Dimethyl Itaconate selectively targets chronic lymphocytic leukemia cells. Dimethyl Itaconate selectively targets chronic lymphocytic leukemia cells

Chronic Lymphocytic Leukemia (CLL) is strictly dependent on the complex interplay between the intrinsic features of the leukemic cells and microenvironmental stimulations including inflammatory stimuli by Toll Like Receptors (TLR) which protect CLL cells from drug induced apoptosis by upregulating NFKBIZ, an atypical co-transcription factor. To face the challenge of targeting transcription factors, we exploited the capacity of Dimethyl Itaconate (DI), an electrophilic synthetic derivative of Itaconate, to block NFKBIZ acting as anti-inflammatory agent. Primary CLL cells isolated from the peripheral blood of patients, leukemic splenocytes isolated from TCL1 transgenic mice, and circulating leukocytes isolated from healthy donors were treated in vitro with increasing concentrations of DI either alone or in combination with the TLR ligands. DI abrogated the induction of NFKBIZ as well as its target genes IL6 and IL10. Remarkably, DI reduced metabolic activation and cell viability of leukemic cells even if added after a robust TLR pre-stimulation; in contrast, no toxic effect was evident in normal leukocytes including T- and B-lymphocytes. DI induced apoptosis of malignant cells by reducing BCL-XL and MCL1 levels while inducing PARP cleavage. RNA sequencing highlighted that DI abrogated the TLR9-mediated upregulation of transcripts related to the metabolism of rRNAs, interferon and cytokine signaling. Notably, in addition to the expected electrophilic stress signature observed after DI treatment, novel pathways emerged including the downregulation of distinct MHC class II complex genes. In conclusion, DI not only abrogated the pro-inflammatory effects of TLR stimulation but also targeted a specific vulnerability in CLL cells. Overall design: CLL primary cells were isolated from peripheral blood of 6 patients with CLL. Cells were plated, treated/stimulated and RNAseq was performed. In each replicate series, one sample was pre-treated with DI overnight followed by 4h of CpG-ODN2006 (human specific) stimulation, one sample was only treated with DI for 20h, one was only stimulated 4h with CpG-ODN2006 and an unstimulated and untreated sample was used as control.

慢性淋巴细胞性白血病（Chronic Lymphocytic Leukemia, CLL）的发生严格依赖于白血病细胞内在特性与微环境刺激之间的复杂互作，其中包括通过Toll样受体（Toll Like Receptors, TLR）介导的炎症刺激——这类刺激可通过上调非典型共转录因子NFKBIZ，使CLL细胞免受药物诱导的细胞凋亡。为破解靶向转录因子的研究难题，我们利用了衣康酸二甲酯（Dimethyl Itaconate, DI）的抗炎活性：其作为衣康酸的亲电性合成衍生物，可通过阻断NFKBIZ发挥作用。我们体外处理了三类实验样本：从CLL患者外周血中分离的原代CLL细胞、从TCL1转基因小鼠体内分离的白血病脾细胞，以及健康供者外周血来源的循环白细胞；处理方案设置为梯度浓度的DI单独给药，或DI与TLR配体联合给药。实验结果显示，DI可有效阻断NFKBIZ及其靶基因IL6、IL10的诱导表达。值得注意的是，即便在强烈的TLR预刺激后再施加DI处理，其仍可抑制白血病细胞的代谢激活并降低细胞存活率；与之形成鲜明对比的是，DI对包括T、B淋巴细胞在内的正常白细胞无明显毒性。DI可通过下调恶性细胞中BCL-XL与MCL1的表达水平、诱导PARP裂解，诱发细胞凋亡。RNA测序分析结果表明，DI可阻断TLR9介导的、与核糖体RNA代谢、干扰素及细胞因子信号通路相关的转录本上调。此外，除了DI处理后预期出现的亲电性应激特征之外，我们还发现了全新的调控通路，包括不同主要组织相容性复合体II类（MHC class II）复合物基因的表达下调。综上，DI不仅可阻断TLR刺激介导的促炎效应，还可靶向CLL细胞的特定脆弱位点。实验整体设计：从6例CLL患者的外周血中分离原代CLL细胞，接种培养后进行处理或刺激，并开展RNA测序。每一组重复实验包含4组样本：一组经DI预处理过夜后，用CpG-ODN2006（人源特异性）刺激4小时；一组仅用DI处理20小时；一组仅用CpG-ODN2006刺激4小时；另有一组未受刺激且未处理的样本作为空白对照。