Synergistic Targeting of DNA-PK and KIT Signaling Pathways in KIT Mutant Acute Myeloid Leukemia

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 (receptor-type tyrosine-protein kinase FLT3) and KIT (mast/stem cell growth factor receptor 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 (DNA-dependent protein kinase), 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 and 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 with 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 extracellular signal-regulated kinase and AKT (RAC-alpha serine/threonine-protein kinase)/MTOR (serine/threonine-protein kinase 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.

Keywords: AML; DNA-PK; c-KIT; leukaemia; phosphoproteomics; synergistic targeted therapies.

Graphical abstract

Fig. 1

Activated KIT signaling increases DNA-PKphosphorylation.A, protein expression. 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 and D816V). N = 3 per experiment. ∗p < 0.05, ∗∗p < 0.01 (ANOVA + Tukey’s test). C, phosphorylation of DNA-PK, assessed by PRM mass spectrometry, in FDC-P1/D816V-KIT cells untreated or treated for 1 h with 4 μM M3814, 2 μM NU7441, 4 μM NU7441, 15 nM dasatinib, 30 nM dasatinib, or their combinations. N ≥ 2. ∗p < 0.05, ∗∗p < 0.01 (ANOVA + Dunnett’s test). A singly phosphorylated DNA-PK peptide from the T2599/T2605/S2608/S2610 cluster was identified; however, the phosphorylation could not be localized to a specific site. DNA-PK, DNA-dependent protein kinase; cs, catalytic subunit; KIT, mast/stem cell growth factor receptor kit.

Fig. 2

The 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 and D816V), were incubated with increasing concentrations of (A) dasatinib, M3814, or their combination; and (B) dasatinib, NU7441, or their combination. Cell viability at 72 h was assessed by resazurin metabolic assay. n ≥ 2. Gray shading = not done. +, synergy, assessed by the fractional product method of Webb. Apoptosis induction at 48 h 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 (supplemental Table S4). DNA-PK, DNA-dependent protein kinase; KIT, mast/stem cell growth factor receptor kit.

Fig. 3

Quantitative phosphoproteomic analysis of mutant KIT-D816V FDC-P1 cells. FDC-P1 D816V-KIT cells were treated with M3814 (4 μM), dasatinib (15 nM), or their combination for 1 h. 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 of M3814 and dasatinib treatment; 238 phosphopeptides were significantly increased and 333 were significantly decreased. A fold-change cutoff of ±1.5 was used. KIT, mast/stem cell growth factor receptor kit.

Fig. 4

Pathway 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 (4 μM), dasatinib (15 nM), or their combination for 1 h. 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, and shading indicates average pathway phosphorylation. The full list of IPA pathways is provided in supplemental Table S3. KIT, mast/stem cell growth factor receptor kit.

Fig. 5

Kinase enrichment analysis. FDC-P1 D816V-mutant KIT cells were treated with M3814 (4 μM), dasatinib (15 nM), or their combination for 1 h. Phosphoproteomes were profiled by mass spectrometry. A, heatmaps of kinase substrates for kinases with more than five substrates significantly increased or decreased in phosphorylation compared with untreated (UT). ∗p < 0.05 compared with UT. Kinase substrates were identified using the Phosphosite Plus database (53). B, kinase enrichment analysis performed using RoKAI (54). 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. KIT, mast/stem cell growth factor receptor kit.

Fig. 6

Quantitative phosphoproteomic analysis of mutant KIT D816V FDC-P1 cells. FDC-P1 D816V-mutant KIT cells were treated with M3814 (4 μM), dasatinib (Das; 15 nM), or their combination for 1 h. Phosphoproteomes were profiled by mass spectrometry. Heatmap of phosphosites identified with (A) synergistic increase or (B) synergistic decrease by the combination of M3814 and Das are shown. Type: ingenuity pathway analysis molecule annotations. KIT, mast/stem cell growth factor receptor kit.

Fig. 7

Schematic 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 (4 μM), dasatinib (15 nM), or their combination for 1 h. 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 with untreated cells (blue = decreased, Red = increased). Underlined phosphosites indicate those with annotated function (PhosphositePlus (53)). 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. KIT, mast/stem cell growth factor receptor kit.

Fig. 8

Phosphorylation of MTOR, BTK, and ERK1/2 in response to M3814, dasatinib, or their combination. FDC-P1 D816V-mutant KIT cells were treated with M3814 (4 μM), dasatinib (15 or 30 nM), or their combination for 1, 24, 48, and 72 h. A, representative Western blot images and (B) densitometry. Bars represent mean ± SEM. N = 2 to 3. BTK, Bruton’s tyrosine kinase; ERK1/2, extracellular signal–regulated kinase 1/2; KIT, mast/stem cell growth factor receptor kit; MTOR, serine/threonine-protein kinase Mtor.

Fig. 9

KIT 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 and 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 h was assessed by resazurin metabolic assay. n ≥ 2. +, synergy, assessed by the fractional product method of Webb (supplemental Table S4). DNA-PK, DNA-dependent protein kinase; KIT, mast/stem cell growth factor receptor kit.