The histone demethylase KDM5C functions as a tumor suppressor in AML by repression of bivalently marked immature genes (ChIP-Seq)

Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). However, epigenetic dysregulation in AML extends beyond recurrently mutated factors, and only little is known about the potential drivers in this context. Identification and characterization of novel epigenetic drivers affecting AML biology will not only improve our basic understanding of AML, but can also uncover novel options for therapeutic intervention. To uncover novel epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of Cebpa mutant AML. This led to the identification of the Histone 3 Lysine 4 (H3K4) demethylase, KDM5C, as a novel tumor suppressor in AML. KDM5C potentially functions as a transcriptional repressor via its demethylase activity at promoters, and dysregulation could therefore have widespread consequences. Here, we found that reduced Kdm5c/KDM5C expression is associated with accelerated growth in both human and murine AML cell lines. In vivo, Kdm5c knockdown in a Cebpa mutant AML mouse model resulted in a more aggressive, immature and short-latency phenotype. Mechanistically, we show that knockdown of Kdm5c increased H3K4me3 globally. This translated into the up-regulation of a group of bivalently marked immature genes, resulting in a de-differentiation phenotype which could be reversed by modulating levels of pro-differentiation factors. Finally, we demonstrated that low levels of KDM5C were associated with a decrease in long-term disease-free survival, specifically in female patients. This emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML. ChIP-seq of H3K4me1, H3K4me3 and H3K27ac in shControl versus shKdm5c Lp30 cells. ChIP-seqs were performed in biological duplicates.

表观遗传调控因子（epigenetic regulators）在包括急性髓系白血病（acute myeloid leukemia，AML）在内的血液系统恶性肿瘤中频发突变。然而，AML中的表观遗传失调并不局限于复发性突变调控因子，目前对此背景下的潜在驱动因子仍知之甚少。鉴定并表征影响AML生物学特性的新型表观遗传驱动因子，不仅可增进我们对AML的基础认知，还能为治疗干预提供新的潜在方向。为揭示AML中的新型表观遗传调控因子，我们在Cebpa突变型AML模型中开展了体内短发夹RNA（short hairpin RNA，shRNA）筛选实验，最终鉴定出组蛋白3赖氨酸4（Histone 3 Lysine 4，H3K4）去甲基化酶KDM5C作为AML的新型肿瘤抑制因子。KDM5C可能通过其在启动子区域的去甲基化酶活性发挥转录抑制功能，因此其失调可能产生广泛的生物学效应。本研究发现，Kdm5c/KDM5C表达降低与人类及小鼠AML细胞系的增殖加速相关。在体内，对Cebpa突变型AML小鼠模型中的Kdm5c进行敲低，会导致疾病表型更具侵袭性、未成熟程度更高且潜伏期更短。从机制层面而言，我们证实Kdm5c敲低会使全基因组范围内的H3K4me3水平升高。这一变化会促使一组二价标记的未成熟基因上调，进而产生去分化表型，该表型可通过调节促分化因子的水平得以逆转。最后，我们发现KDM5C低表达与长期无病生存期缩短相关，这一关联在女性患者中尤为显著。该结果凸显了本研究发现的临床相关性，并将KDM5C鉴定为AML中一种新型的女性偏向性肿瘤抑制因子。本研究对对照组短发夹RNA（shControl）与Kdm5c敲低（shKdm5c）的Lp30细胞开展了H3K4me1、H3K4me3及H3K27ac的染色质免疫共沉淀测序（ChIP-seq）实验，所有测序均设置生物学重复。