Epithelial malignancies are effectively treated by antiangiogenics; however, acquired resistance is a major problem in cancer therapeutics. Epithelial tumors commonly have mutations in the MAPK/Pi3K-AKT pathways, which leads to high-rate aerobic glycolysis. Here, we show how multikinase inhibitor antiangiogenics (TKIs) induce hypoxia correction in spontaneous breast and lung tumor models. When this happens, the tumors downregulate glycolysis and switch to long-term reliance on mitochondrial respiration. A transcriptomic, metabolomic, and phosphoproteomic study revealed that this metabolic switch is mediated by downregulation of HIF1α and AKT and upregulation of AMPK, allowing uptake and degradation of fatty acids and ketone bodies. The switch renders mitochondrial respiration necessary for tumor survival. Agents like phenformin or ME344 induce synergistic tumor control when combined with TKIs, leading to metabolic synthetic lethality. Our study uncovers mechanistic insights in the process of tumor resistance to TKIs and may have clinical applicability.
Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, we use high-throughput phosphoproteomics to build a functional TNBC taxonomy. A cluster of 159 phosphosites is upregulated in relapsed cases of a training set (n = 34 patients), with 11 hyperactive kinases accounting for this phosphoprofile. A mass-spectrometry-to-immunohistochemistry translation step, assessing 2 independent validation sets, reveals 6 kinases with preserved independent prognostic value. The kinases split the validation set into two patterns: one without hyperactive kinases being associated with a >90% relapse-free rate, and the other one showing ≥1 hyperactive kinase and being associated with an up to 9.5-fold higher relapse risk. Each kinase pattern encompasses different mutational patterns, simplifying mutation-based taxonomy. Drug regimens designed based on these 6 kinases show promising antitumour activity in TNBC cell lines and patient-derived xenografts. In summary, the present study elucidates phosphosites and kinases implicated in TNBC and suggests a target-based clinical classification system for TNBC.
FMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.
Background: We hypothesized that the biphasic relapse pattern of TNBC could be explained by a limited number of activation patterns of signaling nodes. In addition, we sought to determine whether the hyperactive signaling nodes, distinguishing the cases with favorable vs adverse outcome, could be potential targets. Methods: Training set of 34 frozen tumor samples divided in two sets, (A) 13 patients, relapsed in <4 years; (B) 21 patients relapse-free >12 years, (mimicking the percentage and relapse patterns of unselected TNBC, but paired for T, N, G and Ki67). TNBC cell lines: 7 indolent (no metastases in 60 weeks) and 3 aggressive, develop metastases and kill recipient mice in <4 months. Shotgun phosphoproteomics and TiO2-IMAC phosphopeptide enrichment coupled with mass spectrometry runs in a Orbitrap Elite Mass Spectrometer was performed. Spectra were processed with MaxQuant software. Differentially expressed phosphopeptides were obtained by applying linear models R limma package. Differential kinase activation driving the profiles segregating cured vs. relapsing cases was done using linear sequence motif analysis. The hyperactivated kinases were validated in an independent set of 113 consecutive TNBC cases with 12+ years of follow-up spotted in TMAs by using an in-house algorithm for immunohistochemistry coupled with computer-aided quantitation using an Ariol scanning, we took the kinases in the upper quartile (high activity). Survival analysis was performed with KM curves and log rank test; and Cox proportionate hazards model was used for multivariate models. Results: 11405 phosphopeptides were identified and quantified in the training set. Supervised clustering of relapsed vs. cured cases showed that 161 and 541 peptides were significantly up-regulated in the A and B groups, respectively (FDR<0.15). After kinase-to-kinase co-linearity was ruled-out , gathering the high activity (upper quartile) of six kinases (a combined variable, herein Var1) showed statistically significant association with relapse, being these: PRKCE, pERK, c-KIT, CDK6, pP70S6K and pPNKP. Cox proportional hazards model of any of the six probes high (var1) vs rest: 9.9 vs. not reached years (P<0.001). Patients that had any of the 6 kinases high have 47% of chance to relapse (only 2 out of 42 relapsed patients have 0/6 active kinases) vs patients with Var1 negative, 7% of chance (29 patients out of 72 have 0/6 active kinases) we also observed constants patterns of activations in the different sets expressions of kinases. We considered the kinases at Var1 as a potentially targets and we developed a pharmacological in vitro assay, testing pairs inhibitors on 10 TNBC cell lines; 99.3% of the combinations were supra-additive. Conclusion: High throughput p-proteomics allows a parsimonious segregation of early TNBC cases, easily detecting the cases with long-term cure vs the remaining while identifying potential therapeutic solutions for the patients falling in the adverse prognostic subgroups. Citation Format: Sara Fernandez Gaitero, Ivana Zagorac, Jose Francisco Lopez-Acosta, Gonzalo Gomez-Lopez, David González Pisano, Javier Muñoz Peralta, Luis Manso, Soledad Alonso, Renske Penning, Maarten Altelaar, Albert JR Heck, Miguel Quintela-Fandino. Triple-negative breast cancer (TNBC) phosphoproteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1221. doi:10.1158/1538-7445.AM2017-1221
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