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Numerous aspects of cellular signaling are regulated by the kinome—the network of over 500 protein kinases that guides and modulates information transfer throughout the cell. The key role played by both individual kinases and assemblies of kinases organized into functional subnetworks leads to kinome dysregulation driving many diseases, particularly cancer. In the case of pancreatic ductal adenocarcinoma (PDAC), a variety of kinases and associated signaling pathways have been identified for their key role in the establishment of disease as well as its progression. However, the identification of additional relevant therapeutic targets has been slow and is further confounded by interactions between the tumor and the surrounding tumor microenvironment. In this work, we attempt to link the state of the human kinome, or kinotype, with cell viability in treated, patient-derived PDAC tumor and cancer-associated fibroblast cell lines. We applied classification models to independent kinome perturbation and kinase inhibitor cell screen data, and found that the inferred kinotype of a cell has a significant and predictive relationship with cell viability. We further find that models are able to identify a set of kinases whose behavior in response to perturbation drive the majority of viability responses in these cell lines, including the understudied kinases CSNK2A1/3, CAMKK2, and PIP4K2C. We next utilized these models to predict the response of new, clinical kinase inhibitors that were not present in the initial dataset for model devlopment and conducted a validation screen that confirmed the accuracy of the models. These results suggest that characterizing the perturbed state of the human protein kinome provides significant opportunity for better understanding of signaling behavior and downstream cell phenotypes, as well as providing insight into the broader design of potential therapeutic strategies for PDAC.

IntroductionApproximately 50% of melanomas harbor an activating BRAFV600E mutation. Standard of care involves a combination of inhibitors targeting mutant BRAF and MEK1/2, the substrate for BRAF in the MAPK pathway. PTEN loss-of-function mutations occur in ~40% of BRAFV600E melanomas, resulting in increased PI3K/AKT activity that enhances resistance to BRAF/MEK combination inhibitor therapy.MethodsTo compare the response of PTEN null to PTEN wild-type cells in an isogenic background, CRISPR/Cas9 was used to knock out PTEN in a melanoma cell line that harbors a BRAFV600E mutation. RNA sequencing, functional kinome analysis, and drug synergy screening were employed in the context of BRAF/MEK inhibition.ResultsRNA sequencing and functional kinome analysis revealed that the loss of PTEN led to an induction of FOXD3 and an increase in expression of the FOXD3 target gene, ERBB3/HER3. Inhibition of BRAF and MEK1/2 in PTEN null, BRAFV600E cells dramatically induced the expression of ERBB3/HER3 relative to wild-type cells. A synergy screen of epigenetic modifiers and kinase inhibitors in combination with BRAFi/MEKi revealed that the pan ERBB/HER inhibitor, neratinib, could reverse the resistance observed in PTEN null, BRAFV600E cells.ConclusionsThe findings indicate that PTEN null BRAFV600E melanoma exhibits increased reliance on ERBB/HER signaling when treated with clinically approved BRAFi/MEKi combinations. Future studies are warranted to test neratinib reversal of BRAFi/MEKi resistance in patient melanomas expressing ERBB3/HER3 in combination with its dimerization partner ERBB2/HER2.

Abstract Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.

Abstract Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.

Abstract Dysregulation of PI3K/Akt signaling is a dominant feature in basal-like or triple-negative breast cancers (TNBC). However, the mechanisms regulating this pathway are largely unknown in this subset of aggressive tumors. Here we demonstrate that the transcription factor SOX4 is a key regulator of PI3K signaling in TNBC. Genomic and proteomic analyses coupled with mechanistic studies identified TGFBR2 as a direct transcriptional target of SOX4 and demonstrated that TGFBR2 is required to mediate SOX4-dependent PI3K signaling. We further report that SOX4 and the SWI/SNF ATPase SMARCA4, which are uniformly overexpressed in basal-like tumors, form a previously unreported complex that is required to maintain an open chromatin conformation at the TGFBR2 regulatory regions in order to mediate TGFBR2 expression and PI3K signaling. Collectively, our findings delineate the mechanism by which SOX4 and SMARCA4 cooperatively regulate PI3K/Akt signaling and suggest that this complex may play an essential role in TNBC genesis and/or progression.

A focused series of substituted 4H-1,2,6-thiadiazin-4-ones was designed and synthesized to probe the anti-cancer properties of this scaffold. Insights from previous kinase inhibitor programs were used to carefully select several different substitution patterns. Compounds were tested on bladder, prostate, pancreatic, breast, chordoma, and lung cancer cell lines with an additional skin fibroblast cell line as a toxicity control. This resulted in the identification of several low single digit micro molar compounds with promising therapeutic windows, particularly for bladder and prostate cancer. A number of key structural features of the 4H-1,2,6-thiadiazin-4-one scaffold are discussed that show promising scope for future improvement.

The first epithelial-to-mesenchymal transition (EMT) occurs in trophoblast stem (TS) cells during implantation. Inactivation of the serine/threonine kinase MAP3K4 in TS cells (TSKI4 cells) induces an intermediate state of EMT, where cells retain stemness, lose epithelial markers, and gain mesenchymal characteristics. Investigation of relationships among MAP3K4 activity, stemness, and EMT in TS cells may reveal key regulators of EMT. Here, we show that MAP3K4 activity controls EMT through the ubiquitination and degradation of HDAC6. Loss of MAP3K4 activity in TSKI4 cells results in elevated HDAC6 expression and the deacetylation of cytoplasmic and nuclear targets. In the nucleus, HDAC6 deacetylates the promoters of tight junction genes, promoting the dissolution of tight junctions. Importantly, HDAC6 knockdown in TSKI4 cells restores epithelial features, including cell-cell adhesion and barrier formation. These data define a role for HDAC6 in regulating gene expression during transitions between epithelial and mesenchymal phenotypes.

Therapeutics that target ERBB2, such as lapatinib, often provide initial clinical benefit, but resistance frequently develops. Adaptive responses leading to lapatinib resistance involve reprogramming of the kinome through reactivation of ERBB2/ERBB3 signaling and transcriptional upregulation and activation of multiple tyrosine kinases. The heterogeneity of induced kinases prevents their targeting by a single kinase inhibitor, underscoring the challenge of predicting effective kinase inhibitor combination therapies. We hypothesized that, to make the tumor response to single kinase inhibitors durable, the adaptive kinome response itself must be inhibited. Genetic and chemical inhibition of BET bromodomain chromatin readers suppresses transcription of many lapatinib-induced kinases involved in resistance, including ERBB3, IGF1R, DDR1, MET, and FGFRs, preventing downstream SRC/FAK signaling and AKT reactivation. Combining inhibitors of kinases and chromatin readers prevents kinome adaptation by blocking transcription, generating a durable response to lapatinib, and overcoming the dilemma of heterogeneity in the adaptive response.

Kinase inhibitors targeting the mitogen/extracellular signal-regulated kinase kinase (MEK)- extracellular signal related kinase (ERK) signaling pathway have limited durability in inhibiting growth of triple-negative breast cancer. We defined genome wide enhancer remodeling following MEK inhibition capable of driving adaptive gene transcription. Targeting positive elongation factor (P-TEFb) transcriptional regulatory complex members can block enhancer remodeling making the response to MEK-ERK inhibition durable.

BackgroundMost novel cancer therapeutics target kinases that are essential to tumor survival. Some of these kinase inhibitors are associated with cardiotoxicity, whereas others appear to be cardiosafe. The basis for this distinction is unclear, as are the molecular effects of kinase inhibitors in the heart. Methods and ResultsWe administered clinically relevant doses of sorafenib, sunitinib (cardiotoxic multitargeted kinase inhibitors), or erlotinib (a cardiosafe epidermal growth factor receptor inhibitor) to mice daily for 2 weeks. We then compared the effects of these 3 kinase inhibitors on the cardiac transcriptome using RNAseq and the cardiac kinome using multiplexed inhibitor beads coupled with mass spectrometry. We found unexpectedly broad molecular effects of all 3 kinase inhibitors, suggesting that target kinase selectivity does not define either the molecular response or the potential for cardiotoxicity. Using in vivo drug administration and primary cardiomyocyte culture, we also show that the cardiosafety of erlotinib treatment may result from upregulation of the cardioprotective signal transducer and activator of transcription 3 pathway, as co‐treatment with erlotinib and a signal transducer and activator of transcription inhibitor decreases cardiac contractile function and cardiomyocyte fatty acid oxidation. ConclusionsCollectively our findings indicate that preclinical kinome and transcriptome profiling may predict the cardiotoxicity of novel kinase inhibitors, and suggest caution for the proposed therapeutic strategy of combined signal transducer and activator of transcription/epidermal growth factor receptor inhibition for cancer treatment.

Using a bisubstituted caspase-3 target sequence: aspartate-glutamate-valine-aspartate, (z-DEVD)2 peptide derivative of the fluorophore, cresyl violet, we have obtained a cell permeant, fluorogenic, caspase substrate capable of detecting the site-specific presence of functionally active, caspase-3 and caspase-7 up-regulation within intact apoptotic cells. Addition of this substrate to induced and noninduced cell culture populations allows for the rapid site-specific detection of caspase up-regulation without the requirement for a wash step. We demonstrate here the use of (z-DEVD)2-cresyl violet substrate for the detection of apoptosis induction in Jurkat, THP-1, and MCF-7 cells using fluorescence microscopy and 96-well fluorescence plate reader analysis. Intracellular up-regulated DEVDase activity, which was clearly visible by fluorescence microscopy and 96-well fluorescence plate reader measurements, showed greater than 6-fold increases in fluorescence output in induced versus noninduced Jurkat cell samples. A simple fluorogenic substrate conversion method is demonstrated here for detecting apoptosis induction within intact living cells.

Background: More than 90 tyrosine kinases have been implicated in the pathogenesis of malignant transformation and tumor angiogenesis. Tyrosine kinase inhibitors (TKIs) have emerged as effective therapies in treating cancer by exploiting this kinase dependency. The TKI erlotinib targets the epidermal growth factor receptor (EGFR), whereas sunitinib targets primarily vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR).TKIs that impact the function of non-malignant cells and have on- and off-target toxicities, including cardiotoxicities. Cardiotoxicity is very rare in patients treated with erlotinib, but considerably more common after sunitinib treatment. We hypothesized that the deleterious effects of TKIs on the heart were related to their impact on cardiac metabolism. Methods: Female FVB/N mice (10/group) were treated with therapeutic doses of sunitinib (40 mg/kg), erlotinib (50 mg/kg), or vehicle daily for two weeks. Echocardiographic assessment of the heart in vivo was performed at baseline and on Day 14. Heart, skeletal muscle, liver and serum were flash frozen and prepped for non-targeted GC-MS metabolomics analysis. Results: Compared to vehicle-treated controls, sunitinib-treated mice had significant decreases in systolic function, whereas erlotinib-treated mice did not. Non-targeted metabolomics analysis of heart identified significant decreases in docosahexaenoic acid (DHA), arachidonic acid (AA)/ eicosapentaenoic acid (EPA), O-phosphocolamine, and 6-hydroxynicotinic acid after sunitinib treatment. DHA was significantly decreased in skeletal muscle (quadriceps femoris), while elevated cholesterol was identified in liver and elevated ethanolamine identified in serum. In contrast, erlotinib affected only one metabolite (spermidine significantly increased). Conclusions: Mice treated with sunitinib exhibited systolic dysfunction within two weeks, with significantly lower heart and skeletal muscle levels of long chain omega-3 fatty acids docosahexaenoic acid (DHA), arachidonic acid (AA)/eicosapentaenoic acid (EPA) and increased serum O-phosphocholine phospholipid. This is the first link between sunitinib-induced cardiotoxicity and depletion of the polyunsaturated fatty acids (PUFAs) and inflammatory mediators DHA and AA/EPA in the heart. These compounds have important roles in maintaining mitochondrial function, and their loss may contribute to cardiac dysfunction.