use for hematologic malignancies including CML and ALL [
      • Brave M.
      • Goodman V.
      • Kaminskas E.
      • Farrell A.
      • Timmer W.
      • Pope S.
      • Harapanhalli R.
      • Saber H.
      • Morse D.
      • Bullock J.
      • et al.
      Sprycel for chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia resistant to or intolerant of imatinib mesylate.
      ]. As expected, the KIT mutant lines were sensitive to dasatinib treatment, with the V560G-KIT cells being more sensitive than the D816V-KIT lines, as previously reported [
      • Shah N.P.
      • Lee F.Y.
      • Luo R.
      • Jiang Y.
      • Donker M.
      • Akin C.
      Dasatinib (BMS-354825) inhibits KITD816V, an imatinib-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis.
      ]; whereas dasatinib had no effect on the EV control cells (Figure S2). The combination of dasatinib with the DNA-PK inhibitors M3814 (Figure 2A) or NU7441 (Figure 2B) led to a synergistic reduction in cell survival, selectively in the mutant KIT lines (V560G, D816V). To investigate the mechanism of cell death, we measured apoptosis following treatment with M3814, NU7441, dasatinib, or their combinations. The combination of either M3814 or NU7441 with dasatinib synergistically increased apoptosis, in both V560G-KIT and D816V-KIT cells (Figure 2C, D).
      Figure thumbnail gr2
      Figure 2The tyrosine kinase inhibitor, dasatinib, in combination with DNA-PK inhibitors, M3814 or NU7441, induces synergistic cell death in KIT mutant cells. FDC-P1 cells expressing an empty vector (EV), or factor-independent mutant forms of KIT (V560G, D816V), were incubated with increasing concentrations of A) dasatinib, M3814, or their combination; and B) dasatinib, NU7441, or their combination. Cell viability at 72 hours was assessed by resazurin metabolic assay. n≥2. Grey shading = not done. +, synergy, assessed by the fractional product method of Webb. Apoptosis induction at 48 hours in C) V560G-KIT and (D) D816V-KIT cells was measured by annexin-V flow cytometry. Points = mean ± SEM, n≥3. +, synergy, assessed by the fractional product method of Webb ().

      Phosphoproteomic analysis of combined DNA-PK and KIT signaling inhibition in D816V-KIT cells reveals co-operative inhibition of ERK and mTOR signaling

      To investigate the mechanism of synergistic cell death observed with combined DNA-PK and KIT signaling inhibitors, we performed a global phosphoproteomic analysis of D816V-KIT cells untreated or treated for 1 hour with 4μM M3814, 15nM dasatinib, or their combination, in biological triplicate. Drug doses were selected that induced no, or low, cytotoxicity as single agents but were synergistically cytotoxic when used in combination (Figure 2A, D). Across the 12 samples, 6,892 phosphopeptides were identified (5,489 unique phosphosites), with expected ratios of serine, threonine, and tyrosine sites [pS:pT:pY 89.1%:10.3%:0.6%] (Table S2).
      Using a fold-change cut off of 1.5 and p-value < 0.05, 322 phosphopeptides were decreased and 616 phosphopeptides were increased in response to M3814 treatment (Figure 3A). In response to single-agent dasatinib treatment, 229 phosphopeptides were decreased and 252 were increased (Figure 3B). Three hundred and thirty-three phosphopeptides were decreased and 238 were increased in response to combination M3814 and dasatinib treatment (Figure 3C). Of these, the expression of 170 phosphopeptides were significantly modulated in all 3 treatment groups (Figure S3).
      Figure thumbnail gr3
      Figure 3Quantitative phosphoproteomic analysis of mutant KIT-D816V FDC-P1 cells. FDC-P1 D816V-KIT cells were treated with M3814 (4μM), dasatinib (15nM), or their combination for 1hr. Phosphoproteomes were profiled by EasyPhos enrichment followed by mass spectrometry. A) Volcano plot of significant fold-changes with M3814 treatment; 616 phosphopeptides were significantly increased and 322 significantly decreased. B) Volcano plot of significant fold-changes with dasatinib treatment; 252 phosphopeptides were significantly increased and 229 significantly decreased. C) Volcano plot of significant fold-changes with combination M3814 and dasatinib treatment; 238 phosphopeptides were significantly increased and 333 significantly decreased. A fold-change cut off of ±1.5 was used.
      Phosphopeptides with the largest fold decrease in response to M3814 single-agent treatment included RPS6 sites (pS235, pS236, pS240, pS244); and IFNGR1 sites (pS370, pT373; Figure 3A, Table S2). The top increased phosphopeptides included sites on NUCKS1 (pS54, pS58, pS61), and HNRNPD (pS80; Figure 3A, Table S2). Pathway analysis revealed decreased phosphorylation of mTOR, ERK/MAPK, and actin cytoskeleton signaling pathways, and increased phosphorylation of BRCA1, ATM, and cell cycle pathways (Figure 4). Concordantly, kinase enrichment analysis revealed decreased phosphorylation of P70S6K, AKT1, mTOR, ERK1/2, and PKACA substrates, and increased phosphorylation of CK2A1 substrates (Figure 5).
      Figure thumbnail gr4
      Figure 4Pathway enrichment analysis of phosphoproteins regulated by M3814, dasatinib, or their combination, in KIT-D816V FDC-P1 cells. FDC-P1 D816V-mutant KIT cells were treated with M3814 (4μM), dasatinib (15nM), or their combination for 1hr. Phosphoproteins with significantly altered phosphorylation were analyzed by Ingenuity Pathway Analysis (IPA), to identify significantly enriched pathways. Bubble size indicates number of proteins with significantly altered phosphorylation in each significantly enriched pathway, shading indicates average pathway phosphorylation. The full list of IPA pathways is provided in .
      Figure thumbnail gr5
      Figure 5Kinase enrichment analysis. FDC-P1 D816V-mutant KIT cells were treated with M3814 (4μM), dasatinib (15nM), or their combination for 1h. Phosphoproteomes were profiled by mass spectrometry. A) Heatmaps of kinase substrates for kinases with more than 5 substrates significantly increased or decreased in phosphorylation compared to untreated (UT). * p<0.05 compared to UT. Kinase substrates were identified using the Phosphosite Plus database [
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      • PhosphoSitePlus
      2014: mutations, PTMs and recalibrations.
      ]. B) Kinase enrichment analysis performed using RoKAI [
      • Yılmaz S.
      • Ayati M.
      • Schlatzer D.
      • Çiçek A.E.
      • Chance M.R.
      • Koyutürk M.
      Robust inference of kinase activity using functional networks.
      ]. Z score indicates inferred kinase or phosphatase (phospha-) activity, with a positive Z score indicating increased activity. Significant (p<0.05) z scores are shown.
      Phosphopeptides decreased in response to dasatinib included sites on IFNGR1 (pS370, pT373), and TRIM28 (pT8; Figure 3B, Table S2). The top increased phosphopeptides included sites on the heat shock protein HSP90AB1 (pS255, pS261); and transcription elongation factor IWS1 (pS458, pS460; Figure 3B, Table S2). Pathways enriched with decreased phosphorylation included p70S6K, mTOR, ERK/MAPK, p38 MAPK, and ERK5 signaling (Figure 4). Consistent with this, phosphorylation of ERK2 substrates were decreased in response to dasatinib, including ERK2 (MAPK1) pY185; indicative of reduced ERK1/2 activity (Figure 5). BRCA1, cell cycle, and DNA methylation and transcriptional repression signaling pathways displayed increased phosphorylation in response to dasatinib (Figure 4).
      Phosphopeptides decreased in response to the combination treatment included sites on RPS6 (pS240, pS244, pS247), and RCSD1 (pT243, pS246; Figure 3C). The top increased phosphopeptides included sites on NUCKS1 (pS73, pS75, pS79), and TJP2 (pS263, pS265, pS267; Figure 3C, Table S2). Pathway analysis identified decreased phosphorylation of p70S6K, mTOR, ERK/MAPK, p38 MAPK, ERK5, actin cytoskeleton, and apoptosis signaling pathways (Figure 4). Consistent with this, phosphorylation of p70S6K, AKT1, mTOR, ERK1/2, and PKACA substrates were decreased, suggesting reduced activity of these kinases (Figure 5). Phosphorylation of cell cycle, BRCA1, and DNA methylation and transcriptional repression signaling pathways was increased (Figure 4), and concordantly CDK1 substrates displayed increased phosphorylation (Figure 5).
      To investigate the crosstalk between M3814 and dasatinib treatments at the peptide level, we filtered for synergistically regulated phosphopeptides. In this context, we defined synergistic inhibition as a fold-change in the M3814+dasatinib treatment group that was ≥1.2 fold lower than the additive fold-change of that in the M3814 and dasatinib single agent groups. Conversely, we defined “synergistic induction” as a fold-change in the M3814+dasatinib treatment group that was ≥1.2 higher than the additive fold-change of the M3814 and dasatinib single-agent groups [
      • Goldstein I.
      • Paakinaho V.
      • Baek S.
      • Sung M.H.
      • Hager G.L.
      Synergistic gene expression during the acute phase response is characterized by transcription factor assisted loading.
      ]. In total, 86 phosphopeptides were synergistically regulated (37 were synergistically induced (Figure 6A), and 49 were synergistically inhibited (Figure 6B)) in response to the combined M3814 and dasatinib treatment. This included synergistic inhibition of the phosphorylation of transcription factors MYC, MYB, and NCOR1, transcription regulator NPM1, and mTOR (Figure 6 and 7D ). The phosphorylation of DNA repair and transcription regulators ATRX, WRN, and SMARCA4 were synergistically increased by the combination treatment (Figure 6 and 7D).
      Figure thumbnail gr6
      Figure 6Quantitative phosphoproteomic analysis of mutant KIT D816V FDC-P1 cells. FDC-P1 D816V-mutant KIT cells were treated with M3814 (4μM), dasatinib (15nM), or their combination for 1h. Phosphoproteomes were profiled by mass spectrometry. Heatmap of phosphosites identified with A) synergistic increase or B) synergistic decrease by the combination of M3814 and dasatinib are shown. Das; dasatinib. Type; Ingenuity Pathway Analysis molecule annotations.
      Figure thumbnail gr7
      Figure 7Schematic of phosphosites increased or decreased by the combination of M3814 and dasatinib treatment, in mutant KIT D816V FDC-P1 cells. FDC-P1 D816V-mutant KIT cells were treated with M3814 (4μM), Dasatinib (15nM), or their combination for 1h. Phosphoproteomes were profiled by EasyPhos enrichment followed by mass spectrometry. A) Curation of key phosphosite changes in the identified significantly enriched pathways. Phosphosite color reflects fold-change in combination treated samples compared to untreated cells (Blue = decreased, Red = increased). Underlined phosphosites indicate those with annotated function (PhosphositePlus [
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      • PhosphoSitePlus
      2014: mutations, PTMs and recalibrations.
      ]). B) Phosphosites selectively decreased by M3814 treatment. C) Phosphosites selectively decreased by dasatinib treatment. D) Phosphosites synergistically regulated by M3814 and dasatinib combination treatment. *p<0.05, **p<0.01
      Taken together, these results suggest that both M3814 and dasatinib treatment inhibit ERK/MAPK and mTOR signaling pathways, with greater inhibition of both pathways when treated in combination (Figure 4, Figure 5). Both treatments increased phosphorylation of cell cycle pathways (Figure 4), consistent with the growth arrest induced by these two inhibitors (Figure 7A). The combination of M3814 and dasatinib potently inhibited both ERK/MAPK and AKT1/mTOR signaling pathways, and also inhibited p90RSK kinase signaling (Figures 4 and 5). Key phosphorylation changes in these pathways, such as RPS6 pS235, pS236, GSK3A pS21, ERK2 pY185, and BTK pY223, are driven by the effect of one inhibitor (Figure 7B, C). Concurrently, key phosphorylation changes such as MYC pS164 and MYB pS528 are synergistically inhibited, while others such as Wrn pS1044, pS1045 and ATRX pT583, pS590 are synergistically induced by the combination treatment (Figure 7D).
      Consistent with these results, treatment with AKT/mTOR inhibitors MK2206 and everolimus, elicited synergy in combination with the MEK/ERK inhibitors, selumetinib and ASTX029 (Figure S4). While cytotoxicity was higher in the KIT mutant lines, EV control cells were also sensitive to these combinations. PI3K/mTOR/AKT and MEK/ERK signaling pathways are involved in diverse cellular processes, therefore it follows that targeting the activated upstreamregulators, KIT and DNA-PK, elicits greater selectivity. Further supporting these findings, M3814 co-treatment with MEK/ERK inhibitors selumetinib and ASTX029 elicits synergy, with higher synergy scores compared to M3814 co-treatment with mTOR/AKT inhibitors everolimus and MK2206 (Figure S5, Table S5).
      Selected phosphoproteins were orthogonally evaluated by western blot, over a time course of 72 hours. Phosphorylation of MTOR at the mitogen-inducible phosphosite S2448 [
      • Chiang G.G.
      • Abraham R.T.
      Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70S6 kinase.
      ] was reduced by 4μM M3814 treatment, at 1, 24, 48, and 72 hours (Figure 8). The combination of 4μM M3814 and either 15 or 30nM dasatinib further reduced MTOR phosphorylation at 24, 48, and 72 hours. Similar to the mass spectrometry results, phosphorylation of BTK at autophosphorylation site Y223 [
      • Wahl M.I.
      • Fluckiger A.C.
      • Kato R.M.
      • Park H.
      • Witte O.N.
      • Rawlings D.J.
      Phosphorylation of two regulatory tyrosine residues in the activation of Bruton's tyrosine kinase via alternative receptors.
      ] was efficiently inhibited by dasatinib treatment, at all time points (Figure 8). Phosphorylation of ERK1/2 at the activating phosphosites T203/Y205 (ERK1) or T183/Y185 (ERK2) [
      • Roskoski Jr., R.
      ERK1/2 MAP kinases: structure, function, and regulation.
      ] was increased in response to M3814 treatment at later time points (24, 48, 72 hours). This effect was opposed by dasatinib, with reduced ERK1/2 phosphorylation observed in response to dasatinib both alone and in combination with M3814, excepting 15nM dasatinib combined with M3814 at 48 hours (Figure 8). Inhibition of KIT downstream signaling synergizes with DNA-PK inhibitors in mutant KIT cells
      Figure thumbnail gr8
      Figure 8Phosphorylation of MTOR, BTK, and ERK1/2 in response to M3814, dasatinib, or their combination. FDC-P1 D816V-mutant KIT cells were treated with M3814 (4μM), dasatinib (15 or 30nM), or their combination for 1, 24, 48, and 72h. A) Representative western blot images, and B) densitometry. Bars = mean ± SEM. N=2-3.
      D816V-KIT mutant cells displayed constitutive BTK phosphorylation, which was inhibited by dasatinib treatment (Figure 7C, Figure 8). This led us to assess cell survival in response to combined BTK and DNA-PK inhibition. Ibrutinib is a covalent inhibitor of BTK, in clinical use for treating lymphomas and chronic lymphocytic leukemia. Ibrutinib leads to inhibition of KIT signaling through BTK [
      • Rushworth S.A.
      • Pillinger G.
      • Abdul-Aziz A.
      • Piddock R.
      • Shafat M.S.
      • Murray M.Y.
      • Zaitseva L.
      • Lawes M.J.
      • MacEwan D.J.
      • Bowles K.M.
      Activity of Bruton's tyrosine-kinase inhibitor ibrutinib in patients with CD117-positive acute myeloid leukaemia: a mechanistic study using patient-derived blast cells.
      ]. Mutant KIT FDC-P1 cell lines (D816V, V560G) were more sensitive to ibrutinib compared to the EV control line (Figure S2). The combination of ibrutinib and DNA-PK inhibitors M3814 (Figure 9A) or NU7441 (Figure 9B) effected synergistic reduction in cell survival in all cell lines, irrespective of KIT signaling dependence. However, the combination treatments were more potent in mutant KIT cell lines (D816V, V560G; Figure 9A,B).
      Figure thumbnail gr9
      Figure 9KIT signaling inhibitors ibrutinib, FTY720, or AALS in combination with DNA-PK inhibitors NU7441 or M3814 induce synergistic cell death in KIT-mutant cells. FDC-P1 cells expressing an empty vector (EV), or factor-independent mutant forms of KIT (V560G, D816V), were incubated with increasing concentrations of A) ibrutinib, M3814, or their combination; B) ibrutinib, NU7441, or their combination; C) FTY720 or AALS, alone or in combination with M3814; and D) FTY720 or AALS, alone or in combination with NU7441. Cell viability at 72 hours was assessed by resazurin metabolic assay. n≥2. +, synergy, assessed by the fractional product method of Webb ()
      Our laboratory has previously shown that oncogenic mutant KIT is associated with inhibition of the serine/threonine protein phosphatase PP2A [
      • Roberts K.G.
      • Smith A.M.
      • McDougall F.
      • Carpenter H.
      • Horan M.
      • Neviani P.
      • Powell J.A.
      • Thomas D.
      • Guthridge M.A.
      • Perrotti D.
      • et al.
      Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers.
      ]. In wildtype cells, PP2A negatively regulates KIT-activated pathways including MAPK, JAK/STAT and PI3K/AKT [
      • Kiely M.
      • Kiely P.A.
      PP2A: The Wolf in Sheep's Clothing?.
      ,
      • Ross J.A.
      • Cheng H.
      • Nagy Z.S.
      • Frost J.A.
      • Kirken R.A.
      Protein phosphatase 2A regulates interleukin-2 receptor complex formation and JAK3/STAT5 activation.
      ]. Activation of PP2A in KIT mutant cells leads to reduced phosphorylation of these pathways, and subsequently apoptosis [
      • Roberts K.G.
      • Smith A.M.
      • McDougall F.
      • Carpenter H.
      • Horan M.
      • Neviani P.
      • Powell J.A.
      • Thomas D.
      • Guthridge M.A.
      • Perrotti D.
      • et al.
      Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers.
      ]. Substrate enrichment analysis of M3814 and dasatinib regulated phosphosites identified dephosphorylation of PP2A substrates (Figure 5B). We therefore tested the interaction between the PP2A activating compounds FTY720 or AAL(S), and DNA-PK inhibitors M3814 or NU7441. As expected, cells dependent on active KIT were more sensitive to FTY720 and AAL(S) compared to EV controls [
      • Roberts K.G.
      • Smith A.M.
      • McDougall F.
      • Carpenter H.
      • Horan M.
      • Neviani P.
      • Powell J.A.
      • Thomas D.
      • Guthridge M.A.
      • Perrotti D.
      • et al.
      Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers.
      ] (Figure 9C, D). DNA-PK inhibitors M3814 or NU7441, in combination with FTY720 or AAL(S), led to a synergistic reduction in cell growth in all FDC-P1 cell lines (Figure 9C, D). However, each DNA-PK inhibitor and PP2A activator combination was more potent in the V560G- and D816V-KIT lines, compared to the EV control line (Figure 9C, D).

      Discussion

      Mutations in genes involved in kinase signaling represent a major proportion of recurrent mutations in AML [
      • Ley T.J.
      • Miller C.
      • Ding L.
      • Raphael B.J.
      • Mungall A.J.
      • Robertson A.
      • Hoadley K.
      • Triche Jr., T.J.
      • Laird P.W.
      • Baty J.D.
      • et al.
      Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia.
      ,
      • Papaemmanuil E.
      • Gerstung M.
      • Bullinger L.
      • Gaidzik V.I.
      • Paschka P.
      • Roberts N.D.
      • Potter N.E.
      • Heuser M.
      • Thol F.
      • Bolli N.
      • et al.
      Genomic Classification and Prognosis in Acute Myeloid Leukemia.
      ]. These include the receptor tyrosine kinases KIT and FLT3, and the mutational activation of these kinases is associated with poor treatment outcome and higher relapse risk [
      • Staudt D.
      • Murray H.C.
      • McLachlan T.
      • Alvaro F.
      • Enjeti A.K.
      • Verrills N.M.
      • Dun M.D.
      Targeting Oncogenic Signaling in Mutant FLT3 Acute Myeloid Leukemia: The Path to Least Resistance.
      ,
      • Wang B.
      • Zhang J.
      • Hua X.
      • Li H.
      • Wang Z.
      • Yang B.
      Clinical heterogeneity under induction with different dosages of cytarabine in core binding factor acute myeloid leukaemia.
      ,
      • Kim H.-J.
      • Ahn H.K.
      • Jung C.W.
      • Moon J.H.
      • Park C.-H.
      • Lee K.-O.
      • Kim S.-H.
      • Kim Y.-K.
      • Kim H.-J.
      • Sohn S.K.
      • et al.
      KIT D816 mutation associates with adverse outcomes in core binding factor acute myeloid leukemia, especially in the subgroup with RUNX1/RUNX1T1 rearrangement.
      ,
      • Yui S.
      • Kurosawa S.
      • Yamaguchi H.
      • Kanamori H.
      • Ueki T.
      • Uoshima N.
      • Mizuno I.
      • Shono K.
      • Usuki K.
      • Chiba S.
      • et al.
      D816 mutation of the KIT gene in core binding factor acute myeloid leukemia is associated with poorer prognosis than other KIT gene mutations.
      ]. Herein, we have shown for the first time that mutant KIT is associated with increased phosphorylation of the DNA double strand break repair protein, DNA-PK. We demonstrate that DNA-PK and KIT signaling inhibitor combinations are synergistic and potent in KIT mutant cell lines, supporting DNA-PK as a novel target for therapy in KIT mutant cancers.
      DNA-PK is the ubiquitously expressed [
      • Uhlen M.
      • Fagerberg L.
      • Hallstrom B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • Sivertsson A.
      • Kampf C.
      • Sjostedt E.
      • Asplund A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      ] core kinase of the non-homologous end joining DNA double strand break repair pathway. The function of DNA-PK is highly regulated by phosphorylation [
      • Neal J.A.
      • Sugiman-Marangos S.
      • VanderVere-Carozza P.
      • Wagner M.
      • Turchi J.
      • Lees-Miller S.P.
      • Junop M.S.
      • Meek K.
      Unraveling the complexities of DNA-dependent protein kinase autophosphorylation.
      ], which is catalyzed by ATM [
      • Chen B.P.
      • Uematsu N.
      • Kobayashi J.
      • Lerenthal Y.
      • Krempler A.
      • Yajima H.
      • Löbrich M.
      • Shiloh Y.
      • Chen D.J.
      Ataxia telangiectasia mutated (ATM) is essential for DNA-PKcs phosphorylations at the Thr-2609 cluster upon DNA double strand break.
      ], ATR [
      • Yajima H.
      • Lee K.J.
      • Chen B.P.
      ATR-dependent phosphorylation of DNA-dependent protein kinase catalytic subunit in response to UV-induced replication stress.
      ], PLK1 [
      • Douglas P.
      • Ye R.
      • Trinkle-Mulcahy L.
      • Neal J.A.
      • De Wever V.
      • Morrice N.A.
      • Meek K.
      • Lees-Miller S.P.
      Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.
      ], and DNA-PK itself [
      • Neal J.A.
      • Sugiman-Marangos S.
      • VanderVere-Carozza P.
      • Wagner M.
      • Turchi J.
      • Lees-Miller S.P.
      • Junop M.S.
      • Meek K.
      Unraveling the complexities of DNA-dependent protein kinase autophosphorylation.
      ]. DNA-PK phosphorylation is also regulated by AKT [
      • Toulany M.
      • Lee K.J.
      • Fattah K.R.
      • Lin Y.F.
      • Fehrenbacher B.
      • Schaller M.
      • Chen B.P.
      • Chen D.J.
      • Rodemann H.P.
      Akt promotes post-irradiation survival of human tumor cells through initiation, progression, and termination of DNA-PKcs-dependent DNA double-strand break repair.
      ], however it is not known whether this regulation is direct. In mouse myeloid progenitor FDC-P1 cells expressing constitutively active mutant KIT, we identified increased phosphorylation of DNA-PKcs at T2599/T2605/S2608/S2610 (homologous to T2603/T2609/S2612 in human DNA-PKcs). These sites are situated within the ABCDE cluster of phosphorylation sites that are required for the DNA repair activity of DNA-PK [
      • Neal J.A.
      • Sugiman-Marangos S.
      • VanderVere-Carozza P.
      • Wagner M.
      • Turchi J.
      • Lees-Miller S.P.
      • Junop M.S.
      • Meek K.
      Unraveling the complexities of DNA-dependent protein kinase autophosphorylation.
      ], indicating that DNA-PK is active in KIT mutant cells. This work adds KIT to a list of growth factor receptors linked with altered DNA-PK activity, including FLT3 [
      • Murray H.C.
      • Enjeti A.K.
      • Kahl R.G.S.
      • Flanagan H.M.
      • Sillar J.
      • Skerrett-Byrne D.A.
      • Al Mazi J.G.
      • Au G.G.
      • de Bock C.E.
      • Evans K.
      • et al.
      Quantitative phosphoproteomics uncovers synergy between DNA-PK and FLT3 inhibitors in acute myeloid leukaemia.
      ], EGFR [
      • Javvadi P.
      • Makino H.
      • Das A.K.
      • Lin Y.F.
      • Chen D.J.
      • Chen B.P.
      • Nirodi C.S.
      Threonine 2609 phosphorylation of the DNA-dependent protein kinase is a critical prerequisite for epidermal growth factor receptor-mediated radiation resistance.
      ], MPL [
      • de Laval B.
      • Pawlikowska P.
      • Barbieri D.
      • Besnard-Guerin C.
      • Cico A.
      • Kumar R.
      • Gaudry M.
      • Baud V.
      • Porteu F.
      Thrombopoietin promotes NHEJ DNA repair in hematopoietic stem cells through specific activation of Erk and NF-kappaB pathways and their target, IEX-1.
      ], and MST1R [
      • Chang H.Y.
      • Chang T.C.
      • Huang W.Y.
      • Lee C.T.
      • Yen C.J.
      • Tsai Y.S.
      • Tzai T.S.
      • Chen S.H.
      • Chow N.H.
      RON Nuclear Translocation under Hypoxia Potentiates Chemoresistance to DNA Double-Strand Break-Inducing Anticancer Drugs.
      ]. Activated growth signaling and DNA damage stress are two hallmarks of cancer, and it is possible that these characteristics are causatively linked [
      • Negrini S.
      • Gorgoulis V.G.
      • Halazonetis T.D.
      Genomic instability--an evolving hallmark of cancer.
      ].
      We investigated the potential therapeutic benefit of targeting KIT signaling in combination with DNA-PK. The TKI, dasatinib, in combination with DNA-PK inhibitors (M3814, NU7441) induced a synergistic and potent reduction in survival in KIT mutant cell lines (D816V, V560G) (Figure 2). To investigate the cellular pathways mediating this synergy, we performed a global phosphoproteomic analysis of D816V-KIT cells treated with single-agent dasatinib or M3814, or their combination (Figure 3). Dasatinib single-agent treatment reduced ERK2 kinase activity (Figure 5), and decreased phosphorylation of p70S6K, mTOR, and ERK/MAPK pathways (Figure 4). D816V-KIT expressing cells display constitutive phosphorylation of ERK and AKT, suggesting their activation [
      • Frost M.J.
      • Ferrao P.T.
      • Hughes T.P.
      • Ashman L.K.
      Juxtamembrane Mutant V560GKit Is More Sensitive to Imatinib (STI571) Compared with Wild-Type c-Kit Whereas the Kinase Domain Mutant D816VKit Is Resistant.
      ]. Dasatinib-induced apoptosis of D816V-KIT expressing cells has been previously correlated with a reduction of ERK2 phosphorylation, with higher doses of dasatinib (>100nM) required to inhibit Akt phosphorylation [
      • Schittenhelm M.M.
      • Shiraga S.
      • Schroeder A.
      • Corbin A.S.
      • Griffith D.
      • Lee F.Y.
      • Bokemeyer C.
      • Deininger M.W.
      • Druker B.J.
      • Heinrich M.C.
      Dasatinib (BMS-354825), a dual SRC/ABL kinase inhibitor, inhibits the kinase activity of wild-type, juxtamembrane, and activation loop mutant KIT isoforms associated with human malignancies.
      ]. Concurrently, single-agent dasatinib treatment increased phosphorylation of cell cycle and BRCA1 DNA damage response pathways (Figure 4), consistent with the induction of growth arrest (Figure 2).
      Treatment with single-agent M3814 decreased mTOR, AKT1, and ERK1/2 activity (Figure 5). This was associated with decreased phosphorylation of mTOR, ERK/MAPK, insulin receptor signaling, and eIF4 and p70S6K signaling pathways (Figure 4). A rebound increase in ERK1/2 phosphorylation was observed at 24, 48, and 72 hours following M3814 treatment (Figure 8). Rebound ERK activation has been observed in response to Raf and MEK inhibitors in numerous studies [
      • Kotani H.
      • Adachi Y.
      • Kitai H.
      • Tomida S.
      • Bando H.
      • Faber A.C.
      • Yoshino T.
      • Voon D.C.
      • Yano S.
      • Ebi H.
      Distinct dependencies on receptor tyrosine kinases in the regulation of MAPK signaling between BRAF V600E and non-V600E mutant lung cancers.
      ,
      • Lito P.
      • Pratilas C.A.
      • Joseph E.W.
      • Tadi M.
      • Halilovic E.
      • Zubrowski M.
      • Huang A.
      • Wong W.L.
      • Callahan M.K.
      • Merghoub T.
      • et al.
      Relief of profound feedback inhibition of mitogenic signaling by RAF inhibitors attenuates their activity in BRAFV600E melanomas.
      ,
      • Gerosa L.
      • Chidley C.
      • Fröhlich F.
      • Sanchez G.
      • Lim S.K.
      • Muhlich J.
      • Chen J.Y.
      • Vallabhaneni S.
      • Baker G.J.
      • Schapiro D.
      • et al.
      Receptor-Driven ERK Pulses Reconfigure MAPK Signaling and Enable Persistence of Drug-Adapted BRAF-Mutant Melanoma Cells.
      ], however this is the first report in response to a DNA-PK inhibitor. DNA-PK has been previously shown to phosphorylate AKT1 (serine 473) in response to insulin stimulation and DNA damage [
      • Park J.
      • Feng J.
      • Li Y.
      • Hammarsten O.
      • Brazil D.P.
      • Hemmings B.A.
      DNA-dependent protein kinase-mediated phosphorylation of protein kinase B requires a specific recognition sequence in the C-terminal hydrophobic motif.
      ,
      • Feng J.
      • Park J.
      • Cron P.
      • Hess D.
      • Hemmings B.A.
      Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase.
      ]; and through AKT-dependent signaling, DNA-PK regulates MYC stability [
      • An J.
      • Yang D.Y.
      • Xu Q.Z.
      • Zhang S.M.
      • Huo Y.Y.
      • Shang Z.F.
      • Wang Y.
      • Wu D.C.
      • Zhou P.K.
      DNA-dependent protein kinase catalytic subunit modulates the stability of c-Myc oncoprotein.
      ]. Indeed, MYC overexpressing cells are sensitive to inhibition of DNA-PK [
      • Zhou Z.
      • Patel M.
      • Ng N.
      • Hsieh M.H.
      • Orth A.P.
      • Walker J.R.
      • Batalov S.
      • Harris J.L.
      • Liu J.
      Identification of synthetic lethality of PRKDC in MYC-dependent human cancers by pooled shRNA screening.
      ], suggesting a role for DNA-PK inhibitors in the treatment of MYC dependent tumors. Consistent with this, M3814 treatment reduced MYC phosphorylation at threonine 58, serine 62, and serine 164 (Table S2); however as a single-agent treatment this did not reach statistical significance.
      While we cannot rule out the possibility that the DNA-PK inhibitors used herein may directly inhibit mTOR, the potent inhibition of DNA-PK phosphorylation observed in response to single agent DNA-PKi treatment (Figure 1) suggests that on-target inhibition of DNA-PK is the major driver of the cellular response to DNA-PKi. A few lines of evidence support that DNA-PK is involved in mTOR signaling. DNA-PK was shown to be incorporated into mTORC2 complexes in keratinocytes [
      • Tu Y.
      • Ji C.
      • Yang B.
      • Yang Z.
      • Gu H.
      • Lu C.C.
      • Wang R.
      • Su Z.L.
      • Chen B.
      • Sun W.L.
      • et al.
      DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-SIN1 association mediates ultraviolet B (UVB)-induced Akt Ser-473 phosphorylation and skin cell survival.
      ]. In a recent study of non-small cell lung carcinoma, DNA-PK was found to be incorporated into an alternative mTOR complex, regulating phosphorylation of mTOR downstream substrates RPS6 and 4EBP1 [
      • Nguyen J.T.
      • Haidar F.S.
      • Fox A.L.
      • Ray C.
      • Mendonça D.B.
      • Kim J.K.
      • Krebsbach P.H.
      mEAK-7 Forms an Alternative mTOR Complex with DNA-PKcs in Human Cancer.
      ]. DNA-PK activity may also be regulated by mTOR signaling, with rapamycin-induced inhibition of mTORC1 leading to increased PP2A activity, consequently increasing DNA-PK activity and AKT phosphorylation [
      • Li Y.
      • Wang X.
      • Yue P.
      • Tao H.
      • Ramalingam S.S.
      • Owonikoko T.K.
      • Deng X.
      • Wang Y.
      • Fu H.
      • Khuri F.R.
      • et al.
      Protein phosphatase 2A and DNA-dependent protein kinase are involved in mediating rapamycin-induced Akt phosphorylation.
      ]. Combined rapamycin and DNA-PK inhibitor treatment was required to suppress AKT phosphorylation, and also decreased 4EBP1 phosphorylation in human lung cancer models [
      • Li Y.
      • Wang X.
      • Yue P.
      • Tao H.
      • Ramalingam S.S.
      • Owonikoko T.K.
      • Deng X.
      • Wang Y.
      • Fu H.
      • Khuri F.R.
      • et al.
      Protein phosphatase 2A and DNA-dependent protein kinase are involved in mediating rapamycin-induced Akt phosphorylation.
      ], supporting that DNA-PK is involved in regulating mTOR signaling. In the current study, M3814 treatment decreased phosphorylation of RPTOR (mTORC1 component), RPS6, 4EBP1, and LARP1 (mTORC1 targets), and reduced AKT1 activity and actin cytoskeleton signaling (mTORC2 targets), suggestive of inhibition of both mTORC1 and 2 activity. In this way, inhibition of DNA-PK may regulate protein translation, glucose metabolism, transcription, and cell growth.
      M3814 single agent treatment of D816V-KIT cells also increased phosphorylation of cell cycle, and ATM and BRCA1 DNA damage response pathways (Figure 4). Increased phosphorylation of homologous recombination repair proteins BRCA1 and FANCM, coupled with increased phosphorylation of cell cycle kinases CDK12 and CDK13 (Table S2), is consistent with the induction of growth arrest (Figure 2).
      Combination M3814 and dasatinib treatment of D816V-KIT cells enhanced the effects of the single agent treatments, with further inhibition of ERK1/2, AKT1, P70S6K, mTOR, and PKACA kinase activity (Figure 5). This was associated with decreased phosphorylation of P70S6K, mTOR, ERK/MAPK, Insulin receptor signaling, and Actin cytoskeleton signaling pathways (Figure 4). The combination of M3814 and dasatinib blocked the rebound increase in ERK phosphorylation observed in response to M3814 single agent treatment (Figure 8). This block is likely an important contributor to the synergistic cytotoxicity observed with the combination. Apoptosis signaling was enriched with the combination treatment (Figure 4), with decreased phosphorylation of anti-apoptotic proteins BAD, ROCK1, and RPS6KA1 (Table S2, Figure 7A). This is indicative of reduced anti-apoptotic signaling, consistent with the synergistic induction of apoptosis effected by M3814 and dasatinib in D816V-KIT cells (Figure 2D). Concurrently, increased phosphorylation of cell cycle and BRCA1 DNA damage signaling pathways (Figure 4), and increased CDK1 activity (Figure 5B) also indicates an induction of growth arrest in response to the combination treatment. Interestingly, phosphorylation of proteins annotated in ATM signaling, including CBX1, CBX3, and KAT5, were increased with M3814 single agent treatment but not in combination with dasatinib (Figure 4, Table S2). Thus it is possible that DNA-PK inhibition by M3814 treatment alone may result in concomitant activation of alternate DNA damage repair pathways, which could be pro-survival. Inhibition of these pathways with the addition of dasatinib could contribute to the synergistic cytotoxicity observed. Along with the additive phosphorylation effects of the combined inhibitors, 86 phosphopeptides were synergistically regulated by the combination treatment (Figure 6). These phosphopeptides map to proteins regulating transcription, translation, and RNA metabolism (Figure 6). This included a synergistic decrease in phosphorylation of transcription factors MYC, MYB, and NCOR1 (Figure 6), suggesting a synergistic inhibition of the activity of these transcription factors. Phosphorylation of DNA repair and transcription regulators ATRX, WRN, and SMARCA4 was co-operatively increased by the combination treatment (Figure 6), suggesting synergistic regulation of DNA damage signaling.
      D816V-KIT cells displayed constitutive phosphorylation of BTK (Figure 7), which was reduced by dasatinib treatment, alone and in combination with M3814 (Figure 7). BTK is activated downstream of a range of receptors, including KIT [
      • Rushworth S.A.
      • Pillinger G.
      • Abdul-Aziz A.
      • Piddock R.
      • Shafat M.S.
      • Murray M.Y.
      • Zaitseva L.
      • Lawes M.J.
      • MacEwan D.J.
      • Bowles K.M.
      Activity of Bruton's tyrosine-kinase inhibitor ibrutinib in patients with CD117-positive acute myeloid leukaemia: a mechanistic study using patient-derived blast cells.
      ] and Fc receptors [
      • Hata D.
      • Kawakami Y.
      • Inagaki N.
      • Lantz C.S.
      • Kitamura T.
      • Khan W.N.
      • Maeda-Yamamoto M.
      • Miura T.
      • Han W.
      • Hartman S.E.
      • et al.
      Involvement of Bruton's tyrosine kinase in FcepsilonRI-dependent mast cell degranulation and cytokine production.
      ,
      • Jongstra-Bilen J.
      • Puig Cano A.
      • Hasija M.
      • Xiao H.
      • Smith C.I.
      • Cybulsky M.I.
      Dual functions of Bruton's tyrosine kinase and Tec kinase during Fcgamma receptor-induced signaling and phagocytosis.
      ]; and leads to signaling through PI3K, PLCy, PKC, and MAPK pathways (reviewed in [
      • Pal Singh S.
      • Dammeijer F.
      • Hendriks R.W.
      Role of Bruton's tyrosine kinase in B cells and malignancies.
      ]). This led us to assess whether DNA-PK inhibitors would synergize with the BTK inhibitor ibrutinib, in cells with activated KIT signaling. Ibrutinib is a covalent inhibitor of BTK, but also functions as an inhibitor of FLT3 [
      • Wu H.
      • Hu C.
      • Wang A.
      • Weisberg E.L.
      • Wang W.
      • Chen C.
      • Zhao Z.
      • Yu K.
      • Liu J.
      • Wu J.
      • et al.
      Ibrutinib selectively targets FLT3-ITD in mutant FLT3-positive AML.
      ,
      • Klaeger S.
      • Heinzlmeir S.
      • Wilhelm M.
      • Polzer H.
      • Vick B.
      • Koenig P.A.
      • Reinecke M.
      • Ruprecht B.
      • Petzoldt S.
      • Meng C.
      • et al.
      The target landscape of clinical kinase drugs.
      ], BLK, BMX, JAK3, and PDGFRα, among others [
      • Klaeger S.
      • Heinzlmeir S.
      • Wilhelm M.
      • Polzer H.
      • Vick B.
      • Koenig P.A.
      • Reinecke M.
      • Ruprecht B.
      • Petzoldt S.
      • Meng C.
      • et al.
      The target landscape of clinical kinase drugs.
      ,
      • Honigberg L.A.
      • Smith A.M.
      • Sirisawad M.
      • Verner E.
      • Loury D.
      • Chang B.
      • Li S.
      • Pan Z.
      • Thamm D.H.
      • Miller R.A.
      • et al.
      The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy.
      ]. Similar to the results observed with dasatinib, the V560G KIT cell line was more sensitive to single-agent ibrutinib compared to D816V-KIT cells (Figure S2). This may be a result of V560G-KIT cells preferentially signaling through JAK regulated pathways, while D816V-KIT cells preferentially signal through mTOR [
      • Chan I.J.
      • Kasprowicz S.
      • Tharp M.D.
      Distinct signalling pathways for mutated KIT(V560G) and KIT(D816V) in mastocytosis.
      ]. Combining DNA-PK inhibitors with ibrutinib led to synergy in all cell lines, irrespective of KIT signaling dependence (Figure 9). This may be due to the role of BTK in GM-CSF signaling [
      • Fiedler K.
      • Sindrilaru A.
      • Terszowski G.
      • Kokai E.
      • Feyerabend T.B.
      • Bullinger L.
      • Rodewald H.R.
      • Brunner C.
      Neutrophil development and function critically depend on Bruton tyrosine kinase in a mouse model of X-linked agammaglobulinemia.
      ], in addition to its role in KIT signaling. However, both DNA-PK inhibitor combinations with ibrutinib were more potent in mutant KIT cell lines compared to the EV control (Figure 9).
      Lastly, we showed that inhibition of KIT signaling by phosphatase activation also synergized with DNA-PK inhibition. The phosphatase PP2A negatively regulates c-KIT [
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      ] activated signaling pathways; but also regulates a number of cellular pathways involved in cell growth signaling, cell cycle, metabolism, and DNA repair and replication [
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      ,
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      ]. PP2A activators FTY720 and AAL(S), used in combination with DNA-PK inhibitors M3814 and NU7441, was synergistic in all cell lines. The PP2A activator and DNA-PK inhibitor combinations however were more potent in cell lines expressing mutant KIT (D816V, V560G; Figure 9).
      Together, our results demonstrate that cells driven by KIT signaling are selectively sensitive to a range of KIT signaling and DNA-PK inhibitor combinations, suggesting this is a class effect. Inhibitors of KIT signaling such as ibrutinib and dasatinib are currently in clinical trial for treatment of AML and are in clinical use for other cancers [
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      ,
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      ]. DNA-PK inhibitors have also recently entered clinical trials for AML, and solid cancers (NCT03983824, NCT03907969), with acceptable toxicity profiles [
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      ] suggesting favorable probability of application in AML therapy. Activating mutations of KIT occur in a range of neoplasms in addition to AML, including melanoma [
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      ]. While further preclinical evaluation is required, the results herein support that treatment combining DNA-PK inhibitors and inhibitors of KIT signaling is a promising strategy for therapy of cancers with activated KIT signaling.

      Funding and additional information

      This work was supported by grants from the National Health and Medical Research Council (NHMRC) (APP1188400), Hunter Medical Research Institute and the Hunter Cancer Research Alliance. H.C.M. was supported by an Australian Government Research Training Program Scholarship and a Cancer Institute NSW Fellowship (ECF1299). M.D.D. was supported by a Cancer Institute NSW Fellowships (13/ECF/1-34, MD00487) and NHMRC Investigator Grant (APP1173892). N.M.V. was supported by an ARC Future Fellowship (FT170100077).

      Data Availability

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [
      • Perez-Riverol Y.
      • Csordas A.
      • Bai J.
      • Bernal-Llinares M.
      • Hewapathirana S.
      • Kundu D.J.
      • Inuganti A.
      • Griss J.
      • Mayer G.
      • Eisenacher M.
      • et al.
      The PRIDE database and related tools and resources in 2019: improving support for quantification data.
      ] with the dataset identifier PXD030005 and DOI 10.6019/ PXD030005 for global phosphoproteomics data or via Panorama Public [
      • Sharma V.
      • Eckels J.
      • Schilling B.
      • Ludwig C.
      • Jaffe J.D.
      • MacCoss M.J.
      • MacLean B.
      Panorama Public: A Public Repository for Quantitative Data Sets Processed in Skyline*.
      ] (https://panoramaweb.org/R7VKXL.url) for targeted PRM data (PXD030214).
      [Reviewer login details: Username: [email protected] Password: c1eQQ2eN Email: [email protected] Password: WYuqgWuY]

      Authorship

      H.C.M., N.M.V. conceptualization; H.C.M. data curation; H.C.M., M.D.D., N.M.V. formal analysis; M.D.D., N.M.V. funding acquisition; H.C.M., K.M., J.S.B., R.G.S.K., N.D.S., S.J.H., M.D.D., N.M.V. investigation; H.C.M., K.M., J.S.B., R.G.S.K., N.D.S., S.J.H., M.D.D., N.M.V. methodology; N.M.V. project administration; M.D.D., N.M.V., resources; M.D.D., N.M.V. supervision; H.C.M., M.D.D., N.M.V. visualization; H.C.M., M.D.D., N.M.V. writing—original draft; H.C.M., R.G.S.K., N.D.S., S.J.H., M.D.D., N.M.V. writing—review and editing.

      Conflict of Interest

      The authors declare no conflict of interest

      Supplementary data

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