The histone-methyltransferase DOT1L cooperates with LSD1 to control cell division in blast-phase MPN [RNA-seq]

Persistence of JAK2-mutated clones that may undergo clonal evolution and malignant transformation remains a challenge in myeloproliferative neoplasms (MPN), Novel therapeutic approaches to attenuate clonal evolution and progression to blast-phase are therefore urgently needed. LSD1 (KDM1A) inhibitors reduce symptoms and clonal burden in MPN, but whether these compounds may be effective in advanced disease stages remained so far elusive. Using a chromatin-focused CRISPR-Cas9 screen, we identified the histone methyltransferase DOT1L as a synthetic lethal target under pharmacologic LSD1 inhibition. DOT1L knockout impaired cellular fitness, reduced proliferation, and prolonged survival in xenografts. Furthermore, genetic inactivation of DOT1L increased LSD1 inhibitor sensitivity up to 100-fold resulting in cell cycle arrest and apoptosis induction in TP53 mutant blast-phase MPN. Mechanistically, we have identified a novel, non-canonical function of DOT1L which co-occupied LSD1-bound enhancers and contributed to the repression of transcriptional programs independent of its enzymatic activity. DOT1L loss cooperated with LSD1 inhibitors to activate tumor suppressive programs, while pharmacologic inhibition of DOT1Ls catalytic activity failed to elicit comparable effects. These findings indicate that leveraging DOT1L targeting via protein degradation or RNA interference, rather than conventional enzymatic inhibition, could enhance the therapeutic efficacy of LSD1 inhibitors in blast-phase MPN. 1. RNAseq of Human Erythroleukemia cells (HEL cell line) with a CRISPR-Cas9-mediated knockout of DOT1L (or non-targeting control) treated with the LSD1-inhibitor Bomedemstat for 24h and 5d. 2. RNAseq of MPN-BP primary patient sample with a shRNA-mediated knockdown of DOT1L (or scrambled control) cultured in methylcellulose for 7 days under puromycine selection for shRNA-expressing cells.

在骨髓增殖性肿瘤（myeloproliferative neoplasms, MPN）中，可发生克隆进化与恶性转化的JAK2突变克隆持续存在仍是临床难题，因此亟需开发可减缓克隆进化并阻止疾病进展至急变期的新型治疗策略。LSD1（KDM1A）抑制剂可缓解MPN患者的症状并降低克隆负荷，但此类药物在疾病晚期阶段是否有效迄今仍不明确。本研究通过染色质靶向CRISPR-Cas9筛选，鉴定出在药物性LSD1抑制条件下的合成致死靶点——组蛋白甲基转移酶DOT1L。在异种移植模型中，DOT1L敲除可损害细胞适应性、抑制细胞增殖，并延长荷瘤小鼠的存活时间。此外，DOT1L的遗传失活可使LSD1抑制剂的敏感性提升最高达100倍，在TP53突变的急变期MPN细胞中诱导细胞周期阻滞与凋亡。机制层面，我们发现了DOT1L的一种全新非经典功能：它可与结合LSD1的增强子区域共占据，并在不依赖其酶活性的情况下参与转录程序的抑制。DOT1L缺失可与LSD1抑制剂协同激活肿瘤抑制程序，而直接抑制DOT1L催化活性的药物处理则无法产生相当的效应。本研究结果提示，相较于传统的酶活性抑制策略，通过蛋白质降解或RNA干扰靶向DOT1L，可提升LSD1抑制剂在急变期MPN中的治疗效果。
1. 对人红白血病细胞（HEL细胞系）进行CRISPR-Cas9介导的DOT1L敲除（或非靶向对照敲除），并用LSD1抑制剂Bomedemstat处理24小时与5天后的转录组测序（RNAseq）。
2. 对MPN急变期（MPN-BP）原代患者样本进行shRNA介导的DOT1L敲低（或乱序对照敲低），将细胞在含嘌呤霉素（用于筛选表达shRNA的细胞）的甲基纤维素培养基中培养7天后的转录组测序（RNAseq）。