In rat adipocytes, insulin provoked rapid increases in (a) endogenous immunoprecipitable combined protein kinase C (PKC)-/ activity in plasma membranes and microsomes and (b) immunoreactive PKC-and PKCin GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-and PKCwere also increased by insulin in situ and phosphatidylinositol 3,4,5-(PO 4 ) 3 (PIP 3 ) in vitro. Because phosphoinositide-dependent kinase-1 (PDK-1) is required for phosphorylation of activation loops of PKC-and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-/ but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-. Also, insulin in situ and PIP 3 in vitro activated and stimulated autophosphorylation of a PKC-mutant, in which threonine 410 is replaced by glutamate (but not by an inactivating alanine) and cannot be activated by PDK-1. Surprisingly, insulin activated a truncated PKC-that lacks the regulatory (presumably PIP 3 -binding) domain; this may reflect PIP 3 effects on PDK-1 or transphosphorylation by endogenous full-length PKC-. Our findings suggest that insulin activates both PKCand PKC-in plasma membranes, microsomes, and GLUT4 vesicles by a mechanism requiring increases in PIP 3 , PDK-1-dependent phosphorylation of activation loop sites in PKC-and , and subsequent autophosphorylation and/or transphosphorylation.Insulin has been reported to activate atypical forms of protein kinase C (PKC), 1 i.e. PKC-and/or PKC-, in 3T3/L1 adipocytes (1, 2), rat adipocytes (3), L6 myotubes (4), and 32D cells (5). These increases in atypical PKC enzyme activity appear to be largely dependent upon activation of phosphatidylinositol (PI) 3-kinase (1-5) and subsequent increases in D3-PO 4 polyphosphoinositides, i.e. PI 3,4,5-(PO 4 ) 3 and PI 3,4-(PO 4 ) 2 (3). Moreover, transfection studies suggest that PKC-and/or PKC-is/are required for and may be sufficient for insulin stimulation of GLUT4 translocation and subsequent glucose transport (1-4). At present, there is only limited information on the mechanism whereby D3-PO 4 polyphosphoinositides activate atypical PKCs and little or no information on the subcellular compartments in which atypical PKCs are activated or, for that matter, whether one or both atypical PKCs are activated by insulin in specific cell types. With respect to the first point, recent findings (6, 7) suggest that PI 3,4,5-(PO 4 ) 3 and PI 3,4-(PO 4 ) 2 activate, or allow access for, 3-phosphoinositide-dependent kinase-1 (PDK-1), which phosphorylates threonine 410 in the activation loop of PKC-, thereby initiating the activation of this atypical PKC. Indeed, in other studies, we have found that PDK-1 action is required for insulin-induced activation of PKC-in rat adipocytes.2 However, it is uncertain whether this requirement reflects a permissive effect of PDK-1 or whether PDK-1 mediates acute a...

The incidence of HCC in the database was 0.4 per 1000 persons. NAFLD/NASH and type 2 diabetes mellitus, along with hepatitis C virus infection, were the major etiologic risk factors associated with HCC. This claims database analysis suggests a gap exists between screening and treatment guidelines and practice patterns, implying a need for greater health care provider awareness and education.

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Patients whose NSCLC tumors become afatinib resistant presently have few effective therapeutic options to extend their survival. Afatinib resistant NSCLC cells were sensitive to clinically relevant concentrations of the irreversible pan-HER inhibitor neratinib, but not by the first generation ERBB1/2/4 inhibitor lapatinib. In multiple afatinib resistant NSCLC clones, HDAC inhibitors reduced the expression of ERBB1/3/4, but activated c-SRC, which resulted in higher total levels of ERBB1/3 phosphorylation. Neratinib also rapidly reduced the expression of ERBB1/2/3/4, c-MET and of mutant K-/N-RAS; K-RAS co-localized with phosphorylated ATG13 and with cathepsin B in vesicles. Combined exposure of cells to [neratinib + HDAC inhibitors] caused inactivation of mTORC1 and mTORC2, enhanced autophagosome and subsequently autolysosome formation, and caused an additive to greater than additive induction of cell death. Knock down of Beclin1 or ATG5 prevented HDAC inhibitors or neratinib from reducing ERBB1/3/4 and K-/N-RAS expression and reduced [neratinib + HDAC inhibitor] lethality. Neratinib and HDAC inhibitors reduced the expression of multiple HDAC proteins via autophagy that was causal in the reduced expression of PD-L1, PD-L2 and ornithine decarboxylase, and increased expression of Class I MHCA. In vivo, neratinib and HDAC inhibitors interacted to suppress the growth of 4T1 mammary tumors, an effect that was enhanced by an anti-PD-1 antibody. Our data support the premises that neratinib lethality can be enhanced by HDAC inhibitors, that neratinib may be a useful therapeutic tool in afatinib resistant NSCLC, and that [neratinib + HDAC inhibitor] exposure facilitates anti-tumor immune responses.

We focused on the ability of the pan-histone deacetylase (HDAC) inhibitors AR42 and sodium valproate to alter the immunogenicity of melanoma cells. Treatment of melanoma cells with HDAC inhibitors rapidly reduced the expression of multiple HDAC proteins as well as the levels of PD-L1, PD-L2 and ODC, and increased expression of MHCA. In a cell-specific fashion, melanoma isolates released the immunogenic protein HMGB1 into the extracellular environment. Very similar data were obtained in ovarian and H&NSCC PDX isolates, and in established tumor cell lines from the lung and kidney. Knock down of HDAC1, HDAC3, HDAC8 and HDAC10, but not HDAC6, recapitulated the effects of the HDAC inhibitors on the immunotherapy biomarkers. Using B16 mouse melanoma cells we discovered that pre-treatment with AR42 or sodium valproate enhanced the anti-tumor efficacy of an anti-PD-1 antibody and of an anti-CTLA4 antibody. In the B16 model, both AR42 and sodium valproate enhanced the anti-tumor efficacy of the multi-kinase inhibitor pazopanib. In plasma from animals exposed to [HDAC inhibitor + anti-PD-1], but not [HDAC inhibitor + anti-CTLA4], the levels of CCL2, CCL5, CXCL9 and CXCL2 were increased. The cytokine data from HDAC inhibitor plus anti-PD-1 exposed tumors correlated with increased activated T cell, M1 macrophage, neutrophil and NK cell infiltration. Collectively, our data support the use of pan-HDAC inhibitors in combination with kinase inhibitors or with checkpoint inhibitor antibodies as novel melanoma therapeutic strategies.

Prior studies demonstrated that the irreversible ERBB1/2/4 inhibitor neratinib caused plasma membrane-associated mutant K-RAS to localize in intracellular vesicles, concomitant with its degradation. Herein, we discovered that neratinib interacted with the chemically distinct irreversible ERBB1/2/4 inhibitor afatinib to reduce expression of ERBB1, ERBB2, K-RAS and N-RAS; this was associated with greater-than-additive cell killing of pancreatic tumor cells. Knock down of Beclin1, ATG16L1, Rubicon or cathepsin B significantly lowered the ability of neratinib to reduce ERBB1 and K-RAS expression, and to cause tumor cell death. Knock down of ATM-AMPK suppressed vesicle formation and knock down of cathepsin B-AIF significantly reduced neratinib lethality. PKG phosphorylates K-RAS and HMG CoA reductase inhibitors reduce K-RAS farnesylation both of which remove K-RAS from the plasma membrane, abolishing its activity. Neratinib interacted with the PKG activator sildenafil and the HMG CoA reductase inhibitor atorvastatin to further reduce K-RAS expression, and to further enhance cell killing. Neratinib is also a Ste20 kinase family inhibitor and in carcinoma cells, and hematopoietic cancer cells lacking ERBB1/2/4, it reduced K-RAS expression and the phosphorylation of MST1/3/4/ Ezrin by ~30%. Neratinib increased LATS1 phosphorylation as well as that of YAP and TAZ also by ~30%, caused the majority of YAP to translocate into the cytosol and reduced YAP/TAZ #

PURPOSE Combination of antiprogrammed cell death protein-1 (PD-1) plus anti–cytotoxic T-cell lymphocyte-4 (anti-CTLA-4) immunotherapy shows greater response rates (RRs) than anti-PD-1 antibody alone in melanoma, but RR after initial anti-PD-1 and programmed death ligand-1 (PD-L1) antibody progression awaits robust investigation. Anti-CTLA-4 antibody alone after anti-PD-1/L1 antibody progression has a historical RR of 13%. We report the results of the first prospective clinical trial evaluating ipilimumab 1 mg/kg plus pembrolizumab following progression on anti-PD-1 immunotherapy. METHODS Patients with advanced melanoma who had progressed on anti-PD-1/L1 antibody as immediate prior therapy (including non–anti-CTLA-4 antibody combinations) were eligible. Patients received pembrolizumab 200 mg plus ipilimumab 1 mg/kg once every 3 weeks for four doses, followed by pembrolizumab monotherapy. The primary end point was RR by irRECIST. After 35 patients, the trial met the primary end point and was expanded to enroll a total of 70 patients to better estimate the RR. RESULTS Prior treatments included 60 on anti-PD-1 antibody alone and 10 on anti-PD-1/L1 antibody–based combinations. Thirteen patients had progressed in the adjuvant setting. The median length of prior treatment with anti-PD-1/L1 antibody was 4.8 months. Response assessments included five complete and 15 partial responses, making the irRECIST RR 29% among the entire trial population. The median progression-free survival was 5.0 months, and the median overall survival was 24.7 months. The median duration of response was 16.6 months. There was no difference in median time on prior anti-PD1/L1 or time to PD1 + CTLA4 initiation between responders and nonresponders. Grade 3-4 drug-related adverse events occurred in 27% of patients. Responses occurred in PD-L1–negative, non-T-cell–inflamed, and intermediate tumor phenotypes. CONCLUSION To our knowledge, this is the first prospective study in melanoma of pembrolizumab plus low-dose ipilimumab after anti-PD-1/L1 immunotherapy failure, demonstrating significant antitumor activity and tolerability.

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