PP2 protects from keratin mutation-associated liver injury and filament disruption via SRC kinase inhibition in male but not female mice.

BACKGROUND AND AIMS: Hepatocyte keratin polypeptides 8/18 (K8/K18) are unique among intermediate filaments proteins (IFs) in that their mutation predisposes to, rather than causes, human disease. Mice that overexpress human K18 R90C manifest disrupted hepatocyte keratin filaments with hyperphosphorylated keratins and predisposition to Fas-induced liver injury. We hypothesized that high-throughput screening will identify compounds that protect the liver from mutation-triggered predisposition to injury. APPROACH AND RESULTS: Using A549 cells transduced with a lentivirus K18 construct and high-throughput screening, we identified the SRC-family tyrosine kinases inhibitor, PP2, as a compound that reverses keratin filament disruption and protects from apoptotic cell death caused by K18 R90C mutation at this highly conserved arginine. PP2 also ameliorated Fas-induced apoptosis and liver injury in male but not female K18 R90C mice. The PP2 male selectivity is due to its lower turnover in male versus female livers. Knockdown of SRC but not another kinase target of PP2, protein tyrosine kinase 6, in A549 cells abrogated the hepatoprotective effect of PP2. Phosphoproteomic analysis and validation showed that the protective effect of PP2 associates with Ser/Thr but not Tyr keratin hypophosphorylation, and differs from the sex-independent effect of the Ser/Thr kinase inhibitor PKC412. Inhibition of RAF kinase, a downstream target of SRC, by vemurafenib had a similar protective effect to PP2 in A549 cells and male K18 R90C mice. CONCLUSIONS: PP2 protects, in a male-selective manner, keratin mutation-induced mouse liver injury by inhibiting SRC-triggered downstream Ser/Thr phosphorylation of K8/K18, which is phenocopied by RAF kinase inhibitor vemurafenib. The PP2/vemurafenib-associated findings, and their unique mechanisms of action, further support the potential role of select kinase inhibition as therapeutic opportunities for keratin and other IF-associated human diseases.

B-Raf inhibitor vemurafenib counteracts sulfur mustard-induced epidermal impairment through MAPK/ERK signaling.

The chemical warfare agent sulfur mustard (SM) causes severe cutaneous lesions characterized by epidermal cell death, apoptosis, and inflammation. At present, the molecular mechanisms underlying SM-induced injury are not well understood, and there is no standard treatment protocol for SM-exposed patients. Here, we conducted a high-content screening of the Food and Drug Administration (FDA)-approved drug library of 1018 compounds against SM injury on an immortal human keratinocyte HaCaT cell line, focusing on cell survival. We found that the B-Raf inhibitor vemurafenib had an apparent therapeutic effect on HaCaT cells and resisted SM toxicity. Other tested B-Raf inhibitors, both type-I (dabrafenib and encorafenib) and type-II (RAF265 and AZ628), also exhibited potent therapeutic effects on SM-exposed HaCaT cells. Both SM and vemurafenib triggered extracellular signal-related kinase (ERK) activation. The therapeutic effect of vemurafenib in HaCaT cells during SM injury was ERK-dependent, indicating a specific role of ERK in keratinocyte regulatory mechanisms. Furthermore, vemurafenib partially improved cutaneous damage in a mouse ear vesicant model. Collectively, our results provide evidence that the B-Raf inhibitor vemurafenib is a potential therapeutic agent against SM injury, and oncogenic B-Raf might be an exciting new therapeutic target following exposure to mustard vesicating agents.

Alternative use of BRAF inhibitors in patients with metastatic melanoma unable to swallow pills.

INTRODUCTION: BRAF and MEK inhibitors have been approved for use in metastatic melanoma therapies. All of them are administered as oral capsules or pills. We report two cases treated applying an alternative method of vemurafenib or debrafenib-trametinib administration in patients unable to swallow. CASE REPORT: The first case involved a 38-year-old man who was referred to a dermatologist for dysphagia and anorexia. After a computerized tomography (CT) scan it was concluded that the dysphagia was due to compression by mediastinal metastasis in a context of metastatic BRAF mutant melanoma. The second case involved a 35-year-old man who was diagnosed in March 2017 with melanoma of the back of the hand. Several months later a positron emission tomography (PET)/CT scan was performed. It revealed multiple disseminated metastasis.Management & Outcome: The first patient presented total dysphagia and was unable to swallow pills. It was decided to dissolve vemurafenib in order to facilitate administration. Dysphagia was improved 48 hours later, and oral feeding was reintroduced. Due to severe tablet phobia, the second patient was unable to swallow pills. Dabrafenib capsules were emptied and trametinib pills were grinded. One month later, we noted improved health associated with reduction of the metastases. DISCUSSION: Our study highlights the possibility of crushing or dissolving BRAF and MEK inhibitors in metastatic melanoma patients for whom it is impossible to swallow pills, eliciting a response and achieving significant if temporary clinical benefit.

BRAFV600E dictates cell survival via c-Myc-dependent induction of Skp2 in human melanoma.

BRAFV600E mutation is frequently observed in melanoma, and contributes to tumor malignancy. Despite inhibition of BRAF causes a profound cell growth inhibition and a strong clinical benefit in BRAFV600E melanoma, acquired drug resistance is still the major hurdle. In this study, we demonstrate that BRAFV600E drives cell growth and glycolysis in melanoma cells but does so by a previously unappreciated mechanism that involves direct induction of Skp2. Skp2 is highly expressed in melanoma tissues and particularly in tissues with BRAFV600E mutation. The inhibition of BRAFV600E by either siRNA or inhibitor vemurafenib suppressed Skp2 expression and cell growth. Mechanistic study shows that BRAFV600E suppression of Skp2 is dependent on c-Myc transcription factor via specifically bounding to the E-box region on SKP2 promoter. Further, the overexpression of Skp2 resulted in a markedly increase in cell growth, cell cycle progression and glycolysis which were repressed by BRAFV600E inhibition. Supporting the biological significance, Skp2 is specifically correlated with poor patient outcome in BRAFV600E but did not in BRAFWT melanomas. Thus, as a downstream target of BRAFV600E, Skp2 is critical for responses to BRAF inhibition, indicating targeting Skp2 might be a promising strategy for the treatment of BRAFi resistant melanomas.

Biochemical Characterization of Full-Length Oncogenic BRAFV600E together with Molecular Dynamics Simulations Provide Insight into the Activation and Inhibition Mechanisms of RAF Kinases.

The most prevalent BRAF mutation, V600E, occurs frequently in melanoma and other cancers. Although extensive progress has been made toward understanding the biology of RAF kinases, little in vitro characterization of full-length BRAFV600E is available. Herein, we show the successful purification of active, full-length BRAFV600E from mammalian cells for in vitro experiments. Our biochemical characterization of intact BRAFV600E together with molecular dynamics (MD) simulations of the BRAF kinase domain and cell-based assays demonstrate that BRAFV600E has several unique features that contribute to its tumorigenesis. Firstly, steady-state kinetic analyses reveal that purified BRAFV600E is more active than fully activated wild-type BRAF; this is consistent with the notion that elevated signaling output is necessary for transformation. Secondly, BRAFV600E has a higher potential to form oligomers, despite the fact that the V600E substitution confers constitutive kinase activation independent of an intact side-to-side dimer interface. Thirdly, BRAFV600E bypasses inhibitory P-loop phosphorylation to enforce the necessary elevated signaling output for tumorigenesis. Together, these results provide new insight into the biochemical properties of BRAFV600E , complementing the understanding of BRAF regulation under normal and disease conditions.

Photosensitization of human skin fibroblasts by vemurafenib promotes pleiotropic effects on membrane-enclosed organelles and apoptosis.

Vemurafenib (VB), a BRAF inhibitor and a first-line treatment for unresectable or metastatic melanoma, is strongly phototoxic towards normal skin cells. Herein, we show that in cultured HS 68 human diploid dermal fibroblasts, low concentrations of VB suffice to promote photosensitization to low doses of UVA (∼ 5 J/cm2), as evidenced by a significant decrease in cell viability. In contrast to data obtained in chemico our results support a role for ROS (reactive oxygen species). Indeed, peroxidation of cellular lipids was observed which could be alleviated by the lipophilic antioxidant BHT (2,6-di-tert-butyl-4-methylphenol). Using in vivo confocal laser scanning microscopy and vital fluorescent probes it was shown at the single cell level that the plasma membrane and lipid-rich organelles, namely mitochondria, endoplasmic reticulum, and lysosomes, as well as actin filaments, were severely damaged by the UVA-induced VB-photosensitization. Finally, we showed that mitochondrial impairment was concurrent with caspase 3/7 activation and cell death by apoptosis.

Vemurafenib improves muscle histopathology in a mouse model of LAMA2-related congenital muscular dystrophy.

Laminin-α2-related congenital muscular dystrophy (LAMA2-CMD) is a neuromuscular disease affecting around 1-9 in 1,000,000 children. LAMA2-CMD is caused by mutations in the LAMA2 gene resulting in the loss of laminin-211/221 heterotrimers in skeletal muscle. LAMA2-CMD patients exhibit severe hypotonia and progressive muscle weakness. Currently, there is no effective treatment for LAMA2-CMD and patients die prematurely. The loss of laminin-α2 results in muscle degeneration, defective muscle repair and dysregulation of multiple signaling pathways. Signaling pathways that regulate muscle metabolism, survival and fibrosis have been shown to be dysregulated in LAMA2-CMD. As vemurafenib is a US Food and Drug Administration (FDA)-approved serine/threonine kinase inhibitor, we investigated whether vemurafenib could restore some of the serine/threonine kinase-related signaling pathways and prevent disease progression in the dyW-/- mouse model of LAMA2-CMD. Our results show that vemurafenib reduced muscle fibrosis, increased myofiber size and reduced the percentage of fibers with centrally located nuclei in dyW-/- mouse hindlimbs. These studies show that treatment with vemurafenib restored the TGF-ß/SMAD3 and mTORC1/p70S6K signaling pathways in skeletal muscle. Together, our results indicate that vemurafenib partially improves histopathology but does not improve muscle function in a mouse model of LAMA2-CMD.

PD-L1-Mediated Immunosuppression in Hepatocellular Carcinoma: Relationship with Macrophages Infiltration and Inflammatory Response Activity.

Immune dysfunction and pro-oncogenic inflammation play critical roles in malignant progression and non-response to immunotherapy for hepatocellular carcinoma (HCC). In particular, PD-1/PD-L1 blockade therapy could induce durable tumor remissions and improve the prognosis of patients to a certain extent. However, PD-L1, as a promising biomarker, has limited knowledge about its relevance to tumor microenvironment (TME) characterization and endogenous inflammatory immune responses. In this study, we systematically investigated and characterized the important intercommunication of PD-L1 with immunosuppressive TME and inflammatory response activity in HCC and predicted promising therapeutic drugs to improve the current therapeutic strategy for specific patients. We identified aberrant expression patterns of PD-L1 in HCC and completely different clinical and molecular characteristics among the PD-L1 subgroups. PD-L1 positively associated with immunosuppressive macrophages and macrophage-derived cytokines, which may contribute to the polarization of macrophages. Moreover, inflammatory response activity exhibited significant differences between high and low PD-L1 expression groups and had robust positive correlativity of the infiltration level of tumor-associated macrophages. Notably, given the immunosuppressive and inflammatory microenvironment in HCC, we screened four candidate drugs, including dasatinib, vemurafenib, topotecan and AZD6482, and corroborated in two pharmacogenomics databases, which might have potential therapeutic implications in specific HCC patients. Our results enhanced the understanding of linkage in PD-L1 expression patterns with macrophages and inflammation, which may provide new insight into the pathogenic mechanisms and potential therapeutic strategy for HCC.

Novel natural inhibitors targeting B-RAF(V600E) by computational study.

The aim of this research was to screen the ZINC15 database to select lead compounds and drug candidates which can inhibit B-RAF (V600E). In order to identify drugs potentially inhibited B-RAF (V600E), numerous modules of Discovery Studio 4.5 were employed. Structure-based screening using LibDock was carried out followed by ADME (absorption, distribution, metabolism, excretion) and toxicity prediction. CDOCKER was performed to demonstrate the binding affinity and mechanism between ligands and B-RAF(V600E). To evaluate whether ligand-receptor complexes were stable, molecular dynamics were employed. Two novel natural compounds (ZINC000100168592 and ZINC000049784088) from ZINC15 database were found binding to B-RAF(V600E) with more favorable interaction energy in comparison with the reference drug Vemurafenib. Also, they were predicted with less ames mutagenicity, rodent carcinogenicity, non-developmental toxic potential and tolerance to cytochrome P450 2D6 (CYP2D6). The molecular dynamics simulation analysis indicated that the compound-B-RAF(V600E) complexes had more favorable potential energy compared with Vemurafenib and they can exist in natural environments stably. The result of this study shows that ZINC000100168592 and ZINC000049784088 are ideal leading potential compounds to inhibit B-RAF(V600E). The findings of this study and these selected drug candidates greatly contributed to the medication design and improvement of B-RAF(V600E) and other proteins.

Discovery of Selective Small Molecule Degraders of BRAF-V600E.

BRAF is among the most frequently mutated oncogenes in human cancers. Multiple small molecule BRAF kinase inhibitors have been approved for treating melanoma carrying BRAF-V600 mutations. However, the benefits of BRAF kinase inhibitors are generally short-lived. Small molecule-mediated targeted protein degradation has recently emerged as a novel pharmaceutical strategy to remove disease proteins through hijacking the cellular ubiquitin proteasome system (UPS). In this study, we developed thalidomide-based heterobifunctional compounds that induced selective degradation of BRAF-V600E, but not the wild-type BRAF. Downregulation of BRAF-V600E suppressed the MEK/ERK kinase cascade in melanoma cells and impaired cell growth in culture. Abolishing the interaction between degraders and cereblon or blocking the UPS significantly impaired the activities of these degraders, validating a mechanistic role of UPS in mediating targeted degradation of BRAF-V600E. These findings highlight a new approach to modulate the functions of oncogenic BRAF mutants and provide a framework to treat BRAF-dependent human cancers.

Resistance mechanism of the oncogenic ß3-αC deletion mutation in BRAF kinase to dabrafenib and vemurafenib revealed by molecular dynamics simulations and binding free energy calculations.

BRAF kinase is an essential target for anti-cancer drug development. Emergence of the ß3-αC loop deletion mutation (ΔNVTAP) in BRAF kinase frequently occurred in human cancers seriously compromises the therapeutic efficacy of some BRAF kinase inhibitors, such as dabrafenib and vemurafenib. However, the mechanism of this resistance is still not well understood. In this study, the influence of the ß3-αC deletion mutation on the binding profiles of three BRAF kinase inhibitors (AZ628, dabrafenib, and vemurafenib) with BRAFV600E or BRAFΔNVTAP was explored by conventional molecular dynamics (MD) simulations and binding free energy calculations. The simulation results indicated that the ß3-αC deletion mutation enhances the flexibility of the αC helix and alters their conformations, which amplify the conformational entropy change (-TΔS) and weaken the interactions between the inhibitors and BRAF. The further per-residue binding free energy decomposition analysis revealed that the ΔNVTAP mutation changed the contributions of a few key residues to the bindings of dabrafenib or vemurafenib, such as L57, L66, W83, C84, F135, G145, and F147, but did not have obvious impact on the contributions of these residues to AZ628. Our results provide valuable clues to understand the mechanisms of drug resistance conferred by the ß3-αC deletion mutation.