HTS hit 7 was modified through hybrid design strategy to introduce a chiral side chain followed by introduction of Michael acceptor group to obtain potent EGFR kinase inhibitors 11 and 19. Both 11 and 19 showed over 3 orders of magnitude enhanced HCC827 antiproliferative activity compared to HTS hit 7 and also inhibited gefitinib-resistant double mutant (DM, T790M/L858R) EGFR kinase at nanomolar concentration. Moreover, treatment with 19 shrinked tumor in nude mice xenograft model.

Phenothiazines (PTZs) have been used for the antipsychotic drugs for centuries. However, some of these PTZs have been reported to exhibit antitumor effects by targeting various signaling pathways in vitro and in vivo. Thus, this study was aimed at exploiting trifluoperazine, one of PTZs, to develop potent antitumor agents. This effort culminated in A4 [10-(3-(piperazin-1-yl)propyl)-2-(trifluoromethyl)-10H-phenothiazine] which exhibited multi-fold higher apoptosis-inducing activity than the parent compound in oral cancer cells. Compared to trifluoperazine, A4 demonstrated similar regulation on the phosphorylation or expression of multiple molecular targets including Akt, p38, and ERK. In addition, A4 induced autophagy, as evidenced by increased expression of the autophagy biomarkers LC3B-II and Atg5, and autophagosomes formation. The antitumor activity of A4 also related to production of reactive oxygen species and adenosine monophosphate-activated protein kinase. Importantly, the antitumor utility of A4 was extended in vivo as it, administrated at 10 and 20 mg/kg intraperitoneally, suppressed the growth of Ca922 xenograft tumors. In conclusion, the ability of A4 to target diverse aspects of cancer cell growth suggests its value in oral cancer therapy.

Key Points• AEB071 demonstrates preclinical in vitro and in vivo activity against CLL independent of survival signaling and stromal cell protection.• AEB071 can either inhibit or activate the WNT pathway emphasizing the importance of pharmacodynamic monitoring in its development.Targeting B-cell receptor (BCR) signaling in chronic lymphocytic leukemia (CLL) has been successful with durable remissions observed with several targeted therapeutics. Protein kinase C-b (PKC-b) is immediately downstream of BCR and has been shown to be essential to CLL cell survival and proliferation in vivo. We therefore evaluated sotrastaurin (AEB071), an orally administered potent PKC inhibitor, on CLL cell survival both in vitro and in vivo. AEB071 shows selective cytotoxicity against B-CLL cells in a dose-dependent manner. Additionally, AEB071 attenuates BCR-mediated survival pathways, inhibits CpG-induced survival and proliferation of CLL cells in vitro, and effectively blocks microenvironment-mediated survival signaling pathways in primary CLL cells. Furthermore, AEB071 alters b-catenin expression, resulting in decreased downstream transcriptional genes as c-Myc, Cyclin D1, and CD44. Lastly, our preliminary in vivo studies indicate beneficial antitumor properties of AEB071 in CLL. Taken together, our results indicate that targeting PKC-b has the potential to disrupt signaling from the microenvironment contributing to CLL cell survival and potentially drug resistance. Future efforts targeting PKC with the PKC inhibitor AEB071 as monotherapy in clinical trials of relapsed and refractory CLL patients are warranted. (Blood. 2014;124(9):1481-1491) IntroductionChronic lymphocytic leukemia (CLL) is one of the most common types of adult leukemia and is currently incurable. Decades of research into the biology of CLL and other B-cell malignancies has brought forth B-cell receptor (BCR) signaling as a common required driving force in the survival and proliferation of these tumor cells. Several survival pathways involved in BCR signaling, including the phosphatidylinositol 3-kinase (PI3K), nuclear factor-kB (NF-kB), and mitogen-activated protein kinase (MAPK)/extracellular signalregulated kinase (ERK), are constitutively active in the lymph node and bone marrow compartment of CLL where disease expansion occurs. [1][2][3] Efforts to target BCR signaling with therapeutic agents which reversibly inhibit PI3K-d 4,5 or irreversibly inhibit Bruton tyrosine kinase (BTK) 6-8 have shown significant clinical activity in CLL, including those with high-risk genomic disease, and are currently in phase 3 studies. Protein kinase C-b (PKC-b) is an immediate downstream target of BTK that has recently been shown to be overexpressed in CLL 9 and is essential to the in vivo development of CLL in Em-TCL1 mice. 10 In B cells, PKC-b is thought to be the predominant PKC isoform mediating BCRdependent NF-kB activation.11-13 Downstream of PKC-b, IkB kinase and caspase recruitment domain-containing protein 11 (CARD11 [also known as CARMA1]) are phosphorylated, le...

Lipotoxicity plays an important role in the progression of chronic kidney damage via various mechanisms, such as endoplasmic reticulum stress. Several studies proposed renal lipotoxicity in glomerular and tubular cells but the effect of lipid on renal erythropoietin (EPO)-producing (REP) cells in the interstitium has not been elucidated. Since renal anemia is caused by derangement of EPO production in REP cells, we evaluated the effect of palmitate, a representative long-chain saturated fatty acid, on EPO production and the endoplasmic reticulum stress pathway. EPO production was suppressed by palmitate (palmitate-conjugated bovine serum albumin [BSA]) or a high palmitate diet, but not oleic acid-conjugated BSA or a high oleic acid diet, especially under cobalt-induced pseudo-hypoxia both in vitro and in vivo. Importantly, suppression of EPO production was not induced by a decrease in transcription factor HIF activity, while it was significantly associated with endoplasmic reticulum stress, particularly transcription factor ATF4 activation, which suppresses 3'-enhancer activity of the EPO gene. ATF4 knockdown by siRNA significantly attenuated the suppressive effect of palmitate on EPO production. Studies utilizing inherited super-anemic mice (ISAM) mated with EPO-Cre mice (ISAM-REC mice) for lineage-labeling of REP cells showed that ATF4 activation by palmitate suppressed EPO production in REP cells. Laser capture microdissection confirmed ATF4 activation in the interstitial area of ISAM-REC mice treated with palmitate-conjugated BSA. Thus, endoplasmic reticulum stress induced by palmitate suppressed EPO expression by REP cells in a manner independent of HIF activation. The link between endoplasmic reticulum stress, dyslipidemia, and hypoxia may contribute to development and progression of anemia in CKD.