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Phosphorylation networks intimately regulate mechanisms of response to therapies. Mapping the phospho-catalytic profile of kinases in cells or tissues remains a challenge. Here, we introduce a practical high-throughput system to measure the enzymatic activity of kinases using biological peptide targets as phospho-sensors to reveal kinase dependencies in tumour biopsies and cell lines. A 228-peptide screen was developed to detect the activity of >60 kinases, including ABLs, AKTs, CDKs and MAPKs. Focusing on BRAF V600E tumours, we found mechanisms of intrinsic resistance to BRAF V600E -targeted therapy in colorectal cancer, including targetable parallel activation of PDPK1 and PRKCA. Furthermore, mapping the phospho-catalytic signatures of melanoma specimens identifies RPS6KB1 and PIM1 as emerging druggable vulnerabilities predictive of poor outcome in BRAF V600E patients. The results show that therapeutic resistance can be caused by the concerted upregulation of interdependent pathways. Our kinase activity-mapping system is a versatile strategy that innovates the exploration of actionable kinases for precision medicine. Coppé and colleagues design a peptide phosphorylation-screening system that simultaneously measures the enzymatic activity of multiple kinases, identifying mechanisms of therapy resistance and druggable targets in colorectal cancer and melanoma.

Background Poly(ADP-ribose) polymerase inhibitors (PARPi), coupled to a DNA damaging agent is a promising approach to treating triple negative breast cancer (TNBC). However, not all patients respond; we hypothesize that non-response in some patients may be due to insufficient drug penetration. As a first step to testing this hypothesis, we quantified and visualized veliparib and carboplatin penetration in mouse xenograft TNBCs and patient blood samples. Methods MDA-MB-231, HCC70 or MDA-MB-436 human TNBC cells were implanted in 41 beige SCID mice. Low dose (20 mg/kg) or high dose (60 mg/kg) veliparib was given three times daily for three days, with carboplatin (60 mg/kg) administered twice. In addition, blood samples were analyzed from 19 patients from a phase 1 study of carboplatin + PARPi talazoparib. Veliparib and carboplatin was quantified using liquid chromatography–mass spectrometry (LC-MS). Veliparib tissue penetration was visualized using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) and platinum adducts (covalent nuclear DNA-binding) were quantified using inductively coupled plasma–mass spectrometry (ICP-MS). Pharmacokinetic modeling and Pearson’s correlation were used to explore associations between concentrations in plasma, tumor cells and peripheral blood mononuclear cells (PBMCs). Results Veliparib penetration in xenograft tumors was highly heterogeneous between and within tumors. Only 35% (CI 95% 26–44%), 74% (40–97%) and 46% (9–37%) of veliparib observed in plasma penetrated into MDA-MB-231, HCC70 and MDA-MB-436 cell-based xenografts, respectively. Within tumors, penetration heterogeneity was larger with the 60 mg/kg compared to the 20 mg/kg dose (RSD 155% versus 255%, P = 0.001). These tumor concentrations were predicted similar to clinical dosing levels, but predicted tumor concentrations were below half maximal concentration values as threshold of response. Xenograft veliparib concentrations correlated positively with platinum adduct formation ( R 2 = 0.657), but no PARPi–platinum interaction was observed in patients’ PBMCs. Platinum adduct formation was significantly higher in five gBRCA carriers (ratio of platinum in DNA in PBMCs/plasma 0.64% (IQR 0.60–1.16%) compared to nine non-carriers (ratio 0.29% (IQR 0.21–0.66%, P <  0.0001). Conclusions PARPi/platinum tumor penetration can be measured by MALDI-MSI and ICP-MS in PBMCs and fresh frozen, OCT embedded core needle biopsies. Large variability in platinum adduct formation and spatial heterogeneity in veliparib distribution may lead to insufficient drug exposure in select cell populations.

HER2-targeted therapies have been the mainstay of treatment of HER2-positive breast cancer. To date, the selection of patients most likely to respond to HER2-targeted agents is based primarily on HER2 amplification and/or overexpression. However, the correlations among current clinical methods of detecting HER2 amplification and/or overexpression are imperfect with regards to both prognostication and the prediction of drug response to many of the HER2-targeted therapies, and therefore, there is a critical need for the discovery and translation of additional biomarkers that predict patient response to a specific HER2-targeted therapy. Here, we evaluated BluePrint molecular subtypes – a gene expression-based molecular subtype classification – as a predictor of response to HER2-targeted therapies using patient data from the I-SPY 2 TRIAL. We demonstrated the potential clinical utility of BluePrint molecular subtyping in identifying a subset of HER2-positive, estrogen receptor-positive (HER2+/HR+) patients who are less likely to benefit from HER2-targeted therapies. In addition, gene expression analysis of this subset of patients reveal lower immune signaling and higher estrogen receptor expression, and thus may potentially benefit from alternative strategies, such as endocrine therapy or immunotherapy. In a second study, we evaluated the baseline activation state of 104 key signaling phosphoproteins/ proteins from prosurvival, mitogenic, apoptotic, and growth regulatory pathways as predictors of response to neratinib – an irreversible pan-HER tyrosine kinase inhibitor of EGFR/HER2 – in HER2-positive breast cancer cell line models with differential neratinib sensitivity. We identified 13 phosphoproteins/ proteins, representing a multitude of pathways, in particular the HER family signaling pathway, that are associated with neratinib sensitivity. We also demonstrated in HER2-positive breast cancer cell line models that acquired resistance to neratinib could potentially be mediated through adaptive kinome reprogramming, and that the combination of neratinib and BET bromodomain inhibitor appears to be a promising therapeutic strategy to overcome such resistance. In conclusion, the work presented here provide insight into mechanisms underlying differential drug responses and resistance to HER2-targeted therapies, and highlight novel genomic and proteomic biomarker candidates that could potentially complement HER2 overexpression and/or amplification in predicting patient response to HER2-targeted therapies.