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

Murray, Heather C; Miller, Kasey; Brzozowski, Joshua S.; Kahl, Richard; Smith, Nathan D.; Humphrey, Sean J.; Dun, Matthew D.; Verrills, Nicole M.

doi:10.1016/j.mcpro.2023.100503

Cited by 8 publications

(14 citation statements)

References 101 publications

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“…The PhosphositePlus human and mouse databases [22] were used to map the upstream kinases for phosphosites in the top quartile of abundance. Kinases were classified as enriched if ≥ 5 substrates were identified [13]. In accordance with the pathway analysis, PKA was enriched, along with mTOR signalling kinases mTOR, P70S6K, P90RSK, and RSK2; with P90RSK displaying the second highest average substrate abundance (Figure 2B).…”

Section: Figurementioning

confidence: 69%

“…Raw files were searched against the UniProt Mus musculus database (25,342 sequences, September 27, 2021), using SEQUEST HT and Proteome Discoverer 2.5 software [12] (Thermo Fisher Scientific) as previously described [13]. Search parameters allowed up to two missed cleavages, with a precursor mass tolerance of 10 ppm and fragment mass tolerance set to 0.02 Da.…”

Section: Mass Spectrometry Analysis Was Performed Using An Orbitrapmentioning

confidence: 99%

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Proteomic and phosphoproteomic characterisation of primary mouse embryonic fibroblasts

Chen,

Roselli,

Panicker

et al. 2023

Proteomics

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Fibroblasts are the most common cell type in stroma and function in the support and repair of most tissues. Mouse embryonic fibroblasts (MEFs) are amenable to isolation and rapid growth in culture. MEFs are therefore widely used as a standard model for functional characterisation of gene knockouts, and can also be used in co‐cultures, commonly to support embryonic stem cell cultures. To facilitate their use as a research tool, we have performed a comprehensive proteomic and phosphoproteomic characterisation of wild‐type primary MEFs from C57BL/6 mice. EIF2/4 and MTOR signalling pathways were abundant in both the proteome and phosphoproteome, along with extracellular matrix (ECM) and cytoskeleton associated pathways. Consistent with this, kinase enrichment analysis identified activation of P38A, P90RSK, P70S6K, and MTOR. Cell surface markers and matrisome proteins were also annotated. Data are available via ProteomeXchange with identifier PXD043244. This provides a comprehensive catalogue of the wild‐type MEF proteome and phosphoproteome which can be utilised by the field to guide future work.

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Section: Figurementioning

confidence: 69%

Section: Mass Spectrometry Analysis Was Performed Using An Orbitrapmentioning

confidence: 99%

Proteomic and phosphoproteomic characterisation of primary mouse embryonic fibroblasts

Chen,

Roselli,

Panicker

et al. 2023

Proteomics

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“…SU-DIPG-XXXVI neurospheres were treated with paxalisib and subjected to proteomic and phosphoproteomics analysis as previously described (41)(42)(43). SU-DIPG-XXXVI neurospheres were treated with paxalisib at IC50 (Supplementary Table 2) for 6 h. Tryptic peptides from each sample were prepared as described (42,44,45) individually labeled using tandem mass tags (TMT) and phosphopeptides were isolated from the proteome using titanium dioxide (44) and immobilized metal affinity chromatography before offline hydrophilic interaction liquid chromatography (HILIC) (26,42,43). Liquid chromatography (LC) tandem mass spectrometry (MS/MS) was performed using a Q-Exactive Plus hybrid quadrupole-Orbitrap MS system, coupled to a Dionex Ultimate 3000RSLC nanoflow HPLC system as described (42,43).…”

Section: High-resolution Quantitative Phosphoproteomic Profilingmentioning

confidence: 99%

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma

Duchatel

Jackson

Parackal

et al. 2023

Preprint

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Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma DIPG), are uniformly fatal brain tumors that lack effective pharmacological treatment. Analysis of pooled CRISPR-Cas9 loss-of-function gene deletion screen datasets, identified PIK3CA and MTOR as targetable molecular dependencies across DIPG patient derived models, highlighting the therapeutic potential of the blood-brain barrier penetrant PI3K/Akt/mTOR inhibitor paxalisib. At the human equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic feedback resulting in increased blood glucose and insulin levels, commensurate with DIPG patients in Phase 1b clinical trials who experienced hyperglycemia/hyperinsulinemia. To exploit genetic dependences, but maintain compliance and benefit, we optimized a paxalisib treatment regimen that employed reduced dosing more frequently, in combination with the anti-hyperglycemic drug, metformin. Combining optimized dosing with metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending the survival of DIPG xenograft models. RNA sequencing and phosphoproteomic profiling of DIPG models treated with paxalisib identified increased calcium-activated PKC signaling. Using the brain penetrant PKC inhibitor, enzastaurin in combination with paxalisib, we synergistically extended the survival of orthotopic xenograft models, benefits further promoted by metformin; thus, identifying a clinically relevant DIPG combinatorial approach.

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“…This yielded >600 µg protein per sample, of which 200µg were digested for each sample. Samples were digested and phospho-enriched using the EasyPhos method [21], as previously described [20]. Label-free proteomes and phosphoproteomes were analysed using an Exploris 480 mass spectrometer.…”

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confidence: 99%

“…Clusters cells (Figure 1B). We have previously identified altered activation of DNA double strand break repair pathways in KIT-mutant AML [20].…”

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confidence: 99%

Pharmaco‐phosphoproteomic analysis of cancer‐associated KIT mutations D816V and V560G

Murray,

Miller,

Dun

et al. 2024

Proteomics

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The CD117 mast/stem cell growth factor receptor tyrosine kinase (KIT) is critical for haematopoiesis, melanogenesis and stem cell maintenance. KIT is commonly activated by mutation in cancers including acute myeloid leukaemia, melanoma and gastrointestinal stromal tumours (GISTs). The kinase and the juxtamembrane domains of KIT are mutation hotspots; with the kinase domain mutation D816V common in leukaemia and the juxtamembrane domain mutation V560G common in GISTs. Given the importance of mutant KIT signalling in cancer, we have conducted a proteomic and phosphoproteomic analysis of myeloid progenitor cells expressing D816V‐ and V560G‐KIT mutants, using an FDCP1 isogenic cell line model. Proteomic analysis revealed increased abundance of proteases and growth signalling proteins in KIT‐mutant cells compared to empty vector (EV) controls. Pathway analysis identified increased oxidative phosphorylation in D816V‐ and V560G‐mutant KIT cells, which was targetable using the inhibitor IACS010759. Dysregulation of RNA metabolism and cytoskeleton/adhesion pathways was identified in both the proteome and phosphoproteome of KIT‐mutant cells. Phosphoproteome analysis further revealed active kinases such as EGFR, ERK and PKC, which were targetable using pharmacological inhibitors. This study provides a pharmaco‐phosphoproteomic profile of D816V‐ and V560G‐mutant KIT cells, which reveals novel therapeutic strategies that may be applicable to a range of cancers.

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

Cited by 8 publications

References 101 publications

Proteomic and phosphoproteomic characterisation of primary mouse embryonic fibroblasts

Proteomic and phosphoproteomic characterisation of primary mouse embryonic fibroblasts

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma

Pharmaco‐phosphoproteomic analysis of cancer‐associated KIT mutations D816V and V560G

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