熊野 恵城

<title>Abstract</title>Although the physiological function of the omentum remains elusive, it has been proposed that it plays an important role in fat storage, immune regulation, and regeneration of injured tissues and organs. Although the omentum undergoes expansion upon activation, reports on the accurate assessment of increased cell types and the origin of the increased cells remain limited. To investigate this aspect, the omenta of parabiotic mice were subjected to activation using distinct fluorescent markers and single-cell RNA sequencing (scRNA-seq) was performed to identify circulation-derived omental cells. We found that a considerable number of circulating cells contributed to the activation of the omentum. The omental cells derived from circulating cells exhibited morphological features similar to those of fibroblasts. scRNA-seq revealed the existence of a novel cell population that co-expressed macrophage and fibroblast markers in the activated omentum, suggesting that it corresponded to circulating macrophage-derived fibroblast-like cells. Lineage tracing experiments revealed that most fibroblasts in the activated omentum were not derived from WT1-positive mesenchymal progenitors. The cell cluster also expressed various chemokine genes, indicating its role in the activation and recruitment of immune cells. These results provide important information regarding the activation of the omentum.

Properties of cancer stem cells involved in drug resistance and relapse have significant effects on clinical outcome. Although tyrosine kinase inhibitors (TKIs) have dramatically improved survival of patients with chronic myeloid leukemia (CML), TKIs have not fully cured CML due to TKI-resistant CML stem cells. Moreover, relapse after discontinuation of TKIs has not been predicted in CML patients with the best TKI response. In our study, a model of CML stem cells derived from CML induced pluripotent stem cells identified ADAM8 as an antigen of TKI-resistant CML cells. The inhibition of expression or metalloproteinase activity of ADAM8 restored TKI sensitivity in primary samples. In addition, residual CML cells in patients with optimal TKI response were concentrated in the ADAM8+ population. Our study demonstrates that ADAM8 is a marker of residual CML cells even in patients with optimal TKI response and would be a predictor of relapse and a therapeutic target of TKI-resistant CML cells.

Abstract Since the emergence of tyrosine kinase inhibitors (TKIs), long-term survival of patients with chronic myelogenous leukemia (CML) has been improved. However, those TKIs have not fully succeeded in curing CML, mainly due to TKI-resistant CML stem cells. CML stem cells are often difficult to analyze because they represent an extremely minor population of CML cells. To overcome this obstacle, we established integration-free induced pluripotent stem cells (iPSCs) from bone marrow (BM) cells of two patients with CML in chronic phase (CML-CP) and obtained CML pre-hematopoietic progenitor cells (CML-pre-HPCs), immature hematopoietic cells phenotypically defined by CD34+/CD45-/CD43+ cells. In semisolid and liquid cultures, CML-pre-HPCs recapitulated the principal features of CML stem cells, multi-potency and the resistance against imatinib. Gene expression enrichment analysis for CML-pre-HPCs demonstrated that several gene sets, including those related to the maintenance of hematopoietic stem cells were enriched. In addition, we found that a disintegrin and metalloprotease 8 (ADAM8), also known as CD156, was highly enhanced in CML-pre-HPCs and the expression level of ADAM8 was even increased after the treatment of imatinib in vitro. To address the significance of ADAM8 in CMP-CP patient, we evaluated purified ADAM8+ cells by fluorescence-activated cell sorting (FACS) in primary samples. First, FACS analysis found that ADAM8+ cells were enriched more in BM samples of patient with newly diagnosed CML-CP than normal or other types of leukemias among CD34+ fraction. ADAM8+ cells were enriched in CD34+/CD38- fraction compaered to CD34+/38+ fraction in BM of CML-CP patients, indicating that ADMA8+ cells represent immature hematopietic cells. In cell viability assays, ADAM8+/CD38+ CML cells in newly diagnosed CML-CP patient enhibited imatinib-resistance and imatinib-induced apoptosis in vitro was strongly suppressed in ADAM8+ CML cells compared to ADAM8- cells. Even in CD34+/CD38+ fraction, which was previously known as TKI-sensitive fraction, ADAM8+ cells exhibited TKI-resistance in both cell viability and apoptosis assay, indicating that ADAM8 would be a useful marker of TKI-resistant CML cells. Finally, to evaluate the significance of ADAM8 as a marker of TKI-resistant CML cells in vivo, we measured the frequency of CML cells in BM samples of CML-CP patients who had achieved major or complete molecular response (MMR; n = 2 or CMR; n = 1) after the administration of TKIs by limiting dilution analysis. In CML patients with MMR, CML cells remained in ADAM8+ cells at higher frequency in spite of steep decline of CML cells in ADAM8- cells The frequency of CML cells was as high in CD34+/CD38+/ADAM8+ fraction as in CD34+/38- CML stem cell fraction. Even in a patient with CMR, residual CML cells were detected in ADAM8+ population among CD34+/CD38+ fraction, whereas CML cells were undetectable in ADAM8- population. In conclusion, we have established a powerful platform with CML-iPSCs to investigate the pathophysiology of TKI-resistant CML stem cells. Using this platform, we have identified ADAM8 as a novel marker of TKI-resistant CML cells. CD34+/CD38+/ADAM8+ fraction, as well as CD34+/CD38- fraction, was an important population that defines residual CML cells even in CML-CP patients with deep molecular response after the treatment of TKI. ADAM8 would become an attractive candidate of novel therapeutic targets against TKI-resistant CML cells. Disclosures Kataoka: Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria.

<p>近年の研究によりNanos2，Ngn3，c-kit等の生殖幹細胞マーカーが同定され，それらの分化段階特異的発現から，これら生殖幹細胞の階層構造およびそれらの分画間のreversibilityが提唱されている．しかし，最も未分化な段階にあるA_single生殖幹細胞については，その動態の直接の解析は行われていなかった．今回我々はA_single生殖幹細胞のマーカーとしてBmi1を同定した．Bmi1陽性生殖幹細胞はGFRα1陽性生殖幹細胞の一部であり，A_single生殖幹細胞におけるBmi1の発現は精上皮stageと関連して増減した．またBmi1の発現減少後にBmi1陽性幹細胞は細胞周期に入り増殖した．Bmi1陽性生殖幹細胞は長期に精子形成を維持し，放射線照射による生殖細胞の障害後に再生を担当する事が判明した．今回の研究においてBmi1の組織サイクル，細胞増殖と連動した長期幹細胞における発現の変化が初めて明らかとなった．</p>

Abstract Abstract 654 Acute myeloid leukemia (AML) is an aggressive hematologic malignancy arising from leukemia initiating cells (LIC), and is comprised of highly heterogeneous groups with different cytogenetic and molecular abnormalities, which makes it difficult to establish a broadly effective therapeutic strategy. Since constitutive NF-kB pathway activation has been reported in different types of AML cells, it is one of the promising candidates which are universally involved in the LIC phenotype. However, the mechanism of activation and its significance in leukemia progression have not been studied well. In this study, we explored NF-kB pathway activity and its role in LICs using various myeloid leukemia mouse models including MLL-ENL, MOZ-TIF2, and BCR-ABL/Nup98-HoxA9 leukemias. A number of NF-kB target genes showed elevated expression in LIC of each model: leukemic granulocyte-monocyte progenitors (L-GMP) in MLL-ENL or MOZ-TIF2 model and lineage- Sca-1+ fraction in BCR-ABL/Nup98-HoxA9 model, compared with normal hematopoietic stem cells (HSC) and GMP. Moreover, in immunofluorescence staining, each type of LIC displayed prominent nuclear translocation of NF-kB subunit p65. On the other hand, p65 was localized mainly in the cytoplasm in normal cells and, interestingly, non-LIC fraction in the bone marrow cells of the leukemia mice. To our surprise, LIC retained NF-kB activity even after serum-free culture, indicating that NF-kB pathway is prevalently activated in LIC in an autonomous fashion. To study the mechanism of activation, we analyzed gene expression profiles of LIC and normal HSC in murine and human AMLs and found that LIC showed distinctly elevated expression of TNF-a, one of the major activators of the NF-kB pathway. Consistent with these results, the culture media conditioned by LIC had higher TNF-a levels than those of normal cells. In all of the three types of leukemia mice, bone marrow extracellular fluid included higher TNF-a than that of control mice. Importantly, TNF-a blockage by the neutralizing antibody significantly attenuated p65 nuclear localization in LIC, and NF-kB inhibition by expression of a super repressor form of IkBa (IkB-SR) suppressed the TNF-a expression in LIC, indicating that LIC maintains its NF-kB activity by autocrine NF-kB/TNF-a positive feedback loop. Disruption of this loop by induction of IkB-SR or shRNA-mediated knockdown of TNF-a significantly reduced colony-forming abilities of leukemia cells and prolonged survival of leukemic mice in all the three models. In contrast, transduction of IkB-SR into normal HSC exerted no influence on their colony-forming ability. These results suggested that the NF-kB/TNF-a positive feedback loop plays a vital role for LIC propagation. We also addressed the mechanism of the difference in NF-kB activity between LIC and non-LIC. Notably, LIC had decreased protein levels of IkBa compared with non-LIC in spite of the same mRNA expression levels between them. In addition, basal 20S proteasome activity and the expression levels of proteasome subunit genes in LIC were higher than those in non-LIC, indicating that enhanced proteasomal degradation of IkBa could lead to selectively high NF-kB activity of LIC. The same propensity was seen in human AML CD34+CD38- cells versus CD34- cells in the analysis of microarray expression data. Proteasome inhibition by bortezomib diminished the differences of IkBa protein level. Moreover, its administration to leukemic mice selectively killed LIC fraction and prolonged survival in the in vivo transplantation model. Finally, forcible maintenance of NF-kB activity in LIC by shRNA-mediated knockdown of IkBa significantly enhanced its self-renewal activity as determined by surface marker profiles after in vitro culture. In the analysis of mice reconstituted with the IkBa-downregulated leukemic cells, bone marrow mononuclear cells had increased colony-forming cell ability and enhanced LIC frequency as determined by in vivo limiting dilution serial transplantation assay. These results indicated that the transition from LIC to non-LIC might be associated with the attenuation of NF-kB activity due to inefficient degradation of IkBa. In summary, these findings elucidate that NF-kB/TNF-a signaling in LIC, under support of the proteasome activity, has a critical role for both maintenance and propagation of LIC and provide a widely applicable approach for targeting LIC in myeloid leukemias. Disclosures: No relevant conflicts of interest to declare.