Iron and leukemia: new insights for future treatments
铁和白血病：未来治疗的新见解

Physiologic iron metabolism
生理铁代谢

Iron homeostasis is a complex and highly regulated process, which involves acquisition, utilization, storage and efflux of iron. Non-heme iron in the diet are mostly presented in the form of ferric iron (Fe³⁺) [17]. The absorption of non-heme iron in the diet involves reduction of Fe³⁺ to Fe²⁺ in the intestinal lumen by ferric reductases, such as duodenal cytochrome b reductase (Dcytb), and subsequent transport of Fe²⁺ into enterocytes by divalent metal transporter 1 (DMT1) [18]. Dietary heme iron can be directly taken up by enterocytes by a yet unknown mechanism [17]. The iron absorbed through enterocytes is either exported across the basolateral membrane into the circulation by ferroportin 1 (FPN1), the only known mammalian iron exporter, or stored in ferritin [19]. On the basolateral membrane, Fe²⁺ is oxidized by ferroxidase hephaestin (HEPH) in order to be associated with transferrin (Tf) in the plasma [20]. Iron is circulated throughout the body in a redox-inert state and is primarily utilized for erythropoiesis [21]. Senescent red blood cells are cleared by macrophages and the iron is released into the systemic iron pool [21]. The balance of whole-body iron is maintained by strictly regulating the absorption of dietary iron in the duodenum, which is mainly achieved by the ferroportin–hepcidin regulatory axis [22]. When whole-body iron levels are high, hepcidin is induced in hepatocytes and secreted into the circulation. Hepcidin binds to FPN1 on enterocytes and macrophages to block the delivery of iron into the circulation [23].
铁稳态是一个复杂且高度受调控的过程，涉及铁的获取、利用、储存和外流。饮食中的非血红素铁主要以三价铁（Fe ³⁺ ）的形式存在[17]。饮食中的非血红素铁的吸收涉及由铁还原酶（如十二指肠细胞色素 b 还原酶（Dcytb））在肠腔中将 Fe ³⁺ 还原为 Fe ²⁺ ，随后由双价金属转运蛋白 1（DMT1）将 Fe ²⁺ 转运至肠细胞[18]。膳食血红素铁可以通过尚不明确的机制直接被肠细胞吸收[17]。通过肠细胞吸收的铁要么通过铁蛋白 1（FPN1，唯一已知的哺乳动物铁输出蛋白）跨越基底侧膜进入循环，要么储存在铁蛋白中[19]。在基底侧膜上，Fe ²⁺ 被铁氧化酶赫菲斯汀（HEPH）氧化，以便与血浆中的转铁蛋白（Tf）结合[20]。铁以氧化还原不活泼的状态在全身循环，并主要用于造血[21]。老化的红细胞被巨噬细胞清除，铁释放到系统铁库中[21]。 整体铁的平衡是通过严格调节十二指肠对膳食铁的吸收来维持的，主要是通过铁转运蛋白-肝铁蛋白调节轴来实现的[22]。当整体铁水平较高时，肝细胞中诱导产生肝铁蛋白并分泌到循环中。肝铁蛋白结合到肠细胞和巨噬细胞上的 FPN1，阻止铁进入循环[23]。

Tf-bound iron in the plasma can be taken up by cells mainly through transferrin receptor 1 (TfR1, 24]. Diferric Tf binds to TfR1 on the plasma membrane and the Tf/TfR1 complex is subsequently taken into the cell by receptor-mediated endocytosis [24]. In the endosome, iron is released from the complex [25], reduced by six-transmembrane epithelial antigen of the prostate (STEAP) proteins to Fe²⁺ and transported into the cytoplasm by DMT1 [26]. Meanwhile, the apo-transferrin (apo-Tf)/TfR1 complex is recycled to the cell surface where apo-Tf is released to the plasma. Certain types of cells can absorb iron in other forms such as non-transferrin bound iron (NTBI), ferritin, heme and hemoglobin [20]. Imported iron enters the cytosolic labile iron pool (LIP), a pool of chelatable and redox-active iron [27]. Iron in the pool is delivered to different parts of the cell for a variety of metabolic needs or stored in ferritin [28]. Excess cellular iron can be exported out of the cell by FPN1 and subsequently oxidized by the ceruloplasmin (Cp) and binded to serum Tf [29]. The cellular iron homeostasis is achieved mainly by the iron responsive elements (IREs)/ iron regulatory proteins (IRPs) system [30]. IRPs regulate the expression of genes involved in iron metabolism by binding to IREs. When cellular iron concentrations are low, the IRPs bind to the IREs, resulting in increased synthesis of TfR1 and decreased synthesis of ferritin and FPN1. This effect allows the cells to absorb iron to the utmost.
血浆中的 Tf 结合铁主要通过转铁蛋白受体 1(TfR1)被细胞摄取[24]。二铁转铁蛋白结合到血浆膜上的 TfR1，形成 Tf/TfR1 复合物，随后通过受体介导的内吞作用被细胞摄入[24]。在内体中，铁从复合物中释放[25]，由前列腺六跨膜上皮抗原（STEAP）蛋白还原为 Fe ²⁺ ，并通过 DMT1 转运到细胞质[26]。同时，无载体转铁蛋白(apo-Tf)/TfR1 复合物被回收到细胞表面，apo-Tf 释放到血浆中。某些类型的细胞可以吸收其他形式的铁，如非转铁蛋白结合铁(NTBI)、铁蛋白、血红素和血红蛋白[20]。进口铁进入细胞质可动态铁库(LIP)，这是一个可螯合和具有氧化还原活性的铁库[27]。铁库中的铁被输送到细胞的不同部位，以满足各种代谢需求，或存储在铁蛋白中[28]。细胞内过量的铁可以通过 FPN1 排出细胞，并随后被铜蓝蛋白(Cp)氧化并结合到血清 Tf[29]。 细胞铁稳态主要通过铁响应元件（IREs）/铁调节蛋白（IRPs）系统实现[30]。IRPs 通过结合 IREs 调节参与铁代谢的基因的表达。当细胞铁浓度较低时，IRPs 结合到 IREs，导致 TfR1 的合成增加，而铁蛋白和 FPN1 的合成减少。这种效应使细胞能够最大限度地吸收铁。

Alternations of iron metabolism in leukemia
白血病铁代谢的变化

Iron metabolism in leukemia is altered, including not only changes in cellular iron uptake, storage and efflux, but also dysregulation of the ferroportin–hepcidin regulatory axis (Fig. 1). Furthermore, multiple red-blood-cell transfusions throughout chemotherapy treatment aggravate systematic iron overload in patients with leukemia. While iron and its catalytic production of ROS are critical to maintain hematopoietic homeostasis, accumulation of iron and subsequent increased oxidative stress are detrimental to normal hematopoiesis. ROS have been implicated as the signal messengers in normal hematopoiesis and participate in controlling the biological activity of HSCs [31]. However, redox dysregulation caused by ROS promotes malignant transformation of HSCs by increasing DNA double strand breaks and repair errors [32, 33]. Besides, iron is essential for the progression of leukemia because maintaining the rapid growth rate of leukemia cells requires the iron-dependent enzyme ribonucleotide reductase for DNA synthesis [7, 34, 35]. Furthermore, iron overload allows leukemia cells immune evasion by triggering apoptosis of adjacent NK cells, CD4⁺ T cells and CD8⁺ T cells, but increasing percentage of regulatory T cells [36, 37].
在白血病中，铁代谢发生改变，不仅包括细胞铁摄取、储存和外流的变化，还包括铁转运蛋白-肝铁蛋白调控轴的失调（图 1）。此外，化疗期间多次输注红细胞加重了患有白血病患者的全身铁超载。虽然铁及其催化产生的 ROS 对于维持造血系统稳态至关重要，但铁的积累和随之增加的氧化应激对正常造血是有害的。ROS 已被认为是正常造血中的信号传递者，并参与控制 HSCs 的生物活性。然而，ROS 引起的氧化还原失调通过增加 DNA 双链断裂和修复错误促进了 HSCs 的恶性转化。此外，铁对于白血病的进展至关重要，因为维持白血病细胞的快速生长需要依赖铁的酶核糖核苷酸还原酶进行 DNA 合成。 此外，铁过载通过诱导相邻 NK 细胞、CD4 T 细胞和 CD8 T 细胞凋亡，使白血病细胞免疫逃避，但增加了调节性 T 细胞的百分比【36, 37】。

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Fig. 1 图 1

Alternations of iron metabolism in leukemia at systemic and cellular levels. a The systematic iron pool and serum ferritin levels are increased which is aggravated by multiple red-blood-cell transfusions. Hepcidin is induced to block the delivery of iron into the circulation from enterocytes, macrophages and some other cells. b Leukemia cells show increased iron uptake and decreased iron efflux, leading to elevated cellular iron levels. Proteins related to iron uptake such as TfR1, TfR2 and STEAP1 are overexpressed and absorption of NTBI is increased. However, the expression of iron export protein FPN1 is decreased. HFE or c-MYC gene variants are also associated with elevated intracellular iron levels in leukemia cells
在系统和细胞水平上，白血病铁代谢的变化。a 系统铁库和血清铁蛋白水平增加，多次红细胞输血加重了这种情况。肝铁素调节蛋白被诱导以阻止铁从肠细胞、巨噬细胞和其他一些细胞进入循环。b 白血病细胞显示铁摄取增加和铁外流减少，导致细胞内铁水平升高。与铁摄取相关的蛋白如转铁蛋白受体 1、转铁蛋白受体 2 和 STEAP1 被过度表达，非转铁蛋白铁的吸收增加。然而，铁输出蛋白 FPN1 的表达减少。HFE 或 c-MYC 基因变异也与白血病细胞内铁水平升高相关。

Alternations of iron metabolism in leukemia at systemic levels
在系统水平上，白血病铁代谢的变化

It has been reported that patients with AML at diagnosis had higher levels of serum ferritin, the routine marker for excess iron [38]. Ferritin promotes the growth of leukemia cells while inhibiting the colony formation of normal progenitor cells, which is identified as leukemia-associated inhibitory activity [39]. Clinical analysis suggests that hyperferritinemia at diagnosis is significantly associated with chemotherapy drug resistance, a higher incidence of relapse as well as poorer overall survival [38, 40]. Furthermore, an elevated pretransplantation serum ferritin level is an adverse prognostic factor for overall survival and nonrelapse mortality for patients with hematologic malignancies undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) [41, 42].
据报道，AML 患者在诊断时血清铁蛋白水平较高，这是过量铁的常规标志物[38]。铁蛋白促进白血病细胞的生长，同时抑制正常祖细胞的克隆形成，这被认为是与白血病相关的抑制活性[39]。临床分析表明，诊断时的高铁蛋白血症与化疗药物抗药性、更高的复发率以及较差的总生存率显著相关[38,40]。此外，移植前血清铁蛋白水平升高是对进行同基因异体造血干细胞移植（allo-HSCT）的血液恶性肿瘤患者的总生存率和非复发死亡率的不利预后因素[41,42]。

Due to the increased systematic iron pool, the ferroportin–hepcidin regulatory axis is also dysregulated. The serum hepcidin levels of AL patients are significantly elevated at the initial of diagnosis and decreased after remission, but still higher than that of the healthy controls [43, 44]. High level of serum hepcidin leads to iron accumulation in leukemia cells which may contribute to leukemogenesis by activating Wnt and nuclear factor kappa-B (NF-κB) signaling pathways [45–48].
由于系统铁库的增加，铁转运蛋白-肝铁素调节轴也失调。AL 患者的血清肝铁素水平在诊断初期明显升高，在缓解后下降，但仍高于健康对照组[43,44]。高水平的血清肝铁素导致白血病细胞中铁的积累，可能通过激活 Wnt 和核因子κB（NF-κB）信号通路促进白血病发生[45-48]。

Meanwhile, the transportation of iron into the circulation from enterocytes and macrophages is blocked, thereby leading to erythropoiesis suppression and iron accumulation in tissues. In addition, patients with AL usually receive multiple red-blood-cell transfusions for hematologic support, which aggravates systematic iron overload. Transfusional iron accumulates in macrophages initially as the senescent red blood cells are eliminated. Then iron accumulates in the liver and later spreads to extrahepatic tissue such as endocrine tissues and the heart [49]. It has been demonstrated that iron overload can cause damage to bone marrow stem cells resulting in iron-correlated hematopoietic suppression, which is mediated by ROS-related signaling pathway [50, 51]. In turn, anemia caused by hematopoiesis inhibition makes further dependence on red-blood-cell transfusions, thus creating a vicious cycle.
同时，从肠细胞和巨噬细胞向循环系统输送铁被阻断，从而导致造血抑制和组织中铁的积累。此外，AL 患者通常接受多次红细胞输血以获得造血支持，这加重了系统性铁过载。输血铁最初在巨噬细胞中积累，随着老化的红细胞被清除。然后铁在肝脏中积累，后来扩散到内分泌组织和心脏等肝外组织[49]。已经证明铁过载会对骨髓干细胞造成损害，导致铁相关的造血抑制，这是通过 ROS 相关的信号通路介导的[50, 51]。反过来，由造血抑制引起的贫血使人更加依赖红细胞输血，从而形成恶性循环。

Alternations of iron metabolism in leukemia at cellular levels
在白血病细胞水平上的铁代谢变化

TfR1, also known as CD71, is essential for iron uptake. Leukemia cells have increased expression of TfR1 compared to their normal counterparts and TfR1 is involved in the clonal development of leukemia [9, 52]. The expression of TfR1 is more prevalent in AML than that in ALL [53]. Moreover, poorly differentiated primary AML blasts tend to express higher levels of TfR1 than partially differentiated AML blasts [52]. TfR1 expression is higher in patients with T-cell ALL than patients with B-cell ALL [11, 54]. Clinical analysis also shows that overexpression of TfR1 in ALL is an adverse prognostic factor [11]. Transferrin receptor 2 (TfR2), another receptor for Tf, is also overexpressed in AML compared with normal counterparts [55]. Although both TfR1 and TfR2 are highly expressed in AML, only TfR2 levels were significantly associated with serum iron [56]. However, elevated mRNA levels of TfR2-α but not TfR1 or TfR2-β contribute to a better prognosis for AML patients [56]. It may be that TfR2-α increases the sensitivity of leukemia cells to chemotherapy drugs through an iron-independent pathway. The interaction of Tf with TfR can be modulated by HFE protein, thereby limiting the amount of internalized iron. Recent research suggests that HFE gene variants confer increased risk of leukemia that is attributed to the toxic effects of higher levels of iron [10, 57, 58]. In addition, the STEAP proteins function as ferric reductases that stimulate cellular uptake of iron through TfR1 [59]. Analysis of publicly available gene expression data shows that the STEAP1 is significantly overexpressed in AML which is associated with poor overall survival [60].
TfR1，也被称为 CD71，对铁的摄取至关重要。白血病细胞相较于其正常对照具有增加的 TfR1 表达，并且 TfR1 参与了白血病的克隆发展[9, 52]。TfR1 的表达在 AML 中比在 ALL 中更为普遍[53]。此外，未分化的原发性 AML 爆发倾向于比部分分化的 AML 爆发表达更高水平的 TfR1[52]。TfR1 的表达在 T 细胞 ALL 患者中高于 B 细胞 ALL 患者[11, 54]。临床分析还显示，在 ALL 中 TfR1 的过度表达是一种不良预后因素[11]。转铁蛋白受体 2（TfR2），另一种 Tf 的受体，与正常对照相比在 AML 中也有过度表达[55]。尽管 TfR1 和 TfR2 在 AML 中均高度表达，但只有 TfR2 水平与血清铁显著相关[56]。然而，TfR2-α的 mRNA 水平升高，而 TfR1 或 TfR2-β没有，有助于 AML 患者的更好预后[56]。也许是 TfR2-α通过一种与铁无关的途径增加了白血病细胞对化疗药物的敏感性。 Tf 与 TfR 的相互作用可以通过 HFE 蛋白调节，从而限制内吞铁的量。最近的研究表明，HFE 基因变体增加了患白血病的风险，这归因于更高水平铁的毒性影响。此外，STEAP 蛋白作为铁还原酶，通过 TfR1 刺激细胞对铁的摄取。对公开可获得的基因表达数据的分析显示，STEAP1 在 AML 中显著过度表达，与整体生存率较差相关。

Transferrin-independent iron is also associated with iron overload in leukemia [61]. Lipocalin 2 (LCN2), also known as neutrophil gelatinase-associated lipocalin, is a less well studied protein that participates in iron uptake [62]. It is reported that overexpression of LCN2 was found in patients with AML, ALL, CML and CLL [63–67]. LCN2 is indispensable for BCR-ABL-induced leukomogenesis in the mouse model and involved in damaging normal hematopoietic cells [67]. Paradoxically, the analysis of whole-genome expression profiles from patients with leukemia (including AML, ALL and CLL) shows that LCN2 is downregulated at both mRNA and protein levels compared with healthy controls [64, 68]. The expression levels of LCN2 in the bone marrow of AML patients are lower than that of normal controls [69]. Importantly, the levels of LCN2 increased when AML patients achieved complete remission (CR), and decreased in patients with refractory disease [69]. Those data suggest that LCN2 expression is associated with better prognosis in AML. Therefore, further research is needed to clarify the specific function of LCN2 in different types of leukemia.
转铁蛋白独立铁也与白血病铁过载相关[61]。脂联素 2（LCN2），又称中性粒细胞明胶酶相关脂联素，是一个研究较少的参与铁摄取的蛋白质[62]。据报道，在 AML、ALL、CML 和 CLL 患者中发现 LCN2 的过表达[63-67]。LCN2 对 BCR-ABL 诱导的小鼠白血病发生至关重要，并参与损害正常造血细胞[67]。矛盾的是，对包括 AML、ALL 和 CLL 在内的白血病患者的全基因组表达谱分析显示，与健康对照组相比，LCN2 在 mRNA 和蛋白水平上均下调[64,68]。AML 患者骨髓中 LCN2 的表达水平低于正常对照组[69]。重要的是，当 AML 患者达到完全缓解（CR）时，LCN2 水平增加，而在难治性疾病患者中下降[69]。这些数据表明 LCN2 表达与 AML 的良好预后相关。因此，需要进一步研究澄清 LCN2 在不同类型白血病中的具体功能。

In addition to the abnormality of iron absorption, dysregulation of the iron-storage protein- ferritin also contributes to the pathogenesis and progression of leukemia. Ferritin is composed of two subunit types, termed ferritin heavy chain (FTH) and ferritin light chain (FTL) subunits. The c-MYC protein encoded by the proto-oncogene c-MYC is a transcription factor that activates the expression of iron regulatory protein-2 (IRP2) and represses ferritin expression [70]. IRP2 can bind to IREs, which results in increased synthesis of TfR1. The consequent increase in iron uptake and reduction in iron storage could raise the intracellular LIP level for metabolic and proliferative purposes¹⁰². It has been suggested that c-MYC gene plays an important role in the pathogenesis of lymphocytic leukemia [71]. T lymphocytic leukemia can be induced by the aberrant expression of c-MYC gene in the zebrafish model [72]. The suppression of c-MYC gene prevents leukemia initiation in mice, and reducing expression levels of c-MYC gene inhibits cell growth in refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL) [73]. FTH is also involved in the NF-κB signaling pathway-mediated cell proliferation, due to that FTH prevents ROS accumulation by iron sequestration, thereby inhibiting the pro-apoptotic c-Jun N-terminal kinase (JNK) signaling pathway [74]. It is reported that FTH and FTL are overexpressed in both AML cells and leukemia stem cells compared with normal HSCs regardless of genetic subgroups [40]. Thus, either downregulation or upregulation of ferritin contributes to the pathogenesis and progression of leukemia.
除了铁吸收异常外，铁储存蛋白-铁蛋白的失调也促成了白血病的发病和进展。铁蛋白由两种亚基类型组成，称为铁蛋白重链（FTH）和铁蛋白轻链（FTL）亚基。由原癌基因 c-MYC 编码的 c-MYC 蛋白是一种转录因子，可激活铁调节蛋白-2（IRP2）的表达并抑制铁蛋白的表达。IRP2 可以结合 IREs，导致 TfR1 的合成增加。随之而来的铁摄取增加和铁储存减少可能提高细胞内 LIP 水平以进行代谢和增殖目的。有人提出 c-MYC 基因在淋巴细胞白血病的发病中起重要作用。在斑马鱼模型中，通过异常表达 c-MYC 基因可以诱导 T 淋巴细胞白血病。抑制 c-MYC 基因可以防止小鼠白血病的发生，并降低 c-MYC 基因表达水平可以抑制难治性和复发性 T 细胞急性淋巴细胞白血病（T-ALL）的细胞生长。 FTH 还参与了 NF-κB 信号通路介导的细胞增殖，因为 FTH 通过铁离子封存防止 ROS 积累，从而抑制促凋亡的 c-Jun N-末端激酶（JNK）信号通路[74]。据报道，与正常 HSCs 相比，无论遗传亚组如何，AML 细胞和白血病干细胞中 FTH 和 FTL 都过度表达[40]。因此，铁蛋白的下调或上调都会促成白血病的发病和进展。

Studies have shown that cancer cells increase metabolically available iron not only by increasing iron uptake and regulating iron storage, but also by reducing iron efflux [7]. Accumulating evidence suggests that iron efflux mediated by FPN1 and controlled by hepcidin is involved in the development and progression of leukemia [43, 75, 76]. The expression level of FPN1 was decreased in the majority of AML cell lines, primary AML samples and leukemia progenitor and stem cells [76]. Low levels of FPN1 in AML are associated with good prognosis, which may occur due to the increased sensitivity to chemotherapy [75]. Of note, leukemia cells may synthesize hepcidin initiating a local autocrine signaling to degrade membrane FPN1, which needs to be confirmed by further research [77].
研究表明，癌细胞不仅通过增加铁的摄取和调节铁的储存来增加代谢可利用铁，还通过减少铁的外流来实现[7]。越来越多的证据表明，由 FPN1 介导的铁外流以及由肝铁蛋白控制的铁外流参与了白血病的发展和进展[43, 75, 76]。FPN1 的表达水平在大多数 AML 细胞系、原发性 AML 样本以及白血病祖细胞和干细胞中降低[76]。AML 中 FPN1 的低水平与良好预后相关，这可能是由于对化疗的增加敏感性[75]。值得注意的是，白血病细胞可能合成肝铁蛋白，启动局部自分泌信号传导以降解膜 FPN1，这需要进一步的研究来证实[77]。

Therapeutic opportunities of targeting iron metabolism in leukemia
在白血病中针对铁代谢的治疗机会

As previously discussed, iron metabolism is dysregulated in patients with AL, which contributes to the development and progression of leukemia. These findings lead to the exploration of therapeutic approaches of targeting iron metabolism, including iron chelators, targeting iron metabolism related proteins and perturbing redox balance based on the high intracellular iron levels (Fig. 2).
正如之前讨论的那样，铁代谢在 AL 患者中失调，这导致了白血病的发展和进展。这些发现促使探索针对铁代谢的治疗方法，包括铁螯合剂、针对铁代谢相关蛋白和扰乱基于高细胞内铁水平的氧化还原平衡（图 2）。

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Fig. 2 图 2

Therapeutic opportunities of targeting iron metabolism in leukemia cells. Iron deprivation by iron chelators or targeting iron metabolism related proteins induces differentiation, apoptosis and cell cycle arrest in leukemia cells. The generation of ROS is involved in the process of inducing cell differentiation. Iron chelators also play anti-leukemia roles through iron-independently regulating multiple signaling pathways or restoring GVL. ADCC is also involved in the anti-leukemia effect of targeting iron metabolism related proteins. Iron metabolism related proteins-targeted delivery systems or iron-based nanoparticles can selectively deliver therapeutic agents into leukemia cells to play enhanced anti-leukemia activity. Furthermore, iron-based nanoparticles elevate iron-catalyzed ROS levels, leading to increased cytotoxicity. Ferroptosis inducers perturb redox balance based on the high intracellular iron levels to induce ferroptosis in leukemia cells
靶向白血病细胞铁代谢的治疗机会。铁螯合剂通过铁离子螯合或靶向铁代谢相关蛋白诱导白血病细胞分化、凋亡和细胞周期停滞。ROS 的产生参与了诱导细胞分化的过程。铁螯合剂还通过独立于铁的调节多条信号通路或恢复 GVL 发挥抗白血病作用。ADCC 也参与了靶向铁代谢相关蛋白的抗白血病效应。铁代谢相关蛋白靶向递送系统或基于铁的纳米颗粒可以选择性地将治疗剂送入白血病细胞，发挥增强的抗白血病活性。此外，基于铁的纳米颗粒提高了铁催化的 ROS 水平，导致细胞毒性增加。诱导铁死亡的剂扰乱基于高胞内铁水平的氧化还原平衡，诱导白血病细胞发生铁死亡。

Iron chelators 铁螯合剂

Iron chelators are natural or synthetic small molecules that can decrease levels of intracellular iron by binding iron with a high affinity and promoting iron excretion. Several iron chelators, such as deferoxamine (DFO) and deferasirox (DFX), are clinically used to treat iron overload including secondary iron overload caused by repeated blood transfusions in patients with leukemia [78, 79]. Application of iron chelators has been proposed as an alternative anti-leukemia therapy in recent years [80]. Iron chelators exert anti-leukemia activity through several mechanisms, including lowering the LIP of leukemia cells by chelating intracellular iron, increasing ROS levels and activating MAPK and some other signaling pathways [14, 81, 82] (Table 1). The application of iron chelators in patients with leukemia and transfusional iron overload has dual effects of anti-leukemia and reducing the complications associated with iron overload.
铁螯合剂是天然或合成的小分子，可以通过高亲和力结合铁并促进铁的排泄来降低细胞内铁的水平。几种铁螯合剂，如去铁胺（DFO）和去铁酸（DFX），在临床上被用于治疗铁过载，包括由白血病患者反复输血引起的继发性铁过载[78, 79]。近年来，铁螯合剂的应用被提议作为一种替代的抗白血病疗法[80]。铁螯合剂通过几种机制发挥抗白血病活性，包括通过螯合细胞内铁降低白血病细胞的 LIP，增加 ROS 水平并激活 MAPK 和其他一些信号通路[14, 81, 82]（表 1）。在白血病患者和输血性铁过载患者中应用铁螯合剂具有抗白血病和减少与铁过载相关并发症的双重效应。

Iron chelators effectively induce cell growth arrest and apoptosis in leukemia cells in a dose- and time-dependent manner [14, 16, 93]. Leukemia cells are more sensitive to iron chelators than their normal counterparts, most probably because their rapid proliferation depends on iron. Moreover, supplementation with iron attenuates the anti-leukemia effect of iron chelators, indicating that iron deprivation is one of the anti-leukemia mechanisms of iron chelators [16, 83]. It has been known for a long time that the rate-limiting step in DNA synthesis is catalyzed by ribonucleotide reductase whose catalytic activity is dependent on the continual presence of iron [94]. Iron deprivation blocks the synthesis of deoxyribonucleotides to inhibit proliferation in leukemia cells [84]. In consistent with the inhibition of DNA synthesis, iron deprivation appears to induce G1/S cell cycle arrest in leukemia cells [95]. Additionally, iron chelation decreases the cyclin-dependent kinase inhibitor p21^CIP1/WAF1 protein through post-transcriptional regulation to achieve G1/S cell cycle arrest and induce apoptosis [96]. The mitogen-activated protein kinase (MAPK) pathway and the caspase pathway are also involved in the cell cycle arrest and apoptosis induced by iron depletion [16, 82].
铁螯合剂以剂量和时间依赖的方式有效地诱导白血病细胞生长停滞和凋亡[14, 16, 93]。白血病细胞对铁螯合剂比它们的正常对应物更敏感，这很可能是因为它们的快速增殖依赖于铁。此外，铁的补充削弱了铁螯合剂的抗白血病作用，表明铁剥夺是铁螯合剂的抗白血病机制之一[16, 83]。长期以来人们已经知道 DNA 合成的速率限制步骤是由核糖核苷酸还原酶催化的，其催化活性依赖于持续存在的铁[94]。铁剥夺阻断了脱氧核苷酸的合成，从而抑制了白血病细胞的增殖[84]。与 DNA 合成的抑制一致，铁剥夺似乎诱导白血病细胞 G1/S 细胞周期停滞[95]。此外，铁螯合剂通过转录后调控降低了细胞周期蛋白依赖激酶抑制剂 p21 蛋白，实现 G1/S 细胞周期停滞并诱导凋亡[96]。 有丝分裂原活化蛋白激酶（MAPK）途径和半胱氨酸蛋白酶途径也参与了铁耗竭诱导的细胞周期停滞和凋亡[16, 82]。

Given the importance of iron in generation of free radicals and the critical role of ROS in HSCs metabolism, the role of ROS in anti-leukemia effects of iron deprivation has been studied [97]. Although iron deprivation by iron chelators may decrease ROS by reducing substrates for Fenton reaction, some iron chelators were shown to induce generation of ROS in a dose and time-dependent manner [85, 98]. Importantly, iron deprivation induces the differentiation of leukemia blasts and normal bone marrow precursors into monocytes/ macrophages by increasing ROS levels [14, 85, 95]. Iron deprivation–induced differentiation depends on activation of the downstream signaling pathways of oxidant stress response, including the MAPK/JNK signaling pathway [14, 86].
考虑到铁在自由基生成中的重要性以及 ROS 在 HSC 代谢中的关键作用，已经研究了铁剥夺对铁剥夺抗白血病作用中 ROS 的作用。尽管铁螯合剂通过减少 Fenton 反应底物可能会减少 ROS，但一些铁螯合剂被证明会以剂量和时间依赖的方式诱导 ROS 的生成。重要的是，铁剥夺通过增加 ROS 水平诱导白血病爆发和正常骨髓前体细胞向单核细胞/巨噬细胞的分化。铁剥夺诱导的分化取决于氧化应激响应的下游信号通路的激活，包括 MAPK/JNK 信号通路。

Iron chelators may play anti-leukemia roles through iron-independently regulating multiple signaling pathways related to cell survival. DFO induces apoptosis in T-ALL cells by reinstating the activation of interferon-γ (IFN-γ) /signal transducer and activator of transcription 1 (STAT1) pathway which is attenuated in T-ALL cells shielding them from the anti-proliferative effect of IFN-γ [99]. DFX also exerts its anti-leukemia activity by inhibiting extracellular signal-regulated kinase (ERK) phosphorylation, repressing the mammalian target of rapamycin (mTOR) and NF-κB signaling pathway [81, 100, 101].
铁螯合剂可能通过独立于铁元素的方式调节与细胞存活相关的多条信号通路，发挥抗白血病作用。DFO 通过恢复干扰素-γ（IFN-γ）/信号转导子和转录激活因子 1（STAT1）通路的激活，在 T-ALL 细胞中诱导凋亡，这一通路在 T-ALL 细胞中被削弱，使其免受 IFN-γ的抗增殖作用[99]。DFX 还通过抑制细胞外信号调节激酶（ERK）磷酸化、抑制哺乳动物雷帕霉素靶蛋白（mTOR）和 NF-κB 信号通路来发挥其抗白血病活性[81,100,101]。

Iron chelators not only have anti-leukemia effects singly, but also exhibit synergistic anti-leukemia effects when combined with traditional chemotherapy drugs. DFO increases the sensitivity of human myeloid leukemia cells to doxorubicin (DOX) and arabinoside cytosine (Ara-C) [102, 103]. DFO combined with arsenic trioxide (ATO) has synergistic effects on anti-proliferation and inducing apoptosis in APL [104]. DFO can be synergized with L-asparaginase or dexamethasone to decrease survival of leukemia cells or associated with DNA-damage inducing agents to increase apoptosis in T-ALL [9]. DFX shows synergistic effect with the DNA methyl transferase inhibitor decitabine (DAC) on apoptosis and cell cycle arrest in leukemia cell lines [88]. However, it has been suggested that DFX creates a synergistic effect combined with Ara-C, while antagonizes the anti-leukemia effect of DOX in the treatment of AML [89]. Therefore, further studies are needed to confirm the effects of iron chelators combined with different traditional chemotherapy drugs to provide information on how to select drug combination for the treatment of leukemia in future clinical trials.
铁螯合剂不仅单独具有抗白血病作用，而且与传统化疗药物结合时还表现出协同的抗白血病效果。DFO 增加人骨髓性白血病细胞对阿霉素（DOX）和阿糖胞苷（Ara-C）的敏感性[102, 103]。DFO 与三氧化二砷（ATO）结合对 APL 的抗增殖和诱导凋亡具有协同效应[104]。DFO 可以与 L-天冬氨酸酶或地塞米松协同作用，降低白血病细胞的存活率，或与诱导 DNA 损伤的药物结合，增加 T-ALL 中的凋亡[9]。DFX 与 DNA 甲基转移酶抑制剂地西他滨（DAC）在白血病细胞系中的凋亡和细胞周期阻滞中显示出协同效应[88]。然而，有人提出 DFX 与阿糖胞苷结合产生协同效应，而与 DOX 结合则拮抗 AML 治疗中的抗白血病效果[89]。因此，需要进一步研究以确认铁螯合剂与不同传统化疗药物结合的效果，为未来临床试验中选择白血病治疗药物组合提供信息。

In addition to traditional iron chelating agents, some new iron chelators have been developed to improve the bioavailability and have also been identified to play anti-leukemia roles. For example, Triapine (3-AP) decreases the DNA synthetic capacity of circulating leukemia cells when administered in patients with refractory leukemia [105]. Salicylaldehyde isonicotinoyl hydrazine analogues (SIHA) is reported to dose-dependently induce apoptosis, cell cycle arrest and dissipation of the mitochondrial membrane potential in AML cells [90]. Additionally, the synthetic chelator di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) shows a significantly high affinity with Fe²⁺ and allows bound iron to participate in redox reactions and free radical formation [91]. Dp44mT has been demonstrated to inhibit the proliferation of leukemia cells with a G1/S phase arrest, accompanied by caspase-mediated induction of apoptosis [106]. Importantly, several agents used in clinical practice for other indications have also been discovered to function as iron chelators. Eltrombopag (EP), a small-molecule nonpeptide thrombopoietin receptor agonist, is reported to block the cell cycle in G1 phase and induce differentiation of leukemia cells through reducing free intracellular iron [15]. The antimicrobial ciclopirox olamine (CPX) has been identified to functionally chelate intracellular iron, which is important for its anti-leukemia cytotoxicity [107]. Further study demonstrates that iron chelation of CPX mediates inhibition of Wnt/β-catenin signaling and thus reduces expression of the Wnt target gene AXIN2 in leukemia cells of patients with AML [87].
除了传统的铁螯合剂外，还开发了一些新的铁螯合剂，以提高生物利用度，并且已被确认具有抗白血病作用。例如，三吡啶（3-AP）在难治性白血病患者中使用时，降低了循环白血病细胞的 DNA 合成能力。据报道，水杨醛异烟肼肼类似物（SIHA）能够剂量依赖性地诱导 AML 细胞凋亡、细胞周期阻滞和线粒体膜电位耗散。此外，合成螯合剂二-2-吡啶酮-4,4-二甲基-3-硫代半胱氨酸酮（Dp44mT）与 Fe ²⁺ 有显著高的亲和力，使结合的铁参与氧化还原反应和自由基形成。Dp44mT 已被证明能够通过 G1/S 期阻滞抑制白血病细胞的增殖，伴随着半胱氨酸介导的凋亡诱导。重要的是，一些用于其他适应症的临床实践药物也被发现具有铁螯合剂的功能。 厄洛替尼（EP）是一种小分子非肽类血小板生成素受体激动剂，据报道能够阻断细胞周期处于 G1 期，并通过减少细胞内游离铁而诱导白血病细胞分化[15]。抗微生物环丙沙星醇胺（CPX）已被确认能够功能性螯合细胞内铁，这对其抗白血病细胞毒性很重要[107]。进一步研究表明，CPX 的铁螯合作用介导了 Wnt/β-连环蛋白信号通路的抑制，从而减少 AML 患者白血病细胞中 Wnt 靶基因 AXIN2 的表达[87]。

Iron chelators have also shown promising anti-leukemia effects in human trials. A 73-year old male patient with relapsed, refractory acute monocytic leukemia achieved hematological and cytogenetic CR after application of DFX with no additional chemotherapy for 12 months [108]. Moreover, a 69-year-old male patient with relapsed AML had decreased peripheral blast counts accompanied by increased monocytic differentiation and partially reversed pancytopenia after DFO and vitamin D therapy [14]. In addition to AML, a six weeks old infant with ALL, who failed to attain remission with induction chemotherapy (IC), had peripheral blast counts significantly reduced accompanied by myelomonocytic differentiation after treatment with DFO and Ara-C [93]. In addition to these sporadic success stories, some clinical trials have also demonstrated the anti-leukemia effect of iron chelators (Table ​(Table2,2, refer to the website: https://clinicaltrials.gov/). A retrospective case control study has shown that DFO administration after allo-HSCT in patients with hematological malignancies reduced relapse incidence and improved disease-free survival [109]. A pilot clinical trial showed that DFO administration prior to allo-HSCT in patients with AL or MDS resulted in good outcomes, with no death or relapse, at a median follow-up of 20 months [110]. Similarly, a retrospective observational study of 339 patients demonstrates that the oral chelator DFX significantly reduces relapse mortality and restores graft-vs-leukemia effects (GVL) after allo-HSCT in AML, which is evidenced by high proportion of NK cells and suppressed regulatory T cells in peripheral blood [111]. Importantly, studies have shown that DFX, at concentrations equal to those clinically used or even at higher ones, has no harm to the viability of normal HSCs [85, 112]. DFX is even reported to have a beneficial effect on the hematopoietic recovery in patients after allo-HSCT [113]. A multicenter prospective cohort study (PCS) on the impact of DFX on relapse after allo-HSCT in patients with AML is recruiting ({"type":"clinical-trial","attrs":{"text":"NCT03659084","term_id":"NCT03659084"}}NCT03659084). Moreover, a randomized controlled trial (RCT) and a single group assignment (SGA) clinical trial have also been registered to clarify the effect of DFX on response rate of AL patients who are not fit for standard chemotherapy regimens ({"type":"clinical-trial","attrs":{"text":"NCT02413021","term_id":"NCT02413021"}}NCT02413021, {"type":"clinical-trial","attrs":{"text":"NCT02341495","term_id":"NCT02341495"}}NCT02341495). Those clinical trials will more strongly demonstrate the effect of DFX on the treatment of leukemia and post-transplant hematopoiesis.
铁螯合剂在人类试验中也显示出有希望的抗白血病效果。一名 73 岁的男性患者患有复发性、难治性的急性单核细胞白血病，在应用 DFX 治疗 12 个月后，在无需额外化疗的情况下达到了血液学和细胞遗传学的完全缓解[108]。此外，一名 69 岁的男性患者患有复发性 AML，在接受 DFO 和维生素 D 治疗后，外周爆发细胞计数减少，单核细胞分化增加，全血细胞减少症部分逆转[14]。除 AML 外，一名六周大的婴儿患有 ALL，在诱导化疗（IC）未能达到缓解的情况下，在接受 DFO 和 Ara-C 治疗后，外周爆发细胞计数显著减少，髓单核细胞分化增加[93]。除了这些零星的成功案例，一些临床试验也证明了铁螯合剂的抗白血病效果（表 2，请参阅网站：https://clinicaltrials.gov/）。一项回顾性病例对照研究显示，在造血干细胞移植后给予 DFO 治疗的患有血液恶性肿瘤的患者，可以降低复发率并改善无病生存率[109]。 一项初步临床试验显示，在 AL 或 MDS 患者中，DFO 在进行异基因造血干细胞移植（allo-HSCT）前的应用取得了良好的效果，在 20 个月的中位随访期内没有死亡或复发[110]。同样，一项回顾性观察性研究涉及 339 名患者表明，口服螯合剂 DFX 显著降低了 AML 患者在 allo-HSCT 后的复发死亡率，并恢复了移植物抗白血病效应（GVL），这一点在外周血中 NK 细胞比例高和调节性 T 细胞受抑制的证据中得到体现[111]。重要的是，研究表明，DFX 在临床使用的浓度甚至更高浓度下对正常造血干细胞的存活没有危害[85,112]。甚至有报道称 DFX 对 allo-HSCT 后患者的造血恢复有益[113]。一项关于 DFX 对 AML 患者进行 allo-HSCT 后复发影响的多中心前瞻性队列研究（PCS）正在招募中（NCT03659084）。 此外，还注册了一项随机对照试验（RCT）和一项单组分配（SGA）临床试验，以澄清对于不适合标准化疗方案的 AL 患者 DFX 对治疗反应率的影响（NCT02413021，NCT02341495）。这些临床试验将更有力地证明 DFX 对白血病治疗和移植后造血的影响。

There are also some clinical trials to study the safety and the anti-leukemia effect of new iron chelators. A dose-escalating phase I study (Ph-I) showed that 4 out of 31 patients (the majority with refractory AL) achieved a CR with a longer median survival after treatment with 3-AP and Ara-C [114]. Dose-limiting toxicities (DLTs) in the study were mucositis, neutropenic colitis, neuropathy and hyperbilirubinemia [114]. In another Ph-I study, similar DLTs were also observed and the toxicities of combination of 3-AP and Ara-C were similar to that of Ara-C singly at the same dose and schedule [115]. 3-AP followed by the adenosine analog fludarabine in adult patients with refractory AL showed controllable drug-related toxicities, including fever, methemoglobinemia and metabolic acidosis [116]. In a single group assignment (SGA) phase II trial in patients with secondary AML (sAML), chronic myeloid leukemia in blast phase (CML-BP) or MPD, 3-AP followed by fludarabine achieved an overall response (OR) rate of 49% (18/37), with a CR rate of 24% (9/37), which further demonstrates the promise of 3-AP to be clinically applied in the treatment of leukemia [117]. A phase I study of CPX showed that once-daily dosing was well tolerated in patients with relapsed or refractory AML and 2 patients had hematologic improvement (HI) while no patients achieved complete remission or partial remission (PR) [107]. The thrombopoietin receptor agonist EP has been approved for the treatment of patients with chronic immune thrombocytopenia and refractory severe aplastic anemia. The role of EP in patients with leukemia has been investigated in several clinical trials. A multicenter RCT reported that EP had an acceptable safety profile in patients with advanced MDS or sAML/MDS (secondary acute myeloid leukemia after myelodysplastic syndrome) and 2 (3%) patients achieved PR [118]. However, data from another multicenter RCT do not support combining EP with IC in patients with AML [119]. The addition of EP didn’t improve the disease response, but there was a shorter OS and a trend for more serious adverse events (AE) in the EP group [119]. Further clinical studies, conducted in larger patient populations with more rigorous design are ongoing to assess the safety and the use of EP in elderly patients with AML, except M3 or acute megakaryocytic leukemia (M7) ({"type":"clinical-trial","attrs":{"text":"NCT03603795","term_id":"NCT03603795"}}NCT03603795; {"type":"clinical-trial","attrs":{"text":"NCT02446145","term_id":"NCT02446145"}}NCT02446145).
还有一些临床试验研究新铁螯合剂的安全性和抗白血病效果。一项逐渐增加剂量的 I 期研究显示，在 31 名患者中有 4 名（大多数为难治性 AL 患者）在接受 3-AP 和 Ara-C 治疗后实现了 CR，并且存活中位数更长[114]。研究中的剂量限制性毒性（DLTs）包括粘膜炎、中性粒细胞减少性结肠炎、神经病和高胆红素血症[114]。在另一项 I 期研究中，也观察到类似的 DLTs，并且 3-AP 和 Ara-C 联合治疗的毒性与相同剂量和方案下的单独 Ara-C 的毒性相似[115]。3-AP 接着腺苷类似物氟达拉滨治疗难治性 AL 成人患者显示出可控制的药物相关毒性，包括发热、高甲红蛋白血症和代谢性酸中毒[116]。在一项单组分配（SGA）II 期试验中，对具有继发 AML（sAML）、慢性髓性白血病爆发期（CML-BP）或 MPD 的患者，3-AP 接着氟达拉滨实现了 49%的总体反应率（18/37），CR 率为 24%（9/37），进一步证明了 3-AP 在白血病治疗中具有临床应用前景[117]。 CPX 的 I 期研究显示，每日一次的剂量在复发或难治性 AML 患者中耐受良好，有 2 名患者出现血液学改善（HI），但没有患者达到完全缓解或部分缓解（PR）[107]。血小板生成素受体激动剂 EP 已获批用于治疗慢性免疫性血小板减少症和难治性重型再生障碍性贫血患者。EP 在白血病患者中的作用已在多项临床试验中进行了研究。一项多中心 RCT 报告称，在晚期 MDS 或 sAML/MDS（骨髓增生异常综合征后继发的继发性急性髓样白血病）患者中，EP 具有可接受的安全性，有 2 名（3%）患者达到 PR[118]。然而，另一项多中心 RCT 的数据不支持将 EP 与 IC 联合用于 AML 患者[119]。EP 的添加并未改善疾病反应，但 EP 组的 OS 较短，且存在更多严重不良事件（AE）的趋势[119]。 进一步的临床研究正在进行中，针对更大规模的患者群体，采用更严格的设计，以评估在老年 AML 患者中使用 EP 的安全性和有效性，除了 M3 或急性巨核细胞白血病（M7）（NCT03603795；NCT02446145）。

Current preclinical and clinical studies have confirmed the anti-leukemia effect of both traditional iron chelating agents and some new iron chelators. Notwithstanding the wide use of traditional iron chelating agents in the treatment of iron overload caused by repeated blood transfusions, the optimal doses for anti-leukemia treatment and their safety remain to be further studied. Systematic studies, which evaluate not only the toxicity but also the anti-leukemia effect of those new iron chelators in different subtypes of leukemia are also needed. More research will focus on the combination effect of iron chelators with different chemotherapeutic agents and the best scheme of their combination to bring to fruition their application in the clinical management of leukemia.
目前的临床前和临床研究已经证实了传统铁螯合剂和一些新型铁螯合剂的抗白血病效果。尽管传统铁螯合剂在治疗由反复输血引起的铁过载方面被广泛使用，但用于抗白血病治疗的最佳剂量及其安全性仍需进一步研究。有必要进行系统研究，评估这些新型铁螯合剂在不同亚型白血病中的毒性和抗白血病效果。更多的研究将集中在铁螯合剂与不同化疗药物的联合效应，以及它们的最佳联合方案，以实现它们在白血病临床管理中的应用。

Targeting iron metabolism related proteins
针对与铁代谢相关的蛋白质

In addition to iron chelators, depletion of intracellular iron can be achieved by targeting iron metabolism-related proteins. As a receptor that is critical for cellular iron uptake, TfR is an attractive target for depleting intracellular iron of leukemia cells. Both inhibitory and non-inhibitory anti-TfR monoclonal antibodies result in decreased Tf binding sites and subsequently inhibit Tf uptake, leading to growth inhibition in leukemia cells by iron deprivation [120]. A24, a monoclonal antibody directed against TfR1, competitively inhibits Tf binding to TfR1 and induces TfR1 endocytosis in lysosomal compartments where the receptor is degraded [121]. A24 inhibits proliferation and induces differentiation of leukemia cells by depleting the intracellular iron [14, 121, 122]. Combinations of two or more anti-TfR monoclonal antibodies can interact synergistically to play anti-leukemia effects, which correlates with their ability to block Tf-mediated iron uptake [123]. When combined with DFO, the monoclonal antibodies against TfR produce greater damage to iron uptake and a rapid depletion of iron pools [83, 124]. In addition to the deprivation of intracellular iron, JST-TfR09, an IgG monoclonal antibody to human TfR1, also plays an anti-leukemia effect through antibody-dependent cell-mediated cytotoxicity (ADCC) [125]. Though anti-TfR monoclonal antibodies show promising effects in the treatment of leukemia in those preclinical studies, there are some limitations for their clinical application. TfR is not specifically expressed in leukemia cells, it is also displayed by a wide variety of normal tissues. Depression of stem cell activity in bone marrow and altered distribution of red blood cell progenitors were observed in leukemia-bearing mice after receiving repeated injections of anti-TfR antibody [126]. A phase I trial of IgA monoclonal anti-TfR antibody 42/6 showed that 42/6 was generally well tolerated, although only transient, mixed antitumor responses were observed in patients with hematological malignancies [92]. Nevertheless, 42/6 also induced apparent down-regulation of TfR display by bone marrow cells, which could impair production of red blood cells [92]. These observations raised major concerns for the use of anti-TfR antibodies that maturing erythroid cells would be severely affected by anti-TfR antibodies, leading to anemia.
除了铁螯合剂外，通过靶向铁代谢相关蛋白可以实现细胞内铁的耗竭。作为细胞铁摄取的关键受体，转铁蛋白受体（TfR）是耗竭白血病细胞内铁的一个理想靶点。抑制和非抑制性的抗 TfR 单克隆抗体均导致 Tf 结合位点减少，随后抑制 Tf 摄取，通过铁剥夺导致白血病细胞生长受阻。针对 TfR1 的单克隆抗体 A24 竞争性地抑制 Tf 与 TfR1 的结合，并诱导 TfR1 在溶酶体区内吞作用，受体在此处被降解。A24 通过耗竭细胞内铁来抑制增殖并诱导白血病细胞分化。两种或更多抗 TfR 单克隆抗体的组合可以协同作用，发挥抗白血病效果，这与它们阻断 Tf 介导的铁摄取能力相关。与 DFO 结合使用时，针对 TfR 的单克隆抗体会对铁摄取造成更大破坏，并迅速耗竭铁库。 除了细胞内铁的缺乏外，JST-TfR09，一种针对人类 TfR1 的 IgG 单克隆抗体，还通过抗体依赖的细胞介导的细胞毒作用（ADCC）发挥抗白血病作用。尽管抗 TfR 单克隆抗体在白血病治疗中显示出有希望的效果，但它们在临床应用中存在一些限制。TfR 不仅在白血病细胞中表达，还在各种正常组织中显示。在接受抗 TfR 抗体重复注射后，观察到白血病小鼠骨髓干细胞活性下降和红细胞祖细胞分布改变。IgA 单克隆抗 TfR 抗体 42/6 的 I 期试验显示，42/6 总体上耐受性良好，尽管在患有血液恶性肿瘤的患者中观察到了暂时的混合抗肿瘤反应。然而，42/6 还明显导致骨髓细胞 TfR 表达下调，可能影响红细胞的产生。 这些观察引起了对抗 TfR 抗体使用的重大关注，即成熟的红细胞会受到抗 TfR 抗体的严重影响，导致贫血。

Taking the upregulation of the TfR on the leukemia cell surface into account, various TfR-targeted delivery systems consisting targeting ligands, carriers, and therapeutic agents have been developed. Not only to mention that TfR expression is significantly upregulated on leukemia cells, the binding of ligands to TfR also elicits very effective receptor-mediated endocytosis [127]. The ligands targeting TfR mainly include Tf, monoclonal antibodies, single-chain antibody fragment (scFv) and targeting peptides. Initially, these ligands are directly linked to some therapeutic agents. Conjugating artemisinin to a TfR targeting peptide shows anti-leukemia activity with a significantly improved leukemia cell selectivity [128]. With the development of technology, some carriers have been developed to link ligands and therapeutic agents for improving the efficacy and safety in therapeutic agent delivery, among which liposomes, dendritic molecules and nanoparticles have been widely used [129, 130]. A human serum albumin based nanomedicine, which is loaded with sorafenib and conjugated ligands for TfR specific delivery, can play enhanced anti-leukemia activity in drug resistant CML patient samples [130]. The sensitivity of leukemia cells to imatinib can also be enhanced by encapsulated with TfR targeted liposomes [131]. It has been reported that anti-TfR-coupled liposomes are more effective for intracellular drug delivery to T-ALL cells than anti-Tac conjugates, a monoclonal antibody directing against the interleukin-2 receptor [129]. Tf conjugated lipopolyplexes carrying G3139, an antisense oligonucleotide for B-cell lymphoma-2 (Bcl-2), induce remarkable pharmacological effect of Bcl-2 inhibition in AML cells and are more effective than free G3139 or non-targeted lipid nanoparticles [132]. Furthermore, iron chelator DFO can up-regulate TfR expression in leukemia cells, resulting in a further increase in anti-leukemia effect of TfR-targeted lipid nanoparticles carrying G3139 [133]. Because traditional chemotherapy drugs are difficult to pass the blood-brain barrier, leukemia cells sheltered in the central nervous system become the source of extramedullary recurrence of leukemia. Accumulating evidences have suggested that TfR-targeted delivery systems are promising strategies in enhancing the blood-brain barrier penetration [134]. More clinical trials of TfR-targeted delivery systems are expected to further improve their therapeutic potential.
考虑到白血病细胞表面 TfR 的上调，已经开发了各种 TfR 靶向递送系统，包括靶向配体、载体和治疗剂。不仅要提到 TfR 在白血病细胞上显著上调，配体与 TfR 的结合还引发非常有效的受体介导的内吞作用。靶向 TfR 的配体主要包括转铁蛋白（Tf）、单克隆抗体、单链抗体片段（scFv）和靶向肽。最初，这些配体直接与一些治疗剂相连。将青蒿素与 TfR 靶向肽结合显示出抗白血病活性，且白血病细胞选择性显著提高。随着技术的发展，一些载体已被开发用于连接配体和治疗剂，以提高治疗剂递送的疗效和安全性，其中脂质体、树突状分子和纳米粒子被广泛使用。 一种基于人血清白蛋白的纳米药物，其中装载了索拉非尼和结合配体以进行 TfR 特异性递送，可以在耐药 CML 患者样本中发挥增强的抗白血病活性[130]。白血病细胞对伊马替尼的敏感性也可以通过包埋 TfR 靶向脂质体来增强[131]。据报道，抗 TfR 偶联脂质体对 T-ALL 细胞的细胞内药物递送更有效，比抗白细胞介素-2 受体的单克隆抗体抗 Tac 结合物更有效[129]。携带 B 细胞淋巴瘤-2（Bcl-2）的反义寡核苷酸 G3139 的 Tf 结合脂质聚合物复合物在 AML 细胞中诱导出 Bcl-2 抑制的显著药理效应，比游离的 G3139 或非靶向脂质纳米粒子更有效[132]。此外，铁螯合剂 DFO 可以上调白血病细胞中的 TfR 表达，导致携带 G3139 的 TfR 靶向脂质纳米粒子的抗白血病效应进一步增加[133]。 由于传统化疗药物难以通过血脑屏障，藏匿在中枢神经系统中的白血病细胞成为白血病骨外复发的来源。越来越多的证据表明，以转铁蛋白受体为靶向的传递系统是增强血脑屏障穿透的有前途的策略。预计更多的转铁蛋白受体靶向传递系统的临床试验将进一步提高其治疗潜力。

In addition to TfR, other iron metabolism related proteins are also promising therapeutic targets. It has been suggested that STEAP can be targeted by specific CD4⁺T cells in non-small-cell lung carcinoma [135]. This provides a basis for STEAP to be used as an immunotherapy target for leukemia. Targeting ferritin results in dramatic anti-leukemia effect, suggesting that the pharmacological modulation of the storage protein of iron could be a new therapeutic target in leukemia [136]. Another consideration is that secreted ferritin can be absorbed by the TfR. Ferritin has also been commonly used for drug targeting because of its nanocage structure, which make it possible to deliver anti-leukemia drugs in the future [137]. Such naturally occurring structure is superior to synthetic ones due to its low toxicity and negligible immune responses. It’s reported that c-MYC contributes to drug resistance in AML and inhibition of c-MYC induces differentiation, apoptosis, and cell cycle arrest in leukemia cells [138, 139].
除了 TfR 之外，其他与铁代谢相关的蛋白质也是有前途的治疗靶点。有人提出 STEAP 可以被非小细胞肺癌中特定的 CD4 ⁺ T 细胞所靶向[135]。这为将 STEAP 用作白血病免疫治疗靶点提供了依据。靶向铁蛋白会产生显著的抗白血病效果，表明通过药理学调节铁的储存蛋白可能成为白血病的新治疗靶点[136]。另一个考虑是分泌的铁蛋白可以被 TfR 吸收。铁蛋白也因其纳米笼结构而常用于药物靶向，这使得未来可以将抗白血病药物传递给白血病患者[137]。这种天然存在的结构由于其低毒性和可忽略的免疫反应而优于合成结构。据报道，c-MYC 在 AML 中促进药物耐药性，抑制 c-MYC 可诱导白血病细胞的分化、凋亡和细胞周期阻滞[138, 139]。

It appears logic to apply approaches targeting iron-associated proteins as therapeutic measures due to their expression differences between normal cells and leukemia cells. However, monoclonal antibodies targeting iron-associated proteins may also damage normal cells, especially those with high iron demand, because iron-associated proteins are not specific in leukemia cells. To conquer the limitations associated with conventional chemotherapy, TfR or ferritin targeted drug delivery systems have been introduced. Furthermore, the combination of those drug delivery systems and molecular targeted drugs brings hope to increase drug efficacy and alleviate the toxicity caused by non-specificity of iron metabolism-related proteins. As prospective clinical data is still missing, approaches to targeting iron-associated proteins are still far from being usable for leukemia treatment.
逻辑上，针对与铁相关的蛋白质作为治疗措施是合理的，因为正常细胞和白血病细胞之间存在表达差异。然而，靶向与铁相关的蛋白质的单克隆抗体也可能损害正常细胞，特别是那些对铁需求高的细胞，因为与白血病细胞不同，铁相关的蛋白质并不具有特异性。为了克服传统化疗的局限性，已经引入了 TfR 或铁蛋白靶向药物传递系统。此外，这些药物传递系统与分子靶向药物的结合为增加药物疗效和减轻铁代谢相关蛋白质非特异性引起的毒性带来了希望。由于前瞻性临床数据仍然缺乏，针对与铁相关的蛋白质的方法仍然远未能用于白血病治疗。

Perturbing redox balance based on the high intracellular iron levels
基于高胞内铁水平扰乱氧化还原平衡

Ferroptosis and Ferritinophagy
铁死亡和铁蛋白自噬

Ferroptosis is a form of oxidative cell death, which is characterized by the production of ROS from accumulated iron and lipid peroxidation to trigger death [1, 140]. As iron is crucially involved in the formation of ROS, iron-catalyzed ROS production is primarily responsible for ferroptosis [1, 141]. Iron chelator DFO and heat shock protein β-1 prevent ferroptosis through reducing intracellular iron, but increasing intracellular iron promotes ferroptosis [140, 142, 143]. Ferritinophagy is an autophagic phenomenon that selectively degrades ferritin to release intracellular free iron and thus promotes ferroptosis [144]. Due to the importance of ROS in ferroptosis, antioxidants are critical regulators of ferroptosis. Glutathione peroxidase 4 (GPX4), which is the only antioxidant enzyme known to directly reduce lipid peroxides produced by ROS, plays a pivotal role in ferroptosis [145, 146]. It has been identified that regulation of GPX4 is a common mechanism shared by multiple ferroptosis inducers [145]. One class of ferroptosis inducers such as RSL3 inhibits GPX4 directly [145]. As glutathione (GSH) is a cofactor essential for GPX4 function, inhibition of GPX4 function by depleting GSH can also induce ferroptosis [146]. Because GSH production is limited by the availability of cystine/cysteine, another class of ferroptosis inducers (such as erastin, sorafenib) reduces GSH production through inhibiting cystine uptake by system X_c⁻, a cell surface cysteine-glutamate antiporter [140, 145, 147]. The well-known tumor suppressor p53 acts as a positive regulator of ferroptosis by inhibiting the expression of SLC7A11, a key component of system X_c⁻[148]. The mechanism of ferroptosis triggered by the multikinase inhibitor sorafenib includes not only inhibition of system X_c⁻, but also iron-dependent induction of oxidative stress [147, 149].
铁死亡是一种氧化性细胞死亡形式，其特点是通过从积累的铁和脂质过氧化物产生 ROS 来触发死亡[1, 140]。由于铁在 ROS 形成中起着至关重要的作用，铁催化的 ROS 产生主要是导致铁死亡的原因[1, 141]。铁螯合剂 DFO 和热休克蛋白β-1 通过减少细胞内铁来预防铁死亡，但增加细胞内铁会促进铁死亡[140, 142, 143]。铁蛋白自噬是一种选择性降解铁蛋白以释放细胞内游离铁并促进铁死亡的现象[144]。由于 ROS 在铁死亡中的重要性，抗氧化剂是铁死亡的关键调节因子。谷胱甘肽过氧化物酶 4（GPX4）是唯一已知能直接减少 ROS 产生的脂质过氧化物的抗氧化酶，它在铁死亡中发挥关键作用[145, 146]。已经确定，调节 GPX4 是多种铁死亡诱导剂共享的常见机制[145]。一类铁死亡诱导剂，如 RSL3，直接抑制 GPX4[145]。 由于谷胱甘肽（GSH）是 GPX4 功能所必需的辅因子，通过耗尽 GSH 抑制 GPX4 功能也可以诱导铁死亡[146]。由于 GSH 的产生受半胱氨酸/半胱氨酸的可用性限制，另一类铁死亡诱导剂（如厄拉斯汀、索拉非尼）通过抑制系统 X _c ⁻ 的半胱氨酸摄取，降低 GSH 的产生[140, 145, 147]。著名的肿瘤抑制基因 p53 通过抑制系统 X _c ⁻ 的关键组分 SLC7A11 的表达，作为铁死亡的正调节因子[148]。多激酶抑制剂索拉非尼引发的铁死亡机制不仅包括抑制系统 X _c ⁻ ，还包括铁依赖性氧化应激的诱导[147, 149]。

Recently, triggering ferroptosis based on the high intracellular iron levels has become a promising therapy to preferentially target leukemia cells (Fig. 3). The tumor suppressing function of ferroptosis has been identified in a wide range of malignancies, including fibrosarcoma, prostate carcinoma, osteosarcoma and so on [140, 145, 150]. Recent studies have indicated that RSL3 or Erastin can trigger death in leukemia cells and even enhance the sensitivity of leukemia cells to chemotherapeutic agents [151–153]. In turn, lipoxygenase inhibitors (such as Ferrostatin-1 and Baicalein) can protect ALL cells from ferroptosis [153]. The ferroptosis inducer sorafenib has been clinically approved for the treatment of FLT3-ITD mutated AML, whose mechanism may include induction of ferroptosis in AML cells [154, 155]. Artemisinin and its derivatives are widely used to treat multidrug-resistant malaria due that they owe the endoperoxide bridge and can induce ROS production in the presence of iron [156]. It has been recently suggested that dihydroartemisinin can induce ferroptosis in leukemia cells through ferritinophagy which increases the cellular LIP and thus promotes accumulation of ROS [157, 158]. The naturally occurring compound ardisiacrispin B and epunctanone have also been identified to induce ferroptosis in ALL cells [159, 160]. Therapies by inducing ferroptosis and ferritinophagy possess great potential in leukemia treatment. In the future, more and more research will focus on disturbing the redox balance to increase sensitivity of leukemia cells to chemotherapeutic agents.
最近，基于高胞内铁水平诱导细胞凋亡已成为一种有前途的治疗方法，可以优先靶向白血病细胞（图 3）。细胞凋亡的肿瘤抑制功能已在广泛的恶性肿瘤中得到确认，包括纤维肉瘤、前列腺癌、骨肉瘤等[140, 145, 150]。最近的研究表明，RSL3 或 Erastin 可以诱导白血病细胞死亡，甚至增强白血病细胞对化疗药物的敏感性[151–153]。反过来，脂氧合酶抑制剂（如 Ferrostatin-1 和 Baicalein）可以保护急性淋巴细胞白血病细胞免受细胞凋亡的影响[153]。细胞凋亡诱导剂索拉非尼已获临床批准用于治疗 FLT3-ITD 突变的 AML，其机制可能包括在 AML 细胞中诱导细胞凋亡[154, 155]。青蒿素及其衍生物被广泛用于治疗多药耐药性疟疾，因为它们具有内过氧化物桥，可以在铁存在的情况下诱导 ROS 产生[156]。 最近有人提出，二氢青蒿素可以通过铁蛋白自噬在白血病细胞中诱导铁死亡，从而增加细胞内游离铁离子，促进 ROS 的积累[157, 158]。天然存在的化合物 ardisiacrispin B 和 epunctanone 也被发现可以诱导所有细胞中的铁死亡[159, 160]。通过诱导铁死亡和铁蛋白自噬的治疗在白血病治疗中具有巨大潜力。未来，越来越多的研究将集中在扰乱氧化还原平衡，增加白血病细胞对化疗药物的敏感性。

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Fig. 3 图 3

Schematic model of ferroptosis in leukemia cells. Ferroptosis occurs as a result of iron-mediated oxidative stress and lipid peroxidation-mediated cytotoxicity. It could be due to elevated intracellular iron concentration or inhibition of GPX4 activity. Dihydroartemisinin induce ferroptosis by ferritinophagy and subsequent accumulation of ROS. RSL3 inhibits GPX4 directly, while erastin, sorafenib and p53 decrease GSH production by inhibiting cysteine transport. Lipoxygenase inhibitors (such as Ferrostatin-1 and Baicalein) suppress ferroptosis through inhibiting lipid peroxidation
白血病细胞中铁死亡的示意模型。铁死亡是由铁介导的氧化应激和脂质过氧化介导的细胞毒性所致。这可能是由于细胞内铁浓度升高或 GPX4 活性受抑制。二氢青蒿素通过铁蛋白吞噬和 ROS 的进一步积累诱导铁死亡。RSL3 直接抑制 GPX4，而厄拉斯汀、索拉非尼和 p53 通过抑制半胱氨酸转运降低 GSH 的产生。脂氧合酶抑制剂（如铁死灵-1 和黄芩素）通过抑制脂质过氧化来抑制铁死亡。

Not applicable. 不适用。