Anti‐tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC‐dependent vulnerability
2018; Springer Nature; Volume: 10; Issue: 6 Linguagem: Inglês
10.15252/emmm.201708289
ISSN1757-4684
AutoresKenichi Iwai, Masahiro Yaguchi, Kazuho Nishimura, Yukiko Yamamoto, Toshiya Tamura, Daisuke Nakata, Ryo Dairiki, Yoichi Kawakita, Ryo Mizojiri, Y. ITO, Moriteru Asano, Hironobu Maezaki, Yusuke Nakayama, Misato Kaishima, Kôzô Hayashi, Mika Teratani, Shuichi Miyakawa, Misa Iwatani, Maki Miyamoto, Michael G. Klein, Wes Lane, G. Snell, Richard Tjhen, Xingyue He, Sai Murali Krishna Pulukuri, Toshiyuki Nomura,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoResearch Article16 May 2018Open Access Source DataTransparent process Anti-tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC-dependent vulnerability Kenichi Iwai Kenichi Iwai Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Masahiro Yaguchi Masahiro Yaguchi Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Kazuho Nishimura Kazuho Nishimura Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Yukiko Yamamoto Yukiko Yamamoto Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Toshiya Tamura Toshiya Tamura Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Daisuke Nakata Daisuke Nakata Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Ryo Dairiki Ryo Dairiki Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Yoichi Kawakita Yoichi Kawakita Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Ryo Mizojiri Ryo Mizojiri Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Yoshiteru Ito Yoshiteru Ito Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Moriteru Asano Moriteru Asano Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Hironobu Maezaki Hironobu Maezaki Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Yusuke Nakayama Yusuke Nakayama Integrated Technology Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Misato Kaishima Misato Kaishima Integrated Technology Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Kozo Hayashi Kozo Hayashi Integrated Technology Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Mika Teratani Mika Teratani Integrated Technology Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Shuichi Miyakawa Shuichi Miyakawa Biomolecular Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Misa Iwatani Misa Iwatani Biomolecular Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Maki Miyamoto Maki Miyamoto Drug Metabolism & Pharmacokinetics Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Michael G Klein Michael G Klein Department of Structural Biology, Takeda California Inc., San Diego, CA, USA Search for more papers by this author Wes Lane Wes Lane Department of Structural Biology, Takeda California Inc., San Diego, CA, USA Search for more papers by this author Gyorgy Snell Gyorgy Snell Department of Structural Biology, Takeda California Inc., San Diego, CA, USA Search for more papers by this author Richard Tjhen Richard Tjhen Department of Structural Biology, Takeda California Inc., San Diego, CA, USA Search for more papers by this author Xingyue He Xingyue He Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, MA, USA Search for more papers by this author Sai Pulukuri Sai Pulukuri Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, MA, USA Search for more papers by this author Toshiyuki Nomura Corresponding Author Toshiyuki Nomura [email protected] orcid.org/0000-0003-4122-0301 Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Kenichi Iwai Kenichi Iwai Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Masahiro Yaguchi Masahiro Yaguchi Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Kazuho Nishimura Kazuho Nishimura Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Yukiko Yamamoto Yukiko Yamamoto Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Toshiya Tamura Toshiya Tamura Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Daisuke Nakata Daisuke Nakata Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Ryo Dairiki Ryo Dairiki Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Yoichi Kawakita Yoichi Kawakita Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Ryo Mizojiri Ryo Mizojiri Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Yoshiteru Ito Yoshiteru Ito Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Moriteru Asano Moriteru Asano Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Hironobu Maezaki Hironobu Maezaki Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Yusuke Nakayama Yusuke Nakayama Integrated Technology Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Misato Kaishima Misato Kaishima Integrated Technology Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Kozo Hayashi Kozo Hayashi Integrated Technology Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Mika Teratani Mika Teratani Integrated Technology Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Shuichi Miyakawa Shuichi Miyakawa Biomolecular Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Misa Iwatani Misa Iwatani Biomolecular Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Maki Miyamoto Maki Miyamoto Drug Metabolism & Pharmacokinetics Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Michael G Klein Michael G Klein Department of Structural Biology, Takeda California Inc., San Diego, CA, USA Search for more papers by this author Wes Lane Wes Lane Department of Structural Biology, Takeda California Inc., San Diego, CA, USA Search for more papers by this author Gyorgy Snell Gyorgy Snell Department of Structural Biology, Takeda California Inc., San Diego, CA, USA Search for more papers by this author Richard Tjhen Richard Tjhen Department of Structural Biology, Takeda California Inc., San Diego, CA, USA Search for more papers by this author Xingyue He Xingyue He Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, MA, USA Search for more papers by this author Sai Pulukuri Sai Pulukuri Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, MA, USA Search for more papers by this author Toshiyuki Nomura Corresponding Author Toshiyuki Nomura [email protected] orcid.org/0000-0003-4122-0301 Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan Search for more papers by this author Author Information Kenichi Iwai1, Masahiro Yaguchi1, Kazuho Nishimura1, Yukiko Yamamoto1, Toshiya Tamura1, Daisuke Nakata1, Ryo Dairiki1, Yoichi Kawakita1, Ryo Mizojiri1, Yoshiteru Ito1, Moriteru Asano1, Hironobu Maezaki1, Yusuke Nakayama2, Misato Kaishima2, Kozo Hayashi2, Mika Teratani2, Shuichi Miyakawa3, Misa Iwatani3, Maki Miyamoto4, Michael G Klein5, Wes Lane5, Gyorgy Snell5, Richard Tjhen5, Xingyue He6, Sai Pulukuri6 and Toshiyuki Nomura *,1 1Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan 2Integrated Technology Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan 3Biomolecular Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan 4Drug Metabolism & Pharmacokinetics Research Laboratories, Takeda Pharmaceutical Company, Limited, Fujisawa, Japan 5Department of Structural Biology, Takeda California Inc., San Diego, CA, USA 6Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, MA, USA *Corresponding author. Tel: +81-466-32-1948; Fax: +81-466-29-4461; E-mail: [email protected] EMBO Mol Med (2018)10:e8289https://doi.org/10.15252/emmm.201708289 See also: F Salvador & RR Gomis (June 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The modulation of pre-mRNA splicing is proposed as an attractive anti-neoplastic strategy, especially for the cancers that exhibit aberrant pre-mRNA splicing. Here, we discovered that T-025 functions as an orally available and potent inhibitor of Cdc2-like kinases (CLKs), evolutionally conserved kinases that facilitate exon recognition in the splicing machinery. Treatment with T-025 reduced CLK-dependent phosphorylation, resulting in the induction of skipped exons, cell death, and growth suppression in vitro and in vivo. Further, through growth inhibitory characterization, we identified high CLK2 expression or MYC amplification as a sensitive-associated biomarker of T-025. Mechanistically, the level of CLK2 expression correlated with the magnitude of global skipped exons in response to T-025 treatment. MYC activation, which altered pre-mRNA splicing without the transcriptional regulation of CLKs, rendered cancer cells vulnerable to CLK inhibitors with synergistic cell death. Finally, we demonstrated in vivo anti-tumor efficacy of T-025 in an allograft model of spontaneous, MYC-driven breast cancer, at well-tolerated dosage. Collectively, our results suggest that the novel CLK inhibitor could have therapeutic benefits, especially for MYC-driven cancer patients. Synopsis MYC oncogene is a highly valuable target for cancer therapy. A newly discovered CLK inhibitor, T-025, exhibited anti-tumor efficacies against cancers with a high CLK2 expression, as well as against MYC-driven cancers, as a result of attacking a MYC-dependent vulnerability. A novel and potent CLK inhibitor, T-025, was discovered and characterized as a new pre-mRNA splicing modulating anti-cancer agent. T-025 exhibited in vivo anti-tumor efficacy in both cell line xenograft tumors and mice spontaneous allograft tumors. The expression level of CLK2 in each cell line was associated with the growth-inhibitory sensitivity and magnitude of alternative skipped exons in response to T-025 treatment. T-025 showed a higher growth inhibition effect on MYC-amplified solid cancer cell lines than on non-MYC-amplified solid cancer cell lines and induced synergistic cell death with ectopic MYC activation. Introduction Pre-mRNA splicing represents a critically important step for various processes such as development, differentiation, and disease (Chabot & Shkreta, 2016; Inoue et al, 2016; Vuong et al, 2016). Recently, there has been an increased interest in mutations of pre-mRNA splicing factors and their roles on oncogenesis in hematological cancers (Yoshida et al, 2011; Dvinge et al, 2016). High-frequency mutations of SF3B1 or SRSF2 have been described in patients with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia, and acute myeloid leukemia (AML) (Meggendorfer et al, 2012; Donaires et al, 2016; Papaemmanuil et al, 2016). In addition, mutations in splicing-related genes have also been found in various solid cancers, including lung, breast, and pancreatic cancers (Dvinge et al, 2016). In parallel with clinical observations, the attractiveness of the pharmacological modulation of pre-mRNA splicing as a cancer therapy strategy has also increased (Bonnal et al, 2012; Lee & Abdel-Wahab, 2016; Salton & Misteli, 2016). Members of the evolutionarily conserved Cdc2-like kinase (CLK) family, which comprises CLK1–4, play biologically important roles in pre-mRNA splicing by regulating serine–arginine-rich (SR) proteins. Upon phosphorylation by CLK, SR proteins relocate from nuclear speckles to the spliceosome, where they interact with pre-mRNA to facilitate exon recognition in the splicing machinery (Colwill et al, 1996; Ghosh & Adams, 2011; Corkery et al, 2015). Both the RNAi-mediated depletion of CLK and chemical inhibition of CLK modulate alternative splicing (AS), particularly the skipped exon (SE) type of AS, resulting in the suppression of cell proliferation (Muraki et al, 2004; Fedorov et al, 2011; Dominguez et al, 2016; Sako et al, 2017). Recently, observations that the well-known proto-oncogene MYC controls pre-mRNA splicing and that MYC-driven cancers are susceptible to spliceosome inhibition have highlighted the use of pharmacological splicing modulators as promising anti-cancer agents for MYC-driven cancers (Hsu et al, 2015; Koh et al, 2015). Aberrant MYC activation, mediated by MYC translocation, amplification, and mutation, is a frequent event in various hematological and solid cancers (Dang, 2012; Kress et al, 2015). Although numerous studies have successfully targeted these cancers, MYC remains a highly significant therapeutic target (Delmore et al, 2011; Kessler et al, 2012; Cermelli et al, 2014; Camarda et al, 2016; Horiuchi et al, 2016). Here, we hypothesized that CLK inhibition might function as a novel pre-mRNA splicing modulation-based anti-cancer strategy, especially for MYC-driven cancers. Our findings, which demonstrate the ability of an orally available CLK inhibitor to effectively target MYC-driven cancers, address a novel biological interaction of CLK inhibition with MYC activation. Results T-025 is a highly potent CLK inhibitor To investigate an anti-tumor efficacy of a CLK inhibitor in animal models, we developed a new class of CLK inhibitors. Specifically, we chemically modified a 7H-pyrrolo[2,3-d]pyrimidine derivative by measuring the CLK2 inhibitory activity and determining the co-crystal structure with CLK2. After optimizing the lead compound, we discovered an orally available and highly potent CLK2 inhibitor, T-025 (N2-methyl-N4-[pyrimidin-2-ylmethyl]-5-[quinolin-6-yl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine; Fig 1A). The co-crystal structural analysis revealed that T-025 inserts into the CLK2 ATP-binding site and interacts with Glu244 and Leu246 in the CLK2 hinge region (Fig 1B). However, a KINOMEScan-based kinase selectivity evaluation identified T-025 as a highly selective inhibitor of CLK (Kd values to CLK1, CLK2, CLK3, and CLK4 were 4.8, 0.096, 6.5, and 0.61 nmol/l, respectively) and dual-specificity tyrosine-phosphorylation-regulated kinase 1 (DYRK1) family proteins (Kd values to DYRK1 and DYRK1B were 0.074 and 1.5 nmol/l, respectively) (Fig 1C). No other kinases outside of the DYRK1 family had Kd values < 30 nmol/l, suggesting that T-025 is a potent CLK/DYRK1 inhibitor with > 300-fold enhanced selectivity for these kinases than to other kinases. Figure 1. T-025 is a novel and potent CLK inhibitor Chemical structure of T-025. X-ray crystal structure of CLK2 with T-025 (see also Appendix Fig S7 for detailed co-crystal structure data). Kd values of T-025 against CLK and DYRK family kinases and the result of a panel of various kinases. N.T., not tested. Binding activities of T-025 at 300 nmol/l were measured against 468 kinases (n = 2). Download figure Download PowerPoint T-025 induced skipping exon, resulting in anti-proliferative effect in MDA-MB-468 in vitro and in vivo Our previous report shows that CLK inhibitors suppress cell proliferation and induce cell death in MDA-MB-468 cells, accompanied by several CLK-associated downstream effects including a global modulation of AS events (Araki et al, 2015). In line with our previous report, treatment of MDA-MB-468 cells with T-025 suppressed the phosphorylation of SR protein detected with 1H4 monoclonal antibody (Fig 2A), resulting in growth inhibition (Fig 2B) with apoptosis as detected by an increase in the sub-G1 population (fluorescence-activated cell sorting analysis; Appendix Fig S1A). To further assess the in vitro cellular inhibition of CLK, we generated a new antibody that recognized phosphorylated Ser98 of CLK2 (pCLK2), which is reported as an auto-phosphorylation of CLK2 (Rodgers et al, 2010), and our in vitro assays also supported this previous finding (Appendix Fig S2A). Immunoblotting with the pCLK2 antibody revealed treatment with T-025 decreased both pCLK2 and CLK2 (Fig 2A), and quantified band intensities showed relative phosphorylation level was reduced in a dose-dependent manner (Appendix Fig S1B). Considering with a previous finding that kinase activity of CLK2 contributed to stability of CLK2 protein (Rodgers et al, 2010), our result suggested that T-025 inhibited the kinase activity of CLK2 in cultured MDA-MB-468 cells, leading to the degradation of CLK2. Figure 2. T-025 exhibited anti-tumor efficacy in MDA-MB-468 xenografts A. MDA-MB-468 cells were treated with T-025 for 6 h, and phosphorylation levels were detected via immunoblotting with phospho-specific antibody. B. T-025 dose–response curve in MDA-MB-468 cells for 72-h treatment. The black dotted line indicates the relative ATP value prior to treatment (Day 0). C. The number of AS events modulated by T-025 treatment for 6 h in MDA-MB-468 cells. The numbers of AS events with a BF > 20 and |ΔPSI| > 0.1 were counted and categorized according to the AS type (SE, skipped exon; RI, retained intron; A5′SS, alternative 5′-splice-site; A3′SS, alternative 3′-splice-site; MEE, mutually exclusive exon; AFE, alternative first exon; ALE, alternative last exon). D–F. MDA-MB-468 xenograft tumors treated with 50 mg/kg of T-025 were sampled and analyzed by immunohistochemistry (F), immunoblotting (E), or RT–PCR (F). Representative images of pCLK2 stained tumors and scale bar (30 μm) are shown. Also shown: The mean unbound plasma T-025 concentration was calculated using protein binding after oral administration. G, H. Anti-tumor efficacy of T-025 in MDA-MB-468 xenograft models. T-025 was administered twice daily on 2 days per week (red arrows). Tumor volume (G) and body weight (H) during the efficacy study are shown as means ± s.e.m. (n = 5). Data information: In (B, D, and F), data are shown as the means ± s.d. of three independent experiments (n = 3). In (G and H), an unpaired Student's t-test was performed. Source data are available online for this figure. Source Data for Figure 2 [emmm201708289-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint We next evaluated a CLK inhibition-mediated AS. As for a specific CLK inhibition-dependent AS event, we confirmed that T-025 induced the skipping exon 7 of RPS6KB1 (Appendix Fig S1C), which is also induced by other CLK inhibitors and RNAi-mediated depletion of CLK2 (Araki et al, 2015), followed by a reduction in the protein level of S6K (Appendix Fig S1D). A whole transcriptome RNA sequencing (RNA-Seq) and its consequent splicing analysis using a mixture of isoforms (MISO) (Katz et al, 2010) revealed that T-025 at the concentrations of 30, 100, and 300 nmol/l (approximate IC50 values for growth inhibition) largely modulated AS via SE in a dose-dependent manner (Fig 2C). Further, when we carefully screened the observed AS events, we found that the skipping of exon 11 of BCLAF1, as an additional downstream AS event, was one of the most sensitive and largest events among the alternative SEs (Appendix Fig S1E). Together, these results in cultured MDA-MB-468 cells indicated that T-025-induced cell death, accompanied by the phenotypes that are previously observed by other CLK inhibitors or RNAi-mediated depletion. Then, we evaluated T-025 in an animal model. The pharmacokinetics evaluation of T-025 in nude mice revealed that the unbound plasma concentrations of T-025 were 554, 97, and 104 nmol/l at 2, 4, and 8 h, respectively, following the oral administration of T-025 at 50 mg/kg (Fig 2D); these concentrations were sufficient to suppress the CLK-dependent phosphorylation and to induce skipping exon in various genes including exon 7 of the RPS6KB1 (Fig 2C and Appendix Fig S1C). Therefore, we performed a pharmacodynamics assessment of T-025 at 50 mg/kg in MDA-MB-468 xenograft tumors, and found that pCLK2 detected with immunohistochemistry and immunoblotting decreased from 2 to 8 h after oral administration (Fig 2D and E), followed by a reduction in the RPS6KB1 exon 7 and BCLAF1 exon 11 percentage splice-in (PSI) values (Fig 2F). An efficacy study in a MDA-MB-468 xenograft model was performed with a regimen of twice daily on 2 days per week schedule. The treatment yielded profound anti-tumor effects, illustrating that the tumor volumes had shrunk relative to the initial volumes at the end of the 3-week treatment cycle (Fig 2G). Additionally, although the T-025 dosage was near the maximum tolerated dose, it was apparently well tolerated with a < 10% nadir body weight loss (Fig 2H). Taken together, these results using MDA-MB-468 xenografts suggested T-025 had an anti-tumor efficacy at tolerable dosage, accompanied by the modulation of downstream markers. Solid cancer cell lines harboring MYC amplification or high CLK2 expression were more sensitive to T-025 For the characterization of T-025 as an anti-tumor agent, we subjected T-025 to a panel of growth inhibition assays in 240 cancer cell lines and a subsequent unbiased bioinformatics analysis by utilizing OncoPanel 240. Consequently, T-025 exerted a broad range of anti-proliferative activities in both hematological and solid cancer cell lines (IC50 values: 30–300 nmol/l), sensitivity to this drug was not organ of origin- or disease type-dependent (Fig 3A). The unbiased bioinformatics analysis flagged several biomarker candidates that were significantly associated with sensitivity; analysis of mRNA expressions identified genes that were significantly expressed higher/lower in the top 25% sensitive cancer cell lines than in the bottom 25% cancer cell lines (Fig EV1A). In the sensitivity-associated mRNAs, we found that the expression of CLK2 was significantly higher in the sensitive cell lines with a P-value of 1.58E-09, which is much lower than the P-value from other CLK family or DYRK family. Considering the primary target of T-025 as well as the oncogenic role of CLK2 in breast cancer (Yoshida et al, 2015), we hypothesized that cancer cells with higher CLK2 expression were dependent on the CLK2 kinase activity for their survival. Another unbiased analysis using mutations and copy number alterations identified 14 statistically significant biomarker candidates, including amplified- or mutated-MYC (Fig EV1B). Recent reports that spliceosome inhibition is more effective against MYC-driven cancer (Hsu et al, 2015; Koh et al, 2015) persuaded us to validate this preliminary analysis result that MYC-mutated or MYC-amplified cancer cell lines were more sensitive. Figure 3. T-025 exhibited a board range of anti-proliferative effect in a panel of cancer cell lines IC50 values of T-025 in 240 cell lines. Each gray circle indicates a single cell line sorted according to its original organ/disease type. Correlation between T-025 sensitivity and MYC amplification status in solid cancer cell lines (n = 150). Each bar indicates a single cell line, and red bar indicate cell lines with amplified MYC. Correlation between T-025 sensitivity and CLK2 expression status in solid cancer cell lines (n = 150). Each bar indicates a single cell line, and red, gray, or blue bar indicate cell lines with high, medium, or low CLK2. Data information: In (B and C), a Mann–Whitney test was performed. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Result of unbiased bioinformatics analysis Genes significantly associated with the sensitivity are shown ranked by their P-value. Also shown are the results of CLK and DYRK family genes. Representative gene alterations and their statistical powers. Correlation between T-025 sensitivity and gene alterations in each cell line. The P-values were determined using Student's t-test and Fisher's exact test. Download figure Download PowerPoint We further analyzed 169 (19 hematological and 150 solid cancer) cell lines of 240 cell lines, whose genomic data such as expression, mutation, and copy number variation (CNV) could be obtained from the Cancer Cell Line Encyclopedia (CCLE) database (Barretina et al, 2012; Fig EV2A). With a careful evaluation of the MYC genetic status to include the role of mutation and to remove passenger mutations, we found that solid cancer cell lines exhibiting MYC alteration (only MYC-amplified cell lines were found) were significantly more sensitive to T-025 than other solid cancer cell lines (P = 0.0042, Fig 3B). Conversely, in the 19 hematological cancer cell lines, we did not observe higher sensitivity associated with MYC alterations (amplified, driver-mutated, translocated; Fig EV2B and C). Since other MYC family proto-oncogenes such as N-Myc or L-Myc share several functions with MYC (Malynn et al, 2000), we additionally considered the gene status of other MYC family; however, we could not find any sensitivity correlating with MYC family gene alteration in hematological cancer cell lines (Fig EV2D). Interestingly, solid cancer cell lines with amplified MYC family genes, such as MYC, MYCN, or MYCL, were more sensitive than those without amplified MYC family genes with P-value at 0.0010 (Fig EV2E), suggesting that common downstream effects of MYC family genes are involved in the sensitivity to T-025. Notably, we found that half of the top 20% sensitive solid cancer cell lines (15 out of 30 cell lines) harbored amplified MYC family genes. Regarding the expression of CLK2, when we divided solid or hematological cancer cell lines into three groups on the basis of CLK2 expression (high, medium, and low), solid cancer cell lines with high CLK2 expression were significantly more sensitive than those with low CLK2 expression (P < 0.0001, Fig 3C), but not hematological cancer cell lines with high CLK2 expression (Fig EV2F). In summary, these analyses revealed that the expression of CLK2 or amplified MYC was statistically associated with the sensitivity to T-025 in the solid cancer cell lines. Click here to expand this figure. Figure EV2. Additional analysis of Oncopanel Analysis flow of the tested cell lines. IC50 value, CLK2 expression, and MYC status of 19 hematological cancer cell lines are shown. Correlation between T-025 sensitivity and MYC status in the hematological cancer cell lines (n = 19). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC. Correlation between T-025 sensitivity and MYC family gene status in the hematological cancer cell lines (n = 19). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC family gene. Correlation between T-025 sensitivity and MYC family gene status in the solid cancer cell lines (n = 150). Each bar indicates a single cell line, and colored bar indicates cell lines with altered MYC family gene. Correlation between T-025 sensitivity and CLK2 expression in the hematological cancer cell lines (n = 19). Each bar indicates a single cell line, and blue, gray, or red bar indicates cell lines with high, medium, or low CLK2. Data information: In (C–F), a Mann–Whitney test was performed. Download figure Download PowerPoint T-025 modulated AS with a magnitude depending on CLK2 expression To evaluate the hypothesis generated from the bioinformatics analysis, we first assessed the protein level of CLK2 in 56 various cancer cell lines because the protein level of CLK2 is also regulated by ubiquitination-dependent degradation (Bidinosti et al, 2016). The findings of the immunoblot analysis of CLK2 revealed that the protein levels of cell lines had a large variation, and MDA-MB-468 cells appeared to express a relatively high CLK2 protein level (11th in 56 cell lines, Appendix Fig S3); furthermore, the protein expression level of CLK2 significantly correlated with sensitivity (Fig 4A). In addition, we found that T-025 showed high in vitro growth suppressive effect in an additional cancer cell line with higher CLK2 protein, that is, lung cancer NCI-H1048; also, T-025 caused moderate an
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