Proteomic Characterization of Acute Myeloid Leukemia for Precision Medicine.

Acute myeloid leukemia (AML) is a highly heterogeneous cancer of the hematopoietic system with no cure for most patients. In addition to chemotherapy, treatment options for AML include recently approved therapies that target proteins with roles in AML pathobiology, such as FLT3, BLC2, and IDH1/2. However, due to disease complexity, these therapies produce very diverse responses, and survival rates are still low. Thus, despite considerable advances, there remains a need for therapies that target different aspects of leukemic biology and for associated biomarkers that define patient populations likely to respond to each available therapy. To meet this need, drugs that target different AML vulnerabilities are currently in advanced stages of clinical development. Here, we review proteomics and phosphoproteomics studies that aimed to provide insights into AML biology and clinical disease heterogeneity not attainable with genomic approaches. To place the discussion in context, we first provide an overview of genetic and clinical aspects of the disease, followed by a summary of proteins targeted by compounds that have been approved or are under clinical trials for AML treatment and, if available, the biomarkers that predict responses. We then discuss proteomics and phosphoproteomics studies that provided insights into AML pathogenesis, from which potential biomarkers and drug targets were identified, and studies that aimed to rationalize the use of synergistic drug combinations. When considered as a whole, the evidence summarized here suggests that proteomics and phosphoproteomics approaches can play a crucial role in the development and implementation of precision medicine for AML patients

Reconstructing kinase network topologies from phosphoproteomics data reveals cancer-associated rewiring

Understanding how oncogenic mutations rewire regulatory-protein networks is important for rationalizing the mechanisms of oncogenesis and for individualizing anticancer treatments. We report a chemical phosphoproteomics method to elucidate the topology of kinase-signaling networks in mammalian cells. We identified >6,000 protein phosphorylation sites that can be used to infer >1,500 kinase–kinase interactions and devised algorithms that can reconstruct kinase network topologies from these phosphoproteomics data. Application of our methods to primary acute myeloid leukemia and breast cancer tumors quantified the relationship between kinase expression and activity, and enabled the identification of hitherto unknown kinase network topologies associated with drug-resistant phenotypes or specific genetic mutations. Using orthogonal methods we validated that PIK3CA wild-type cells adopt MAPK-dependent circuitries in breast cancer cells and that the kinase TTK is important in acute myeloid leukemia. Our phosphoproteomic signatures of network circuitry can identify kinase topologies associated with both phenotypes and genotypes of cancer cells

Phosphoproteomics data classify hematological cancer cell lines according to tumor type and sensitivity to kinase inhibitors

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

eEF2K Activity Determines Synergy to Cotreatment of Cancer Cells With PI3K and MEK Inhibitors

PI3K-mammalian target of rapamycin and MAPK/ERK kinase (MEK)/mitogen-activated protein kinase (MAPK) are the most frequently dysregulated signaling pathways in cancer. A problem that limits the success of therapies that target individual PI3K-MAPK members is that these pathways converge to regulate downstream functions and often compensate each other, leading to drug resistance and transient responses to therapy. In order to overcome resistance, therapies based on cotreatments with PI3K/AKT and MEK/MAPK inhibitors are now being investigated in clinical trials, but the mechanisms of sensitivity to cotreatment are not fully understood. Using LC-MS/MS-based phosphoproteomics, we found that eukaryotic elongation factor 2 kinase (eEF2K), a key convergence point downstream of MAPK and PI3K pathways, mediates synergism to cotreatment with trametinib plus pictilisib (which target MEK1/2 and PI3Kα/δ, respectively). Inhibition of eEF2K by siRNA or with a small molecule inhibitor reversed the antiproliferative effects of the cotreatment with PI3K plus MEK inhibitors in a cell model–specific manner. Systematic analysis in 12 acute myeloid leukemia cell lines revealed that eEF2K activity was increased in cells for which PI3K plus MEKi cotreatment is synergistic, while PKC potentially mediated resistance to such cotreatment. Together, our study uncovers eEF2K activity as a key mediator of responses to PI3Ki plus MEKi and as a potential biomarker to predict synergy to cotreatment in cancer cells

Computational Analysis of Cholangiocarcinoma Phosphoproteomes Identifies Patient-Specific Drug Targets

Cholangiocarcinoma is a form of hepatobiliary cancer with an abysmal prognosis. Despite advances in our understanding of cholangiocarcinoma pathophysiology and its genomic landscape, targeted therapies have not yet made a significant impact on its clinical management. The low response rates of targeted therapies in cholangiocarcinoma suggest that patient heterogeneity contributes to poor clinical outcome. Here we used mass spectrometry–based phosphoproteomics and computational methods to identify patient-specific drug targets in patient tumors and cholangiocarcinoma-derived cell lines. We analyzed 13 primary tumors of patients with cholangiocarcinoma with matched nonmalignant tissue and 7 different cholangiocarcinoma cell lines, leading to the identification and quantification of more than 13,000 phosphorylation sites. The phosphoproteomes of cholangiocarcinoma cell lines and patient tumors were significantly correlated. MEK1, KIT, ERK1/2, and several cyclin-dependent kinases were among the protein kinases most frequently showing increased activity in cholangiocarcinoma relative to nonmalignant tissue. Application of the Drug Ranking Using Machine Learning (DRUML) algorithm selected inhibitors of histone deacetylase (HDAC; belinostat and CAY10603) and PI3K pathway members as high-ranking therapies to use in primary cholangiocarcinoma. The accuracy of the computational drug rankings based on predicted responses was confirmed in cell-line models of cholangiocarcinoma. Together, this study uncovers frequently activated biochemical pathways in cholangiocarcinoma and provides a proof of concept for the application of computational methodology to rank drugs based on efficacy in individual patients. SIGNIFICANCE: Phosphoproteomic and computational analyses identify patient-specific drug targets in cholangiocarcinoma, supporting the potential of a machine learning method to predict personalized therapies

Integrating Phosphoproteome and Transcriptome Reveals New Determinants of Macrophage Multinucleation

This research was originally published in Molecular and Cellular Proteomics. Rotival M, Ko JH, Srivastava PK, Kerloc'h A, Montoya A, Mauro C, Faull P, Cutillas PR, Petretto E, Behmoaras J. Integrating phosphoproteome and transcriptome reveals new determinants of macrophage multinucleation. Molecular and Cellular Proteomics. 2014. Vol:pp-pp. © the American Society for Biochemistry and Molecular Biology.File embargoed until 22 Dec 2015

Characterisation of preproendothelin-1 derived peptides identifies Endothelin-Like Domain Peptide as a modulator of Endothelin-1

These studies were supported by the Medical Research Council (UK) (Grant G0801509), Community Fund of the National Lottery Charities Board, William Harvey Research Foundation and British Cardiac Research Trust. The LC-MS/MS facility was supported by Barts and the London Charity (297/2249)

Characterization of four subtypes in morphologically normal tissue excised proximal and distal to breast cancer

Widespread mammographic screening programs and improved self-monitoring allow for breast cancer to be detected earlier than ever before. Breast-conserving surgery is a successful treatment for select women. However, up to 40% of women develop local recurrence after surgery despite apparently tumor-free margins. This suggests that morphologically normal breast may harbor early alterations that contribute to increased risk of cancer recurrence. We conducted a comprehensive transcriptomic and proteomic analysis to characterize 57 fresh-frozen tissues from breast cancers and matched histologically normal tissues resected proximal to (<2 cm) and distant from (5–10 cm) the primary tumor, using tissues from cosmetic reduction mammoplasties as baseline. Four distinct transcriptomic subtypes are identified within matched normal tissues: metabolic; immune; matrisome/epithelial–mesenchymal transition, and non-coding enriched. Key components of the subtypes are supported by proteomic and tissue composition analyses. We find that the metabolic subtype is associated with poor prognosis (p < 0.001, HR6.1). Examination of genes representing the metabolic signature identifies several genes able to prognosticate outcome from histologically normal tissues. A subset of these have been reported for their predictive ability in cancer but, to the best of our knowledge, these have not been reported altered in matched normal tissues. This study takes an important first step toward characterizing matched normal tissues resected at pre-defined margins from the primary tumor. Unlocking the predictive potential of unexcised tissue could prove key to driving the realization of personalized medicine for breast cancer patients, allowing for more biologically-driven analyses of tissue margins than morphology alone