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Papers - Kagoya, Yuki

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Plasma membrane-coated nanoparticles and membrane vesicles to orchestrate multimodal antitumor immunity.

Yusuke Ito, Hitomi Kasuya, Mirei Kataoka, Noriko Nakamura, Toshiaki Yoshikawa, Takahiro Nakashima, Haosong Zhang, Yang Li, Tetsuya Matsukawa, Satoshi Inoue, Chitose Oneyama, Seiichi Ohta, Yuki Kagoya

Journal for immunotherapy of cancer 13 ( 1 ) 2025.01

Last author, Corresponding author, Accepted

BACKGROUND: A number of immunotherapeutic approaches have been developed and are entering the clinic. Bispecific antibodies (BsAbs) are one of these modalities and induce robust efficacy by endogenous T cells in several hematological malignancies. However, most of the treated patients experience only a temporary benefit. Currently available BsAbs provide only anti-CD3 antibody-mediated T-cell stimulation, but not the costimulation or cytokine signaling essential for full T-cell activation. Here, we hypothesized that the simultaneous input of more comprehensive signals would elicit more robust and durable effector T-cell functions. METHODS: We genetically engineered the leukemia cell line K562 to express BsAbs, costimulatory ligands, cytokines, and blocking antibodies against immune checkpoint molecules on the cell surface, from which we obtained plasma membrane fractions by mechanical homogenization and subsequent isolation steps. Plasma membranes were reconstituted on the poly (lactic-co-glycolic acid) surface to generate membrane-coated nanoparticles (NPs). Alternatively, nano-sized membrane vesicles (MVs) were generated by ultrasonic dispersion of the isolated membranes. The antitumor function of NPs and MVs loaded with various immunomodulatory factors was evaluated in vitro and in vivo. RESULTS: Both membrane-coated NPs and MVs induced BsAb-mediated antigen-specific cytotoxic activity in non-specific T cells, with MVs inducing a slightly better response in vivo. Importantly, T-cell activation was elicited only in the presence of target tumor cells, providing a safety advantage for clinical use. NPs and MVs expressing costimulatory molecules (CD80/4-1BBL) and cytokines (interleukin (IL)-7/IL-15) further enhanced effector T-cell function and induced therapeutic efficacy in vivo. In addition, MVs expressing immune checkpoint antibodies and inflammatory cytokines IL-12 and IL-18 induced objective antitumor responses in solid tumor models partly by converting immunosuppressive macrophages to proinflammatory phenotypes and inducing cytotoxic T-cell infiltration into the tumor. Finally, we showed that MVs were also engineered to activate natural killer (NK) cells by loading multiple ligands. MVs loaded with BsAbs, 4-1BBL, IL-15, and IL-21 induced NK-cell cytotoxic activity in an antigen-specific manner. CONCLUSIONS: We developed antitumor NPs and MVs that efficiently induced antitumor immune responses in vivo by simultaneously delivering multiple immunostimulatory signals to endogenous T cells. This platform enables the delivery of desired combinations of antitumor immune signals into T cells and NK cells.

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Protocol to measure human IL-6 secretion from CAR T cell-primed macrophage and monocyte lineage cells in vitro and in vivo using humanized mice.

Thao Nguyen, Toshiaki Yoshikawa, Yusuke Ito, Hitomi Kasuya, Takahiro Nakashima, Sachiko Okamoto, Yasunori Amaishi, Haosong Zhang, Yang Li, Tetsuya Matsukawa, Satoshi Inoue, Yuki Kagoya

STAR protocols 5 ( 4 ) 103423 - 103423 2024.12

Last author, Corresponding author, Accepted

Chimeric antigen receptor (CAR) T cell therapy often causes serious toxicities, such as cytokine release syndrome (CRS), mainly due to interleukin-6 (IL-6) secreted by monocyte lineage cells. Here, we describe a protocol to generate anti-CD19 CAR T cells and quantify human monocyte-derived IL-6 cocultured with CAR T cells and target tumor cells in vitro. We further describe a protocol to generate a humanized mouse model and evaluate CAR T cell-associated plasma IL-6 levels in vivo. For complete details on the use and execution of this protocol, please refer to Yoshikawa et al.1.

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Gene editing technology to improve antitumor T-cell functions in adoptive immunotherapy

Ito Y., Inoue S., Kagoya Y.

Inflammation and Regeneration (Inflammation and Regeneration) 44 ( 1 ) 2024.12

Last author, Corresponding author, Accepted

Adoptive immunotherapy, in which tumor-reactive T cells are prepared in vitro for adoptive transfer to the patient, can induce an objective clinical response in specific types of cancer. In particular, chimeric antigen receptor (CAR)-redirected T-cell therapy has shown robust responses in hematologic malignancies. However, its efficacy against most of the other tumors is still insufficient, which remains an unmet medical need. Accumulating evidence suggests that modifying specific genes can enhance antitumor T-cell properties. Epigenetic factors have been particularly implicated in the remodeling of T-cell functions, including changes to dysfunctional states such as terminal differentiation and exhaustion. Genetic ablation of key epigenetic molecules prevents the dysfunctional reprogramming of T cells and preserves their functional properties. Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based gene editing is a valuable tool to enable efficient and specific gene editing in cultured T cells. A number of studies have already identified promising targets to improve the therapeutic efficacy of CAR-T cells using genome-wide or focused CRISPR screening. In this review, we will present recent representative findings on molecular insights into T-cell dysfunction and how genetic modification contributes to overcoming it. We will also discuss several technical advances to achieve efficient gene modification using the CRISPR and other novel platforms.

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Current progress of CAR-T-cell therapy for patients with multiple myeloma

Nakashima T, Kagoya Y

Int J Hematol 120 ( 1 ) 15 - 22 2024.05

Last author, Corresponding author, Accepted, ISSN 09255710

Currently available chimeric antigen receptor (CAR)-engineered T-cell therapies targeting B-cell maturation antigen (BCMA), namely, idecabtagene vicleucel and ciltacabtagene autoleucel, have shown marked efficacy against relapsed and refractory multiple myeloma. However, further improvement in CAR-T-cell function is warranted as most patients treated with these products eventually relapse due to various mechanisms such as antigen loss and T-cell dysfunction or disappearance. Strategies for improving CAR-T-cell function include targeting of dual antigens, enhancing cell longevity through genetic modification, and eliminating the immunosuppressive tumor microenvironment. Serious side effects can also occur after CAR-T-cell infusions. Although understanding of the molecular pathogenesis of cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome is growing, the unique movement disorder caused by BCMA-targeted therapy is less understood, and its molecular mechanisms must be further elucidated to establish better management strategies. In this article, we will review the current status of BCMA-targeting CAR-T-cell therapy. We will also highlight progress in the development of CAR-T cells targeting other antigens, as well as universal allogeneic CAR-T cells and bispecific antibodies.

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Development of a chimeric cytokine receptor that captures IL-6 and enhances the antitumor response of CAR-T cells

Toshiaki Yoshikawa, Yusuke Ito, Zhiwen Wu, Hitomi Kasuya, Takahiro Nakashima, Sachiko Okamoto, Yasunori Amaishi, Haosong Zhang, Yang Li, Tetsuya Matsukawa, Satoshi Inoue, Yuki Kagoya

Cell Reports Medicine (Elsevier BV) 5 ( 5 ) 101526 - 101526 2024.05

Last author, Corresponding author, Accepted, ISSN 2666-3791

The efficacy of chimeric antigen receptor (CAR)-engineered T cell therapy is suboptimal in most cancers, necessitating further improvement in their therapeutic actions. However, enhancing antitumor T cell response inevitably confers an increased risk of cytokine release syndrome associated with monocyte-derived interleukin-6 (IL-6). Thus, an approach to simultaneously enhance therapeutic efficacy and safety is warranted. Here, we develop a chimeric cytokine receptor composed of the extracellular domains of GP130 and IL6RA linked to the transmembrane and cytoplasmic domain of IL-7R mutant that constitutively activates the JAK-STAT pathway (G6/7R or G6/7R-M452L). CAR-T cells with G6/7R efficiently absorb and degrade monocyte-derived IL-6 in vitro. The G6/7R-expressing CAR-T cells show superior expansion and persistence in vivo, resulting in durable antitumor response in both liquid and solid tumor mouse models. Our strategy can be widely applicable to CAR-T cell therapy to enhance its efficacy and safety, irrespective of the target antigen.

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High CD62L expression predicts the generation of chimeric antigen receptor T cells with potent effector functions

Hitomi Kasuya, Haosong Zhang, Yusuke Ito, Toshiaki Yoshikawa, Takahiro Nakashima, Yang Li, Tetsuya Matsukawa, Satoshi Inoue, Yuki Kagoya

International Immunology (Oxford University Press (OUP)) 36 ( 7 ) 353 - 364 2024.03

Last author, Corresponding author, Accepted, ISSN 09538178

Abstract

The efficient generation of chimeric antigen receptor (CAR) T cells is highly influenced by the quality of apheresed T cells. Healthy donor-derived T cells usually proliferate better than patients-derived T cells and are precious resources to generate off-the-shelf CAR-T cells. However, relatively little is known about the determinants that affect the efficient generation of CAR-T cells from healthy donor-derived peripheral blood mononuclear cells (PBMCs) compared with those from the patients’ own PBMCs. We here examined the efficiency of CAR-T cell generation from multiple healthy donor samples and analyzed its association with the phenotypic features of the starting peripheral blood T cells. We found that CD62L expression levels within CD8+ T cells were significantly correlated with CAR-T cell expansion. Moreover, high CD62L expression within naïve T cells was associated with the efficient expansion of T cells with a stem cell-like memory phenotype, an indicator of high-quality infusion products. Intriguingly, genetic disruption of CD62L significantly impaired CAR-T cell proliferation and cytokine production upon antigen stimulation. Conversely, ectopic expression of a shedding-resistant CD62L mutant augmented CAR-T cell effector functions compared to unmodified CAR-T cells, resulting in improved antitumor activity in vivo. Collectively, we identified the surface expression of CD62L as a concise indicator of potent T-cell proliferation. CD62L expression is also associated with the functional properties of CAR-T cells. These findings are potentially applicable to selecting optimal donors to massively generate CAR-T cell products.

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Cytokine signaling in chimeric antigen receptor T-cell therapy

Yuki Kagoya

International Immunology (Oxford University Press (OUP)) 36 ( 2 ) 49 - 56 2024.02

Lead author, Last author, Corresponding author, Accepted, ISSN 09538178

Abstract

Adoptive immunotherapy using chimeric antigen-receptor (CAR)-engineered T cells can induce robust antitumor responses against hematologic malignancies. However, its efficacy is not durable in the majority of the patients, warranting further improvement of T-cell functions. Cytokine signaling is one of the key cascades regulating T-cell survival and effector functions. In addition to cytokines that use the common γ chain as a receptor subunit, multiple cytokines regulate T-cell functions directly or indirectly. Modulating cytokine signaling in CAR-T cells by genetic engineering is one promising strategy to augment their therapeutic efficacy. These strategies include ectopic expression of cytokines, cytokine receptors, and synthetic molecules that mimic endogenous cytokine signaling. Alternatively, autocrine IL-2 signaling can be augmented through reprogramming of CAR-T cell properties through transcriptional and epigenetic modification. On the other hand, cytokine production by CAR-T cells triggers systemic inflammatory responses, which mainly manifest as adverse events such as cytokine-release syndrome (CRS) and neurotoxicity. In addition to inhibiting direct inflammatory mediators such as IL-6 and IL-1 released from activated macrophages, suppression of T-cell-derived cytokines associated with the priming of macrophages can be accomplished through genetic modification of CAR-T cells. In this review, I will outline recently developed synthetic biology approaches to exploit cytokine signaling to enhance CAR-T cell functions. I will also discuss therapeutic target molecules to prevent or alleviate CAR-T cell-related toxicities.

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Introduction: Synthetic Biology for Cancer Immunology Special Issue

Kagoya Y.

International Immunology (International Immunology) 36 ( 2 ) 45 - 48 2024.02

Last author, Corresponding author, Accepted, ISSN 09538178

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AKT2 inhibition accelerates the acquisition of phagocytic ability in induced pluripotent stem cell-derived neutrophils.

Toshiya Hino, Fumio Nakahara, Masashi Miyauchi, Yusuke Ito, Yosuke Masamoto, Ken Morita, Yuki Kagoya, Hirotatsu Kojima, Mineo Kurokawa

Experimental hematology (Experimental Hematology) 130 104137 - 104137 2024.02

Last author, Corresponding author, Accepted, ISSN 0301472X

Neutrophils are key components of the immune system that inhibit bacterial infections. Systemic bacterial infections can cause lethal conditions, especially in patients with neutropenia associated with chemotherapy or other systemic illnesses; hence, early detection of the symptoms and prompt management are crucial in such cases. Previously, we established expandable engineered neutrophil-primed progenitors (NeuPs-XL) using human-induced pluripotent stem cells (iPSCs), which can produce neutrophil-like cells at a clinically suitable scale within 4 days of inducing myeloid differentiation. In this study, using small-molecule compound-based screening, we detected that MK-2206, a selective pan-AKT inhibitor, can accelerate this differentiation process, promote phagocytic ability in neutrophils, and enhance cytokine and chemokine expression in response to lipopolysaccharides. The inhibition of AKT2 has been identified as the key mechanism underlying this acceleration. These results can make a substantial contribution to the development of strategies for the prompt production of clinically applicable iPSC-derived neutrophils, which can potentially lead to the management of severe infections associated with life-threatening neutropenia and the effective treatment of related health conditions in the future.

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Epigenetic profiles guide improved CRISPR/Cas9-mediated gene knockout in human T cells

Yusuke Ito, Satoshi Inoue, Takahiro Nakashima, Haosong Zhang, Yang Li, Hitomi Kasuya, Tetsuya Matsukawa, Zhiwen Wu, Toshiaki Yoshikawa, Mirei Kataoka, Tetsuo Ishikawa, Yuki Kagoya

Nucleic Acids Research (Oxford University Press (OUP)) 52 ( 1 ) 141 - 153 2024.01

Last author, Corresponding author, Accepted, ISSN 0305-1048

Abstract

Genetic modification of specific genes is emerging as a useful tool to enhance the functions of antitumor T cells in adoptive immunotherapy. Current advances in CRISPR/Cas9 technology enable gene knockout during in vitro preparation of infused T-cell products through transient transfection of a Cas9-guide RNA (gRNA) ribonucleoprotein complex. However, selecting optimal gRNAs remains a major challenge for efficient gene ablation. Although multiple in silico tools to predict the targeting efficiency have been developed, their performance has not been validated in cultured human T cells. Here, we explored a strategy to select optimal gRNAs using our pooled data on CRISPR/Cas9-mediated gene knockout in human T cells. The currently available prediction tools alone were insufficient to accurately predict the indel percentage in T cells. We used data on the epigenetic profiles of cultured T cells obtained from transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Combining the epigenetic information with sequence-based prediction tools significantly improved the gene-editing efficiency. We further demonstrate that epigenetically closed regions can be targeted by designing two gRNAs in adjacent regions. Finally, we demonstrate that the gene-editing efficiency of unstimulated T cells can be enhanced through pretreatment with IL-7. These findings enable more efficient gene editing in human T cells.

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CD83 expression characterizes precursor exhausted T cell population

Zhiwen Wu, Toshiaki Yoshikawa, Satoshi Inoue, Yusuke Ito, Hitomi Kasuya, Takahiro Nakashima, Haosong Zhang, Saki Kotaka, Waki Hosoda, Shiro Suzuki, Yuki Kagoya

Communications Biology (Springer Science and Business Media LLC) 6 ( 1 ) 258 2023.03

Research paper (scientific journal), Last author, Corresponding author, Accepted

T cell exhaustion is a main obstacle against effective cancer immunotherapy. Exhausted T cells include a subpopulation that maintains proliferative capacity, referred to as precursor exhausted T cells (TPEX). While functionally distinct and important for antitumor immunity, TPEX possess some overlapping phenotypic features with the other T-cell subsets within the heterogeneous tumor-infiltrating T-lymphocytes (TIL). Here we explore surface marker profiles unique to TPEX using the tumor models treated by chimeric antigen receptor (CAR)-engineered T cells. We find that CD83 is predominantly expressed in the CCR7+PD1+ intratumoral CAR-T cells compared with the CCR7-PD1+ (terminally differentiated) and CAR-negative (bystander) T cells. The CD83+CCR7+ CAR-T cells exhibit superior antigen-induced proliferation and IL-2 production compared with the CD83- T cells. Moreover, we confirm selective expression of CD83 in the CCR7+PD1+ T-cell population in primary TIL samples. Our findings identify CD83 as a marker to discriminate TPEX from terminally exhausted and bystander TIL.

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Dissecting the heterogeneity of exhausted T cells at the molecular level

Yuki Kagoya

International Immunology (Oxford University Press (OUP)) 34 ( 11 ) 547 - 553 2022.10

Lead author, Last author, Corresponding author, Accepted, ISSN 09538178

Abstract

Our understanding of mechanisms underlying T-cell exhaustion has been refined by analysis of exhausted T cells at the molecular level. The development and functions of exhausted T cells are regulated by a number of transcription factors, epigenetic factors and metabolic enzymes. In addition, recent work to dissect exhausted T cells at the single-cell level has enabled us to discover a precursor exhausted T-cell subset equipped with long-term survival capacity. Starting from the analysis of mouse models, the existence of precursor exhausted T cells has also been documented in human T cells in the context of chronic virus infections or tumors. Clinical data suggest that evaluating the quality of exhausted T cells on the basis of their differentiation status may be helpful to predict the therapeutic response to inhibition of programmed death 1 (PD1). Moreover, beyond immune-checkpoint blockade, novel therapeutic approaches to re-invigorate exhausted T cells have been explored based on molecular insights into T-cell exhaustion. Here I will discuss key molecular profiles associated with the development, maintenance and differentiation of exhausted T cells and how these findings can be applicable in the field of cancer immunotherapy.

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Epigenetic engineering for optimal chimeric antigen receptor T cell therapy.

Yusuke Ito, Yuki Kagoya

Cancer science (Cancer Science) 113 ( 11 ) 3664 - 3671 2022.08

Last author, Corresponding author, Accepted, ISSN 13479032

Recent advancements in cancer immunotherapy, such as chimeric antigen receptor (CAR)-engineered T cell therapy and immune checkpoint therapy, have significantly improved the clinical outcomes of patients with several types of cancer. To broaden its applicability further and induce durable therapeutic efficacy, it is imperative to understand how antitumor T cells elicit cytotoxic functions, survive as memory T cells, or are impaired in their effector functions (exhausted) at the molecular level. T cell properties are regulated by their gene expression profiles, which are further controlled by epigenetic architectures, such as DNA methylation and histone modifications. Multiple studies have elucidated specific epigenetic genes associated with T-cell phenotypic changes. Conversely, exogenous modification of these key epigenetic factors can significantly alter T cell functions by extensively altering the transcription network, which can be applied in cancer immunotherapy by improving T cell persistence or augmenting effector functions. As CAR-T cell therapy involves a genetic engineering step during the preparation of the infusion products, it would be a feasible strategy to additionally modulate specific epigenetic genes in CAR-T cells to improve their quality. Here, we review recent studies investigating how individual epigenetic factors play a crucial role in T-cell biology. We further discuss future directions to integrate these findings for optimal cancer immunotherapy.

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Molecular Profiles of Exhausted T Cells and Their Impact on Response to Immune Checkpoint Blockade

Kagoya Y.

Gan to kagaku ryoho. Cancer & chemotherapy (Gan to kagaku ryoho. Cancer & chemotherapy) 49 ( 6 ) 609 - 614 2022.06

Last author, Corresponding author, Accepted, ISSN 03850684

T cell exhaustion is induced in the context of chronic virus infection and tumor microenvironment, in which cytotoxic T cells are repeatedly exposed to the target antigen and deprived of their effector functions. Multiple studies have already shown the significant impact of immune checkpoint molecules such as PD1 on functional properties of exhausted T cells. In addition to these signals, exhausted T cells possess distinct transcriptional and epigenetic profiles compared with conventional effector and memory T cells. Importantly, most of these features are not affected by immune checkpoint blockade, suggesting that genetic and epigenetic remodeling of T cells is an underlying molecular mechanism essential for T cell exhaustion. Moreover, it has now been evident that exhausted T cells are a heterogeneous cell population composed of distinct T cell subsets, and these functional differences profoundly affect therapeutic efficacy of cancer immunotherapy. In this review, I will discuss recent studies investigating molecular mechanisms of T cell exhaustion, including novel key molecules essentially associated with T cell exhaustion. These findings are potentially applicable to reinvigorate effector functions of exhausted T cells.

Genetic ablation of PRDM1 in antitumor T cells enhances therapeutic efficacy of adoptive immunotherapy

Toshiaki Yoshikawa, Zhiwen Wu, Satoshi Inoue, Hitomi Kasuya, Hirokazu Matsushita, Yusuke Takahashi, Hiroaki Kuroda, Waki Hosoda, Shiro Suzuki, Yuki Kagoya

Blood (American Society of Hematology) 139 ( 14 ) 2156 - 2172 2022.04

Last author, Corresponding author, Accepted, ISSN 0006-4971

Adoptive cancer immunotherapy can induce objective clinical efficacy in patients with advanced cancer; however, a sustained response is achieved in a minority of cases. The persistence of infused T cells is an essential determinant of a durable therapeutic response. Antitumor T cells undergo a genome-wide remodeling of the epigenetic architecture upon repeated antigen encounters, which inevitably induces progressive T-cell differentiation and the loss of longevity. In this study, we identified PR domain zinc finger protein 1 (PRDM1) ie, Blimp-1, as a key epigenetic gene associated with terminal T-cell differentiation. The genetic knockout of PRDM1 by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) supported the maintenance of an early memory phenotype and polyfunctional cytokine secretion in repeatedly stimulated chimeric antigen receptor (CAR)-engineered T cells. PRDM1 disruption promoted the expansion of less differentiated memory CAR-T cells in vivo, which enhanced T-cell persistence and improved therapeutic efficacy in multiple tumor models. Mechanistically, PRDM1-ablated T cells displayed enhanced chromatin accessibility of the genes that regulate memory formation, thereby leading to the acquisition of gene expression profiles representative of early memory T cells. PRDM1 knockout also facilitated maintaining an early memory phenotype and cytokine polyfunctionality in T-cell receptor-engineered T cells as well as tumor-infiltrating lymphocytes. In other words, targeting PRDM1 enabled the generation of superior antitumor T cells, which is potentially applicable to a wide range of adoptive cancer immunotherapies.

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CAMK2G is identified as a novel therapeutic target for myelofibrosis

Masashi Miyauchi, Ken Sasaki, Yuki Kagoya, Kazuki Taoka, Yosuke Masamoto, Sho Yamazaki, Shunya Arai, Hideaki Mizuno, Mineo Kurokawa

Blood Advances (American Society of Hematology) 6 ( 5 ) 1585 - 1597 2022.03

Accepted, ISSN 2473-9529

Although JAK1/2 inhibition is effective in alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here, we established induced pluripotent stem cells (iPSCs) derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were used for chemical screening, and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutation recurrently detected in MF patients, were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 γ subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515L decreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes, such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.

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Development of next-generation chimeric antigen receptor-engineered T-cell therapy

Yuki Kagoya

臨床血液 63 ( 9 ) 1290 - 1297 2022

Lead author, Last author, Corresponding author, Accepted

Development of universal CAR-T cells

Yuki Kagoya, Naoto Hirano

臨床血液 63 ( 7 ) 782 - 789 2022

Lead author, Last author, Corresponding author, Accepted

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CD62L expression level determines the cell fate of myeloid progenitors

Yusuke Ito, Fumio Nakahara, Yuki Kagoya, Mineo Kurokawa

Stem Cell Reports (Elsevier BV) 16 ( 12 ) 2871 - 2886 2021.12

Accepted, ISSN 2213-6711

Hematopoietic cells differentiate through several progenitors in a hierarchical manner, and recent single-cell analyses have revealed substantial heterogeneity within each progenitor. Although common myeloid progenitors (CMPs) are defined as a multipotent cell population that can differentiate into granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythrocyte progenitors (MEPs), and GMPs generate neutrophils and monocytes, these myeloid progenitors must contain some lineage-committed progenitors. Through gene expression analysis at single-cell levels, we identified CD62L as a marker to reveal the heterogeneity. We confirmed that CD62L-negative CMPs represent "bona fide" CMPs, whereas CD62L-high CMPs are mostly restricted to GMP potentials both in mice and humans. In addition, we identified CD62L-negative GMPs as the most immature subsets in GMPs and Ly6C+CD62L-intermediate and Ly6C+CD62L-high GMPs are skewed to neutrophil and monocyte differentiation in mice, respectively. Our findings contribute to more profound understanding about the mechanism of myeloid differentiation.

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Evi1 upregulates Fbp1 and supports progression of acute myeloid leukemia through pentose phosphate pathway activation

Hideaki Mizuno, Junji Koya, Yosuke Masamoto, Yuki Kagoya, Mineo Kurokawa

Cancer Science (Wiley) 112 ( 10 ) 4112 - 4126 2021.10

Accepted, ISSN 1347-9032

Evi1 is a transcription factor essential for the development as well as progression of acute myeloid leukemia (AML) and high Evi1 AML is associated with extremely poor clinical outcome. Since targeting metabolic vulnerability is the emerging therapeutic strategy of cancer, we herein investigated a novel therapeutic target of Evi1 by analyzing transcriptomic, epigenetic, and metabolomic profiling of mouse high Evi1 leukemia cells. We revealed that Evi1 overexpression and Evi1-driven leukemic transformation upregulate transcription of gluconeogenesis enzyme Fbp1 and other pentose phosphate enzymes with interaction between Evi1 and the enhancer region of these genes. Metabolome analysis using Evi1-overexpressing leukemia cells uncovered pentose phosphate pathway upregulation by Evi1 overexpression. Suppression of Fbp1 as well as pentose phosphate pathway enzymes by shRNA-mediated knockdown selectively decreased Evi1-driven leukemogenesis in vitro. Moreover, pharmacological or shRNA-mediated Fbp1 inhibition in secondarily transplanted Evi1-overexpressing leukemia mouse significantly decreased leukemia cell burden. Collectively, targeting FBP1 is a promising therapeutic strategy of high Evi1 AML.

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Efficient production of human neutrophils from iPSCs that prevent murine lethal infection with immune cell recruitment.

Miyauchi Masashi, Yusuke Ito, Fumio Nakahara, Toshiya Hino, Fumi Nakamura, Yuki Iwasaki, Taiki Kawagoshi, Junji Koya, Akihide Yoshimi, Shunya Arai, Yuki Kagoya, Mineo Kurokawa

Blood (Blood) 138 ( 24 ) 2555 - 2569 2021.09

Accepted, ISSN 00064971

Neutrophils play an essential role in innate immune responses to bacterial and fungal infections and loss of neutrophil function can increase the risk of acquiring lethal infections in clinical settings. Here, we show that engineered neutrophil-primed progenitors derived from human induced pluripotent stem cells (iPSCs) can produce functional neutrophil-like cells at a clinically applicable scale that can act rapidly in vivo against lethal bacterial infections. Using five different mouse models, we systematically demonstrated that these neutrophil-like cells migrate to sites of inflammation and infection and increase survival against bacterial infection. In addition, we found that these human neutrophil-like cells can recruit murine immune cells. This system potentially provides a straight-forward solution for patients with neutrophil deficiency-an off-the-shelf neutrophil transfusion. This platform should facilitate the administration of human neutrophils for a broad spectrum of physiological and pathological conditions.

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Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4+ T cells

Kenji Sugata, Yukiko Matsunaga, Yuki Yamashita, Munehide Nakatsugawa, Tingxi Guo, Levon Halabelian, Yota Ohashi, Kayoko Saso, Muhammed A Rahman, Mark Anczurowski, Chung-Hsi Wang, Kenji Murata, Hiroshi Saijo, Yuki Kagoya, Dalam Ly, Brian D Burt, Marcus O Butler, Tak W Mak, Naoto Hirano

Nature Biotechnology (Nature Biotechnology) 39 ( 8 ) 958 - 967 2021.08

Research paper (scientific journal), Accepted, ISSN 1087-0156

Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses.

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Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy

Yuki Kagoya, Tingxi Guo, Brian Yeung, Kayoko Saso, Mark Anczurowski, Chung-Hsi Wang, Kenji Murata, Kenji Sugata, Hiroshi Saijo, Yukiko Matsunaga, Yota Ohashi, Marcus O. Butler, Naoto Hirano

Cancer Immunology Research (American Association for Cancer Research (AACR)) 8 ( 7 ) 926 - 936 2020.07

Lead author, Accepted, ISSN 2326-6066

Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.

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Landscape mapping of shared antigenic epitopes and their cognate TCRs of tumor-infiltrating T lymphocytes in melanoma

Kenji Murata, Munehide Nakatsugawa, Muhammed A Rahman, Linh T Nguyen, Douglas G Millar, David T Mulder, Kenji Sugata, Hiroshi Saijo, Yukiko Matsunaga, Yuki Kagoya, Tingxi Guo, Mark Anczurowski, Chung-Hsi Wang, Brian D Burt, Dalam Ly, Kayoko Saso, Alexandra Easson, David P Goldstein, Michael Reedijk, Danny Ghazarian, Trevor J Pugh, Marcus O Butler, Tak W Mak, Pamela S Ohashi, Naoto Hirano

eLife (eLife Sciences Publications, Ltd) 9 2020.04

Accepted

HLA-restricted T cell responses can induce antitumor effects in cancer patients. Previous human T cell research has largely focused on the few HLA alleles prevalent in a subset of ethnic groups. Here, using a panel of newly developed peptide-exchangeable peptide/HLA multimers and artificial antigen-presenting cells for 25 different class I alleles and greater than 800 peptides, we systematically and comprehensively mapped shared antigenic epitopes recognized by tumor-infiltrating T lymphocytes (TILs) from eight melanoma patients for all their class I alleles. We were able to determine the specificity, on average, of 12.2% of the TILs recognizing a mean of 3.1 shared antigen-derived epitopes across HLA-A, B, and C. Furthermore, we isolated a number of cognate T cell receptor genes with tumor reactivity. Our novel strategy allows for a more complete examination of the immune response and development of novel cancer immunotherapy not limited by HLA allele prevalence or tumor mutation burden.

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Chaperones of the class I peptide-loading complex facilitate the constitutive presentation of endogenous antigens on HLA-DP84GGPM87

Mark Anczurowski, Kenji Sugata, Yukiko Matsunaga, Yuki Yamashita, Chung-Hsi Wang, Tingxi Guo, Kenji Murata, Hiroshi Saijo, Yuki Kagoya, Kayoko Saso, Marcus O. Butler, Naoto Hirano

Journal of Autoimmunity 102 114 - 125 2019.08

Lead author, Accepted, ISSN 0896-8411

Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPβ chain (DP84GGPM87). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP84GGPM87 for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP84GGPM87 to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4+ T cells.

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Arginine methylation of FOXP3 is crucial for the suppressive function of regulatory T cells

Yuki Kagoya, Hiroshi Saijo, Yukiko Matsunaga, Tingxi Guo, Kayoko Saso, Mark Anczurowski, Chung-Hsi Wang, Kenji Sugata, Kenji Murata, Marcus O. Butler, Cheryl H. Arrowsmith, Naoto Hirano

Journal of Autoimmunity (Elsevier BV) 97 10 - 21 2019.02

Lead author, Last author, Corresponding author, Accepted, ISSN 0896-8411

Forkhead box transcription factor 3 (FOXP3) plays a pivotal role in the suppressive function of regulatory T cells. In addition to mRNA levels, FOXP3 activity can also be controlled by posttranslational mechanisms, which have not been studied in a comprehensive manner. Through extensive screening using selective inhibitors, we demonstrate that the inhibition of type I protein arginine methytransferases (PRMTs) attenuates the suppressive functions of regulatory T cells. FOXP3 undergoes methylation on arginine residues at positions 48 and 51 by interacting with protein arginine methyltransferase 1 (PRMT1). The inhibition of arginine methylation confers gene expression profiles representing type I helper T cells to FOXP3+ T cells, which results in attenuated suppressive activity. A methylation-defective mutant of FOXP3 displays less potent activity to suppress xenogeneic graft-versus-host disease in vivo. These results elucidate an important role of arginine methylation to enhance FOXP3 functions and are potentially applicable to modulate regulatory T cell functions.

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DOT1L inhibition attenuates graft-versus-host disease by allogeneic T cells in adoptive immunotherapy models

Yuki Kagoya, Munehide Nakatsugawa, Kayoko Saso, Tingxi Guo, Mark Anczurowski, Chung-Hsi Wang, Marcus O. Butler, Cheryl H. Arrowsmith, Naoto Hirano

Nature Communications (Springer Science and Business Media LLC) 9 ( 1 ) 2018.12

Lead author, Accepted

Adoptive T-cell therapy is a promising therapeutic approach for cancer patients. The use of allogeneic T-cell grafts will improve its applicability and versatility provided that inherent allogeneic responses are controlled. T-cell activation is finely regulated by multiple signaling molecules that are transcriptionally controlled by epigenetic mechanisms. Here we report that inhibiting DOT1L, a histone H3-lysine 79 methyltransferase, alleviates allogeneic T-cell responses. DOT1L inhibition reduces miR-181a expression, which in turn increases the ERK phosphatase DUSP6 expression and selectively ameliorates low-avidity T-cell responses through globally suppressing T-cell activation-induced gene expression alterations. The inhibition of DOT1L or DUSP6 overexpression in T cells attenuates the development of graft-versus-host disease, while retaining potent antitumor activity in xenogeneic and allogeneic adoptive immunotherapy models. These results suggest that DOT1L inhibition may enable the safe and effective use of allogeneic antitumor T cells by suppressing unwanted immunological reactions in adoptive immunotherapy.

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Mechanisms underlying the lack of endogenous processing and CLIP-mediated binding of the invariant chain by HLA-DP84Gly

Mark Anczurowski, Yuki Yamashita, Munehide Nakatsugawa, Toshiki Ochi, Yuki Kagoya, Tingxi Guo, Chung-Hsi Wang, Muhammed A. Rahman, Kayoko Saso, Marcus O. Butler, Naoto Hirano

Scientific Reports (Springer Science and Business Media LLC) 8 ( 1 ) 2018.12

Accepted

While the principles of classical antigen presentation via MHC class II are well-established, the mechanisms for the many routes of cross-presentation by which endogenous antigens become associated with class II molecules are not fully understood. We have recently demonstrated that the single amino acid polymorphism HLA-DPβ84Gly (DP84Gly) is critical to abrogate class II invariant chain associated peptide (CLIP) region-mediated binding of invariant chain (Ii) to DP, allowing endoplasmic reticulum (ER)-resident endogenous antigens to constitutively associate with DP84Gly such as DP4. In this study, we demonstrate that both the CLIP and N-terminal non-CLIP Ii regions cooperatively generate an Ii conformation that cannot associate with DP84Gly via the CLIP region. We also demonstrate the ability of DP4 to efficiently process and present antigens encoded in place of CLIP in a chimeric Ii, regardless of wild type Ii and HLA-DM expression. These data highlight the complex interplay between DP polymorphisms and the multiple Ii regions that cooperatively regulate this association, ultimately controlling the presentation of endogenous antigens on DP molecules. These results may also offer a mechanistic explanation for recent studies identifying the differential effects between DP84Gly and DP84Asp as clinically relevant in human disease.

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A novel chimeric antigen receptor containing a JAK–STAT signaling domain mediates superior antitumor effects

Yuki Kagoya, Shinya Tanaka, Tingxi Guo, Mark Anczurowski, Chung-Hsi Wang, Kayoko Saso, Marcus O Butler, Mark D Minden, Naoto Hirano

Nature Medicine 24 ( 3 ) 352 - 359 2018.03

Lead author, Accepted, ISSN 1078-8956

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A Subset of Human Autoreactive CD1c-Restricted T Cells Preferentially Expresses TRBV4-1+TCRs

Tingxi Guo, Ming Yin Koo, Yuki Kagoya, Mark Anczurowski, Chung-Hsi Wang, Kayoko Saso, Marcus O. Butler, Naoto Hirano

The Journal of Immunology (The American Association of Immunologists) 200 ( 2 ) 500 - 511 2018.01

Accepted, ISSN 0022-1767

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HLA-DP84Gly constitutively presents endogenous peptides generated by the class I antigen processing pathway

Yuki Yamashita, Mark Anczurowski, Munehide Nakatsugawa, Makito Tanaka, Yuki Kagoya, Ankit Sinha, Kenji Chamoto, Toshiki Ochi, Tingxi Guo, Kayoko Saso, Marcus O. Butler, Mark D. Minden, Thomas Kislinger, Naoto Hirano

Nature Communications (Springer Science and Business Media LLC) 8 ( 1 ) 2017.08

Last author, Corresponding author, Accepted

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Key Residues at Third CDR3β Position Impact Structure and Antigen Recognition of Human Invariant NK TCRs

Kenji Chamoto, Tingxi Guo, Stephen W. Scally, Yuki Kagoya, Mark Ancruzowski, Chung-Hsi Wang, Muhammed A. Rahman, Kayoko Saso, Marcus O. Butler, Priscilla P. L. Chiu, Jean-Philippe Julien, Naoto Hirano

The Journal of Immunology (The American Association of Immunologists) 198 ( 3 ) 1056 - 1065 2017.02

Accepted, ISSN 0022-1767

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Transient stimulation expands superior antitumor T cells for adoptive therapy

Yuki Kagoya, Munehide Nakatsugawa, Toshiki Ochi, Yuchen Cen, Tingxi Guo, Mark Anczurowski, Kayoko Saso, Marcus O. Butler, Naoto Hirano

JCI Insight (American Society for Clinical Investigation) 2 ( 2 ) 2017.01

Lead author, Accepted

Adoptive cell therapy is a potentially curative therapeutic approach for patients with cancer. In this treatment modality, antitumor T cells are exponentially expanded in vitro prior to infusion. Importantly, the results of recent clinical trials suggest that the quality of expanded T cells critically affects their therapeutic efficacy. Although anti-CD3 mAb-based stimulation is widely used to expand T cells in vitro, a protocol to generate T cell grafts for optimal adoptive therapy has yet to be established. In this study, we investigated the differences between T cell stimulation mediated by anti-CD3/CD28 mAb-coated beads and cell-based artificial antigen-presenting cells (aAPCs) expressing CD3/CD28 counter-receptors. We found that transient stimulation with cell-based aAPCs, but not prolonged stimulation with beads, resulted in the superior expansion of CD8+ T cells. Transiently stimulated CD8+ T cells maintained a stem cell-like memory phenotype and were capable of secreting multiple cytokines significantly more efficiently than chronically stimulated T cells. Importantly, the chimeric antigen receptor-engineered antitumor CD8+ T cells expanded via transient stimulation demonstrated superior persistence and antitumor responses in adoptive immunotherapy mouse models. These results suggest that restrained stimulation is critical for generating T cell grafts for optimal adoptive immunotherapy for cancer.

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Generating <em>De Novo</em> Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Tingxi Guo, Toshiki Ochi, Munehide Nakatsugawa, Yuki Kagoya, Mark Anczurowski, Chung-Hsi Wang, Muhammed A. Rahman, Kayoko Saso, Marcus O. Butler, Naoto Hirano

Journal of Visualized Experiments (MyJove Corporation) ( 116 ) 2016.10

Accepted

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BET bromodomain inhibition enhances T cell persistence and function in adoptive immunotherapy models

Yuki Kagoya, Munehide Nakatsugawa, Yuki Yamashita, Toshiki Ochi, Tingxi Guo, Mark Anczurowski, Kayoko Saso, Marcus O. Butler, Cheryl H. Arrowsmith, Naoto Hirano

Journal of Clinical Investigation (American Society for Clinical Investigation) 126 ( 9 ) 3479 - 3494 2016.08

Lead author, Accepted, ISSN 0021-9738

Adoptive immunotherapy is a potentially curative therapeutic approach for patients with advanced cancer. However, the in vitro expansion of antitumor T cells prior to infusion inevitably incurs differentiation towards effector T cells and impairs persistence following adoptive transfer. Epigenetic profiles regulate gene expression of key transcription factors over the course of immune cell differentiation, proliferation, and function. Using comprehensive screening of chemical probes with defined epigenetic targets, we found that JQ1, an inhibitor of bromodomain and extra-terminal motif (BET) proteins, maintained CD8+ T cells with functional properties of stem cell-like and central memory T cells. Mechanistically, the BET protein BRD4 directly regulated expression of the transcription factor BATF in CD8+ T cells, which was associated with differentiation of T cells into an effector memory phenotype. JQ1-treated T cells showed enhanced persistence and antitumor effects in murine T cell receptor and chimeric antigen receptor gene therapy models. Furthermore, we found that histone acetyltransferase p300 supported the recruitment of BRD4 to the BATF promoter region, and p300 inhibition similarly augmented antitumor effects of the adoptively transferred T cells. These results demonstrate that targeting the BRD4-p300 signaling cascade supports the generation of superior antitumor T cell grafts for adoptive immunotherapy.

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CD4+ and CD8+ TCRβ repertoires possess different potentials to generate extraordinarily high-avidity T cells

Munehide Nakatsugawa, Muhammed A. Rahman, Yuki Yamashita, Toshiki Ochi, Piotr Wnuk, Shinya Tanaka, Kenji Chamoto, Yuki Kagoya, Kayoko Saso, Tingxi Guo, Mark Anczurowski, Marcus O. Butler, Naoto Hirano

Scientific Reports (Springer Science and Business Media LLC) 6 ( 1 ) 2016.07

Accepted

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BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and KLF4

K Morita, Y Masamoto, K Kataoka, J Koya, Y Kagoya, H Yashiroda, T Sato, S Murata, M Kurokawa

Leukemia (Springer Science and Business Media LLC) 29 ( 11 ) 2248 - 2256 2015.11

Accepted, ISSN 0887-6924

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Targeted gene correction of RUNX1 in induced pluripotent stem cells derived from familial platelet disorder with propensity to myeloid malignancy restores normal megakaryopoiesis

Hiromitsu Iizuka, Yuki Kagoya, Keisuke Kataoka, Akihide Yoshimi, Masashi Miyauchi, Kazuki Taoka, Keiki Kumano, Takashi Yamamoto, Akitsu Hotta, Shunya Arai, Mineo Kurokawa

Experimental Hematology (Elsevier BV) 43 ( 10 ) 849 - 857 2015.10

Accepted, ISSN 0301-472X

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Central nervous system relapse in patients with diffuse large B-cell lymphoma: analysis of incidence and prognostic factors

Masahiro Uni, Yuki Kagoya, Yasuhito Nannya, Fumihiko Nakamura, Mineo Kurokawa

Leukemia & Lymphoma (Informa UK Limited) 56 ( 6 ) 1869 - 1871 2015.06

Accepted, ISSN 1042-8194

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NF-κB activity in myeloid leukemia stem cells

Yuki Kagoya

臨床血液 56 ( 4 ) 384 - 391 2015.04

Lead author, Last author, Corresponding author, Accepted

Thrombopoietin/MPL signaling confers growth and survival capacity to CD41-positive cells in a mouse model of Evi1 leukemia

Satoshi Nishikawa, Shunya Arai, Yosuke Masamoto, Yuki Kagoya, Takashi Toya, Naoko Watanabe-Okochi, Mineo Kurokawa

Blood (American Society of Hematology) 124 ( 24 ) 3587 - 3596 2014.12

Accepted, ISSN 0006-4971

AML1-ETO (AE) is a fusion product of translocation (8;21) that accounts for 40% of M2 type acute myeloid leukemia (AML). In addition to its role in promoting preleukemic hematopoietic cell self-renewal, AE represses DNA repair genes, which leads to DNA damage and increased mutation frequency. Although this latter function may promote leukemogenesis, concurrent p53 activation also leads to an increased baseline apoptotic rate. It is unclear how AE expression is able to counterbalance this intrinsic apoptotic conditioning by p53 to promote survival and self-renewal. In this report, we show that Bcl-xL is up-regulated in AE cells and plays an essential role in their survival and self-renewal. Further investigation revealed that Bcl-xL expression is regulated by thrombopoietin (THPO)/MPL-signaling induced by AE expression. THPO/MPL-signaling also controls cell cycle reentry and mediates AE-induced self-renewal. Analysis of primary AML patient samples revealed a correlation between MPL and Bcl-xL expression specifically in t(8;21) blasts. Taken together, we propose that survival signaling through Bcl-xL is a critical and intrinsic component of a broader self-renewal signaling pathway downstream of AML1-ETO–induced MPL.

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JAK2V617F+ myeloproliferative neoplasm clones evoke paracrine DNA damage to adjacent normal cells through secretion of lipocalin-2

Yuki Kagoya, Akihide Yoshimi, Takako Tsuruta-Kishino, Shunya Arai, Takashi Satoh, Shizuo Akira, Mineo Kurokawa

Blood (American Society of Hematology) 124 ( 19 ) 2996 - 3006 2014.11

Lead author, Accepted, ISSN 0006-4971

Genetic instability is strongly involved in cancer development and progression, and elucidating the mechanism could lead to novel therapeutics for preventing carcinogenesis. Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal myeloid disorders with a high prevalence of JAK2V617F mutation, and transformation to acute myeloid leukemia through accumulation of additional mutations is a major complication in MPNs. Here, we showed that JAK2V617F+ cells conferred paracrine DNA damage to neighboring normal cells as well as to themselves through increased reactive oxygen species (ROS). We screened candidate factors responsible for the effect and found that lipocalin-2 (Lcn2) is overexpressed in JAK2V617F+ cells and that short hairpin RNA–mediated knockdown of Lcn2 significantly alleviated the paracrine DNA damage. Normal hematopoietic cells showed elevated ROS levels through increased intracellular iron levels when treated with lipocalin-2, which led to p53 pathway activation, increased apoptosis, and decreased cellular proliferation. In contrast, JAK2V617F+ cells did not suffer from lipocalin-2–induced growth suppression resulting from attenuated p53 pathway activation, which conferred a relative growth advantage to JAK2V617F+ clones. In summary, we demonstrated that JAK2V617F-harboring cells cause paracrine DNA damage accumulation through secretion of lipocalin-2, which gives proliferative advantage to themselves and an increased risk for leukemic transformation to both JAK2V617F+ and JAK2V617F– clones.

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Evi1 defines leukemia-initiating capacity and tyrosine kinase inhibitor resistance in chronic myeloid leukemia

T Sato, S Goyama, K Kataoka, R Nasu, T Tsuruta-Kishino, Y Kagoya, A Nukina, K Kumagai, N Kubota, M Nakagawa, S Arai, A Yoshimi, H Honda, T Kadowaki, M Kurokawa

Oncogene (Springer Science and Business Media LLC) 33 ( 42 ) 5028 - 5038 2014.10

Accepted, ISSN 0950-9232

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Gene expression profiles of central nervous system lymphoma predict poor survival in patients with diffuse large B-cell lymphoma

Yuki Kagoya, Yasuhito Nannya, Fumihiko Nakamura, Mineo Kurokawa

British Journal of Haematology (Wiley) 166 ( 5 ) 794 - 797 2014.09

Lead author, Accepted, ISSN 0007-1048

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Inhibition of histone methyltransferase EZH 2 depletes leukemia stem cell of mixed lineage leukemia fusion leukemia through upregulation of p16

Koki Ueda, Akihide Yoshimi, Yuki Kagoya, Satoshi Nishikawa, Victor E. Marquez, Masahiro Nakagawa, Mineo Kurokawa

Cancer Science (Wiley) 105 ( 5 ) 512 - 519 2014.05

Accepted, ISSN 1347-9032

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Single-cell gene expression analysis reveals clonal architecture of blast-phase chronic myeloid leukaemia

Arika Nukina, Yuki Kagoya, Naoko Watanabe-Okochi, Shunya Arai, Koki Ueda, Akihide Yoshimi, Yasuhito Nannya, Mineo Kurokawa

British Journal of Haematology (Wiley) 165 ( 3 ) 414 - 416 2014.05

Lead author, Accepted, ISSN 0007-1048

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Positive feedback between NF-κB and TNF-α promotes leukemia-initiating cell capacity

Yuki Kagoya, Akihide Yoshimi, Keisuke Kataoka, Masahiro Nakagawa, Keiki Kumano, Shunya Arai, Hiroshi Kobayashi, Taku Saito, Yoichiro Iwakura, Mineo Kurokawa

Journal of Clinical Investigation (American Society for Clinical Investigation) 124 ( 2 ) 528 - 542 2014.02

Lead author, Accepted, ISSN 0021-9738

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy that originates from leukemia-initiating cells (LICs). The identification of common mechanisms underlying LIC development will be important in establishing broadly effective therapeutics for AML. Constitutive NF-κB pathway activation has been reported in different types of AML; however, the mechanism of NF-κB activation and its importance in leukemia progression are poorly understood. Here, we analyzed myeloid leukemia mouse models to assess NF-κB activity in AML LICs. We found that LICs, but not normal hematopoietic stem cells or non-LIC fractions within leukemia cells, exhibited constitutive NF-κB activity. This activity was maintained through autocrine TNF-α secretion, which formed an NF-κB/TNF-α positive feedback loop. LICs had increased levels of active proteasome machinery, which promoted the degradation of IκBα and further supported NF-κB activity. Pharmacological inhibition of the proteasome complex markedly suppressed leukemia progression in vivo. Conversely, enhanced activation of NF-κB signaling expanded LIC frequency within leukemia cell populations. We also demonstrated a strong correlation between NF-κB activity and TNF-α secretion in human AML samples. Our findings indicate that NF-κB/TNF-α signaling in LICs contributes to leukemia progression and provide a widely applicable approach for targeting LICs.

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Thalidomide maintenance therapy for patients with multiple myeloma: Meta-analysis

Yuki Kagoya, Yasuhito Nannya, Mineo Kurokawa

Leukemia Research (Elsevier BV) 36 ( 8 ) 1016 - 1021 2012.08

Lead author, Accepted, ISSN 0145-2126

We performed a meta-analysis of randomized controlled trials comparing thalidomide maintenance with other regimens after induction chemotherapy for multiple myeloma. Overall, 6 trials including 2786 patients were identified. Patients treated with thalidomide maintenance had marginally better overall survival (hazard ratio HR 0.83, P=0.07). The improvement was especially prominent in a subgroup of studies using corticosteroids with thalidomide (HR 0.70, P=0.02). Thalidomide improved progression-free survival (HR 0.65, P<0.01), but had more frequent venous thrombosis (risk difference 0.024, P<0.05) and peripheral neuropathy (risk difference 0.072, P<0.01). These results suggest that thalidomide maintenance with corticosteroids is effective in prolonging survival for multiple myeloma.

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Hyperlipidemia after allogeneic stem cell transplantation: prevalence, risk factors, and impact on prognosis

Yuki Kagoya, Sachiko Seo, Yasuhito Nannya, Mineo Kurokawa

Clinical Transplantation (Wiley) 26 ( 2 ) E168 - E175 2012.03

Lead author, Last author, Corresponding author, Accepted, ISSN 0902-0063

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Hyperbilirubinemia after hematopoietic stem cell transplantation: comparison of clinical and pathologic findings in 41 autopsied cases

Yuki Kagoya, Tsuyoshi Takahashi, Yasuhito Nannya, Aya Shinozaki, Satoshi Ota, Masashi Fukayama, Mineo Kurokawa

Clinical Transplantation (Wiley) 25 ( 5 ) E552 - E557 2011.09

Lead author, Accepted, ISSN 0902-0063

Hyperbilirubinemia is often associated with morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Diagnosis of its etiology is usually made clinically among various possible causes, and analysis of histological findings as compared with the clinical diagnosis has not been performed sufficiently. We retrospectively analyzed clinical and pathological findings in 41 autopsied patients who died with hyperbilirubinemia (>2 mg/dL). Overall, liver graft-versus-host disease (GVHD) showed the most prominent discordance between clinical and pathological diagnoses. Only 11 of the 22 patients, considered to have liver GVHD clinically, had GVHD findings at autopsy. Serum gamma-glutamyl transpeptidase (GGT), GGT/aspartate aminotransferase (AST) ratio, and alkaline phosphatase (ALP)/AST ratio in GVHD patients were significantly higher compared with those without GVHD (p = 0.02, <0.01, and 0.03, respectively), which was useful in clinical diagnosis of liver GVHD. Other major findings include liver invasion of the primary malignancies in 8 patients, post-transplant lymphoproliferative disorder of the liver in two patients, and disseminated liver invasion by fungus or varicella-zoster virus in one patient, respectively. Although analysis of clinical data is useful for narrowing diagnosis, histological analysis by liver biopsy is crucially important, especially in cases suspected of having GVHD.

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Pretransplant Predictors and Posttransplant Sequels of Acute Kidney Injury after Allogeneic Stem Cell Transplantation

Yuki Kagoya, Keisuke Kataoka, Yasuhito Nannya, Mineo Kurokawa

Biology of Blood and Marrow Transplantation (Elsevier BV) 17 ( 3 ) 394 - 400 2011.03

Lead author, Accepted, ISSN 1083-8791

Acute kidney injury (AKI) is a common complication after allogeneic stem cell transplantation (SCT). Although various risk factors for AKI have been reported, the influence of pretransplant comorbidity on the incidence of AKI has not been well investigated. We performed a retrospective analysis of 207 consecutive patients undergoing myeloablative or nonmyeloablative SCT between 2001 and 2009, using the hematopoietic cell transplantation-specific comorbidity index (HCT-CI) as a representative of pretransplant comorbidities. According to Risk, Injury, Failure, Loss, and End-stage kidney disease (RIFLE) criteria, 158 patients (76.3%) developed AKI, and 92 patients (44.4%) developed severe AKI (RIFLE class I or class F) within 100 days after SCT. The cumulative incidence of severe AKI within 100 days in patients with an HCT-CI score 0, 1-2, and ≥3 was 21.3%, 48.8%, and 73.9%, respectively. In multivariate analysis, the HCT-CI was independently associated with severe AKI (HCT-CI 1-2: adjusted hazard ratio [HR] 2.42, P < .01; HCT-CI ≥3: adjusted HR 4.69, P < .01). In a landmark analysis, patients with severe AKI had a lower 3-year overall survival (OS) (39.3% versus 61.4%, P < .01), and a higher 3-year nonrelapse mortality (NRM) (40.8% versus 5.6%, P < .01) than those without AKI. Multivariate analysis showed that severe AKI was a significant risk factor for worse OS (HR: 2.10, P = .01) and NRM (HR: 6.15, P < .01). Thus, it is important to assess the HCT-CI to predict the incidence of AKI, which is a strong indicator of worse prognosis after SCT.

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Recurrent pericardial effusion after treatment for primary effusion lymphoma-like lymphoma: an autopsied case

Yuki Kagoya, Tsuyoshi Takahashi, Toyoki Yoshimoto, Motoshi Ichikawa, Akira Hangaishi, Masashi Fukayama, Mineo Kurokawa

Annals of Hematology (Springer Science and Business Media LLC) 90 ( 2 ) 219 - 220 2011.02

Lead author, Accepted, ISSN 0939-5555

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A Case of Primary Bone Marrow B-Cell Non Hodgkin’s Lymphoma with Severe Thrombocytopenia: Case Report and A Review of the Literature

Yuki Kagoya, Naohi Sahara, Takashi Matsunaga, Toshimasa Uekusa, Seiji Irie, Kazuhito Hatanaka

Indian Journal of Hematology and Blood Transfusion (Springer Science and Business Media LLC) 26 ( 3 ) 106 - 108 2010.09

Lead author, Corresponding author, Accepted, ISSN 0971-4502

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High-dose dexamethasone therapy for severe thrombocytopenia and neutropenia induced by EBV infectious mononucleosis

Yuki Kagoya, Akira Hangaishi, Tsuyoshi Takahashi, Yoichi Imai, Mineo Kurokawa

International Journal of Hematology (Springer Science and Business Media LLC) 91 ( 2 ) 326 - 327 2010.03

Lead author, Accepted, ISSN 0925-5710

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