PIK3CA (which encodes the phosphoinositide-3 kinase (PI3K) alpha isoform) is the most frequently mutated oncogene in breast cancer. Small-molecule PI3K inhibitors have shown promise in clinical trials; however, intrinsic and acquired resistance limits their utility. We used a systematic gain-of-function approach to identify genes whose upregulation confers resistance to the PI3K inhibitor BYL719 in breast cancer cells. Among the validated resistance genes, PIM kinases conferred resistance by maintaining downstream PI3K effector activation in an AKT-independent manner. Concurrent pharmacological inhibition of PIM and PI3K overcame this resistance mechanism. We also observed upregulated PIM expression and activity in a subset of breast cancer biopsies with clinical resistance to PI3K inhibitors. PIM1 overexpression is mutually exclusive with PIK3CA mutation in treatment-naïve breast cancers, suggesting downstream functional redundancy. Together, these results offer new insights into resistance to PI3K inhibitors and support clinical studies of combined PIM/PI3K inhibition in a subset of PIK3CA-mutant cancers.

Retinal pigment epithelial (RPE) cells, derived from the neuroectoderm, biosynthesize the novel lipid mediator neuroprotectin D1 (NPD1) from docosahexaenoic acid (DHA) in response to oxidative stress or to neurotrophins, and in turn, elicits cytoprotection. Here, we report the identification of a 16,17-epoxide-containing intermediate in the biosynthesis of NPD1 in ARPE-19 cells from 17S-hydro-(peroxy)-docosahexaenoic acid. We prepared and isolated tritium-labeled NPD1 ([ 3 H]-NPD1) and demonstrate specific and high-affinity stereoselective binding to ARPE-19 cells (K d = 31.3 ± 13.1 pmol/mg of cell protein). The stereospecific NPD1 interactions with these cells in turn gave potent protection against oxidative stress-induced apoptosis, and other structurally related compounds were weak competitors of NPD1 specific binding. This [ 3 H]-NPD1/PD1 also displayed specific and selective high affinity binding with isolated human neutrophils (K d = ~25 nM). Neither resolvin E1 nor lipoxin A 4 competed for [ 3 H]-NPD1/PD1 specific binding with human neutrophils. Together, these results provide evidence for stereoselective specific binding of NPD1/PD1 with retinal pigment epithelial cells as well as human neutrophils. Moreover, they suggest specific receptors for this novel mediator in both the immune and visual systems.

Retinal pigment epithelial (RPE) cell integrity is critical for the survival of photoreceptor cells. Bcl-x L is a major anti-apoptotic Bcl-2 protein required for RPE cell survival, and phosphorylation of Bcl-x L at residue Ser-62 renders this protein pro-apoptotic. In this study, we identify serine/threonine protein phosphatase 2A (PP2A) as a key regulator of Bcl-x L phosphorylation at residue Ser-62 in ARPE-19 cells, a spontaneously arising RPE cell line in which Bcl-x L is highly expressed. We found that either PP2A inhibitor okadaic acid or depletion of catalytic subunit ␣ of PP2A (PP2A/C␣) by small interfering RNA enhanced Bcl-x L phosphorylation when activated with hydrogen peroxide and tumor necrosis factor ␣-induced oxidative stress. Disruption of PP2A/C␣ exacerbated oxidative stressinduced apoptosis. PP2A/C␣ colocalized and interacted with S62Bcl-x L in cells stressed with H 2 O 2 /tumor necrosis factor ␣. By contrast, the omega-3 fatty acid docosahexaenoic acid derivative, neuroprotectin D1 (NPD1), a potent activator of survival signaling, down-regulated oxidative stress-induced phosphorylation of Bcl-x L by increasing protein phosphatase activity. NPD1 also attenuated the oxidative stress-induced apoptosis by knockdown of PP2A/C␣ and increased the association of PP2A/C␣ with S62Bcl-x L as well as total Bcl-x L . NPD1 also enhanced the heterodimerization of Bcl-x L with its counterpart, pro-apoptotic protein Bax. Thus, NPD1 modulates the activation of this Bcl-2 family protein by dephosphorylating in a PP2A-dependent manner, suggesting a coordinated, NPD1-mediated regulation of cell survival in response to oxidative stress. Retinal pigment epithelial (RPE)2 cell integrity is necessary for the survival of rod and cone photoreceptors, and these cells accomplish a myriad of functions, including transport of retinol and of the essential omega-3 fatty acid, docosahexaenoic acid, and also transport of nutrients between photoreceptors and the choriocapillaries (1, 2). Our laboratory has shown that RPE cells, when induced with oxidative stress, produce and release to the media a stereospecific oxygenation product of docosahexaenoic acid, named neuroprotectin D1 (NPD1) (3-6). Hence, NPD1 is a pleiotropic modulator of inflammation resolution (7). NPD1 up-regulates anti-apoptotic proteins (Bcl-2, Bcl-x L ) and down-regulates pro-apoptotic proteins (Bax, Bad) in ARPE-19 cells upon exposure to hydrogen peroxide/tumor necrosis factor ␣ (H 2 O 2 /TNF␣)-induced oxidative stress (3, 4). Under these conditions, NPD1 inhibits cytokine-mediated pro-inflammatory gene induction (3, 8) and oxidative stressinduced apoptosis and also promotes RPE cell survival (9, 10). Oxidative stress (leading to apoptosis), neovascularization, and lipid peroxidation are involved in neurodegenerative diseases, including age related-macular degeneration (11)(12)(13)(14).Apoptotic pathway activation comprises well orchestrated interactions between the Bcl-2 anti-apoptotic (Bcl-2 and Bcl-x L ) and pro-apoptotic (Bax and Bad) proteins. Bcl-2 and B...

Melanomas that contain B-RAFV600E mutations respond transiently to RAF and MEK inhibitors; however, resistance to these agents remains a formidable challenge. Although B- or C-RAF dysregulation represents prominent resistance mechanisms, resistance-associated point mutations in RAF oncoproteins are surprisingly rare. To gain insights herein, we conducted random mutagenesis screens to identify B- or C-RAF mutations that confer resistance to RAF inhibitors. Whereas bona fide B-RAFV600E resistance alleles were rarely observed, we identified multiple C-RAF mutations that produced biochemical and pharmacologic resistance. Potent C-RAF resistance alleles localized to a 14-3-3 consensus binding site or a separate site within the P loop. These mutations elicited paradoxical upregulation of RAF kinase activity in a dimerization-dependent manner following exposure to RAF inhibitors. Knowledge of resistance-associated C-RAF mutations may enhance biochemical understanding of RAF-dependent signaling, anticipate clinical resistance to novel RAF inhibitors, and guide the design of “next-generation” inhibitors for deployment in RAF- or RAS-driven malignancies. Cancer Res; 73(15); 4840–51. ©2013 AACR.