Mutsumi Hayashi

Nuclear localization of CD26 induced by a humanized monoclonal antibody inhibits tumor cell growth by modulating of POLR2A transcription.

CD26 is a type II glycoprotein known as dipeptidyl peptidase IV and has been identified as one of the cell surface markers associated with various types of cancers and a subset of cancer stem cells. Recent studies have suggested that CD26 expression is involved in tumor growth, tumor invasion, and metastasis. The CD26 is shown in an extensive intracellular distribution, ranging from the cell surface to the nucleus. We have previously showed that the humanized anti-CD26 monoclonal antibody (mAb), YS110, exhibits inhibitory effects on various cancers. However, functions of CD26 on cancer cells and molecular mechanisms of impaired tumor growth by YS110 treatment are not well understood. In this study, we demonstrated that the treatment with YS110 induced nuclear translocation of both cell-surface CD26 and YS110 in cancer cells and xenografted tumor. It was shown that the CD26 and YS110 were co-localized in nucleus by immunoelectron microscopic analysis. In response to YS110 treatment, CD26 was translocated into the nucleus via caveolin-dependent endocytosis. It was revealed that the nuclear CD26 interacted with a genomic flanking region of the gene for POLR2A, a subunit of RNA polymerase II, using a chromatin immunoprecipitation assay. This interaction with nuclear CD26 and POLR2A gene consequently led to transcriptional repression of the POLR2A gene, resulting in retarded cell proliferation of cancer cells. Furthermore, the impaired nuclear transport of CD26 by treatment with an endocytosis inhibitor or expressions of deletion mutants of CD26 reversed the POLR2A repression induced by YS110 treatment. These findings reveal that the nuclear CD26 functions in the regulation of gene expression and tumor growth, and provide a novel mechanism of mAb-therapy related to inducible translocation of cell-surface target molecule into the nucleus.

Ordering of mutations in acute myeloid leukemia with partial tandem duplication of MLL (MLL-PTD)

Partial tandem duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated, whereas NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS), typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.

Localization of CD26/DPPIV in nucleus and its nuclear translocation enhanced by anti-CD26 monoclonal antibody with anti-tumor effect

BackgroundCD26 is a type II, cell surface glycoprotein known as dipeptidyl peptidase (DPP) IV. Previous studies have revealed CD26 expression in T cell leukemia/lymphoma and malignant mesothelioma, and an inhibitory effect of anti-CD26 monoclonal antibody (mAb) against the growth of CD26+ cancer cells in vitro and in vivo. The function of CD26 in tumor development is unknown and the machinery with which the CD26 mAb induces its anti-tumor effect remains uncharacterized.ResultsThe localization of CD26 in the nucleus of T cell leukemia/lymphoma cells and mesothelioma cells was shown by biochemical and immuno-electron microscopic analysis. The DPPIV enzyme activity was revealed in the nuclear fraction of T cell leukemia/lymphoma cells. These expressions of intra-nuclear CD26 were augmented by treatment with the CD26 mAb, 1F7, with anti-tumor effect against the CD26+ T cell leukemia/lymphoma cells. In contrast, the CD26 mAb, 5F8, without anti-tumor effect, did not augment CD26 expressions in the nucleus. Biotin-labeled, cell surface CD26 translocated into the nucleus constantly, and this translocation was enhanced with 1F7 treatment but not with 5F8.ConclusionThese results indicate that the intra-nuclear CD26 which moves from plasma membrane may play certain roles in cell growth of human cancer cells.

Blockade of CD26 signaling inhibits human osteoclast development

Bone remodeling is maintained by the delicate balance between osteoblasts (OBs) and osteoclasts (OCs). However, the role of CD26 in regulating bone remodeling has not yet been characterized. We herein show that CD26 is preferentially expressed on normal human OCs and is intensely expressed on activated human OCs in osteolytic bone alterations. Macrophage‐colony stimulating factor (M‐CSF) and soluble receptor activator of NF‐κB ligand (sRANKL) induced human OC differentiation, in association with CD26 expression on monocyte‐macrophage lineage cells. CD26 expression was accompanied by increased phosphorylation of p38 mitogen‐activated protein kinase (p38 MAPK), which is crucial for early human OC differentiation. The humanized anti‐CD26 monoclonal antibody, huCD26mAb, impaired the formation and function of tartrate‐resistant acid phosphatase (TRAP)/CD26 positive multi‐nucleated (nuclei > 3) OCs with maturation in the manner of dose‐dependency. It was revealed that huCD26mAb inhibits early OC differentiation via the inactivation of MKK3/6, p38 MAPK and subsequent dephosphorylation of microphthalmia‐associated transcription factor (mi/Mitf). These inhibitions occur immediately after RANKL binds to RANK on the human OC precursor cells and were demonstrated using the OC functional assays. huCD26mAb subsequently impaired OC maturation and bone resorption by suppressing the expression of TRAP and OC fusion proteins. In addition, p38 MAPK inhibitor also strongly inhibited OC formation and function. Our results suggest that the blockade of CD26 signaling impairs the development of human functional OCs by inhibiting p38 MAPK‐mi/Mitf phosphorylation pathway and that targeting human OCs with huCD26mAb may have therapeutic potential for the treatment of osteolytic lesions following metastasis to alleviate bone destruction and reduce total skeletal‐related events (SREs).

Altered cellular immunity in transgenic mice with T cell-specific expression of human D4-guanine diphosphate-dissociation inhibitor (D4-GDI)

D4-GDI, a Rho guanosine diphosphate (GDP) dissociation inhibitor, is preferentially expressed in hematopoietic tissues and binds to a small GTP-binding protein, Rho, and inhibits GDP dissociation from Rho. We identified point mutations in the D4-GDI gene in human leukemic cells. We therefore investigated the functions of D4-GDI and mutated D4-GDI in T cells. Transgenic mice (Tg) harboring human wild-type and mutant D4-GDI transgenes driven by the lck promoter were generated. Cellular immunity responses against cytozoic pathogens were examined. The cytoskeletal organization in the CD3+T cells and the proliferation of splenocytes by Con A were investigated in both Tg and littermates (LMs). Granuloma formation by bacille Calmette-Guerin was impaired in the wild-type D4-GDI Tg. On the other hand, the number of granulomas of the mutated D4-Tg was significantly higher. Infection with Listeria was more rapidly fatal to wild-type D4-GDI Tg than to LMs, while the survival of mutated D4-GDI Tg was prolonged. The CD3+T cells in wild-type D4-GDI Tg showed an impairment in the formation of stress fibers on anti-CD3 antibody-coated plates, whereas the cytoskeletal organization in CD3+T cells of the mutated D4-GDI Tg was augmented. The proliferation of splenocytes after Con A stimulation was higher in the mutated D4-GDI Tg than in the LMs. D4-GDI may have important functions, such as induction of T cell migration, adhesion and/or proliferation in inflammatory foci, in cellular immunity responses to cytozoic pathogens.

Establishment of a novel childhood acute myeloid leukaemia cell line, KOPM-88, containing partial tandem duplication of the MLL gene and an in vivo model for childhood acute myeloid leukaemia using NOD/SCID mice

MLL gene rearrangement is common in both adult and childhood acute myeloid leukaemia (AML), and its role in oncogenesis has been investigated. While over 50 translocated‐partner genes have been identified so far, few studies have detailed the molecular mechanism of partial tandem duplication (PTD) of the MLL gene. The prognostic impact and contribution to leukaemogenesis of MLL‐PTD, especially in childhood cases, remain unknown. We have established a novel cell line containing MLL‐PTD derived from an 11‐year‐old patient with AML and designated as KOPM‐88. KOPM‐88 cells exhibited certain characteristics associated with the myeloid lineage including abundant primary granules in the cytoplasm and the expression of myeloperoxidase. The cell growth of KOPM‐88 was cytokine independent but was accelerated by granulocyte colony‐stimulating factor and granulocyte‐macrophage colony‐stimulating factor. MLL‐PTD of exon 2 to exon 6 and exon 2 to exon 8 was revealed using Southern blotting, fluorescence in situ hybridisation, and reverse transcription polymerase chain reaction/DNA sequencing. Furthermore, non‐obese diabetic/severe combined immunodeficient mice inoculated with KOPM‐88 cells exhibited leukaemic infiltrations in the bone marrow and hemiparalysis because of compression myelopathy. This is the first report of an in vivo animal model exhibiting the systemic involvement of childhood AML containing MLL‐PTD. KOPM‐88 cells and our murine model may be useful for investigating the pathogenesis of childhood AML associated with MLL gene rearrangement.

CD26 is a potential therapeutic target by humanized monoclonal antibody for the treatment of multiple myeloma

CD26, a 110-kDa transmembrane glycoprotein that is expressed on several tumor cells including malignant lymphoma, has been implicated in tumorigenesis: however, little is known regarding its role in multiple myeloma (MM). Recently, we identified CD26 expression on human osteoclasts (OCs) and demonstrated that humanized IgG1 monoclonal antibody targeting CD26, huCD26mAb, inhibits human OC differentiation. Herein, we show that CD26 expression was present on plasma cells in the bone marrow tissues of MM patients. In vitro immunostaining studies revealed that although CD26 expression was low or absent on MM cell lines cultured alone, it was intensely and uniformly expressed on MM cell lines co-cultured with OCs. The augmented CD26 expression in MM cells was exploited to enhance anti-MM efficacy of huCD26mAb via a substantial increase in antibody-dependent cytotoxicity (ADCC) but not complement-dependent cytotoxicity (CDC). Moreover, huCD26mAb in combination with novel agents synergistically enhanced huCD26mAb induced ADCC activity against CD26+ MM cells compared with each agent alone. huCD26mAb additionally reduced the ratio of the side population (SP) fraction in CD26+ MM cells by ADCC. Finally, huCD26mAb significantly reduced the MM tumor burden and OC formation in vivo. These results suggest that CD26 is a potential target molecule in MM and that huCD26mAb could act as a therapeutic agent.

Abstract 2450: Mutational profiling of MLL-PTD acute myeloid leukemia

In this study, we performed whole-exome and targeted sequencing on 85 MLL-PTD AML patients. These AMLs have oncogenic tandem duplication of the MLL gene. At least one well-known oncogenic driver mutation was identified in over 90% of the MLL-PTD patients. In line with earlier sequencing studies of other AML subtypes and the TCGA-AML-sequencing project, DNMT3A was the most often mutated epigenetic regulator (25%); IDH1/2 hotspot mutations were identified in 31% of patients. TET family was the third most prominently mutated epigenetic regulator (TET1 (5%), TET2 (16.3%). Mutations of epigenetic regulators also occurred in polycomb-associated proteins (EZH2, ASXL family members), chromatin remodelers (ARID2, ARID1A), genes associated with histone acetylation (CREBBP, EP300, KAT6A, KAT6B) and histone methylation (MLL2, MLL3). Proliferation-related pathway was extensively mutated, with 54 of 80 MLL-PTD patients (67.5%) carrying at least one mutation of proliferative genes. Specifically, FLT3 mutations were found in 46% of patient samples. Notably, some FLT3-ITD patients had more than one type of internal tandem duplication (ITD) insertion, probably reflecting existence of multiple subclones in these leukemias. We found highly prevalent mutations of cohesin genes: STAG2 (16%), SMC1A (6%), SMC3 (1%), RAD21 (1%) and CTCF (6%). Cohesin pathway is more frequently mutated in MLL-PTD patients (26%) than the AML samples from either TCGA (13%) or a meta-analysis of 1000 AML (9.1%). Remarkably, an extremely high proportion of the mutations had a strong tendency to disrupt the coding sequence in STAG2, emphasizing their crucial tumor-suppressor role in this AML subtype (16% in MLL-PTD vs 3% in TCGA-AML. RNA processing pathway was also strikingly altered in MLL-PTD patients. The most prominently mutated genes within this category were the splicing factors. They included U2AF1 (13%, S34F/Y), SRSF2 (3%), SF3A1 (5%), ZRSR2 (3%), DHX15 (1%) and CWC22 (1%). Multiple mutations co-occur with MLL-PTD which are usually acquired in a sequential manner. A potential ordering for acquisition of many mutations include IDH2/DNMT3A/U2AF1/TET2→MLL-PTD→RAS-receptor tyrosine kinase based on the following reasons: #1, real-time-PCR showed that MLL-PTD was absent in remission while mutations of IDH2, DNMT3A, TET2 and U2AF1 were still retained with a high VAF. This suggests that MLL-PTD was acquired after mutations of IDH2, DNMT3A, TET2 and U2AF1; #2, MLL-PTD is highly stable during disease progression as compared with mutations of the RAS-RTK. On the other hand, RAS-RTK mutations frequently exist as subclonal mutations and tend to be unstable during disease progression. These observations support a notion that MLL-PTD was acquired prior to RAS-RTK. Taken together, MLL-PTD is acquired after those remission-persisting, initiating mutations (IDH2, DNMT3A, TET2 and U2AF1), but prior to lesions of the proliferation-related drivers. Citation Format: Lingwen Ding, Qiaoyang Sun, Kar-Tong Tan, Wenwen Chien, Anand Mayakonda, Dechen Lin, Xinyi Loh, Jinfen Xiao, Manja Meggendorfer, Tamara Alpermann, Manoj Garg, Su-Lin Lim, Vikas Madan, Norimichi Hattori, Yasunobu Nagata, Satoru Miyano, Allen Yeoh Eng Juh, Hsin-An Hou, Yan-Yi Jiang, Yan-Yi Jiang, Sumiko Takao, Li-Zhen Liu, Siew-Zhuan Tan, Siew-Zhuan Tan, Michael Lill, Mutsumi Hayashi, Akitoshi Kinoshita, Hagop M. Kantarjian, Steven M. Kornblau, Seishi Ogawa, Torsten Haferlach, Henry Yang, H. Phillip Koeffler. Mutational profiling of MLL-PTD acute myeloid leukemia [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 2450. doi:10.1158/1538-7445.AM2017-2450