Chemoproteomics Reveals Novel Protein and Lipid Kinase Targets of Clinical CDK4/6 Inhibitors in Lung Cancer

Several selective CDK4/6 inhibitors are in clinical trials for non-small cell lung cancer (NSCLC). Palbociclib (PD0332991) is included in the phase II/III Lung-MAP trial for squamous cell lung carcinoma (LUSQ). We noted differential cellular activity between palbociclib and the structurally related ribociclib (LEE011) in LUSQ cells. Applying an unbiased mass spectrometry-based chemoproteomics approach in H157 cells and primary tumor samples, we here report distinct proteome-wide target profiles of these two drug candidates in LUSQ, which encompass novel protein and, for palbociclib only, lipid kinases. In addition to CDK4 and 6, we observed CDK9 as a potent target of both drugs. Palbociclib interacted with several kinases not targeted by ribociclib, such as casein kinase 2 and PIK3R4, which regulate autophagy. Furthermore, palbociclib engaged several lipid kinases, most notably, PIK3CD and PIP4K2A/B/C. Accordingly, we observed modulation of autophagy and inhibition of AKT signaling by palbociclib but not ribociclib

Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase targets

The BCR-ABL tyrosine kinase inhibitor imatinib represents the current frontline therapy in chronic myeloid leukemia. Because many patients develop imatinib resistance, 2 second-generation drugs, nilotinib and dasatinib, displaying increased potency against BCR-ABL were developed. To predict potential side effects and novel medical uses, we generated comprehensive drug-protein interaction profiles by chemical proteomics for all 3 drugs. Our studies yielded 4 major findings: (1) The interaction profiles of the 3 drugs displayed strong differences and only a small overlap covering the ABL kinases. (2) Dasatinib bound in excess of 30 Tyr and Ser/Thr kinases, including major regulators of the immune system, suggesting that dasatinib might have a particular impact on immune function. (3) Despite the high specificity of nilotinib, the receptor tyrosine kinase DDR1 was identified and validated as an additional major target. (4) The oxidoreductase NQO2 was bound and inhibited by imatinib and nilotinib at physiologically relevant drug concentrations, representing the first nonkinase target of these drugs

Dabrafenib inhibits the growth of BRAF‐WT cancers through CDK16 and NEK9 inhibition

Although the BRAF inhibitors dabrafenib and vemurafenib have both proven successful against BRAF‐mutant melanoma, there seem to be differences in their mechanisms of action. Here, we show that dabrafenib is more effective at inhibiting the growth of NRAS‐mutant and KRAS‐mutant cancer cell lines than vemurafenib. Using mass spectrometry‐based chemical proteomics, we identified NEK9 and CDK16 as unique targets of dabrafenib. Both NEK9 and CDK16 were highly expressed in specimens of advanced melanoma, with high expression of both proteins correlating with a worse overall survival. A role for NEK9 in the growth of NRAS‐ and KRAS‐mutant cell lines was suggested by siRNA studies in which silencing was associated with decreased proliferation, cell cycle arrest associated with increased p21 expression, inhibition of phospho‐CHK1, decreased CDK4 expression, and the initiation of a senescence response. Inhibition of CDK4 but not CHK1 recapitulated the effects of NEK9 silencing, indicating this to be the likely mechanism of growth inhibition. We next turned our attention to CDK16 and found that its knockdown inhibited the phosphorylation of the Rb protein at S780 and increased expression of p27. Both of these effects were phenocopied in NRAS‐ and KRAS‐mutant cancer cells by dabrafenib, but not vemurafenib. Combined silencing of NEK9 and CDK16 was associated with enhanced inhibition of melanoma cell proliferation. In summary, we have identified dabrafenib as a potent inhibitor of NEK9 and CDK16, and our studies suggest that inhibition of these kinases may have activity against cancers that do not harbor BRAF mutations

Structural basis for the cytoskeletal association of Bcr-Abl/c-Abl

The Bcr-Abl tyrosine kinase causes different forms of leukemia in humans. Depending on its position within the cell, Bcr-Abl differentially affects cellular growth. However, no structural and molecular details for the anticipated localization determinants are available. We present the NMR structure of the F-actin binding domain (FABD) of Bcr-Abl and its cellular counterpart c-Abl. The FABD forms a compact left-handed four-helix bundle in solution. We show that the nuclear export signal (NES) previously reported in this region is part of the hydrophobic core and nonfunctional in the intact protein. In contrast, we could identify the critical residues of helix alphaIII that are responsible for F-actin binding and cytoskeletal association. We propose that these interactions represent a major determinant for both Bcr-Abl and c-Abl localization

Proteome-wide Profiling of Clinical PARP Inhibitors Reveals Compound-Specific Secondary Targets

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a promising class of targeted cancer drugs, but their individual target profiles beyond the PARP family, which could result in differential clinical use or toxicity, are unknown. Using an unbiased, mass spectrometry-based chemical proteomics approach, we generated a comparative proteome-wide target map of the four clinical PARPi, olaparib, veliparib, niraparib, and rucaparib. PARPi as a class displayed high target selectivity. However, in addition to the canonical targets PARP1, PARP2, and several of their binding partners, we also identified hexose-6-phosphate dehydrogenase (H6PD) and deoxycytidine kinase (DCK) as previously unrecognized targets of rucaparib and niraparib, respectively. Subsequent functional validation suggested that inhibition of DCK by niraparib could have detrimental effects when combined with nucleoside analog pro-drugs. H6PD silencing can cause apoptosis and further sensitize cells to PARPi, suggesting that H6PD may be, in addition to its established role in metabolic disorders, a new anticancer target

Dual Targeting of WEE1 and PLK1 by AZD1775 Elicits Single Agent Cellular Anticancer Activity

Inhibition of the WEE1 tyrosine kinase enhances anticancer chemotherapy efficacy. Accordingly, the WEE1 inhibitor AZD1775 (previously MK-1775) is currently under evaluation in clinical trials for cancer in combination with chemotherapy. AZD1775 has been reported to display high selectivity and is therefore used in many studies as a probe to interrogate WEE1 biology. However, AZD1775 also exhibits anticancer activity as a single agent although the underlying mechanism is not fully understood. Using a chemical proteomics approach, we here describe a proteome-wide survey of AZD1775 targets in lung cancer cells and identify several previously unknown targets in addition to WEE1. In particular, we observed polo-like kinase 1 (PLK1) as a new target of AZD1775. Importantly, <i>in vitro</i> kinase assays showed PLK1 and WEE1 to be inhibited by AZD1775 with similar potency. Subsequent loss-of-function experiments using RNAi for <i>WEE1</i> and <i>PLK1</i> suggested that targeting PLK1 enhances the pro-apoptotic and antiproliferative effects observed with <i>WEE1</i> knockdown. Combination of RNAi with AZD1775 treatment suggested WEE1 and PLK1 to be the most relevant targets for mediating AZD1775’s anticancer effects. Furthermore, disruption of <i>WEE1</i> by CRISPR-Cas9 sensitized H322 lung cancer cells to AZD1775 to a similar extent as the potent PLK1 inhibitor BI-2536 suggesting a complex crosstalk between PLK1 and WEE1. In summary, we show that AZD1775 is a potent dual WEE1 and PLK1 inhibitor, which limits its use as a specific molecular probe for WEE1. However, PLK1 inhibition makes important contributions to the single agent mechanism of action of AZD1775 and enhances its anticancer effects

GSK3 Alpha and Beta Are New Functionally Relevant Targets of Tivantinib in Lung Cancer Cells

Tivantinib has been described as a potent and highly selective inhibitor of the receptor tyrosine kinase c-MET and is currently in advanced clinical development for several cancers including non-small cell lung cancer (NSCLC). However, recent studies suggest that tivantinib’s anticancer properties are unrelated to c-MET inhibition. Consistently, in determining tivantinib’s activity profile in a broad panel of NSCLC cell lines, we found that, in contrast to several more potent c-MET inhibitors, tivantinib reduces cell viability across most of these cell lines. Applying an unbiased, mass-spectrometry-based, chemical proteomics approach, we identified glycogen synthase kinase 3 (GSK3) alpha and beta as novel tivantinib targets. Subsequent validation showed that tivantinib displayed higher potency for GSK3α than for GSK3β and that pharmacological inhibition or simultaneous siRNA-mediated loss of GSK3α and GSK3β caused apoptosis. In summary, GSK3α and GSK3β are new kinase targets of tivantinib that play an important role in its cellular mechanism-of-action in NSCLC