Osamu Nakanishi

Reversal of multidrug resistance by a novel quinoline derivative, MS-209

MS-209, a novel quinoline derivative, was examined for its reversing effect on multidrug-resistant tumor cells. MS-209 at 1–10 μM completely reversed resistance against vincristine (VCR) in vitro in multidrug-resistant variants of mouse leukemia P388 cells (VCR-resistant P388/VCR and Adriamycin (ADM)-resistant P388/ADM) and human leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM). MS-209 at 1–10 μM also completely reversed resistance against ADM in vitro in P388/VCR cells, K562/VCR cells, and K562/ADM cells. In ADM-resistant P388 (P388/ADM) cells, however, ADM resistance was only partially reversed at the MS-209 concentrations tested. MS-209 enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice. When MS-209 was given p.o. at 80 mg/kg twice a day (total dose, 160 mg/kg per day) with 100 μg/kg VCR, a treated/control (T/C) value of 155% was obtained. MS-209 also enhanced the chemotherapeutic effect of ADM in P388/ADM-bearing mice. The most prominent effects were obtained when MS-209 was given with 2 mg/kg ADM, yielding T/C values of 150%–194% for the combined treatment at an MS-209 dose of 200–450 mg/kg. MS-209 inhibited [3H]-azidopine photolabeling of P-glycoprotein efficiently. Furthermore, the accumulation of ADM in K562/ADM cells was increased more eficiently by MS-209 than by verapamil. These results indicate that MS-209, like verapamil, directly interacts with P-glycoprotein and inhibits the active efflux of antitumor agents, thus overcoming multidrug resistance in vitro and in vivo.

Inhibitors of CLK Protein Kinases Suppress Cell Growth and Induce Apoptosis by Modulating Pre-mRNA Splicing

Accumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.

TAK-441, a novel investigational smoothened antagonist, delays castration-resistant progression in prostate cancer by disrupting paracrine hedgehog signaling

Hedgehog (Hh) signaling is a highly conserved intercellular and intracellular communication mechanism that governs organogenesis and is dysregulated in cancers of numerous tissues, including prostate. Up‐regulated expression of the Hh ligands, Sonic (Shh) and Desert (Dhh), has been reported in androgen‐deprived and castration‐resistant prostate cancer (CRPC). In a cohort of therapy naive, short‐ and long‐term neoadjuvant hormone therapy‐treated (NHT), and CRPC specimens, we observed elevated Dhh expression predominantly in long‐term NHT specimens and elevated Shh expression predominantly in CRPC specimens. Together with previously demonstrated reciprocal signaling between Shh‐producing prostate cancer (PCa) cells and urogenital mesenchymal fibroblasts, these results suggest that castration‐induced Hh expression promotes CRPC progression through reciprocal paracrine signaling within the tumor microenvironment. We tested whether the orally available Smoothened (Smo) antagonist, TAK‐441, could impair castration‐resistant progression of LNCaP PCa xenografts by disrupting paracrine Hh signaling. Although TAK‐441 or cyclopamine did not affect androgen withdrawal‐induced Shh up‐regulation or viability of LNCaP cells, castration‐resistant progression of LNCaP xenografts was significantly delayed in animals treated with TAK‐441. In TAK‐441‐treated xenografts, expression of murine orthologs of the Hh‐activated genes, Gli1, Gli2 and Ptch1, was substantially suppressed, while expression of the corresponding human orthologs was unaffected. As androgen‐deprived LNCaP cells up‐regulate Shh expression, but are not sensitive to Smo antagonists, these studies indicate that TAK‐441 leads to delayed castration‐resistant progression of LNCaP xenografts by disrupting paracrine Hh signaling with the tumor stroma. Thus, paracrine Hh signaling may offer unique opportunities for prognostic biomarker development, drug targeting and therapeutic response monitoring of PCa progression.

Relationship between multidrug resistant gene expression and multidrug resistant-reversing effect of MS-209 in various tumor cells

MS-209 is a novel quinoline compound which can overcome multidrug resistance (MDR) both in vitro and in vivo, while having a low level of side effects, and is now being evaluated in a clinical phase II study. Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantitative the expression levels ofMDR genes in various mouse and human tumor cell lines. TheMDR gene and the βactin gene, as the internal reference standard, were coamplified separately, and the relative expression of theMDR gene was represented by the MDR/β actin ratio. The in vitro MDR-reversing effect of MS-209 was then compared with theMDR gene expression (MDR/β actin ratio). We found a significant correlation between these two parameters. Moreover, a significant correlation was also observed between the level of expression of theMDR1 gene and that of P-glycoprotein in human cell lines. Therefore, the efficacy of MS-209 seems to specifically depend on the level ofMDR gene expression (P-glycoprotein). From these observations, it is suggested that RT-PCR assays ofMDR1 gene in tumor biopsy specimens might be an effective means to predict the response of tumor cells to combination therapy with MS-209.

CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3′-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription.

A novel synthetic DNA minor groove binder, MS-247: antitumor activity and cytotoxic mechanism

Purpose: MS-247 is a novel synthetic compound possessing a DNA-binding moiety and a DNA-alkylating residue, chlorambucil. In this study, we evaluated the antitumor activity of MS-247 against murine tumor cell lines and its effects on DNA molecules in both cell-free and cellular systems. Methods: The in vitro cytotoxic activity of MS-247 was evaluated against four murine tumor cell lines, P388, L1210, Colon26 and B16, and its in vivo antitumor activity was also tested in comparison with Adriamycin (ADM), cisplatin (CDDP) and paclitaxel. The ability of MS-247 to associate with the DNA minor groove was assessed by measuring quenching of Hoechst 33342 fluorescence. DNA-DNA interstrand crosslinks (ICL) were detected by an alkaline elution assay for cellular DNA and a band-shift assay using the plasmid pBR322. The effects of MS-247 on macromolecule synthesis (DNA, RNA and proteins) were examined by measuring incorporation of the radiolabeled precursors. Results: MS-247 exhibited in vitro cytotoxicity with IC50 values ranging 11 to 500 nM, and MS-247 given i.v. showed strong in vivo antitumor activity against i.p.-implanted L1210 leukemia cells and s.c.-implanted Colon26 carcinoma cells, and moderate activity against i.p.-implanted P388 leukemia cells but no apparent activity against s.c.-implanted B16 melanoma cells. MS-247 reversibly displaced Hoechst 33342 bound to DNA within a few minutes, and irreversibly formed ICL within 1–6 h in both the cell-free system and the cellular system. These results suggest that an association of MS-247 with the DNA minor groove occurred more quickly than ICL formation. The inhibition of DNA synthesis was more prominent than the inhibition of RNA and protein synthesis in L1210 cells exposed to MS-247, and a 6-h incubation with MS-247, which formed apparent ICL in the cellular system, strongly inhibited DNA synthesis. This result suggests that impairment of DNA replication preceded the inhibition of RNA and protein synthesis and that ICL formation greatly contributed to the inhibition of macromolecule synthesis. Conclusion: The results of this study suggest that MS-247 exerts its cytotoxic effect through impairment of DNA function by getting into the minor groove of DNA and subsequently forming ICL. MS-247 has potent antitumor activity with a different spectrum from the activity of clinically proven antitumor agents such as paclitaxel, ADM and CDDP against several murine tumor cell lines. This result suggests that MS-247 may be useful for the treatment of human cancers.

Abstract 4952: A novel smoothened antagonist, TAK-441, delays castration-resistant progression in prostate cancer by disrupting paracrine hedgehog signaling.

Hedgehog (Hh) signaling is a highly conserved inter- and intracellular communication mechanism that governs organogenesis and is dysregulated in cancers of numerous tissues, including prostate. Up-regulated expression of the Hh ligands, Sonic (Shh) and Desert (Dhh), has been reported in androgen-deprived and castration-resistant prostate cancer (CRPC). In a cohort of therapy naive, short- and long-term neoadjuvant hormone therapy-treated (NHT), and CRPC specimens, we observed elevated Dhh expression predominantly in long-term NHT specimens and elevated Shh expression predominantly in CRPC specimens. Together with previously demonstrated reciprocal signaling between Shh-producing prostate cancer (PCa) cells and urogenital mesenchymal fibroblasts, these results suggest that castration-induced Hh expression promotes CRPC progression through reciprocal paracrine signaling within the tumor microenvironment. We tested whether the orally available Smoothened (Smo) antagonist, TAK-441, could impair castration-resistant progression of LNCaP PCa xenografts by disrupting paracrine Hh signaling. While TAK-441 or cyclopamine did not affect androgen withdrawal-induced Shh up-regulation or viability of LNCaP cells, castration-resistant progression of LNCaP xenografts was significantly delayed in animals treated with TAK-441. In TAK-441-treated xenografts, expression of murine orthologues of the Hh-activated genes, Gli1, Gli2, and Ptch1, was substantially suppressed, while expression of the corresponding human orthologues was unaffected. Since androgen-deprived LNCaP cells up-regulate Shh expression, but are not sensitive to Smo antagonists, these studies indicate that TAK-441 leads to delayed castration-resistant progression of LNCaP xenografts by disrupting paracrine Hh signaling with the tumor stroma. Thus, paracrine Hh signaling may offer unique opportunities for prognostic biomarker development, drug targeting and therapeutic response monitoring of PCa progression. Citation Format: Mazyar Ghaffari, Naokazu Ibuki, Irene Iu, Mitali Pandey, Ladan Fazli, Masahide Kashiwagi, Hideaki Tojo, Osamu Nakanishi, Martin E. Gleave, Michael E. Cox. A novel smoothened antagonist, TAK-441, delays castration-resistant progression in prostate cancer by disrupting paracrine hedgehog signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4952. doi:10.1158/1538-7445.AM2013-4952