Masaaki Okajima

Human T-cell leukemia virus type 1 Tax induces an aberrant clustering of the tumor suppressor Scribble through the PDZ domain-binding motif dependent and independent interaction

Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia. HTLV-1 Tax1 transforming protein interacts with several PDZ domain-containing proteins, and the interaction is associated with the transforming activities of Tax1 as well as persistent HTLV-1 infection. In this study, we show that Tax1 interacts with the tumor suppressor Scribble containing PDZ domains. Unlike other Tax1-interacting PDZ domain proteins, the PDZ domain-binding motif (PBM) of Tax1 was not required for the interaction with transiently expressed Scribble in 293T cells, but it was essential for the interaction with endogenous Scribble. Endogenous Scribble in 293T cells was primarily localized at the plasma membrane and colocalized with Tax1 but not Tax1∆C lacking PBM, whereas transiently expressed Scribble was localized in the cytoplasm and colocalized with Tax1∆C as well as Tax1, thus suggesting that Tax1 is recruited to the site of endogenous Scribble, such as the plasma membrane, in a PBM-dependent manner, and thereafter it interacts with Scribble in a PBM-independent and PBM-dependent manner. Endogenous Scribble was diffusely localized at the plasma membrane of HTLV-1-uninfected T-cell lines, whereas it colocalized with Tax1 as small and large aggregate at the plasma membranes. These results suggest that Tax1 through two binding sites induce aberrant clustering of Scribble, thereby altering the functions in HTLV-1-infected cells, which may thus play a role in persistent HTLV-1 infection and the pathogenesis.

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.

Recurrent Interstitial Lung Disease Induced By Various Therapies forNon-Small Cell Lung Cancer

Erlotinib is a human epidermal growth factor receptor type 1 tyrosine kinase inhibitor which is used for non-small cell lung cancer treatment. Interstitial lung disease has been reported as an adverse event of erlotinib. We report the case of a 39-year-old man with erlotinib-induced interstitial lung disease in a non-small cell lung cancer patient. Although interstitial lung disease had improved by steroid therapy, palliative radiotherapy recalled the pneumonitis beyond the radiation fields. After the pneumonitis was well controlled, the patient was started on irinotecan, but the interstitial lung disease recurred shortly thereafter. We may have to abandon further cytotoxic therapies to avoid the recurrence of interstitial lung disease in patients who develop erlotinib-induced interstitial lung disease once.

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.

Abstract 3206: Programmed death receptor-1/programmed death receptor ligand-1 blockade improves priming of antitumor effector T cells after cytotoxic therapies

Cytotoxic lymphodepletion therapies, such as chemotherapy and radiotherapy, have been established to augment antitumor immunity. Naive T cells elicit effector-like phenotypes and functions during recovery from lymphopenia. We and others have repeatedly demonstrated that transfer of naive T cells into lymphopenic-tumor bearing mice delayed tumor growth. This enhancement of naive T cells is required an activation through T-cell receptor (TCR). Programmed death receptor-1 (PD-1)/programmed death receptor ligand-1 (PD-L1) blockade therapy has been demonstrated to augment antitumor immunity. Previous studies have shown that PD-1/PD-L1 blockade therapy stimulates or restores the function of antitumor T cells in the effector phase. Because engagement of programmed death receptor-1 (PD-1) by ligand suppresses TCR signaling and inhibits T cell activation and function, there is a possibility that PD-1 regulates activation of T cells during recovery from lymphopenia after cytotoxic therapies. In the current study, we transferred naive T cells into tumor-bearing mice following whole-body irradiation for lymphodepletion. These mice were further treated with anti-PD-1 antibodies. PD-1/PD-L1 blockade therapy after lymphodepletion significantly suppressed tumor progression. Next, we tested several kinds of cytotoxic agents to induce lymphopenia in mice. We found that the kind of cytotoxic agents affected the augmentation of antitumor efficacies of PD-1/PD-L1 blockade. Analyses of tumor-draining lymph-nodes (TDLNs) revealed that the number of tumor-specific effector T cells was significantly increased in mice treated with anti-PD-1 antibodies. By contrast, the number of effector T cells in spleens was not increased by PD-1/PD-L1 blockade therapy. These results indicate that PD-1 regulates a priming of antitumor effector T cells in TDLNs after cytotoxic lymphodepletion therapies. PD-1/PD-L1 blockade therapy is able to enhance antitumor T cell responses not only in the effector phase but also in the priming phase. Citation Format: Miho Takahashi, Satoshi Watanabe, Ko Sato, Tomohiro Tanaka, Yu Saida, Junko Baba, Aya Ohtsubo, Miyuki Sato, Rie Kondo, Masaaki Okajima, Junta Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Toshiaki Kikuchi. Programmed death receptor-1/programmed death receptor ligand-1 blockade improves priming of antitumor effector T cells after cytotoxic therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3206.

Abstract 3153: Critical roles of chemo-resistant effector and regulatory T cells in cancer immunotherapy during hemostatic proliferation

Antitumor immunity has been well established to be augmented by cytotoxic lymphodepletion therapies. Adoptively transferred naive and effector T cells proliferate extensively and show enhanced antitumor effects during homeostatic proliferation when they were adoptively transferred into tumor-bearing hosts that were lymphodepleted with cytotoxic agents or by whole body irradiation. 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, such as fludarabine, cisplatin, etoposide, paclitaxel or gemcitabine; however, the percentage of CD4 + CD25 + Foxp3 + Tregs was significantly increased in CPA-treated lymphopenic mice. Depletion of these chemo-resistant 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. Citation Format: Ko Sato, Satoshi Watanabe, Yu Saida, Tomohiro Tanaka, Junko Baba, Aya Ohtsubo, Satoshi Shoji, Daisuke Ishikawa, Rie Kondo, Masaaki Okajima, Satoru Miura, Junta Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Ichiei Narita. Critical roles of chemo-resistant effector and regulatory T cells in cancer immunotherapy during hemostatic proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3153. doi:10.1158/1538-7445.AM2015-3153

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