Project description: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.
Project description:Acute Myeloid Leukaemia (AML) carries a 5 year survival rate of just 24%. Toxic chemotherapy regimens remain the backbone of standard of care for AML. The KIT tyrosine kinase is a recognised AML oncogene, associated with poor outcome. We recently identified DNA-PK as a novel therapeutic target in FLT3 mutant AML. The similarity between KIT and FLT3 regulated signalling pathways led us to investigate DNA-PK in KIT-mutant AML.
Project description:A flexible fiberoptic bronchoscope was inserted trans-orally and advanced through the vocal cords of subjects. Brochoalveolar lavage (BAL) fluid was obtained from tertiary airways in the right upper and lower lobes. The BAL procedure was performed sequentially in the right upper lobe (RUL) and right lower lobe (RLL) with 20 ml of sterile saline followed by 10 ml of air and this was repeated for a total of 5 times per airway. BAL fluid was then filtered through 2-layer gauze, centrifuged, and washed twice in 0.9% NaCl. Cells were counted with a T10 automated cell counter. Cytospins were performed using a Shandon Cytospin 3 centrifuge. 75,000 cells resuspended in 200 l of 0.9% NaCl were loaded into a cytology funnel and centrifuged for 10 mins. Cells were allowed to air dry and processed for viewing via Hema 3 Stat pack. One pair of samples (GEO accessions GSM3610354 [H_1_RLL] and GSM3610362 [H_1_RUL]) from our previous publication, collected and measured in the same manner, was also included in our present analysis. DNA was extracted from lung macrophages via Qiagen DNeasy Blood and Tissue Kit. DNA was quantitated on a Qubit 3.0 Fluorometer. Bisulfite conversion of DNA was performed using the Zymo EZ DNA methylation kit. The Illumina MethylationEPIC array hybridization and scanning were performed at the University of Southern California Molecular Genomics Core.