Introduction
Intensive multi-agent treatment has boosted survival up to 80% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Nevertheless, relapsed patients have a poor prognosis due to acquired therapy resistance while most survivors have detrimental chemotherapy-induced side effects. Therefore, novel targeted therapies are urgently needed since further intensification of the current standard treatment regimen is not feasible for refractory/relapsed cases.
Protein kinase inhibitors (PKIs) are amongst the most successful cancer treatments. Targetable kinases activated by gene fusions are rare in T-ALL and include subclonal NUP214-ABL1 fusion in 6% of cases or other rare clonal ABL1 fusions. Nevertheless, leukemic blasts rely on enhanced kinase signaling to sustain their dysregulated proliferation. Protein kinases can be hyper-activated even in the absence of defects in their genes. Thus, together with the identification of genomic aberrations, phospho-proteomics can provide information on pathway activation, signaling networks and aberrant kinase activities that offer important opportunities for targeted therapies.
Aim
Here, we aimed to identify and quantify kinase activation in T-ALL cell lines that may yield differential sensitivity to PKIs in vitro. This approach could pinpoint targetable leukemia vulnerabilities and provide effective (combination) treatment strategies.
Methods
Protein extracts from 11 T-ALL cell lines were enriched for phospho-peptides by titanium dioxide enrichment and anti-phospho tyrosine immunoprecipitation followed by liquid chromatography - tandem mass spectrometry (MS). Subsequently, the Integrative Inferred Kinase Activity (INKA) pipeline was used to rank activated kinases in our panel (Beekhof et al., 2019). Based on these data, selected kinase inhibitors were tested in vitro as single treatment or in combinations. Eventually, drugs of interest were further tested ex vivo in a cohort of T-ALL patient-derived xenografts (PDXs).
Results
MS-based phospho-proteome profiling of 11 T-ALL cell lines identified about 3700 tyrosine phospho-sites and more than 13300 serine/threonine phosphorylation sites. We found SRC-family kinases including LCK, SRC, FYN, and YES1 as most activated kinases in many T-ALL cell lines while ABL1, ZAP70, LYN, and FGR were detected only in specific lines. Additionally, other kinases including CDK1/2 and PAK1/2 were found to be activated in all the cell lines while activation of the INSR/IGF-1R axis was detected only in a subset of lines. We then tested cellular response to multiple clinically relevant PKIs based on predicted kinase activities. The in vitro drug screening showed an effective response and G1-arrest following treatment with the CDK1/2 inhibitor milciclib in all the cell lines tested, with IC50 values between 10nM and 1uM.
Despite a general SRC-family kinases activation profile, dual SRC/ABL inhibitors like dasatinib reduced cellular viability only in cell lines with ABL1 fusions or LCK translocations (IC50 < 10nM) while other lines lacking ABL or LCK rearrangements were affected at much higher drug concentration (IC50 > 3uM, which is beyond the clinical achievable plasma concentration). Thus, PKIs were tested in combination with other relevant inhibitors based on additional kinase activities detected. Interestingly, the concomitant inhibition of the SRC-family kinases by dasatinib and the INSR/IGF-1R axis by BMS-754807 led to a drastic reduction of cell viability at nanomolar concentrations even in cell lines that did not respond to dasatinib, identifying a novel possible effective combination strategy for T-ALL.
Eventually, we tested clinically relevant PKIs in 50 PDXs ex vivo and identified various T-ALL samples with a high sensitivity to dasatinib single treatment (IC50 < 100nM) as previously reported by others (Frismantas et al., 2017). Moreover, 70% of our PDXs efficiently responded to the broad spectrum kinase inhibitor midostaurin (IC50 < 1uM), highlighting the importance of targeting multiple signaling nodes simultaneously to tackle T-ALL vulnerabilities.
Conclusions
Ranking kinase activities and signaling networks from phospho-proteomic data can guide the use of PKIs as treatment option for T-ALL patients. Moreover, kinase activity profiling can provide insights for efficient treatment combination strategies to develop personalized medicine.
Disclosures
No relevant conflicts of interest to declare.
Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics
