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Response to "Selective PKC# inhibitor B106 elicits uveal melanoma growth inhibitory effects independent of activated PKC isoforms" Douglas V. Faller ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00959 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 13, 2018
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ACS Chemical Biology
Response to "Selective PKCδ inhibitor B106 elicits uveal melanoma growth inhibitory effects independent of activated PKC isoforms" Complete List of Authors: Faller, Douglas V.; Boston University, Cancer Center 80 East Concord St, Boston, MA 02118
We have previously reported that the compound known as B106, a PKCδ inhibitory small molecule, is cytotoxic to a variety of tumor cells bearing mutationally- activated Ras alleles or constitutive activation of certain Ras effector pathways.1,2 Constitutive activation of Ras signaling confers dependency upon PKCδ signaling for survival.3,4 The “Ras-dependency” of the cytotoxic effect of B106 and related PKCδ inhibitors was also confirmed using cell lines isogenic for activated Ras. The consequent dependency of these cells upon PKCδ activity was established using highly-specific siRNA genetic downregulation of PKCδ. Heijkants, et al 5 report in their correspondence that three uveal melanoma cells with activating GNAQ/11 mutations, which have been reported to render them dependent upon PKCδ, are sensitive to B106 at sub-M concentrations. These investigators also reproduced the B106-induced cytotoxicity we had previously reported using the FM melanoma cell line, which contains a mutationally-activated NRAS allele, at sub-M concentrations.1 They confirmed activation of the stress-responsive JNK pathway by B106 (and by a pan PKC inhibitor) in these cells, as we have previously reported. They further report that two uveal melanoma cell lines without GNAQ/11 mutations are also sensitive to B106. It is not clear from their report how the dependence or independence of these cell lines specifically on PKCδ was independently established. (For example, the line MEL 202 was only modestly sensitive to two pan-PKC inhibitors, which seems at odds with the assertion that this cell line is dependent upon PKCδ activity.) The major discrepancy between our reports and that of Heijkants, et al is in the assay of PKCδ inhibitor activity for the B106 molecule, and their conclusion that B106 does not inhibit PKCδ at the concentrations tested. It is difficult for us to address the differences in findings between the reports, as Heijkants, et al had their enzymatic assays carried out by a contract laboratory, using apparently proprietary technology. As one example, we could no locate any positive control data within the supplemental data provided; it is therefore not possible for us to determine what signal a known enzyme inhibitor would deliver in these proprietary assays. We have observed one possible technical cause of issues with in vitro enzymatic assays of PKC activity when we established these assays in our laboratory. B106 is an extremely hydrophobic molecule, as noted in our reports. Dilution of B106 from 100% DMSO solutions into aqueous solutions for enzymatic assays resulted in its immediate precipitation. This precipitation could be abrogated if the diluent contained serum albumin or lipids; in our studies, we included the diacylglycerol and the phospholipids,
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used in the assay to activate the PKC for analysis, in the diluent. This ensured consistently reproducible analyses of the compounds, and did not affect the positive and negative controls. Heijkants, et al speculate that B106 may have anti-tumor properties other than inhibition of PKC-delta. While we cannot exclude that possibility, we note that we have exposed a number of normal human and murine primary cells and non-transformed cell lines to concentrations of B106 which are cytotoxic to tumor cells with Ras pathway activation, without generating cytotoxic effects.1,2 B106 can also be infused subcutaneously in animals at concentrations which produce systemic activity without causing local tissue damage (unpublished observations).
1. Takashima, A., Chen, Z., English, B., Williams, R.A., Faller, D.V. (2014) Protein kinase C δ is a therapeutic target in malignant melanoma with NRas mutation or BRaf inhibitor-resistance, ACS Chem Biol 9, 1003-1014. 2. Chen, Z., Forman, L.W., Williams, R.M. Faller, D.V. (2014) Protein Kinase C-delta Inactivation Inhibits the Proliferation and survival of Cancer Stem Cells in culture and in vivo, BMC Cancer 14, 90-98. 3. Xia, S., Forman, L.W., Chen, Z., and Faller, D.V. (2009) PKCδ survival signaling in cells containing an activated p21Ras oncoprotein, Cell Signal 21, 502-508. 4. Xia, S., Forman, L.W. and Faller, D.V. (2007) PKCδ is required for survival of cells expressing activated p21Ras. J Biol Chem 282, 13199-210. 5. Heijkants, R., Teunisse, A., de Vries, J., Ovaa, H., and Jochemsen, A. (2018) Selective PKCδ inhibitor B106 elicits uveal melanoma growth inhibitory effects independent of activated PKC isoforms. ACS Chem Biol, ___
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