U of Newcastle Verrills Lab - DNAPK and KIT 2021

Synergistic targeting of DNA-PK and KIT signaling pathways in KIT mutant acute myeloid leukemia
Data License: CC BY 4.0 | ProteomeXchange: PXD030214 | doi: https://doi.org/10.6069/q8d2-gm38
  • Organism: Mus musculus
  • Instrument: Orbitrap Exploris 480
  • SpikeIn: No
  • Keywords: Phosphoproteomics, AML, KIT
  • Lab head: Nikki Verrills Submitter: Heather Murray
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 myeloid progenitor cell lines transduced with an empty vector or oncogenic mutant KIT (V560G, D816V). Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK at threonine 2599 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 via using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Discovery phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib single-agent treatment inhibited ERK1 activity, and M3814 single-agent treatment inhibited Akt/mTOR activity. The combination of dasatinib and M3814 treatment inhibited both ERK/MAPK and Akt/mTOR activity, and induced synergistic inhibition of phosphorylation of transcription regulators including 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 novel therapeutic target for KIT mutant cancers.
Experiment Description
FDC-P1 cells were transduced with either an empty vector, or oncogenic mutant KIT (D816V, V560G). Parallel reaction monitoring (PRM) for phospho-DNA-PK was performed following phosphopeptide enrichment using EasyPhos (Humphrey et al, 2018). Samples were separated over 60 minutes on an Exploris 480 with FAIMS (CV -60). Full MS scans were acquired using a resolution of 60,000, with an automatic gain control of 1e6 and maximum injection time 50ms. MS/MS scans were acquired using a resolution of 15,000, with an automatic gain control of 1e6 and a maximum injection time of 120ms. We undertook discovery phosphoproteomic analysis of D816V-KIT FDC-P1 cells, untreated or treated for 1 hour with 4uM M3814, 15nM dasatinib, or their combination, in biological triplicate. Phosphopeptide enrichment was performed using EasyPhos. Samples were separated over 90 minutes on an Exploris 480 with FAIMS (CV -60). Full MS scans 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 normalised collision energy of 36, and maximum injection time of 100ms. Skyline 20.2 (MacCoss Lab) was used to analyse PRM data files. Proteome Discoverer version 2.5 was used to search DDA data files with the Sequest HT search engine. Files were searched against the Uniprot Mus Musculus database (Sequest, downloaded 27.09.2021).
Sample Description
FDC-P1 myeloid progenitor cell lines transduced with an empty vector or oncogenic mutant KIT (V560G, D816V) were used. Cells were untreated or treated for 1 h with 4µM M3814, 15nM dasatinib, or their combination, as indicated
Created on 12/12/22, 10:26 PM
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REP2_NU_310822_Phos_inclusion_Eclipse_quan_121222.sky.zip2022-12-12 22:26:141111112
REP1_NU_FDCP1_310822_Phos_inclusion_261022_rep1_quan_120222.sky.zip2022-12-12 22:26:141111512
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D816V PhosphoDNAPK quan 240621_2021-12-05_18-30-37.sky.zip2022-12-12 22:26:141111112
FDCP1 NM DNAPK quan 240621_2021-12-05_18-17-59.sky.zip2022-12-12 22:26:14333239
D816V NM DNAPK quan 240621_2021-12-05_18-15-00.sky.zip2022-12-12 22:26:143332512