Takashi Ushiki

CIS is a potent checkpoint in NK cell–mediated tumor immunity

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish−/− mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell–mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

The Helix-Loop-Helix Protein ID2 Governs NK Cell Fate by Tuning Their Sensitivity to Interleukin-15

The inhibitor of DNA binding 2 (Id2) is essential for natural killer (NK) cell development with its canonical role being to antagonize E-protein function and alternate lineage fate. Here we have identified a key role for Id2 in regulating interleukin-15 (IL-15) receptor signaling and homeostasis of NK cells by repressing multiple E-protein target genes including Socs3. Id2 deletion in mature NK cells was incompatible with their homeostasis due to impaired IL-15 receptor signaling and metabolic function and this could be rescued by strong IL-15 receptor stimulation or genetic ablation of Socs3. During NK cell maturation, we observed an inverse correlation between E-protein target genes and Id2. These results shift the current paradigm on the role of ID2, indicating that it is required not only to antagonize E-proteins during NK cell commitment, but constantly required to titrate E-protein activity to regulate NK cell fitness and responsiveness to IL-15.

In vivo imaging of HIF-active tumors by an oxygen-dependent degradation protein probe with an interchangeable labeling system.

Hypoxia-inducible factor (HIF) functions as a master transcriptional regulator for adaptation to hypoxia by inducing adaptive changes in gene expression for regulation of proliferation, angiogenesis, apoptosis and energy metabolism. Cancers with high expression of the alpha subunit of HIF (HIFα) are often malignant and treatment-resistant. Therefore, the development of a molecular probe that can detect HIF activity has great potential value for monitoring tumor hypoxia. HIF prolyl hydroxylases (HPHDs) act as oxygen sensors that regulate the fate of HIFα protein through its oxygen-dependent degradation (ODD) domain. We constructed a recombinant protein PTD-ODD-HaloTag (POH) that is under the same ODD regulation as HIFα and contains protein transduction domain (PTD) and an interchangeable labeling system. Administration of near-infrared fluorescently labeled POH (POH-N) to mouse models of cancers allowed successful monitoring of HIF-active regions. Immunohistochemical analysis for intratumoral localization of POH probe revealed its specificity to HIF-active cells. Furthermore, lack of the PTD domain or a point mutation in the ODD domain abrogated the specificity of POH-N to HIF-active cells. Overall results indicate that POH is a practical probe specific to HIF-active cell in cancers and suggest its large potential for imaging and targeting of HIF-related diseases.

Rapid Inflammation in Mice Lacking Both SOCS1 and SOCS3 in Hematopoietic Cells.

The Suppressors of Cytokine Signalling (SOCS) proteins are negative regulators of cytokine signalling required to prevent excess cellular responses. SOCS1 and SOCS3 are essential to prevent inflammatory disease, SOCS1 by attenuating responses to IFNγ and gamma-common (γc) cytokines, and SOCS3 via regulation of G-CSF and IL-6 signalling. SOCS1 and SOCS3 show significant sequence homology and are the only SOCS proteins to possess a KIR domain. The possibility of overlapping or redundant functions was investigated in inflammatory disease via generation of mice lacking both SOCS1 and SOCS3 in hematopoietic cells. Loss of SOCS3 significantly accelerated the pathology and inflammatory disease characteristic of SOCS1 deficiency. We propose a model in which SOCS1 and SOCS3 operate independently to control specific cytokine responses and together modulate the proliferation and activation of lymphoid and myeloid cells to prevent rapid inflammatory disease.

The Glasgow Prognostic Score as a pre-transplant risk assessment for allogeneic hematopoietic cell transplantation

Evaluation methods, such as scoring systems for predicting complications in advance, are necessary for determining the adaptation of allogeneic hematopoietic cell transplantation (HCT) and selecting appropriate conditioning regimens. The Hematopoietic Cell Transplantation‐specific Comorbidity Index (HCT‐CI), which is based on functions of main organs, is a useful tool for pre‐transplant risk assessments and has been widely applied in determining treatment strategies for patients with hematological diseases. However, as allogeneic HCT is performed on patients with diverse backgrounds, another factor, which reinforces the HCT‐CI, is required to evaluate pre‐transplant risk assessments. The Glasgow Prognostic Score (GPS), which assesses the combined C‐reactive protein and albumin, was reported to predict survival of patients with solid‐organ malignancies independently of receiving chemo/radiotherapy and stages of cancer. In this study, we applied the GPS for pre‐transplant risk assessments for allogeneic HCT. The GPS successfully stratified the patients into three risk groups of overall survival (OS) and non‐relapse mortality (NRM). Moreover, the GPS could predict outcomes independently of the HCT‐CI for OS and NRM in multivariate analysis. The GPS is considered to be a useful tool and reinforces the HCT‐CI for determining adaptation of allogeneic HCT for patients with hematopoietic neoplasms.

Refinement of the Glasgow Prognostic Score as a pre-transplant risk assessment for allogeneic hematopoietic cell transplantation

The Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) is a widely used tool for pre-transplant risk assessment. Allogeneic hematopoietic cell transplantation (HCT) is performed on patients with diverse backgrounds, highlighting the need for other predictors to complement the HCT-CI and support bedside decision-making. There is a strong body of evidence supporting the use of pre-transplant serum ferritin (SF) in risk assessments of allogeneic HCT. We additionally found that the Glasgow Prognostic Score (GPS), which assesses inflammatory biomarkers and predicts survival of patients with solid organ malignancies, is a useful predictive marker for overall survival (OS) and non-relapse mortality (NRM) in allogeneic HCT, independent of HCT-CI and SF. In this study, we refined the GPS by adding pre-transplant SF to improve its prognostic ability and enable better stratification; we call this revised index the HCT-specific revised Glasgow Prognostic Score (HCT-GPS). We observed that the HCT-GPS more accurately predicted NRM and early-term OS than the GPS. Moreover, the HCT-GPS provides an independent prognostic factor adjusted for the HCT-CI and disease status, and stratifies patients into four risk groups by OS and NRM. Thus, the HCT-GPS is a useful index for predicting early-term complications after allogeneic HCT in patients with hematopoietic diseases.

Genetically Reduced Chondroitin Sulfate Prevents the Progression of Diabetic Neuropathy

Background: The extracellular matrix is associated with the pathophysiology of diabetic complications; however, the role of chondroitin sulfate (CS) remains unclear. To clarify the effects of CS on diabetic neuropathy (DN), we assessed the effect of genetically reducing CS in mice through disruption of a gene encoding the rate-limiting CS-synthesizing enzyme, i.e., CS N-acetylgalactosaminyltransferase-1 (T1). Methods: T1 knockout (T1KO) mice were generated from the C57BL/6N strain and C57BL/6N were prepared as wild type (WT). Diabetes was induced through streptozotocin injection in 6-week-old male mice. All data were obtained 3 weeks after streptozotocin injection. Results: In the heat radiant test, while thermal nociception in nondiabetic WT and T1KO mice were normal and significantly disrupted in diabetic WT mice, that in diabetic T1KO mice was preserved. The number of plantar peripheral nerve fibers was also significantly decreased in diabetic WT mice; however, that in diabetic T1KO mice were relatively normal. Furthermore, immunohistochemistry revealed loss of calcitonin gene-related peptide-positive neurons in the dorsal root ganglia (DRG) in diabetic WT mice, which contains a cluster of sensory neuron bodies. In contrast, those neurons were protected in diabetic T1KO mice. Hence, nociception and thermoception are preserved in diabetic T1KO mice. To investigate the mechanisms underlying these events, we analyzed gene expression in DRG through real-time polymerase chain reaction and confirmed the suppression of caspase-3 and caspase-9 in diabetic T1KO mice, compared to those in diabetic WT mice. However, levels of Bcl2, TNF-α, MMP9, and reactive oxygen species-related enzymes (HO-1, NOX) did not differ significantly between WT and T1KO diabetic mice. Conclusions: Reduced CS production is suggested to have potentially beneficial effects on preventing DN by suppressing apoptotic signaling and could be a cutting-edge target of clinical application. Disclosure H. Ishiguro: Research Support; Self; MSD K.K., Sanofi K.K., Eli Lilly and Company. T. Ushiki: None. A. Kawasaki: None. K. Cho: None. M. Masuko: None. K. Sango: None. M. Igarashi: None. H. Sone: Research Support; Self; Novo Nordisk Inc., Eli Lilly and Company, MSD K.K., Chugai Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Development Center Asia, Pte. Ltd., Daiichi Sankyo Company, Limited, Ono Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Sanofi, Kowa Pharmaceuticals America, Inc., Eisai Inc..

Successful 5-azacytidine treatment of myeloid sarcoma and leukemia cutis associated with myelodysplastic syndrome: A case report and literature review

Rationale: Myeloid sarcoma (MS) and leukemia cutis (LC) are extramedullary tumors comprising myeloid blasts. They can occur de novo or concurrently with hematological disorders, usually acute myeloid leukemia (AML). AML chemotherapy is generally the initial therapy for MS and LC, and hematopoietic stem cell transplantation (HSCT) can be considered as additional therapy. However, treatment for older patients who are unable to continue intensive chemotherapy is not currently standardized. Patient concerns: A 71-year-old Japanese woman was diagnosed with multiple MSs associated with myelodysplastic syndrome (MDS), using bone marrow aspiration and lymph node biopsy. Diagnoses: Additionally, LC was diagnosed by skin biopsy. Extramedullary MS and LC lesions were formed by massive infiltration of myeloblastic cells. Interventions: Twenty courses of 5-azacytidine (5-Aza) were administrated as maintenance therapy after induction therapy with daunorubicin and cytarabine. Outcomes: Myeloblasts decreased in the bone marrow and the LC disappeared after induction therapy. The MSs completely disappeared, except for the palatine tonsil lesion, after 5-Aza maintenance therapy. 5-Aza treatment provided long-term partial response for more than 21 months. Lessons: 5-Aza was well tolerated and may be a good option for the treatment of MS and LC associated with MDS, especially in older patients who cannot receive HSCT.

Abstract 5230: In vivo imaging of tumor malignancy with near-infrared fluorescence probes specific to HIF-active cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC In solid tumors, the distribution of oxygen pressure is not homogenous because of the uncontrolled tumor growth and immature blood vessels during angiogenesis, which generate a hypoxic microenvironment. Tumor hypoxia causes resistance to radiotherapy and chemotherapy, and malignant progression Hypoxia-inducible factor-1 (HIF-1) is a master transcriptional regulator for adaptation to hypoxia by inducing more than 100 genes, which are closely associated with malignant phenotype. Thus, HIF-1-active cells are hallmark of malignant tumors. We have been developing fusion protein probes specific to HIF-1-active microenvironment for imaging and targeting malignant tumors. We recently constructed a fusion protein POH, which consisted of Protein Transduction Domain (PTD), Oxygen-dependent Degradation Domain (ODD) and HaloTag. The PTD was a membrane-permeable peptide, which efficiently delivered fusion proteins into cells. The ODD was responsible for the oxygen-dependent regulation of the probe, which stabilized in a hypoxic environment and degraded immediately under normoxic conditions. In addition, HaloTag was used to covalently conjugate with its specific ligand labeled with near-infrared fluorescence (NIRF) dye. POH-NIRF was examined for its target-specificity and in vivo dynamic status by in vivo and ex vivo fluorescence imaging with IVIS-SPECTRUM. To visualize the targets in vivo, we used bioluminescence imaging of tumor cells, which stably retained a HIF-1-dependent luciferase reporter gene. POH-NIRF probes successfully imaged HIF-1-active regions defined by bioluminescence imaging in subcutaneously implanted human xenograft tumors and orthotopic pancreatic cancers 9 to 24 hrs after POH-NIRF injection. Furthermore, immunohistochemical analysis of tumor sections from the mice 6h after POH-NIRF injection revealed the regions imaged with POH-NIFR was overlapped with HIF-1α-positive regions, while the intracellular localization of POH-NIRF was in cytoplasm. Overall results demonstrated that POH-NIRF was a specific probe for HIF-1-active/hypoxic cells and suggested that a PTD-ODD probe would become a unique imaging probe for tumor malignancy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5230.