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Background: Chromosomal inversions involving Anaplastic lymphoma kinase (ALK) and Echinoderm Microtubule Associated Protein Like 4 (EML4) generate a fusion protein EML4-ALK with the constitutive ALK kinase activity in non-small cell lung carcinoma (NSCLC). The basic understanding of EML4-ALK remains insufficient due to the lack of functional studies using normal human cells. Material and Method: We investigate the activities of EML4-ALK in mortal and immortalized normal human cells. Results: The expression of EML4-ALK in normal, mortal human fibroblasts caused, through its ALK kinase activity, the early induction of cellular senescence with senescence-associated β-galactosidase activity, upregulation of p16INK4A and p21WAF1, telomere shortening and fusions, and accumulated DNA damage. In contrast, when EML4-ALK was expressed in telomerase reverse transcriptase (hTERT)-immortalized normal human fibroblasts, the cells showed accelerated proliferation and anchorage-independent growth, revealing its transformation activity. No chromosome aberration, no mutation or loss of p53, nor impairment of the p16INK4A response was associated with this transformation, likely reflecting clinical features of EML4-ALK-positive NSCLC. In both mortal and immortalized cells, EML4-ALK induced the phosphorylation of STAT3, which is involved in both cellular senescence and transformation. EML4-ALK was also able to transform hTERT-immortalized human bronchial epithelial cells, a cell type relevant to NSCLC. Conclusions: Our data not only validate that EML4-ALK functions as an oncogene in human cells, but also suggest that telomerase-mediated immortalization manifests the oncogenic activity of EML4-ALK, switching from its senescence-inducing activity. This study also provides the isogenic pairs of human cell lines with and without EML4-ALK as an in vitro model for screening and testing candidate drugs. Citation Format: Akihiko Miyanaga, Izumi Horikawa, Masaru Matsumoto, Takahiro Oike, Jessica Beck, Hiromi Tanaka, Ana I. Robles, Masahiro Seike, Akihiko Gemma, Curtis C. Harris. Cellular senescence and transformation induced by an oncogenic EML4-ALK fusion gene in normal and immortalized human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4648.

The occurrence of inactivating mutations in SWI/SNF chromatin-remodeling genes in common cancers has attracted a great deal of interest. However, mechanistic strategies to target tumor cells carrying such mutations are yet to be developed. This study proposes a synthetic-lethality therapy for treating cancers deficient in the SWI/SNF catalytic (ATPase) subunit, BRG1/SMARCA4. The strategy relies upon inhibition of BRM/SMARCA2, another catalytic SWI/SNF subunit with a BRG1-related activity. Immunohistochemical analysis of a cohort of non–small-cell lung carcinomas (NSCLC) indicated that 15.5% (16 of 103) of the cohort, corresponding to preferentially undifferentiated tumors, was deficient in BRG1 expression. All BRG1-deficient cases were negative for alterations in known therapeutic target genes, for example, EGFR and DDR2 gene mutations, ALK gene fusions, or FGFR1 gene amplifications. RNA interference (RNAi)–mediated silencing of BRM suppressed the growth of BRG1-deficient cancer cells relative to BRG1-proficient cancer cells, inducing senescence via activation of p21/CDKN1A. This growth suppression was reversed by transduction of wild-type but not ATPase-deficient BRG1. In support of these in vitro results, a conditional RNAi study conducted in vivo revealed that BRM depletion suppressed the growth of BRG1-deficient tumor xenografts. Our results offer a rationale to develop BRM-ATPase inhibitors as a strategy to treat BRG1/SMARCA4–deficient cancers, including NSCLCs that lack mutations in presently known therapeutic target genes. Cancer Res; 73(17); 5508–18. ©2013 AACR.

Liver cancer is third leading cause of cancer-related deaths worldwide. A tremendous burden of this disease is evident in that there is an estimated rise in liver cancer incidence from 39,239 in 2016 to 95,374 by 2020. This bleak statistic serves as a motivation to search for early diagnosis biomarkers, especially considering that the 5-year relative survival rate for people with localized disease is ~30.5%, and for regional stage is only 10.7%. Hepatocellular Carcinoma (HCC) is the most common primary malignant tumor of the liver, and is considered to be the third leading cause of all cancer-related deaths and fifth common cancer worldwide. Hepatitis virus B and C infections, fatty and alcoholic liver disease are established risk factors for developing HCC. Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy. Liver fluke, a chronic parasitic disease of the bile ducts, is a risk factor for developing CCA; otherwise not enough is known about the etiology of this form of liver cancer. We initially conducted a pilot study in the NCI-MD cohort comprising 98 HCC cases, 101 high risk subjects and 95 controls, to evaluate whether previously identified urinary metabolite biomarkers of lung cancer were also predictive of liver cancer, given preliminary evidence that they were universally elevated in several cancer types. Thus, we leveraged ultraperformance liquid chromatography coupled to tandem mass-spectrometry (UPLC-MS/MS) for quantitation of creatine riboside, N-acetylneuraminic acid, cortisol sulfate, and a lipid molecule designated as 561+. This study resulted in findings that all four aforementioned metabolites are significantly increased in HCC cases compared to both, population controls and, more importantly, high risk subjects. We next evaluated whether our previously described findings from the NCI-MD liver case-control study validate in the TIGER-LC cohort (n=370 cases, 471 high risk subjects, 251 controls), where CCA is highly prevalent. We confirmed that indeed the four metabolites were significantly increased in HCC cases compared to both, controls and high risk subjects in TIGER-LC. Most importantly, we also showed that these metabolites are also deregulated in CCA, as their levels are significantly elevated in CCA cases when compared to both, controls and high risk subjects. Furthermore, we showed in the Receiver Operating Characteristic (ROC) analysis a robust ability of the four-metabolite profile to classify both, HCC (AUC=0.75) and CCA (AUC=0.81) compared to high risk subjects. Additionally, the four-metabolite profile performed better in classifying CCA than a clinically utilized CCA tumor marker, CA19-9, and their combination led to a significantly improved classifier (AUC=0.88, P=9.4E-7). Currently, we are investigating a number of pesticides and herbicides used in the areas of Thailand from where TIGER-LC subjects were recruited, to investigate whether these putative carcinogens participate in the etiology of CCA. Citation Format: Majda Haznadar, Kristopher W. Krausz, Christopher M. Diehl, Elise D. Bowman, Anuradha S. Budhu, Jittiporn Chaisaingmongkol, Siritida Rabibhadana, Marshonna Forgues, Takahiro Oike, Mathuros Ruchirawat, Frank J. Gonzalez, Xin W. Wang, Curtis C. Harris. Urinary metabolites are diagnostic biomarkers of liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2500. doi:10.1158/1538-7445.AM2017-2500

We thank Dr. Thompson and colleagues for their insightful comments about our article ([1][1]). We have now reevaluated our finding based on their comments. Recently, two studies sought to identify synthetic lethal partners for BRG1 using the same (and other) cancer cell lines as used by us ([2, 3][

Background: The histone acetyltransferases (HATs) including p300, CBP and TIP60 are transcriptional co-activators implicated in many gene regulatory pathways and protein acetylation events. We have previously shown that siRNA-mediated ablation of p300 and CBP sensitized cancer cells to ionizing radiation (IR) (Ogiwara, Oncogene 2011), and that garcinol, curcumin and anacardic acid, natural compounds with multi-HATs inhibitory activity, had radiosensitizing effect in cancer cells (Oike, Int J Radiat Oncol Biol Phys 2012). However, radiosensitization by selective small molecule inhibitors targeting activity of a specific HAT, that can have much potential for clinical use, have not been well investigated. C646 is a selective small molecule inhibitor of p300 identified by structure-based and active site-directed in silico screening (Bowers, Chem Biol 2010). In this study, we investigated the in vitro effect of C646 on radiosensitization and cell death in human cancer cells. Materials and Methods: The radiosensitizing effect in A549 (lung adenocarcinoma), H460 (lung squamous cell carcinoma) and H157 (lung large cell carcinoma) cells were assessed by colony formation assay, while that in MRC-5 (fibroblast) cells lacking in colony-forming ability was examined by ATP assay. Cell cycle distribution in A549 cells was analyzed using flow cytometry. The effect on mitotic catastrophe, apoptosis and senescence in A549 cells was evaluated by DAPI, annexin V and senescence-associated β-galactosidase staining, respectively. C646 was used at concentrations less than IC50. Results: C646 radiosensitized A549, H460 and H157 cancer cells with dose enhancement ratio at 10% surviving fraction of 1.4, 1.2 and 1.2, respectively. Meanwhile, C646 unaffected the radiosensitivity of MRC-5 cells. Cell cycle analysis showed that the hyperploid cell population (> 4N DNA cells) increased in C646-treated A549 cells after IR. Analyses of mode of cell death revealed that C646 enhanced mitotic catastrophe, but not apoptosis nor senescence, induced by IR in A549 cells. Conclusion: These data indicate that C646 is a radiosensitizer which enhances mitotic catastrophe induced by IR. Citation Format: Takahiro Oike, Mayumi Komachi, Hideaki Ogiwara, Jun Yokota, Takashi Nakano, Takashi Kohno. C646, a selective small molecule inhibitor of p300, radiosensitizes cancer cells through enhancement of mitotic catastrophe. [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 1597. doi:10.1158/1538-7445.AM2013-1597

Purpose: Non-homologous end joining (NHEJ), a major pathway to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through acetylation of histones by histone acetyltransferases (HATs). However, effects of compounds with HAT inhibitory activities on DNA damage response (DDR), including NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells remain largely unclear. Here, we investigated whether garcinol, an HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR and whether it can be used for radiosensitization. Methods and Materials: The ability of garcinol to inhibit DSB repair, including NHEJ, was examined by two assays; the conventional neutral comet assay and a cell-based assay recently developed by us, in which the activity of NHEJ against DSBs on chromosomal DNA was evaluated. The effects on cell cycle checkpoint after IR were assessed by phosphorylation levels of checkpoint kinases CHK1 and CHK2, and histone H3. The radiosensitizing effect was assessed by clonogenic survival assay. The effects on apoptosis and senescence were examined by annexin V and Senescence-associated β-galactosidase (SA-β-Gal) staining, respectively. Results: Garcinol was shown to inhibit DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with a dose enhancement ratio at 10% surviving fraction of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that inhibits NHEJ and facilitates senescence, without impairing activation of cell cycle checkpoint. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5721. doi:1538-7445.AM2012-5721