Taking the STING out of acute myeloid leukemia through macrophage-mediated phagocytosis

Macrophages within the bone marrow (BM) microenvironment take on unexpected roles in acute myeloid leukemia (AML) as reported by Moore and colleagues in this issue of the JCI. In contrast to solid tumors, where tumor-associated macrophages frequently assume an immunosuppressive phenotype that promotes tumor progression, this study revealed that BM macrophages repressed leukemia expansion in AML through a pathway called LC3-associated phagocytosis (LAP). After phagocytosis of dead and dying leukemic cells, including the mitochondria within the leukemic blasts, mitochondrial DNA activated stimulator of IFN genes (STING), leading to inflammatory signals that enhanced phagocytosis and restrained leukemic cell expansion. These findings unveil the modulation of macrophage-mediated phagocytosis via LAP as a potential therapeutic strategy directed at the BM microenvironment in AML.     

干扰素刺激基因、巨噬细胞移动抑制因子在肿瘤细胞和肿瘤微环境中淋巴细胞的表达水平

目的探讨干扰素刺激基因(STING)、巨噬细胞移动抑制因子(MIF)在肿瘤细胞和肿瘤微环境中淋巴细胞的表达水平。方法回顾性分析80例乳腺癌患者临床资料,酶联免疫吸附测定法检测STING、MIF在肿瘤细胞和肿瘤微环境中淋巴细胞的表达水平。结果肿瘤微环境淋巴细胞Th1、Th2、Th17及CD8中STING、TBK1和IRF3、MIF及TNF-α的表达均显著高于肿瘤细胞中的表达,而IL-2和IL-6的表达显著低于肿瘤细胞表达(P0.05)。结论 STING、MIF在肿瘤细胞中表达显著高于肿瘤微环境淋巴细胞中的表达。     

骨髓增生异常综合征克隆细胞生物学特征的初步研究

目的 研究骨髓增生异常综合征（MDS）克隆细胞和急性髓系白血病（AML）克隆细胞的部分生物学差异。方法 对经核型分析证实有克隆标志的51例MDS和11例AML患者骨髓中克隆细胞（荧光原位杂交分析）和原始细胞百分比进行比较；检测MDS患者骨髓中晚期红系（血型糖蛋白A阳性的晚幼红细胞）、粒系（DAPI复染可分辨的分叶核细胞）和巨核系（CD61阳性磁珠分选，CD41阳性的形态学成熟的巨核细胞）中克隆细胞比例；同步检测MDS患者骨髓及外周血中克隆细胞比例；用流式细胞术检测含有克隆细胞的MDS患者外周血中性粒细胞的吞噬和氧化功能并与正常人和AML患者标本比较。结果 MDS患者骨髓克隆细胞百分比（平均48．2％）均高于原始细胞（平均6．7％）（P〈0．01）。进展型MDS与非进展型相比，克隆细胞与原始细胞数更接近。单纯染色体＋8异常与单纯5q-患者比较，克隆细胞与原始细胞数量更接近。而在11例AML患者，克隆细胞与原始细胞百分比的平均差距接近零。在MDS患者骨髓细胞中检出相当数量带有克隆标志的晚期造血细胞：分叶核细胞中平均为45．9％；晚幼红细胞中为46．0％；成熟巨核细胞中为38．0％。MDS患者外周血中检出与骨髓中核型一致的克隆细胞，数量与骨髓中克隆细胞数相关（骨髓中平均为48．6％，外周血中为37．3％）。功能性检测显示MDS患者外周血中的中性粒细胞具有与正常对照者几乎一致的对DHR的摄取和氧化功能（P〉0．05）。结论 MDS克隆细胞与AML克隆细胞具有明显的生物学差异。     

Persistence and Efficacy of Second Generation CAR T Cell Against the LeY Antigen in Acute Myeloid Leukemia

In a phase I study of autologous chimeric antigen receptor (CAR) anti-LeY T-cell therapy of acute myeloid leukemia (AML), we examined the safety and postinfusion persistence of adoptively transferred T cells. Following fludarabine-containing preconditioning, four patients received up to 1.3 × 109 total T cells, of which 14–38% expressed the CAR. Grade 3 or 4 toxicity was not observed. One patient achieved a cytogenetic remission whereas another with active leukemia had a reduction in peripheral blood (PB) blasts and a third showed a protracted remission. Using an aliquot of In111-labeled CAR T cells, we demonstrated trafficking to the bone marrow (BM) in those patients with the greatest clinical benefit. Furthermore, in a patient with leukemia cutis, CAR T cells infiltrated proven sites of disease. Serial PCR of PB and BM for the LeY transgene demonstrated that infused CAR T cells persisted for up to 10 months. Our study supports the feasibility and safety of CAR–T-cell therapy in high-risk AML, and demonstrates durable in vivo ­persistence.     

SOX30 confers a tumor suppressive effect in acute myeloid leukemia through inactivation of Wnt/β-catenin signaling

Recent studies suggested SRY-related high mobility group box 30 (SOX30) as a candidate tumor-promoter or tumor-inhibitor in multiple tumor types. Yet, the detailed role of SOX30 in acute myeloid leukemia (AML) has not been well studied. The present research was designed to investigate the detailed relevance of SOX30 in AML. The data of our study indicated that SOX30 expression was markedly downregulated in AML cells, a pattern associated with its hypermethylation. SOX30 overexpression caused a marked reduction in AML cell proliferation and colony formation, but it promoted AML cell apoptosis. By contrast, SOX30 depletion by small interfering RNA (siRNA)-mediated gene silencing had the opposite effect. Moreover, SOX30 overexpression markedly decreased β-catenin expression, a change that led to inactivation of Wnt/β-catenin pathway. Notably, restoration of β-catenin expression partially reversed SOX30-mediated tumor suppressive effect in AML cells. In an AML-derived mouse xenograft model, SOX30 overexpression remarkably retarded the tumor growth in vivo. Overall, these data of the study suggest a tumor-inhibition role of SOX30 in AML, and highlight a key role of SOX30/Wnt/β-catenin axis in the progression of AML.     

AML1-ETO inhibits maturation of multiple lymphohematopoietic lineages and induces myeloblast transformation in synergy with ICSBP deficiency

The translocation (8;21), generating the AML1-ETO fusion protein, is one of the most frequent chromosomal abnormalities associated with acute myelogenous leukemia (AML). To elucidate its role in oncogenesis, bone marrow (BM) cells were infected with a retroviral vector carrying AML1-ETO and transplanted into mice. In contrast to previous transgenic mouse models, we show that AML1-ETO directly stimulates granulopoiesis, suppresses erythropoiesis, and impairs the maturation of myeloid, B, and T lymphoid cells in vivo. To determine the significance of earlier findings that expression of the tumor suppressor ICSBP is often downregulated in AML myeloblasts, AML1-ETO was introduced into BM cells derived from mice lacking the interferon regulatory factor ICSBP. Our findings demonstrate that AML1-ETO synergizes with an ICSBP deficiency to induce myeloblastic transformation in the BM, reminiscent of AML.     

CD47与白血病干细胞

CD47,又称整联蛋白相关蛋白（integrin—associated protein,IAP）,是一种免疫球蛋白样蛋白质,可通过与巨噬细胞上的抑制性受体信号调节蛋白alpha链（signal regulatory protein α chain,SIRPa）结合,降低吞噬细胞的吞噬活性。正常造血干细胞（HSC）上CD47的表达有助于保持其在机体内的相对稳定,但是另一方面CD47在AML患者的白血病千细胞（LSC）中高表达,LSC通过CD47的高表达来降低自身被巨噬细胞的吞噬,从而降低机体固有免疫系统对LSC的清除作用。本文将就CD47在HSC和LSC上的表达、功能以及白血病预后和靶向治疗中的作用加以综述。     

CD47 blockade enhances the efficacy of intratumoral STING-targeting therapy by activating phagocytes

Activation of STING signaling plays an important role in anti-tumor immunity, and we previously reported the anti-tumor effects of STING through accumulation of M1-like macrophages in tumor tissue treated with a STING agonist. However, myeloid cells express SIRPα, an inhibitory receptor for phagocytosis, and its receptor, CD47, is overexpressed in various cancer types. Based on our findings that breast cancer patients with highly expressed CD47 have poor survival, we evaluated the therapeutic efficacy and underlying mechanisms of combination therapy with the STING ligand cGAMP and an antagonistic anti-CD47 mAb using E0771 mouse breast cancer cells. Anti-CD47 mAb monotherapy did not suppress tumor growth in our setting, whereas cGAMP and anti-CD47 mAb combination therapy inhibited tumor growth. The combination therapy enhanced phagocytosis of tumor cells and induced systemic anti-tumor immune responses, which rely on STING and type I IFN signaling. Taken together, our findings indicate that coadministration of cGAMP and an antagonistic anti-CD47 mAb may be promising for effective cancer immunotherapy.     

Inhibition of inflammatory mediator secretion by (−)-DHMEQ in mouse bone marrow-derived macrophages

Previously, we designed and synthesized a new NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), and found that racemic DHMEQ inhibited cytokine secretion and phagocytosis by cells of the macrophage cell line RAW264.7. In the present research, we looked into the effect of optically active (−)-DHMEQ on the NO production, inflammatory cytokine secretion, and prostaglandin secretion in mouse bone marrow-derived macrophages (BMMs). We also studied the effect of (−)-DHMEQ on the differentiation of macrophages. DHMEQ inhibited lipopolysaccharide (LPS)-induced NF-κB activation. It also inhibited the expression of inducible NO synthase (iNOS) and NO production induced by LPS. Using enzyme-linked immunosorbent assays, we showed DHMEQ to inhibit LPS-induced secretion of IL-6 and TNF-α. It also inhibited COX-2 expression and prostaglandin E₂ production and secretion. It did not inhibit the phagocytosis of fluorescently labeled Escherichia coli by BMMs treated with LPS, unlike in the case of RAW264.7 cells. Next we examined the effect of the inhibitor on M-CSF-induced differentiation of bone marrow cells to macrophages. DHMEQ showed no effect on the differentiation in terms of reactive oxygen species production and F4/80 expression. However, although BMM incorporated oxidized LDL to give rise to foam cells, the (−)-DHMEQ-treated bone marrow cells did not take up oxidized LDL. Taken together, our data show that (−)-DHMEQ inhibited LPS-induced activation of BMM in terms of NO and cytokine secretion, but its effect on phagocytosis differed between BMMs and RAW264.7 cells. We also found that the functional differentiation into macrophages was inhibited by (−)-DHMEQ.     

ANGPTL2-containing small extracellular vesicles from vascular endothelial cells accelerate leukemia progression

Small extracellular vesicles (SEVs) are functional messengers of certain cellular niches that permit noncontact cell communications. Whether niche-specific SEVs fulfill this role in cancer is unclear. Here, we used 7 cell type–specific mouse Cre lines to conditionally knock out Vps33b in Cdh5⁺ or Tie2⁺ endothelial cells (ECs), Lepr⁺ BM perivascular cells, Osx⁺ osteoprogenitor cells, Pf4⁺ megakaryocytes, and Tcf21⁺ spleen stromal cells. We then examined the effects of reduced SEV secretion on progression of MLL-AF9–induced acute myeloid leukemia (AML), as well as normal hematopoiesis. Blocking SEV secretion from ECs, but not perivascular cells, megakaryocytes, or spleen stromal cells, markedly delayed the leukemia progression. Notably, reducing SEV production from ECs had no effect on normal hematopoiesis. Protein analysis showed that EC-derived SEVs contained a high level of ANGPTL2, which accelerated leukemia progression via binding to the LILRB2 receptor. Moreover, ANGPTL2-SEVs released from ECs were governed by VPS33B. Importantly, ANGPTL2-SEVs were also required for primary human AML cell maintenance. These findings demonstrate a role of niche-specific SEVs in cancer development and suggest targeting of ANGPTL2-SEVs from ECs as a potential strategy to interfere with certain types of AML.     

mTORC1 is essential for leukemia propagation but not stem cell self-renewal

Although dysregulation of mTOR complex 1 (mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse hematopoiesis using a mouse model of conditional deletion of Raptor, an essential component of mTORC1. Raptor deficiency impaired granulocyte and B cell development but did not alter survival or proliferation of hematopoietic progenitor cells. In a mouse model of acute myeloid leukemia (AML), Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. mTORC1 did not control cell cycle or cell growth in undifferentiated AML cells in vivo. Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. We further demonstrated that the reactivation of mTORC1 in those cells restored their leukemia-initiating capacity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells. Our findings provide mechanistic insight into how residual tumor cells circumvent anticancer therapies and drive tumor recurrence.     

急性髓系白血病骨髓“前ALIP”样结构的特征研究

本研究提出骨髓组织"前ALIP"样结构的概念,采用计算机图像处理技术研究其特征,并探讨其与急性髓系白血病(AML)的相关性。以抽吸-活检一步取材法采集化疗后完全缓解(CR)的AML患者骨髓,常规方法制作骨髓病理切片、光学显微镜成像系统摄片;比较4种计算机图像分割技术,筛选最佳方法对前体细胞进行识别、定位;结合形态学及面积法分割骨小梁;计算AML患者骨髓图像中单个及2个聚集前体细胞数量并以欧式距离变换法检测其与骨小梁的相对距离,与正常骨髓比较;分析在ALIP出现之前前体细胞定位、定量异常与AML的关系,探讨"前ALIP"的结构特征。结果表明,与8-sobel算子、canny算子、分水岭法相比,本研究创建的基于形态学的计算机图像分割方法能准确识别骨髓切片中前体细胞,同时结合面积法实现了骨小梁的分割。检测CR组骨髓切片中单个前体细胞为(19.27±11.60)个/mm2,2个聚集前体细胞为(1.77±1.76)簇/mm2,均明显高于正常对照组(p<0.05)。欧式距离变换法检测前体细胞与骨小梁间相对距离的结果显示,CR组单个前体细胞与骨小梁相距(230.12±97.68)μm(523±222像素),与正常对照组(260.92±99.88)μm(593±227像素)相比明显靠近骨小梁(p<0.05);而CR组两个聚集细胞与骨小梁相距(274.56±139.48)μm(624±317像素),与正常对照组相比无统计学差异(p>0.05),但与CR组单个前体细胞相比明显向骨小梁间区迁移(p<0.05)。结论:前体细胞从正常定位、定量到ALIP1结构出现之前存在更早期的骨髓异常状态,即"前ALIP"状态,其结构特征为定位或定量异常的单个及2个聚集前体细胞。     

Targeting of mTOR is associated with decreased growth and decreased VEGF expression in acute myeloid leukaemia cells

Background  The mammalian target of rapamycin (mTOR) has recently been implicated in leukaemic cell growth, tumour-associated angiogenesis and expression of vascular endothelial growth factor (VEGF). We examined whether mTOR plays a role as regulator of growth and VEGF-expression in acute myeloid leukaemia (AML). Three mTOR-targeting drugs, rapamycin, everolimus (RAD001) and CCI-779, were applied. The effects of these drugs on growth, survival, apoptosis and VEGF expression in primary AML cells and various AML cell lines were examined.
Materials and methods  Growth of AML cells and AML-derived cell lines was assessed by ³H-thymidine incorporation, survival was examined by light- and electron microscopy, by Tunel assay and by AnnexinV-staining, and the expression of VEGF by Northern blotting, RT-PCR and ELISA.
Results  Rapamycin was found to counteract growth in the AML cell lines U937 and KG1a as well as in primary AML cells in 14/18 patients examined. The effects of rapamycin and its derivatives were dose-dependent (IC₅₀: 10 pM–100 nM). It was also found that exposure to mTOR-targeting drugs resulted in apoptosis and in decreased expression of VEGF in leukaemic cells.
Conclusions  mTOR-targeting drugs exert antileukaemic effects on AML cells in vitro through multiple actions, including direct inhibition of proliferation, induction of apoptosis and suppression of VEGF. Based on this study and other studies, mTOR can be regarded as a potential drug target in AML.     

急性髓系白血病中uPA和uPAR的表达及意义

目的观察急性髓系白血病人骨髓单个核细胞uPAR（CD87）的表达及血浆uPA的水平,并探讨其临床意义。方法用ELISA法测定22例急性髓系白血病患者（观察组）及34例良性血液病患者（对照组）血浆中uPA表达水平,用流式细胞学技术测定骨髓单个核细胞uPAR水平,比较两组之间的表达差异。结果血浆中uPA水平和骨髓单个核细胞uPAR水平急性髓系白血病组均高于良性血液病组,差异具有显著性（P〈0.05）;pearson相关分析显示uPA表达水平及uPAR水平之间存在正相关性（观察组r1=0.614,对照组r2=0.921;P〈0.05）。结论急性髓系白血病患者血浆uPA水平及骨髓单个核细胞uPAR表达均升高,二者具有协同表达关系,联合检测对于急性髓系白血病的病情监控和预后分析有临床指导意义。     

BM mesenchymal stromal cell–derived exosomes facilitate multiple myeloma progression

BM mesenchymal stromal cells (BM-MSCs) support multiple myeloma (MM) cell growth, but little is known about the putative mechanisms by which the BM microenvironment plays an oncogenic role in this disease. Cell-cell communication is mediated by exosomes. In this study, we showed that MM BM-MSCs release exosomes that are transferred to MM cells, thereby resulting in modulation of tumor growth in vivo. Exosomal microRNA (miR) content differed between MM and normal BM-MSCs, with a lower content of the tumor suppressor miR-15a. In addition, MM BM-MSC–derived exosomes had higher levels of oncogenic proteins, cytokines, and adhesion molecules compared with exosomes from the cells of origin. Importantly, whereas MM BM-MSC–derived exosomes promoted MM tumor growth, normal BM-MSC exosomes inhibited the growth of MM cells. In summary, these in vitro and in vivo studies demonstrated that exosome transfer from BM-MSCs to clonal plasma cells represents a previously undescribed and unique mechanism that highlights the contribution of BM-MSCs to MM disease progression.     

中期因子在急性髓系白血病患者的表达及临床意义

为了探讨中期因子(midkine,MK)与急性髓系白血病(AML)患者预后的关系,本研究采用半定量RT-PCR方法检测65例AML患者与15例正常人骨髓单个核细胞(MNC)MK、bcl-2mRNA的表达,并对20例AML患者和5例正常人采用Westernblot检测MNC中MK基因的表达。结果显示:初治组、复发组和化疗缓解组MK基因表达水平分别为0.331±0.436,0.374±0.463和0.067±0.190,正常人MK基因表达阴性;MK基因表达阳性与表达阴性患者的首次完全缓解(CR)率分别为63.16%和93.55%(P=0.01),MK基因表达阳性患者的复发率(100%)明显高于表达阴性者(40%)(P=0.019);多药耐药组与药物敏感组MK基因的表达率分别为57.69%和25.64%(P<0.01);MK与bcl-2基因表达呈正相关(r=0.556,P<0.001)。结论:AML患者白血病细胞可以产生MK,且MK阳性率的高低与AML病期相关,是影响AML患者近期预后的重要因素之一。MK可能通过上调bcl-2的表达抑制白血病细胞凋亡。     

Prognostic potential of morphological and cytogenetic indices in patients with myelodysplastic syndrome

AIM: To examine prognostic potential of the number of bone marrow (BM) blasts and cell karyotype as risk factors of transformation of myelodysplastic syndrome (MDS) in acute myeloblastic leukemia AML. MATERIAL AND METHOD: The analysis of examination was made for 72 patients with primary MDS in the groups formed by number of blasts in BM, karyotype and IPSS variant. MDS was diagnosed by WHO criteria. Transformation into AML was established in blastosis > 20% in peripheral blood and/or BM. The karyotype was studied according to GTG technique. RESULTS: More frequent progression of MDS was seen in patients with blastosis > 10%, unfavourable karyotype and high IPSS risk. The least number of leukemic transformations occurred in karyotype of intermediate prognosis while disease-free survival in patients with karyotype of good prognosis was similar to that of patients with unfavourable karyotype. The number of blasts in BM and IPSS variant appeared to be prognostic markers of duration of leukemia-free survival in one-factor analysis. The multifactorial analysis found out one factor of MDS transformation in AML: number of blasts in BM puncture biopsy. CONCLUSION: Prognostic priority of the number of BM blasts as a risk factor of MDS progression compared to karyotype is explained by biological heterogenicity of MDS.     

Retinoic acid in mono- or combined differentiation therapy of myelodysplasia and acute promyelocytic leukemia

Myelodysplastic preleukemic syndromes (MDPS) and acute promyelocytic leukemia (APL) share a surprising in vivo sensitivity to the hormonally acting 13 cis or all trans retinoic acids (transRA). Here we show that transRA as a monotherapeutic agent induced a stable remission in APL at the third relapse. In MDPS, treatment with prednisone and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25D3) 13 cis RA induced a long-lasting hematological remission. Initially both patients had an impaired BM microenvironment which regenerated on retinoid therapy as judged by reappearance of the Hematon fraction in the BM aspirates. Our preclinical experiments using long-term liquid BM cultures (LTBMC) indicated that several individual patterns of growth and differentiation responses can be induced by combinations of transRA, 1 alpha,25D3 and hemopoietic growth factors (HGFs). The biological responses may vary from complete clonal extinction to a significant growth stimulation of the leukemic blast cell populations. These results further support the importance of preclinical studies in selecting "good" responders for, and excluding "poor" responders from protocols using differentiation therapy.     

Oxidized phosphatidylserine-CD36 interactions play an essential role in macrophage-dependent phagocytosis of apoptotic cells

The phagocytosis of apoptotic cells within an organism is a critical terminal physiological process in programmed cell death. Evidence suggests that apoptotic cell engulfment and removal by macrophages is facilitated by phosphatidylserine (PS) displayed at the exofacial surface of the plasma membrane; however, neither the macrophage receptors responsible for PS recognition, nor characterization of the PS molecular species potentially involved, have been clearly defined. We show that the class B scavenger receptor CD36 plays an essential role in macrophage clearance of apoptotic cells in vivo. Further, macrophage recognition of apoptotic cells via CD36 is shown to occur via interactions with membrane-associated oxidized PS (oxPS) and, to a lesser extent, oxidized phosphatidylcholine, but not nonoxidized PS molecular species. Mass spectrometry analyses of oxPS species identify structures of candidate ligands for CD36 in apoptotic membranes that may facilitate macrophage recognition. Collectively, these results identify oxPS-CD36 interactions on macrophages as potential participants in a broad range of physiologic processes where macrophage-mediated engulfment of apoptotic cells is involved.