Gerges Steve, Rohde Katharina, Fulda Simone
Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstr. 3a, 60528 Frankfurt, Germany.
Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstr. 3a, 60528 Frankfurt, Germany; German Cancer Consortium (DKTK), Heidelberg, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany.
Cancer Lett. 2016 May 28;375(1):127-132. doi: 10.1016/j.canlet.2016.02.040. Epub 2016 Mar 2.
Treatment resistance in acute lymphoblastic leukemia (ALL) is often caused by defects in programmed cell death, e.g. by overexpression of Inhibitor of Apoptosis (IAP) proteins. Here, we report that small-molecule Smac mimetics (i.e. BV6, LCL161, birinapant) that neutralize x-linked IAP (XIAP), cellular IAP (cIAP)1 and cIAP2 cooperate with demethylating agents (i.e. 5-azacytidine (5AC) or 5-aza-2'-deoxycytidine (DAC)) to induce cell death in ALL cells. Molecular studies reveal that induction of cell death is preceded by BV6-mediated depletion of cIAP1 protein and involves tumor necrosis factor (TNF)α autocrine/paracrine signaling, since the TNFα-blocking antibody Enbrel significantly reduces BV6/5AC-induced cell death. While BV6/5AC cotreatment induces caspase-3 activation, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) only partly rescues ALL cells from BV6/5AC-induced cell death. This indicates that BV6/5AC cotreatment engages non-apoptotic cell death upon caspase inhibition. Indeed, genetic silencing of key components of necroptosis such as Receptor-Interacting Protein (RIP)3 or mixed lineage kinase domain-like (MLKL) in parallel with administration of zVAD.fmk provides a significantly better protection against BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. Similarly, concomitant administration of pharmacological inhibitors of necroptosis (i.e. necrostatin-1s, GSK'872, dabrafenib, NSA) together with zVAD.fmk is superior in rescuing cells from BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. These findings demonstrate that in ALL cells BV6/5AC-induced cell death is mediated via both apoptotic and necroptotic pathways. Importantly, BV6/5AC cotreatment triggers necroptosis in ALL cells that are resistant to apoptosis due to caspase inhibition. This opens new perspectives to overcome apoptosis resistance with important implications for the development of new treatment strategies for ALL.
急性淋巴细胞白血病（ALL）中的治疗耐药性通常由程序性细胞死亡缺陷引起，例如凋亡抑制蛋白（IAP）的过表达。在此，我们报告称，可中和X连锁IAP（XIAP）、细胞IAP（cIAP）1和cIAP2的小分子Smac模拟物（即BV6、LCL161、birinapant）与去甲基化剂（即5-氮杂胞苷（5AC）或5-氮杂-2'-脱氧胞苷（DAC））协同作用，可诱导ALL细胞死亡。分子研究表明，细胞死亡的诱导在BV6介导的cIAP1蛋白耗竭之前发生，并且涉及肿瘤坏死因子（TNF）α自分泌/旁分泌信号传导，因为TNFα阻断抗体恩利可显著降低BV6/5AC诱导的细胞死亡。虽然BV6/5AC联合治疗可诱导半胱天冬酶-3激活，但广谱半胱天冬酶抑制剂N-苄氧羰基-Val-Ala-Asp-氟甲基酮（zVAD.fmk）只能部分挽救ALL细胞免于BV6/5AC诱导的细胞死亡。这表明BV6/5AC联合治疗在半胱天冬酶抑制后会引发非凋亡性细胞死亡。事实上，与单独使用zVAD.fmk相比，在给予zVAD.fmk的同时对坏死性凋亡关键成分（如受体相互作用蛋白（RIP）3或混合谱系激酶结构域样蛋白（MLKL））进行基因沉默，能显著更好地保护细胞免受BV6/5AC诱导的细胞死亡。同样，与单独使用zVAD.fmk相比，将坏死性凋亡的药理学抑制剂（即坏死素-1s、GSK'872、达拉非尼、NSA）与zVAD.fmk联合使用，在挽救细胞免于BV6/5AC诱导的细胞死亡方面更具优势。这些发现表明，在ALL细胞中，BV6/5AC诱导的细胞死亡是通过凋亡和坏死性凋亡途径介导的。重要的是，BV6/5AC联合治疗可在因半胱天冬酶抑制而对凋亡具有抗性的ALL细胞中引发坏死性凋亡。这为克服凋亡抗性开辟了新的前景，对ALL新治疗策略的开发具有重要意义。
