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General control nonderepressible 2 (GCN2) plays a major role in the cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, the contribution of GCN2 to cancer cell survival and proliferation is poorly understood. In this study, we generated GCN2 inhibitors and demonstrated that inhibition of GCN2 sensitizes cancer cells with low basal-level expression of asparagine synthetase (ASNS) to the antileukemic agent l-asparaginase (ASNase) in vitro and in vivo. We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of ASNS, resulting in reduced levels of de novo protein synthesis. Comprehensive gene-expression profiling revealed that combined treatment with ASNase and GCN2 inhibitors induced the stress-activated MAPK pathway, thereby triggering apoptosis. By using cell-panel analyses, we also showed that acute myelogenous leukemia and pancreatic cancer cells were highly sensitive to the combined treatment. Notably, basal ASNS expression at protein levels was significantly correlated with sensitivity to combined treatment. These results provide mechanistic insights into the role of GCN2 in the amino acid response and a rationale for further investigation of GCN2 inhibitors for the treatment of cancer.

Studies in circulating tumor cells (CTCs) have proceeded to be accepted as prognostic markers in several types of cancers. But they are still limited because many are mainly from enumeration of CTCs. Here, we tried to evaluate the tumorigenicity of CTCs from advanced gastric cancer patients (n = 42). Peripheral blood mononuclear cells (PBMC) from the patients were separated into CD45 negative and positive fractions and both were subcutaneously injected into immunodeficient mice. Within 5 months nine tumor-like-structures from six patients but not from healthy volunteers were established. They were durable for passages and all had been confirmed human origin. Eight of the nine tumor-like-structures were from nonauthorized CTC containing cells expressing CD45 and B-cell markers. On the contrary, one of them was developed from CD45(-) PBMC fraction of a patient with bone marrow metastasis reflecting authorized CTCs. Histopathology showed common features with that of original gastric tumor. The cells isolated from the tumor-like-structure expressed EpCAM and CEA further supporting they were from the original tumor. Moreover the cells were CD44 positive to varying degree and a limiting dilution study showed that the CD44(+/high) fraction had tumorigenicity. The CD44 was dominantly in the form of CD44 variant 8-10. The CD44(+/high) cells had higher expression of the glutamate/cysteine transporter xCT compared with the CD44(-/low) cells. Our results showed the existence of tumor-initiating cells in blood of advanced gastric cancer patients and they could be a therapeutic target and prospective tool for further investigations.

Background: The clinical significance of circulating tumour cell (CTC) detection in gastrointestinal (GI) cancer remains controversial and the molecular biological characteristics of CTCs are poorly understood. Methods: A total of 87 patients with metastatic or recurrent GI cancer were prospectively enrolled. Circulating tumour cells and their HER2 status were assessed using the CellSearch System. Results: Among the 62 CTC-positive cases, we found 22 discordant cases (35.5%). Among the HER2-negative primary tumours, 17 of 54 developed HER2-positive CTCs. Five of eight had HER2-negative CTCs among the HER2-positive primary tumours. Conclusion: The findings in the current study suggest that it is critical to evaluate the HER2 status of not only the primary tumour but also the CTCs because the metastasising tumour cells are the primary target of systemic therapy.

Dysregulation of lysine (K)-specific demethylase 1A (LSD1), also known as KDM1A, has been implicated in the development of various cancers, including leukemia. Here, we describe the antileukemic activity and mechanism of action of T-3775440, a novel irreversible LSD1 inhibitor. Cell growth analysis of leukemia cell lines revealed that acute erythroid leukemia (AEL) and acute megakaryoblastic leukemia cells (AMKL) were highly sensitive to this compound. T-3775440 treatment enforced transdifferentiation of erythroid/megakaryocytic lineages into granulomonocytic-like lineage cells. Mechanistically, T-3775440 disrupted the interaction between LSD1 and growth factor–independent 1B (GFI1B), a transcription factor critical for the differentiation processes of erythroid and megakaryocytic lineage cells. Knockdown of LSD1 and GFI1B recapitulated T-3775440–induced transdifferentiation and cell growth suppression, highlighting the significance of LSD1–GFI1B axis inhibition with regard to the anti-AML effects of T-3775440. Moreover, T-3775440 exhibited significant antitumor efficacy in AEL and AMKL xenograft models. Our findings provide a rationale for evaluating LSD1 inhibitors as potential treatments and indicate a novel mechanism of action against AML, particularly AEL and AMKL. Mol Cancer Ther; 16(2); 273–84. ©2016 AACR.

General control nonderepressible 2 (GCN2) plays a major role in cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, little is known concerning the contribution of GCN2 to cancer cell proliferation and its potential as a therapeutic target. In this study, we demonstrate that inhibition of GCN2 with small-molecule compounds sensitizes cancer cells to anti-leukemic agent asparaginase (ASNase). We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of asparagine synthetase (ASNS). The prevention of ASNS expression suppresses asparagine synthesis and thus inhibits protein translation. In addition, comprehensive gene expression profiling revealed that the combined treatment induced stress-activated MAPK pathway and thereby triggering apoptosis. Using cell-panel analyses, we also identified a clear synergy between GCN2 inhibitors and ASNase in acute myelogenous leukaemia (AML) and pancreatic cancer cells besides ALL. Notably, basal ASNS expression levels were significantly correlated with sensitivity to combinatorial treatment. Furthermore, the combination displayed robust antitumor activity in mouse xenograft models of ALL, AML, and pancreatic cancer. These results demonstrate a novel mechanistic insight into a role of GCN2 in amino acid response and provide a rationale for further investigating GCN2 inhibitors for the treatment of cancer. Citation Format: Akito Nakamura, Tadahiro Nambu, Shunsuke Ebara, Yuka Hasegawa, Kosei Toyoshima, Yasuko Tsuchiya, Daisuke Tomita, Jun Fujimoto, Osamu Kurasawa, Chisato Takahara, Ayumi Kawamura, Ryuichi Nishigaki, Yoshinori Satomi, Akito Hata, Takahito Hara. Inhibition of GCN2 sensitizes ASNS-downregulated cancer cells to asparaginase by disrupting amino acid response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3847.

Lysine-specific demethylase 1A (LSD1, KDM1A) specifically demethylates di- and monomethylated histones H3K4 and K9, resulting in context-dependent transcriptional repression or activation. We previously identified an irreversible LSD1 inhibitor T-3775440, which exerts antileukemic activities in a subset of acute myeloid leukemia (AML) cell lines by inducing cell transdifferentiation. The NEDD8-activating enzyme inhibitor pevonedistat (MLN4924, TAK-924) is an investigational drug with antiproliferative activities in AML, and is also reported to induce cell differentiation. We therefore tested the combination of these two agents in AML models. The combination treatment resulted in synergistic growth inhibition of AML cells, accompanied by enhanced transdifferentiation of an erythroid leukemia lineage into granulomonocytic-like lineage cells. In addition, pevonedistat-induced rereplication stress during the S phase was greatly augmented by concomitant treatment with T-3775440, as reflected by the increased induction of apoptosis. We further demonstrated that the combination treatment was markedly effective in subcutaneous tumor xenograft models as well as in a disseminated model of AML, leading to tumor eradication or prolonged survival in T-3775440/pevonedistat cotreated mice. Our findings indicate the therapeutic potential of the combination of LSD1 inhibitors and pevonedistat for the treatment of AML.

Studies in circulating tumor cells (CTCs) have proceeded to be accepted as prognostic markers in several types of cancers. But they are still limited because many are mainly from enumeration of CTCs. Here, we tried to evaluate the tumorigenicity of CTCs from advanced gastric cancer patients (n = 42). Peripheral blood mononuclear cells (PBMC) from the patients were separated into CD45 negative and positive fractions and both were subcutaneously injected into immunodeficient mice. Within 5 months nine tumor-like-structures from six patients but not from healthy volunteers were established. They were durable for passages and all had been confirmed human origin. Eight of the nine tumor-like-structures were from nonauthorized CTC containing cells expressing CD45 and B-cell markers. On the contrary, one of them was developed from CD45(-) PBMC fraction of a patient with bone marrow metastasis reflecting authorized CTCs. Histopathology showed common features with that of original gastric tumor. The cells isolated from the tumor-like-structure expressed EpCAM and CEA further supporting they were from the original tumor. Moreover the cells were CD44 positive to varying degree and a limiting dilution study showed that the CD44(+/high) fraction had tumorigenicity. The CD44 was dominantly in the form of CD44 variant 8-10. The CD44(+/high) cells had higher expression of the glutamate/cysteine transporter xCT compared with the CD44(-/low) cells. Our results showed the existence of tumor-initiating cells in blood of advanced gastric cancer patients and they could be a therapeutic target and prospective tool for further investigations.

The suppression of gene expression of vascular endothelial growth factor (VEGF) which regulates tumor angiogenesis in vivo and is an important factor in tumor growth represents a novel approach to cancer treatment. Although small interfering RNA (siRNA) has rapidly become a major tool in gene therapy and is a key inhibitory factor of gene expression, its effect is temporary. The present study investigates the preparation of long-term sustained release poly (dl-lactic/glycolic acid) (PLGA) microspheres encapsulating anti-VEGF siRNA with a carrier (arginine or branched polyethylenimine) using the w/o/w in-water drying method and their anti-tumor activities. The ratio (%) of encapsulated siRNA increased when arginine or PEI was added to the inner water phase during preparation. The release of siRNA from microspheres in phosphate buffer (pH 7.4) was sustained for over one month. The anti-tumor effects of microspheres in vivo were evaluated in mice bearing S-180 tumors. An intra-tumor injection of microspheres with encapsulated siRNA obviously suppressed tumor growth. These results indicate that the microspheres of anti-VEGF siRNA with a transfection agent (carrier) have achieved a higher and sustained suppressive effect on VEGF gene expression and should be a practically useful preparation.