Polak Anna, Kiliszek Przemysław, Sewastianik Tomasz, Szydłowski Maciej, Jabłońska Ewa, Białopiotrowicz Emilia, Górniak Patryk, Markowicz Sergiusz, Nowak Eliza, Grygorowicz Monika A, Prochorec-Sobieszek Monika, Nowis Dominika, Gołąb Jakub, Giebel Sebastian, Lech-Marańda Ewa, Warzocha Krzysztof, Juszczyński Przemysław
Dept. of Experimental Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.
Dept. of Diagnostic Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.
PLoS One. 2016 May 19;11(5):e0155893. doi: 10.1371/journal.pone.0155893. eCollection 2016.
Resistance to glucocorticosteroids (GCs) is a major adverse prognostic factor in B-ALL, but the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular background of GC resistance in B-ALL and characterize the therapeutic potential of targeted intervention in these mechanisms. Using exploratory bioinformatic approaches, we found that resistant cells exhibited significantly higher expression of MEK/ERK (MAPK) pathway components. We found that GC-resistant ALL cell lines had markedly higher baseline activity of MEK and small-molecule MEK1/2 inhibitor selumetinib increased GCs-induced cell death. MEK inhibitor similarly increased in vitro dexamethasone activity in primary ALL blasts from 19 of 22 tested patients. To further confirm these observations, we overexpressed a constitutively active MEK mutant in GC-sensitive cells and found that forced MEK activity induced resistance to dexamethasone. Since recent studies highlight the role GC-induced autophagy upstream of apoptotic cell death, we assessed LC3 processing, MDC staining and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Unlike either drug alone, only their combination markedly increased these markers of autophagy. These changes were associated with decreased mTOR activity and blocked 4E-BP1 phosphorylation. In cells with silenced beclin-1 (BCN1), required for autophagosome formation, the synergy of DEX and SEL was markedly reduced. Taken together, we show that MEK inhibitor selumetinib enhances dexamethasone toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTOR signaling pathway and modulation of autophagy markers, likely reflecting induction of this process and required for cell death. Thus, our data demonstrate that modulation of MEK/ERK pathway is an attractive therapeutic strategy overcoming GC resistance in B-ALL patients.
对糖皮质激素（GCs）耐药是B淋巴细胞白血病（B-ALL）的一个主要不良预后因素，但导致GC耐药的分子机制尚未完全明确。在此，我们试图阐明B-ALL中GC耐药的分子背景，并描述针对这些机制进行靶向干预的治疗潜力。通过探索性生物信息学方法，我们发现耐药细胞中MEK/ERK（丝裂原活化蛋白激酶）信号通路成分的表达显著更高。我们发现GC耐药的ALL细胞系中MEK的基础活性明显更高，小分子MEK1/2抑制剂司美替尼可增加GC诱导的细胞死亡。MEK抑制剂同样增强了22例受试患者中19例原发性ALL原始细胞的体外地塞米松活性。为进一步证实这些观察结果，我们在GC敏感细胞中过表达组成型活性MEK突变体，发现强制激活MEK活性可诱导对地塞米松的耐药。由于最近的研究强调了GC诱导的自噬在凋亡性细胞死亡上游的作用，我们评估了用DEX、SEL或药物组合处理的细胞中的LC3加工、MDC染色和GFP-LC3重新定位。与单独使用任何一种药物不同，只有它们的组合显著增加了这些自噬标志物。这些变化与mTOR活性降低和4E-BP1磷酸化受阻有关。在自噬体形成所需的beclin-1（BCN1）沉默的细胞中，DEX和SEL的协同作用明显降低。综上所述，我们表明MEK抑制剂司美替尼可增强GC耐药的B-ALL细胞对地塞米松的毒性。这种相互作用的潜在机制涉及抑制mTOR信号通路和调节自噬标志物，可能反映了该过程的诱导，并且是细胞死亡所必需的。因此，我们的数据表明，调节MEK/ERK信号通路是克服B-ALL患者GC耐药的一种有吸引力的治疗策略。
