Cell death induction in resting lymphocytes by pan-Cdk inhibitor, but not by Cdk4/6 selective inhibitor.

Immunosuppression is one of the common side effects of many anti-tumor agents targeting proliferating cells. We previously reported the development of a new class of pan-cyclin-dependent kinase (Cdk) inhibitor compounds that induce immunosuppression in rodents. Here, we demonstrated that a pan-Cdk inhibitor, Compound 1 very rapidly reduced white blood cells in mice, only 8 h after administration. Compound 1 induced death of peripheral blood cells or purified resting (non-stimulated) lymphocytes ex vivo. Cell death was induced very rapidly, after 4 h of incubation, suggesting that acute immunosuppression observed in rodents might be, at least in part, due to direct cytotoxic effects of Compound 1 on resting lymphocytes. While cell cycle-related Cdks were not activated, the carboxyl terminal domain (CTD) of the largest subunit of RNA polymerase II was phosphorylated, indicating activation of Cdk7 or Cdk9, which phosphorylates this domain, in resting lymphocytes. Indeed, the pan-Cdk inhibitor suppressed CTD phosphorylation in resting cells at the dose required for cell death induction. Inhibition of Cdk7 or Cdk9 by Compound 1 was also confirmed by suppression of nuclear factor-kappa B (NF-κB)-dependent transcription activity in the human cancer cell line U2OS. Interestingly, a Cdk4/6 inhibitor with selectivity against Cdk7 and Cdk9 did not induce cell death in resting lymphocytes. These results suggest that CTD phosphorylation possibly by Cdk7 or Cdk9 might be important for survival of resting lymphocytes and that Cdk inhibitors without inhibitory activity on these kinases might be an attractive agent for cancer chemotherapy.

Potent anti-tumor activity of a macrocycle-quinoxalinone class pan-Cdk inhibitor in vitro and in vivo.

Deregulation of cell-cycle control is a hallmark of cancer. Thus, cyclin-dependent kinases (Cdks) are an attractive target for the development of anti-cancer drugs. Here, we report the biological characterization of a highly potent pan-Cdk inhibitor with a macrocycle-quinoxalinone structure. Compound M inhibited Cdk1, 2, 4, 5, 6, and 9 with equal potency in the nM range and was selective against kinases other than Cdks. This compound inhibited multiple events in the cell cycle in vitro, including retinoblastoma protein (pRb) phosphorylation, E2F-dependent transcription, DNA replication (determined by bromodeoxyuridine incorporation), and mitosis completion (assayed by flow cytometry) in the 10 nM range. Moreover, this compound induced cell death, as determined by induction of the subG1 fraction, activated caspase-3, and anexin V. In vivo, Compound M showed anti-tumor efficacy at a tolerated dose. In a nude rat xenograft tumor model, an 8-h constant infusion of Compound M inhibited pRb phosphorylation and induced apoptosis in tumor cells at ~ 30 nM, which led to the inhibition of tumor growth. Immunosuppression was the only liability observed at this dose, but immune function returned to normal after 10 days. Suppression of pRb phosphorylation in tumor cells was clearly correlated with tumor cell growth inhibition and cell death in vitro and in vivo. In vivo, Compound M inhibited pRb phosphorylation in both tumor and gut crypt cells. Rb phosphorylation may be a suitable pharmacodynamic biomarker in both tumors and normal tissues for monitoring target engagement and predicting the efficacy of Compound M.

The ATM-related Tel1 protein of Saccharomyces cerevisiae controls a checkpoint response following phleomycin treatment.

MEC1 and TEL1 encode ATR- and ATM-related proteins in the budding yeast Saccharomyces cerevisiae, respectively. Phleomycin is an agent that catalyzes double-strand breaks in DNA. We show here that both Mec1 and Tel1 regulate the checkpoint response following phleomycin treatment. MEC1 is required for Rad53 phosphorylation and cell-cycle progression delay following phleomycin treatment in G1, S or G2/M phases. The tel1Delta mutation confers a defect in the checkpoint responses to phleomycin treatment in S phase. In addition, the tel1Delta mutation enhances the mec1 defect in activation of the phleomycin-induced checkpoint pathway in S phase. In contrast, the tel1Delta mutation confers only a minor defect in the checkpoint responses in G1 phase and no apparent defect in G2/M phase. Methyl methanesulfonate (MMS) treatment also activates checkpoints, inducing Rad53 phosphorylation in S phase. MMS-induced Rad53 phosphorylation is not detected in mec1Delta mutants during S phase, but occurs in tel1Delta mutants similar to wild-type cells. Finally, Xrs2 is phosphorylated after phleomycin treatment in a TEL1-dependent manner during S phase, whereas no significant Xrs2 phosphorylation is detected after MMS treatment. Together, our results support a model in which Tel1 contributes to checkpoint control in response to phleomycin-induced DNA damage in S phase.

The RAIG family member, GPRC5D, is associated with hard-keratinized structures.

Retinoic acid-inducible gene-1 was originally identified as an orphan G-protein coupled receptor induced by retinoic acid. Three highly homologous oGPCR (GPRC5B, GPRC5C, and GPRC5D) have since been classified into the RAIG1 family. We describe here, the unique tissue distribution of GPRC5D and its mechanism of expression. Hybridization in situ has shown that GPRC5D is expressed in differentiating cells that produce hard keratin, including cortical cells of the hair shaft, the keratogenous zone of the nail, and in a central region of the filiform papillae of the tongue. The GPRC5D transcript is expressed in hair follicles during mid- and late anagen, and catagen but not at telogen and early anagen phases. The differentiation-inducer, all-trans retinoic acid, induces GPRC5D expression in cultured hair bulb cells. Because the tissue distribution of GPRC5D indicates a relationship with hard keratins that constitute the major structural proteins of hard epithelial tissues, we investigated the effect of GPRC5D on acid hard keratins. Analyses of cultured cells showed that transient overexpression resulted in suppression of Ha3 and stimulation of Ha4 hair keratin gene expression. The expression was maintained in the hair follicles of whn-deficient (nude) mice, suggesting that this gene is regulated by a signal pathway different from that of hair keratin synthesis. Collectively, these data provide a framework for understanding the molecular mechanisms of GPRC5D function in hard keratinization.

Selective and potent Akt inhibition triggers anti-myeloma activities and enhances fatal endoplasmic reticulum stress induced by proteasome inhibition.

The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM.

MK-5108, a highly selective Aurora-A kinase inhibitor, shows antitumor activity alone and in combination with docetaxel.

Aurora-A kinase is a one of the key regulators during mitosis progression. Aurora-A kinase is a potential target for anticancer therapies because overexpression of Aurora-A, which is frequently observed in some human cancers, results in aberrant mitosis leading to chromosomal instability and possibly tumorigenesis. MK-5108 is a novel small molecule with potent inhibitory activity against Aurora-A kinase. Although most of the Aurora-kinase inhibitors target both Aurora-A and Aurora-B, MK-5108 specifically inhibited Aurora-A kinase in a panel of protein kinase assays. Inhibition of Aurora-A by MK-5108 in cultured cells induced cell cycle arrest at the G(2)-M phase in flow cytometry analysis. The effect was confirmed by the accumulation of cells with expression of phosphorylated Histone H3 and inhibition of Aurora-A autophosphorylation by immunostaining assays. MK-5108 also induced phosphorylated Histone H3 in skin and xenograft tumor tissues in a nude rat xenograft model. MK-5108 inhibited growth of human tumor cell lines in culture and in different xenograft models. Furthermore, the combination of MK-5108 and docetaxel showed enhanced antitumor activities compared with control and docetaxel alone-treated animals without exacerbating the adverse effects of docetaxel. MK-5108 is currently tested in clinical trials and offers a new therapeutic approach to combat human cancers as a single agent or in combination with existing taxane therapies.

Biological characterization of 2-aminothiazole-derived Cdk4/6 selective inhibitor in vitro and in vivo.

Abnormalities in the p16INK4a/ cyclin-dependent kinase (Cdk)4, 6/ Retinoblastoma (Rb) pathway frequently occur in various human cancers. Thus, Cdk4/6 is an attractive target for cancer therapy. Here we report the biological characterization of a 2-aminothiazole-derived Cdk4/6 selective inhibitor, named Compound A in vitro and in vivo. Compound A potently inhibits Cdk4 and Cdk6 with high selectivity (more than 57-fold) against other Cdks and 45 serine/threonine and tyrosine kinases. Compound A inhibits Rb protein (pRb) phosphorylation at Ser780, inhibits E2F-dependent transcription, and induces cell-cycle arrest at G1 in the T98G human glioma cell line. Among 82 human cells derived from various tissues, cell lines derived from hematological cancers (leukemia/lymphoma) tended to be more sensitive to Compound A in cell proliferation assay. Rb-negative cells tended to be insensitive to Compound A, as we had expected. In a nude rat xenograft model, Compound A inhibited pRb phosphorylation and bromodeoxyuridine (BrdU) incorporation in Eol-1 xenograft tumor at plasma concentration of 510 nM. Interestingly Compound A only moderately inhibited those pharmacodynamic and cell cycle parameters of normal crypt cells in small intestine even at 5 times higher plasma concentration. In F344 rats, Compound A did not cause immunosuppression even at 17 times higher plasma conc. These results suggest that Cdk4/6 selective inhibitors only moderately affects on the cell cycle of normal proliferating tissues and has a safer profile than pan-Cdk inhibitor in vivo.

Expression levels of p18INK4C modify the cellular efficacy of cyclin-dependent kinase inhibitors via regulation of Mcl-1 expression in tumor cell lines.

Because cyclin-dependent kinases (CDK) play a pivotal role in cancer progression, the development of CDK inhibitors has attracted attention in antitumor therapy. However, despite significant preclinical and clinical developments, CDK inhibition biomarkers for predicting efficacy against certain cancers in individual patients have not been identified. Here, we characterized a macrocyclic quinoxalin-2-one CDK inhibitor, compound A, and identified a gene biomarker for predicting its efficacy. Compound A showed 100-fold selectivity for CDK family proteins over other kinases and inhibited both E2F transcriptional activity and RNA polymerase II phosphorylation. Compound A treatment resulted in decreased proliferation in various tumor cell lines; however, the apoptosis induction rate differed significantly among the cell lines examined, which was consistent with roscovitine. By comparing the mRNA expression profiles of sensitive and resistant cell lines, we found that expression levels of an endogenous CDK inhibitor, p18(INK4C), showed a strong negative correlation to the sensitivity. In fact, p18 status was correlated with the response to CDK inhibitor in an independent data set of multiple myeloma cell lines and silencing p18 expression increased the susceptibility of resistant cells to CDK inhibitors. The analysis of molecular mechanisms revealed that cells with lowered p18 had aberrant CDK6 and E2F activities, which resulted in a transcriptional down-regulation of Mcl-1, a key molecule associated with flavopiridol-induced apoptosis, thereby leading to susceptibility to therapeutic intervention with CDK inhibitors. These results identified a molecular basis for CDK inhibitors to exert an antitumor effect in p18-deficient cancers and support the clinical use of CDK inhibitors.