PO.ET03.04 · 实验与分子治疗
LEDGF/p75 通过调节 JAK/STAT 通路驱动 CML 的化疗耐药
LEDGF/p75 drives chemoresistance in CML via JAK/STAT pathway modulation
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
晶状体上皮衍生生长因子/p75（LEDGF/p75，PSIP1）是一种 H3K36me2/3 阅读器，与多种恶性肿瘤相关。在 MLL/KMT2A 融合复合体中，它将融合蛋白锚定于染色质。KMT2A 重排（KMT2A-r）发生于约 10% 的急性白血病患者中，由于复发和化疗耐药，与不良预后相关。我们实验室已证明 LEDGF/p75 对 KMT2A-r 白血病发生至关重要，但对造血过程可有可无。高 LEDGF/p75 水平已被证实与 AML 原始细胞、前列腺癌和乳腺癌的耐药相关。作为 KMT2A-r 白血病的依赖因子，LEDGF/p75 是一个有吸引力的治疗靶点，也是针对 LEDGF/p75 驱动的化疗耐药癌症的联合策略的潜在组成部分。然而，依赖 LEDGF/p75 的白血病亚型仍不明确。我们此前证明，LEDGF/p75 缺失可通过鞘氨醇-1 通路使 KMT2A-r AML 细胞对阿糖胞苷敏感。在此，我们旨在评估 LEDGF/p75 在多种白血病模型中在治疗耐药中的作用。使用表达 miRNA 的载体生成稳定的 LEDGF/p75 敲低（LEDGF/p75 KD）细胞系，并对十余种白血病细胞系进行了 LEDGF/p75 依赖性化疗耐药的筛选。在 KMT2A 野生型（WT）AML 细胞系 U-937 和 Kasumi-1 中，LEDGF/p75 缺失未改变阿糖胞苷敏感性，在 WT T-ALL 细胞系 Jurkat 和 SupT-1 中也观察到相同趋势。然而，在 KMT2A-r T-ALL 细胞系 Karpas-45 中，LEDGF/p75 KD 显著增强了细胞对阿糖胞苷的敏感性（IC 50 Karpas 对照/KD：76.32 ± 8.32 μM / 53.75 ± 12.46 μM）。出乎意料的是，SEM 细胞（B-ALL，KMT2A-AF4）中的 LEDGF/p75 KD 在阿糖胞苷处理下增加了增殖并减少了凋亡（IC 50 SEM 对照/KD：0.12 ± 0.02 μM / 0.53 ± 0.06 μM）。后者伴随较低水平的 caspase3。有趣的是，两种 WT KMT2A CML 细胞系 K-562 和 JUR-MK1 中的 LEDGF/p75 KD 在长春新碱处理下导致增殖显著降低（IC 50 K-562 对照/KD：2.61 ± 0.10 nM / 0.81 ± 0.09 nM；IC 50 JUR-MK1 对照/KD：3.93 ± 0.35 nM / 2.37 ± 0.30 nM），并伴随 caspase3 升高、凋亡增加。用 BCR-ABL 抑制剂伊马替尼（Imatinib）和普纳替尼（Ponatinib）处理 K-562 细胞，同样显示 LEDGF/p75 缺失后增殖减少。对 LEDGF/p75 KD 的 K-562 和 JUR-MK1 细胞进行 RNA-seq 分析显示，LEDGF/p75 缺失影响与造血细胞分化和免疫反应调节相关的通路。抗凋亡基因如 BCL-XL 和 BCL2A1 下调，JAK/STAT 通路的关键因子 STAT5 和 STAT2 也一同下调。Western blot 证实 LEDGF/p75 缺失的 CML 细胞中总 STAT5 及活化 STAT5 减少。综上所述，我们的结果表明 LEDGF/p75 在治疗耐药中的作用具有情境依赖性，并因白血病亚型而异。我们发现了 LEDGF/p75 一个此前未知的功能，即通过 JAK/STAT 通路介导 CML 细胞的耐药。
查看英文原文 English abstract
Lens Epithelium Derived Growth Factor/p75 (LEDGF/p75,PSIP1) is a H3K36me2/3 reader implicated in several malignancies. Within the MLL/KMT2A fusion complex, it tethers the fusion protein to chromatin. KMT2A rearrangement (KMT2A-r) occurs in ~10% of acute leukemia patients and is associated with poor prognosis due to relapse and chemoresistance. Our lab has shown that LEDGF/p75 is essential for KMT2A-r leukemogenesis but dispensable for hematopoiesis. High LEDGF/p75 levels have been linked to drug resistance in AML blasts, prostate and breast cancer. As a dependency factor in KMT2A-r leukemia, LEDGF/p75 is an attractive therapeutic target and a potential component of combinatorial strategies for LEDGF/p75 driven chemoresistance cancers. However, the leukemic subtypes dependent on LEDGF/p75 remains unclear. We previously showed that LEDGF/p75 depletion sensitizes KMT2A-r AML cells to cytarabine via the sphingosine-1 pathway. Here, we aimed to assess the role of LEDGF/p75 in therapy resistance across multiple leukemia models. Stable LEDGF/p75 knockdown (LEDGF/p75 KD) cell lines were generated using miRNA-expressing vectors, and more than ten leukemia cell lines were screened for LEDGF/p75 dependent chemoresistance. In KMT2A-wild-type (WT) AML cell lines U-937 and Kasumi-1, LEDGF/p75 depletion did not alter cytarabine sensitivity and the same trend was observed in WT T-ALL cell lines Jurkat and SupT-1. However, in the KMT2A-r T-ALL cell line, Karpas-45, LEDGF/p75 KD significantly sensitized the cells to cytarabine (IC 50 Karpas Mock/KD: 76.32 ± 8.32 μM / 53.75 ± 12.46 μM). Unexpectedly, LEDGF/p75 KD in SEM cells (B-ALL, KMT2A-AF4) increased proliferation and reduced apoptosis upon cytarabine treatment (IC 50 SEM Mock/KD: 0.12 ± 0.02 μM / 0.53 ± 0.06 μM). The latter was accompanied by a lower level of caspase3. Interestingly, LEDGF/p75 KD in two WT KMT2A CML cell lines, K-562 and JUR-MK1, led to a significant reduction in proliferation upon vincristine treatment (IC 50 K-562 Mock/KD: 2.61 ± 0.10 nM / 0.81 ± 0.09 nM and IC 50 JUR-MK1 Mock/KD: 3.93 ± 0.35 nM / 2.37 ± 0.30 nM) and increased apoptosis with elevated caspase3. Treatment of K-562 cells with BCR-ABL inhibitors Imatinib and Ponatinib also showed decreased proliferation upon LEDGF/p75 depletion. RNA-seq analysis of LEDGF/p75 KD K-562 and JUR-MK1 cells showed that LEDGF/p75 loss affects pathways associated with hematopoietic cell differentiation and immune response regulation. Anti apoptotic genes such as BCL-XL and BCL2A1 were downregulated, along with STAT5 and STAT2, key players in the JAK/STAT pathway. Western blotting confirmed reduced total and activated STAT5 in LEDGF/p75 depleted CML cells. Taken together, our result indicates that the role of LEDGF/p75 in therapy resistance is context dependent and varies across leukemic subtypes. We identify a previously unknown function of LEDGF/p75 in mediating drug resistance in CML cells acting through the JAK/STAT Pathway.
利益披露 Disclosure
T. Akele, None..
C. Iglesias-Herrero, None..
F. Christ, None..
Z. Debyser, None.