Naoya Tatsumi

CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia.

Permanent cure of acute myeloid leukemia (AML) by chemotherapy alone remains elusive for most patients because of the inability to effectively eradicate leukemic stem cells (LSCs), the self-renewing component of the leukemia. To develop therapies that effectively target LSC, one potential strategy is to identify cell surface markers that can distinguish LSC from normal hematopoietic stem cells (HSCs). In this study, we employ a signal sequence trap strategy to isolate cell surface molecules expressed on human AML-LSC and find that CD96, which is a member of the Ig gene superfamily, is a promising candidate as an LSC-specific antigen. FACS analysis demonstrates that CD96 is expressed on the majority of CD34+CD38− AML cells in many cases (74.0 ± 25.3% in 19 of 29 cases), whereas only a few (4.9 ± 1.6%) cells in the normal HSC-enriched population (Lin−CD34+CD38−CD90+) expressed CD96 weakly. To examine whether CD96+ AML cells are enriched for LSC activity, we separated AML cells into CD96+ and CD96− fractions and transplanted them into irradiated newborn Rag2−/− γc−/− mice. In four of five samples, only CD96+ cells showed significant levels of engraftment in bone marrow of the recipient mice. These results demonstrate that CD96 is a cell surface marker present on many AML-LSC and may serve as an LSC-specific therapeutic target.

Antiapoptotic function of 17AA(+)WT1 (Wilms' tumor gene) isoforms on the intrinsic apoptosis pathway.

The WT1 gene is overexpressed in human primary leukemia and a wide variety of solid cancers. The WT1 gene is alternatively spliced at two sites, yielding four isoforms: 17AA(+)KTS(+), 17AA(+)KTS(−), 17AA(−)KTS(+), and 17AA(−)KTS(−). Here, we showed that 17AA(+)WT1-specific siRNA induced apoptosis in three WT1-expressing leukemia cell lines (K562, HL-60, and Kasumi-1), but not in WT1-non-expressing lymphoma cell line (Daudi). 17AA(+)WT1-specific siRNA activated caspase-3 and -9 in the intrinsic apoptosis pathway but not caspase-8 in the extrinsic one. On the other hand, 17AA(−)WT1-specific siRNA did not induce apoptosis in the three WT1-expressing cell lines. The apoptosis was associated with activation of proapoptotic Bax, which was activated upstream of the mitochondria. Constitutive expression of 17AA(+)WT1 isoforms inhibited apoptosis of K562 leukemia cells induced by apoptosis-inducing agents, etoposide and doxorubicin, through the protection of mitochondrial membrane damages, and DNA-binding zinc-finger region of 17AA(+)WT1 isoform was essential for the antiapoptotic functions. We further studied the gene(s) whose expression was altered by the expression of 17AA(+)WT1 isoforms and showed that the expression of proapoptotic Bak was decreased by the expression of 17AA(+)KTS(−)WT1 isoform. Taken together, these results indicated that 17AA(+)WT1 isoforms played antiapoptotic roles at some points upstream of the mitochondria in the intrinsic apoptosis pathway.

Wilms’ tumor gene WT1 17AA(–)/KTS(–) isoform induces morphological changes and promotes cell migration and invasion in vitro

The wild‐type Wilms’ tumor gene WT1 is overexpressed in human primary leukemia and in a wide variety of solid cancers. All of the four WT1 isoforms are expressed in primary cancers and each is considered to have a different function. However, the functions of each of the WT1 isoforms in cancer cells remain unclear. The present study demonstrated that constitutive expression of the WT1 17AA(–)/KTS(–) isoform induces morphological changes characterized by a small‐sized cell shape in TYK‐nu.CP‐r (TYK) ovarian cancer cells. In the WT1 17AA(–)/KTS(–) isoform‐transduced TYK cells, cell–substratum adhesion was suppressed, and cell migration and in vitro invasion were enhanced compared to that in mock vector‐transduced TYK cells. Constitutive expression of the WT1 17AA(–)/KTS(–) isoform also induced morphological changes in five (one gastric, one esophageal, two breast and one fibrosarcoma) of eight cancer cell lines examined. No WT1 isoforms other than the WT1 17AA(–)/KTS(–) isoform induced the phenotypic changes. A decrease in α‐actinin 1 and cofilin expression and an increase in gelsolin expression were observed in WT1 17AA(–)/KTS(–) isoform‐transduced TYK cells. In contrast, co‐expression of α‐actinin 1 and cofilin or knockdown of gelsolin expression by small interfering RNA restored WT1 17AA(–)/KTS(–) isoform‐transduced TYK cells to a phenotype that was comparable to that of the parent TYK cells. These results indicated that the WT1 17AA(–)/KTS(–) isoform exerted its oncogenic functions through modulation of cytoskeletal dynamics. The present results may provide a novel insight into the signaling pathway of the WT1 gene for its oncogenic functions. (Cancer Sci 2006; 97: 259–270)

CD138-negative clonogenic cells are plasma cells but not B cells in some multiple myeloma patients

Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19+ clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19+ B cells. We found that in vitro MM colony-forming cells were enriched in CD138−CD19−CD38++ plasma cells, while CD19+ B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138−CD19−CD38++ plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19+ B cells was detected. In 4 out of 9 cases, CD138+ plasma cells propagated MM, although more slowly than CD138− cells. Finally, we transplanted CD19+ B cells from 13 MM patients into NOD/SCID IL2Rγc−/− mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138− and CD138+ cells have the potential to propagate MM clones in vivo in the absence of CD19+ B cells.

Identification and characterization of a WT1 (Wilms Tumor Gene) protein-derived HLA-DRB1*0405-restricted 16-mer helper peptide that promotes the induction and activation of WT1-specific cytotoxic T lymphocytes.

Effective tumor vaccine may be required to induce both cytotoxic T lymphocyte (CTL) and CD4+ helper T-cell responses against tumor-associated antigens. CD4+ helper T cells that recognize HLA class II-restricted epitopes play a central role in the initiation and maintenance of antitumor immune responses. The Wilms tumor gene WT1 is overexpressed in both leukemias and solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for cancer immunotherapy. In this study, we identified a WT1 protein-derived 16-mer peptide, WT1332 (KRYFKLSHLQMHSRKH), which was restricted with HLA-DRB1*0405, one of the most common HLA class II types in Japanese, as a helper epitope that could elicit WT1-specific CD4+ T-cell responses. We established a WT1332-specific CD4+ helper T-cell clone (E04.1), which could respond to both HLA-DRB1*0405-positive, WT1-expressing transformed hematopoietic cells and autologous dendritic cells pulsed with apoptosis-induced WT1-expressing cells, indicating that the WT1332 was a naturally processed helper epitope. Stimulation of peripheral blood mononuclear cells with both the CTL epitope (WT1235) and the helper epitope (WT1332) in the presence of WT1332-specific TH1-type CD4+ T cell clone strikingly enhanced the induction and the functional activity of WT1235-specific CTLs compared with that of peripheral blood mononuclear cells with the WT1235 alone. These results indicated that a helper epitope, WT1332 should be useful for improvement of the efficacy of CTL epitope-based cancer vaccine targeting WT1 in the clinical setting.

Maintenance of complete remission after allogeneic stem cell transplantation in leukemia patients treated with Wilms tumor 1 peptide vaccine

The prognosis of patients after allogeneic hematopoietic stem cell transplantation (HSCT) is still not satisfactory because, while treatment-related mortalities have decreased, relapse after HSCT remains a major concern. The effectiveness of allogeneic HSCT for hematological malignancies is the result of immunologic rejection of recipient leukemia cells by donor T cells, known as the graft-versus-leukemia (GVL) effect.1 It is thus obviously important to be able to exploit the GVL effect while minimizing graft-versus-host disease (GVHD). A targeted anti-leukemic immunotherapy, such as use of a leukemia vaccine,2 is a promising strategy to boost the GVL effect. Wilms tumor1 (WT1) protein is one of the best targets for leukemia vaccines. Overexpression of the wild-type WT1 gene has been detected in all types of human leukemia.3, 4, 5 We performed a phase I clinical study of immunotherapy targeting the WT1 protein in patients with leukemia, and were able to show that WT1 vaccination was safe and could induce WT1-specific cytotoxic T lymphocyte (CTL).6 Furthermore, reduction of minimal residual disease and long-lasting complete remission (CR) was observed in some leukemia patients who were given the WT1 vaccine.7 This report presents the results of phase I clinical study of WT1 vaccination for HLA-A*2402-positivie post-HSCT patients who were at high risk of relapse (HSCT in non-CR and 2nd HSCT for post-transplant relapse) or had already relapsed. The HLA-A*2402-restricted modified 9-mer WT1 peptide (amino acids 235–243 CYTWNQMNL)8 was emulsified with Montanide ISA51 adjuvant. Patients were intradermally injected with 1.0 mg (three patients: UPNs 1, 4 and 6) or 3.0 mg (other six patients) of WT1 peptide four times weekly. When no adverse effects and no obvious disease progression were observed after the fourth injection, further WT1 vaccinations at 2-week intervals were administered. Nine patients (five with acute myeloid leukemia (AML), one each with acute lymphoblastic leukemia, chronic myelomonocytic leukemia, multiple myeloma and T-cell lymphoblastic lymphoma) were enrolled in this study (Supplementary Tables 1 and 2). Local inflammatory response was observed at the vaccine injection sites of all patients. One patient (UPN5) suffered mild hypoxia (PaO2 65 mm Hg at room air) and restrictive pulmonary dysfunction (FEV1.0 40%) 65 days after the start of WT1 vaccination (day 199 after HSCT; Figure 1a). He was diagnosed with bronchioleitis obliterans (BO), which was a symptom of chronic GVHD. The patient recovered soon after administration of inhaled steroids. While early and sudden discontinuation of prednisolone and tacrolimus (day 103 after HSCT) were considered to be the reason for development of BO, the possibility of an association between BO and WT1 vaccination cannot be entirely ruled out. In other eight patients, no severe toxicities related to WT1 vaccine were observed (Table1). Figure 1 Clinical course of patients who attained CR after the start of WT1 peptide vaccination. (a) Clinical course of UPN5 who achieved CR after administration of WT1 vaccine but stopped vaccination because of the development of bronchioleitis obliterans. ( ... Table 1 Patient outcomes Three AML patients (UPN1–3), who had undergone HSCT in non-CR, started WT1 vaccine in CR (Supplementary Tables 1 and 2). They started WT1 vaccination on post-HSCT days 141, 76 and 93 and have remained in CR for 1038, 973 and 662 days, respectively (as of 8 April 2013; Table1), suggesting the potential of WT1 vaccination as a maintenance therapy after HSCT. Six patients started WT1 vaccination in non-CR and two of them became CR after WT1 vaccination. One B-ALL patient (UPN4) with MLL-AF4 underwent bone marrow transplantation from an HLA-matched unrelated donor during the first CR. On post-HSCT day 111, MLL-AF4 and WT1 mRNA in peripheral blood (PB) had increased to 16 000 and 15 000 copies/μg RNA, indicating that the disease had relapsed. Tacrolimus and prednisolone doses were tapered off to induce GVL effects. The expression levels of MLL-AF4 and WT1 mRNA in PB had decreased to 2700 and 190 copies/μg RNA by day 132, and WT1 vaccination was started on day 133. MLL-AF4 mRNA had become undetectable by day 146, and had never appeared until post-HSCT day 1312 (day 1179 after the start of WT1 vaccination as of 8 April 2013; Figure 1b). Skin tumors appeared in UPN5 (AML-M5) on post-HSCT day 103 and was diagnosed by biopsy as leukemia relapse. Tacrolimus was discontinued on day103, and WT1 vaccination was started on day 130. Cutaneous tumors had regressed 2 weeks after the start of WT1 vaccination, but vaccination was terminated after the second injection because of the development of BO as described earlier (Figure 1a). This patient has been remained in CR until post-HSCT day 972 (day 842 after the start of WT1 vaccination at 8 April 2013). While the exact contribution of the vaccination effect to the disease remission in addition to the GVL effect was unclear, the fact that both of these two patients still have remained in CR until now is encouraging to continue this trial. In the following phase II trials, the enumeration of WT1-specific CTLs should be performed more frequently after the start of vaccination to clarify the relationship between the effect of WT1 peptide vaccination and leukemia regression. WT1 (a natural 9-mer WT1 peptide) HLA-A*2402 tetramer assays could be performed with peripheral blood mononuclear cell in seven of the nine patients to determine whether WT1235 peptide-specific CD8+ T cells had increased after WT1 vaccination. The gates for WT1 tetramer+ cells were drawn as <0.1% of CD8+ T cells were included in the tetramer-positive gate in multiple healthy individuals (Supplementary Figure 1A). WT1235 tetramer+ cells increased after the start of vaccination in three (UPNs1, 2 and 4) of the four patients who have remained in CR (Figure 1b and Supplementary Figure 1B). In the cases with progressive disease, continuous increase in the frequencies of WT1235 tetramer+ cells was not observed (Supplementary Figure 1B). Our results suggest that WT1 vaccination should be started when the leukemia burden is minimal. The timing of the start of WT1 vaccination may be also important. For the cases with good outcomes, WT1 vaccination was started 76–140 days after transplantation (UPNs1–5), and at later times (days 299–1815) for PD cases (UPNs 6–9). A lymphopenic environment a few months after transplantation may be favorable for rapid and extensive expansion of tumor antigen-specific CTLs. In summary, this report suggests that WT1 vaccine can be safely administrated for post-HSCT patients with hematological malignancies and has potential as a maintenance therapy. Clinical benefit of WT1 vaccination for post-HSCT patients will be evaluated in the subsequent phase II trials.

The translation elongation factor eEF2 is a novel tumor‑associated antigen overexpressed in various types of cancers

Recent studies have shown that cancer immunotherapy could be a promising therapeutic approach for the treatment of cancer. In the present study, to identify novel tumor-associated antigens (TAAs), the proteins expressed in a panel of cancer cells were serologically screened by immunoblot analysis and the eukaryotic elongation factor 2 (eEF2) was identified as an antigen that was recognized by IgG autoantibody in sera from a group of patients with head and neck squamous cell carcinoma (HNSCC) or colon cancer. Enzyme-linked immunosorbent assay showed that serum eEF2 IgG Ab levels were significantly higher in colorectal and gastric cancer patients compared to healthy individuals. Immunohistochemistry experiments showed that the eEF2 protein was overexpressed in the majority of lung, esophageal, pancreatic, breast and prostate cancers, HNSCC, glioblastoma multiforme and non-Hodgkin’s lymphoma (NHL). Knockdown of eEF2 by short hairpin RNA (shRNA) significantly inhibited the growth in four eEF2-expressing cell lines, PC14 lung cancer, PCI6 pancreatic cancer, HT1080 fibrosarcoma and A172 glioblastoma cells, but not in eEF2-undetectable MCF7 cells. Furthermore, eEF2-derived 9-mer peptides, EF786 (eEF2 786–794 aa) and EF292 (eEF2 292–300 aa), elicited cytotoxic T lymphocyte (CTL) responses in peripheral blood mononuclear cells (PBMCs) from an HLA-A*24:02- and an HLA-A*02:01-positive healthy donor, respectively, in an HLA-A-restricted manner. These results indicated that the eEF2 gene is overexpressed in the majority of several types of cancers and plays an oncogenic role in cancer cell growth. Moreover, the eEF2 gene product is immunogenic and a promising target molecule of cancer immunotherapy for several types of cancers.

Enhanced tumor immunity of WT1 peptide vaccination by interferon-β administration.

To induce and activate tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) for cancer immunity, it is important not only to select potent CTL epitopes but also to combine them with appropriate immunopotentiating agents. Here we investigated whether tumor immunity induced by WT1 peptide vaccination could be enhanced by IFN-β. For the experimental group, C57BL/6 mice were twice pre-treated with WT1 peptide vaccine, implanted with WT1-expressing C1498 cells, and treated four times with WT1 peptide vaccine at one-week intervals. During the vaccination period, IFN-β was injected three times a week. Mice in control groups were treated with WT1 peptide alone, IFN-β alone, or PBS alone. The mice in the experimental group rejected tumor cells and survived significantly longer than mice in the control groups. The overall survival on day 75 was 40% for the mice treated with WT1 peptide+IFN-β, while it was 7, 7, and 0% for those treated with WT1 peptide alone, IFN-β alone or PBS alone, respectively. Induction of WT1-specific CTLs and enhancement of NK activity were detected in splenocytes from mice in the experimental group. Furthermore, administration of IFN-β enhanced expression of MHC class I molecules on the implanted tumor cells. In conclusion, our results showed that co-administration of WT1 peptide+IFN-β enhanced tumor immunity mainly through the induction of WT1-specific CTLs, enhancement of NK activity, and promotion of MHC class I expression on the tumor cells. WT1 peptide vaccination combined with IFN-β administration can thus be expected to enhance the clinical efficacy of WT1 immunotherapy.

HLA-DPB1*05: 01-restricted WT1332-specific TCR-transduced CD4+ T lymphocytes display a helper activity for WT1-specific CTL induction and a cytotoxicity against leukemia cells.

Wilms tumor gene 1 (WT1) is overexpressed in various malignant neoplasms, and has been demonstrated as an attractive target for cancer immunotherapy. We previously reported the identification of a WT1 protein-derived, 16-mer helper peptide WT1332 that could elicit Th1-type CD4+ T-cell response and bind to multiple HLA class II molecules. In this study, we examined the feasibility of adoptive therapy using CD4+ T cells that were transduced an HLA-DPB1*05:01-restricted, WT1332-specific T-cell receptor (TCR). HLA-DPB1*05:01-restricted, WT1332-specific TCR-transduced CD4+ T cells were successfully generated using lentiviral vector and exhibited strong proliferative response and Th1-type cytokine production in response to WT1332 peptide, WT1 protein, or WT1-expressing tumor cell lysate. Furthermore, the WT1332-specific TCR-transduced CD4+ T cells lysed HLA-DPB1*05:01-positive, WT1-expressing human leukemia cells through granzyme B/perforin pathway. Furthermore, stimulation of peripheral blood mononuclear cells with both HLA-A*24:02-restricted cytotoxic T lymphocytes-epitope peptide (modified 9-mer WT1235 peptide, WT1235m) and WT1332 helper peptide in the presence of WT1332-specific TCR-transduced CD4+ T cells strikingly enhanced the induction of WT1235m-specific cytotoxic T lymphocytes. Thus, these results demonstrated the feasibility of immunotherapy based on adoptive transfer of WT1332-specific TCR-transduced CD4+ T cells for the treatment of leukemia.

Deficiency in WT1-targeting microRNA-125a leads to myeloid malignancies and urogenital abnormalities

The Wilms’ tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3’UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (−/−) mice than in miR-125a (+/−) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.