Koichiro Nozaki

DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked is an immunogenic target of cancer stem cells

Abstract Accumulating evidence suggests that most solid malignancies consist of heterogeneous tumor cells and that a relatively small subpopulation, which shares biological features with stem cells, survives through potentially lethal stresses such as chemotherapy and radiation treatment. Since the survival of this subpopulation of cancer stem cells (CSC) plays a critical role in recurrence, it must be eradicated in order to cure cancer. We previously reported that vaccination with CD133+ murine melanoma cells exhibiting biological CSC features induced CSC-specific effector T cells. These were capable of eradicating CD133+ tumor cells in vivo, thereby curing the parental tumor. In the current study, we indicated that DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked (DDX3X) is an immunogenic protein preferentially expressed in CD133+ tumor cells. Vaccination with DDX3X primed specific T cells, resulting in protective and therapeutic antitumor immunity. The DDX3X-primed CD4+ T cells produced CD133+ tumor-specific IFNγ and IL-17 and mediated potent antitumor therapeutic efficacy. DDX3X is expressed in various human cancer cells, including lung, colon, and breast cancer cells. These results suggest that anti-DDX3X immunotherapy is a promising treatment option in efforts to eradicate CSC in the clinical setting.

Critical Roles of Chemoresistant Effector and Regulatory T Cells in Antitumor Immunity after Lymphodepleting Chemotherapy.

Antitumor immunity is augmented by cytotoxic lymphodepletion therapies. Adoptively transferred naive and effector T cells proliferate extensively and show enhanced antitumor effects in lymphopenic recipients. Although the impact of lymphodepletion on transferred donor T cells has been well evaluated, its influence on recipient T cells is largely unknown. The current study demonstrates that both regulatory T cells (Tregs) and effector CD8+ T cells from lymphopenic recipients play critical roles in the development of antitumor immunity after lymphodepletion. Cyclophosphamide (CPA) treatment depleted lymphocytes more efficiently than other cytotoxic agents; however, the percentage of CD4+CD25+ Foxp3+ Tregs was significantly increased in CPA-treated lymphopenic mice. Depletion of these chemoresistant Tregs following CPA treatment and transfer of naive CD4+ T cells augmented the antitumor immunity and significantly suppressed tumor progression. Further analyses revealed that recipient CD8+ T cells were responsible for this augmentation. Using Rag2−/− mice or depletion of recipient CD8+ T cells after CPA treatment abrogated the augmentation of antitumor effects in CPA-treated reconstituted mice. The transfer of donor CD4+ T cells enhanced the proliferation of CD8+ T cells and the priming of tumor-specific CD8+ T cells originating from the lymphopenic recipients. These results highlight the importance of the recipient cells surviving cytotoxic regimens in cancer immunotherapies.

DDX3X Induces Primary EGFR-TKI Resistance Based on Intratumor Heterogeneity in Lung Cancer Cells Harboring EGFR-Activating Mutations

The specific mechanisms how lung cancer cells harboring epidermal growth factor receptor (EGFR) activating mutations can survive treatment with EGFR-tyrosine kinase inhibitors (TKIs) until they eventually acquire treatment-resistance genetic mutations are unclear. The phenotypic diversity of cancer cells caused by genetic or epigenetic alterations (intratumor heterogeneity) confers treatment failure and may foster tumor evolution through Darwinian selection. Recently, we found DDX3X as the protein that was preferentially expressed in murine melanoma with cancer stem cell (CSC)-like phenotypes by proteome analysis. In this study, we transfected PC9, human lung cancer cells harboring EGFR exon19 deletion, with cDNA encoding DDX3X and found that DDX3X, an ATP-dependent RNA helicase, induced CSC-like phenotypes and the epithelial-mesenchymal transition (EMT) accompanied with loss of sensitivity to EGFR-TKI. DDX3X expression was associated with upregulation of Sox2 and increase of cancer cells exhibiting CSC-like phenotypes, such as anchorage-independent proliferation, strong expression of CD44, and aldehyde dehydrogenase (ALDH). The EMT with switching from E-cadherin to N-cadherin was also facilitated by DDX3X. Either ligand-independent or ligand-induced EGFR phosphorylation was inhibited in lung cancer cells that strongly expressed DDX3X. Lack of EGFR signal addiction resulted in resistance to EGFR-TKI. Moreover, we found a small nonadherent subpopulation that strongly expressed DDX3X accompanied by the same stem cell-like properties and the EMT in parental PC9 cells. The unique subpopulation lacked EGFR signaling and was highly resistant to EGFR-TKI. In conclusion, our data indicate that DDX3X may play a critical role for inducing phenotypic diversity, and that treatment targeting DDX3X may overcome primary resistance to EGFR-TKI resulting from intratumor heterogeneity.

Advanced Thymic Cancer Treated with Carboplatin and Paclitaxel in a Patient Undergoing Hemodialysis

A 53-year-old man with an asymptomatic anterior mediastinal tumor undergoing hemodialysis was referred to our institution. He was diagnosed with thymic basaloid carcinoma based on the findings of a chest tomography-guided biopsy and successfully treated with carboplatin (300 mg/m(2)/day) and paclitaxel (200 mg/m(2)/day) on day 1 for six three-week cycles. To our knowledge, this is the first report regarding the efficiency of a carboplatin dose-definition method based on the body surface area with paclitaxel in a hemodialysis patient. This report may therefore be useful for treating hemodialysis patients who are candidates for carboplatin and paclitaxel therapy.

Transfer of in vitro-expanded naïve T cells after lymphodepletion enhances antitumor immunity through the induction of polyclonal antitumor effector T cells

The adoptive transfer of effector T cells combined with lymphodepletion has demonstrated promising antitumor effects in mice and humans, although the availability of tumor-specific T cells is limited. We and others have also demonstrated that the transfer of polyclonal naïve T cells induces tumor-specific effector T cells and enhances antitumor immunity after lymphodepletion. Because tumors have been demonstrated to induce immunosuppressive networks and regulate the function of T cells, obtaining a sufficient number of fully functional naïve T cells that are able to differentiate into tumor-specific effector T cells remains difficult. To establish culture methods to obtain a large number of polyclonal T cells that are capable of differentiating into tumor-specific effector T cells, naïve T cells were activated with anti-CD3 mAbs in vitro. These cells were stimulated with IL-2 and IL-7 for the CD8 subset or with IL-7 and IL-23 for the CD4 subset. Transfer of these hyperexpanded T cells after lymphodepletion showed significant antitumor efficacy, and tumor-specific effector T cells were primed from these expanded T cells in tumor-bearing hosts. Moreover, these ex vivo—expanded T cells maintained T cell receptor diversity and showed long-term persistence of memory against specific tumors. Further analyses revealed that combination therapy consisting of vaccination with dendritic cells that were co-cultured with irradiated whole tumor cells and the transfer of ex vivo—expanded T cells significantly enhanced antitumor immunity. These results indicate that the transfer of ex vivo—expanded polyclonal T cells can be combined with other immunotherapies and augment antitumor effects.

Abstract 200: DDX3X induces signal switching to stem cell-specific Wnt/β-catenin signaling, resulting in EGFR-TKI resistance in lung cancer cells harboring EGFR activating mutation

[Background] The treatment targeting signal addiction caused by oncogenic driver mutation has led to unprecedented results in clinical setting; however, it is still difficult to achieve cure. Tumor diversity based on both genetic and non-genetic influences give rise of survivors as treatment-resistant cells. Most of the acquired resistance reflects the selection of the cancer cells harboring stochastic resistance-conferring genetic alterations. However, the mechanisms how the cancer cells survive until acquisition of additional mutations are unclear. We identified DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked (DDX3X), an ATP-dependent RNA helicase, was preferentially expressed in a cancer stem cell (CSC)-like population of murine melanoma. DDX3X is believed to be involved in epigenetic regulation of gene expression based on metabolism of mRNA and miRNA. [Objectives] In the current study, we examine that DDX3X affects phenotype and signal status of PC9 cells, a lung adenocarcinoma harboring EGFR exon19 deletion as an oncogenic driver mutation. [Results] DDX3X plays a role in acquisition of CSC-like properties. PC9 A-1 cells that were engineered to overexpress DDX3X acquired ability to proliferate as tumor spheres and showed up-regulated Sox2 and ALDH expression. Epithelial mesenchymal transition with cadherin switching from E-cadherin to N-cadherin accompanied with vimentin expression was detected in non-adherent A-1 population. E-cadherin+ non-adherent cells exhibited strong CD44 expression, a putative CSC marker. Surprisingly, A-1 cells exhibited almost no EGFR phosphorylation even in the presence of EGF. Knocking-down of DDX3X restored EGFR phosphorylation. On the other hand, activated nuclear translocation of β-catenin was observed. Loss of EGFR signal addiction resulted in resistance to EGFR-TKI. On contrast, PC9-A1 cells were sensitive to ICG-001, a β-catenin signal inhibitor. Further, we identified a minor non-adherent subpopulation of parental PC9 cells strongly expressed DDX3X and that they were resistant to EGFR-TKI because they did not addict to EGFR signaling. Survivor cells of parental PC9 after long term-exposure to EGFR-TKI strongly expressed DDX3X. [Conclusion] DDX3X plays a critical role in a novel EGFR-TKI resistance mechanism, signal switching, correlating with CSC transformation. DDX3X and β-catenin are likely promising target molecules to overcome EGFR-TKI resistance. Citation Format: Satoshi Shoji, Hiroshi Kagamu, Koichiro Nozaki, Natsue Igarashi, Masaaki Okajima, Satoru Miura, Satoshi Watanabe, Hrohisa Yoshizawa, Ichiei Narita. DDX3X induces signal switching to stem cell-specific Wnt/β-catenin signaling, resulting in EGFR-TKI resistance in lung cancer cells harboring EGFR activating mutation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 200. doi:10.1158/1538-7445.AM2014-200

Abstract 2554: DDX3X-specific effector T cells in small cell lung cancer patients reflect disease stage

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA [Background] Small cell lung cancer (SCLC) possesses high tendency to disseminate. However, SCLC patients with paraneoplastic syndrome mediated by immunity against onconeural antigens remain in limited-stage disease (LD) without distant metastases. We previously demonstrated that effector-dominant CD4+ T cell balance was observed only in LD-SCLC but not in extended-stage disease (ED)-SCLC. CD4+ T cell balance in peripheral blood of SCLC patients reflected disease stage and recurrence. Accumulating evidence suggests that most solid malignancies consist of heterogeneous tumor cells and that a relatively small subpopulation, which shares biological features with stem cells, is responsible for distant metastases and recurrence. We previously reported that DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked (DDX3X) is an immunogenic protein preferentially expressed in CD133+ murine melanoma cells exhibiting biological CSC features and that DDX3X-primed CD4+ T cells mediated potent antitumor therapeutic efficacy. We found that DDX3X is strongly expressed in small cell lung cancer (SCLC). [Objectives] In the current study, we examine that DDX3X-specific effector T cells exist in SCLC patients. [Patients and Methods] The present study comprised 20 consecutive SCLC patients, 5 long-term survivors, and 10 healthy volunteers from a single institution (Niigata University Medical and Dental Hospital). Specimens were collected after obtaining written informed consent approved by the Niigata University Ethical Committee. [Results] Six of 12 LD-SCLC patients possessed DDX3X-responsive effector T cells. CD62Llow CD4+ effector T cells obtained from peripheral blood of 5 LD-SCLC secreted significantly more IFNγ upon DDX3X antigen stimulation in the presence of CD11c+ autologous dendritic cells. CD62Llow CD8+ effector T cells from one LD-SCLC patient responded to DDX3X. In contrast, effector T cells obtained from ED-SCLC patients or healthy volunteers never responded to DDX3X. [Conclusion] DDX3X is likely one of major antigens that is expressed in SCLC and is recognized by T cell immune system. Antitumor immunotherapy to induce effector-dominant CD4+ T-cell immunity on DDX3X antigen may be a promising therapy to eliminate SCLC cells that mediate distant metastases. Citation Format: Natsue Igarashi, Hiroshi Kagamu, Koichiro Nozaki, Satoshi Shoji, Masaaki Okajima, Satoru Miura, Satoshi Watanabe, Hirohisa Yoshizawa, Ichiei Narita. DDX3X-specific effector T cells in small cell lung cancer patients reflect disease stage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2554. doi:10.1158/1538-7445.AM2014-2554

Abstract 2818: Dendritic cell vaccination and regulatory T-cell depletion augment antitumor immunity after cytotoxic therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The ability of lymphodepleting cytotoxic regimens to augment antitumor immune responses has been well established. We and others have previously reported that naive T cells, as well as effector T cells, transferred into lymphopenic tumor-bearing mice acquire memory-like phenotypes and show antitumor effects. The transfer of naive T cells into irradiated lymphopenic mice inhibited tumor progression. Further analyses revealed that the transfer of CD4+ naive T cells was essential for this augmentation of antitumor immunity after lymphodepletion. To enhance the antitumor efficacy of lymphodepletion and transfer of naive T cells, we have tested anti-CTLA-4 mAbs and anti-CD25 mAbs for depletion of regulatory T cells. Mice were irradiated with 500 cGy to deplete lymphocytes and were transferred i.v. with T cells (2 x 107). On the same day, mice were inoculated with MCA205 tumor cells (1 x 105) followed by the injection of anti-CTLA-4 mAbs (UC10) or the injection of anti-CD25 mAbs (PC61). Although the injection of anti-CTLA-4 mAbs showed minimal augmentation of the antitumor effects of the combination of lymphodepletion and naive T cell transfer, anti-CD25 mAbs significantly enhanced antitumor immune responses. Next, we evaluated whether dendritic cell vaccination augments this combination therapy. Different from previous experiments, 3-day tumor-bearing mice were irradiated and reconstituted with T cells. Dendritic cells were generated from bone marrow of naive mice and were co-cultured with irradiated MCA205 tumor cells followed by stimulation with agonistic anti-CD40 mAbs. We found that dendritic cell vaccination significantly inhibited tumor growth in irradiated reconstituted mice. Previously, we reported that a significant increase of CD4+CD25+Foxp3+ regulatory T cells in irradiated mice. We demonstrated that these radio-resistant regulatory T cells from irradiated recipient mice suppressed the development of antitumor immunity. To clarify the roles of other recipient cells in antitumor immunity during recovery from lymphopenia, Rag-2 knockout mice were irradiated and reconstituted with T cells. These mice were then inoculated MCA205 tumor cells. In Rag-2 knockout mice, antitumor effects were not observed after lymphodepletion and reconstitution. These findings indicate that recipient T cells or B cells were required to augment antitumor immunity in lymphopenic mice in our model. Citation Format: Tomohiro Tanaka, Satoshi Watanabe, Ko Sato, Yu Saida, Junko Baba, Koichiro Nozaki, Daisuke Ishikawa, Natsue Igarashi, Satoshi Shoji, Masaaki Okajima, Satoru Miura, Junta Tanaka, Hiroshi Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Ichiei Narita. Dendritic cell vaccination and regulatory T-cell depletion augment antitumor immunity after cytotoxic therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2818. doi:10.1158/1538-7445.AM2014-2818

Abstract 3716: DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked plays oncogenic roles to induce cancer stem cell-like properties and resistance to EGFR-TKI.

Background. DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked (DDX3X) is a member of the DEAD-box family of ATP-dependent RNA helicases. DEAD-box helicases perform multiple functions, including RNA splicing, mRNA export, transcriptional and translational regulation, RNA decay, and ribosome biogenesis. Using two-dimensional electrophoretic and MS/MS spectrometric analyses, we demonstrated that DDX3X is one of the proteins that are specifically expressed in CD133+ B16 melanoma cells. These cells possess cancer stem cell (CSC)-like properties, such as high tumorigenicity, spheroid formation ability, and anchorage independent growth. DDX3X is evolutionarily well conserved from yeast to humans, suggesting that it is essential for cell survival. In humans, DDX3X deletion or dysfunction results in genetically related primary amenorrhea and impaired female fertility. Although originally DDX3X was reported to suppress growth by modulating the p21waf/cip1 gene expression, recent studies have shown direct correlations between DDX3X and oncogenesis. Furthermore, DDX3X overexpression in breast cancer cells has been shown to facilitate β-catenin signal transduction and induce epithelial mesenchymal transition (EMT), a known feature of CSCs. However, the mechanism by which DDX3X plays an oncogenic role to induce CSC features remains unclear. Results. Immunoblotting analyses revealed that all of the examined cancer cells, including lung cancer, colon cancer, and breast cancer cells, strongly expressed DDX3X, whereas normal human epidermal keratinocytes, human microvascular endothelial cells, and normal human bronchial epithelial cells faintly expressed DDX3X. Furthermore, putative CSC marker-positive cancer cells, such as 87.5, HCT116, and MCF7, strongly expressed DDX3X. PC9 cells, which are lung adenocarcinoma cells harboring EGFR exon 19 deletion, showed relatively weak expression of DDX3X and did not express CD133 or other CSC markers. Parental PC9 cells were ALDH−, CD44low, and E-cadherinmid and proliferated in an anchorage dependent manner. Overexpression of DDX3X in PC9 cells led to increase in the number of ALDH+ CD44high E-cadherinhigh and ALDH+ CD44low E-cadherin− cancer cells, both of which proliferated in an anchorage independent manner. Moreover, PC9 cells that overexpressed DDX3X (PC9DDX3X) acquired EGFR-TKI resistance. In the presence of erlotinib at 100 times the concentration at IC50, apoptosis was not induced in PC9DDX3X. Conclusion. DDX3X expression is correlated with CSC-like properties in human cancer cells. DDX3X plays a role in the EGFR-TKI resistance mechanism, and it could be used as a possible target molecule to modulate EGFR-TKI resistance in lung cancer cells harboring EGFR-activating mutation. Citation Format: Koichiro Nozaki, Hiroshi Kagamu, Satoshi Shoji, Natsue Igarashi, Msaaki Okajima, Satoru Miura, Satoshi Watanabe, Hirohisa Yoshizawa, Ichiei Narita. DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked plays oncogenic roles to induce cancer stem cell-like properties and resistance to EGFR-TKI. [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 3716. doi:10.1158/1538-7445.AM2013-3716

Abstract 1550: Successful treatment of advanced tumors with chemo-immunotherapy: a combination of cyclophosphamide and inhibition of chemo-resistant immune suppressor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL It is well known that naive T cells transferred into lymphopenic hosts develop into memory like T cells and acquire some effector functions. We and others have been shown that transfer of naive T cells following sublethal whole body irradiation augmented antitumor immunity and inhibited tumor progression. Further analyses showed that antitumor effector T cells were primed from transferred naive T cells. Although the exact mechanism underlying this enhancement of antitumor immune responses remains unclear, possible explanations have been proposed; depletion of suppressor cells, improvement of tumor-antigen presentation, and elimination of lymphocytes competing activation cytokines. Combination of lymphodepletion by cytotoxic regimens, such as chemotherapy or radiotherapy, and transfer of naive T cells seems to be a promising strategy. Although whole body irradiation induces lymphopenia to tumor-bearing animals and enhances antitumor immunity, it has not been routinely used in clinical settings. To test whether cytotoxic agents deplete lymphocytes and enhance antitumor immune responses, we infused cyclophosphamide (CPA), fludarabine, cisplatin and etoposide at the sublethal doses to mice. CPA treatment depleted 95% of lymphocytes in mice, and enhanced antitumor effects of transferred naive T cells. Other cytotoxic drugs failed to augment antitumor immunity in combination with transfer of T cells. Previously, we have demonstrated that CD4+CD25+Foxp3+ regulatory T cells (Treg) and CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) inhibited priming of antitumor effector T cells. Depletion of these suppressor cells increased the number of tumor-specific T cells and augmented antitumor immune responses. To examine the effect of sublethal doses of cyclophosphamide on immune suppressor cells, we injected cyclophosphamide into mice and harvested lymph-nodes and spleens for FACS analyses. Unexpectedly, we found a significant increase in the frequency of Treg and MDSC after CPA treatment. Magnetically isolated Treg and MDSC from cyclophosphamide treated mice suppressed tumor-specific responses of effector T cells in vitro. Depletion of Treg with anti-CD25 monoclonal antibodies following CPA administration and transfer of naive T cells increased the number of antitumor effector T cells. Further, the combination of Treg depletion, CPA treatment and transfer of naive T cells succeeded to cure 20-day established skin tumors in mice. We have also been testing whether inhibition of MDSC after CPA treatment augments antitumor immunity. Our results showed that CPA treatment efficiently depletes lymphocytes and enhanced antitumor immune responses despite an increase of immune suppressor cells. Further inhibition of suppressor cells after CPA administration successfully treated advanced tumors. 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 1550. doi:1538-7445.AM2012-1550