Venetoclax (VEN) inhibits the prosurvival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL but with eventual loss of efficacy. A spectrum of subclonal genetic changes associated with VEN resistance has now been described. To fully understand clinical resistance to VEN, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired VEN resistance. These appear to be multilayered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the prosurvival family members. This includes previously described mutations in BCL2 and amplification of the MCL1 gene but is heterogeneous across and within individual patient leukemias. Changes in the proapoptotic genes are notably uncommon, except for single cases with subclonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF-κB (nuclear factor kappa B) activation, which persisted in circulating cells during VEN therapy. We discovered that MCL1 could be a direct transcriptional target of NF-κB. Both the switch to alternative prosurvival factors and NF-κB activation largely dissipate following VEN discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade VEN-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent VEN resistance and provide a specific biological justification for the strategy of VEN discontinuation once a maximal response is achieved rather than maintaining long-term selective pressure with the drug.

中文翻译：

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单细胞多组学揭示了多层适应的规模，使 CLL 在 Venetoclax 治疗期间复发。

Venetoclax (VEN) 抑制促生存蛋白 BCL2 以诱导细胞凋亡，是慢性淋巴细胞白血病 (CLL) 的标准疗法，可为复发性 CLL 提供较高的完全缓解率和延长的无进展生存期，但最终会失去疗效。现在已经描述了与 VEN 抗性相关的一系列亚克隆遗传变化。为了充分了解 VEN 的临床耐药性，我们结合了单细胞短读长和长读长 RNA 测序，揭示了支撑获得性 VEN 耐药性的遗传和表观遗传变化的规模，此前未被认识到。这些似乎是多层的。一层包含凋亡调节因子 BCL2 家族的变化，尤其是促存活家族成员。这包括之前描述的 BCL2 突变和 MCL1 基因扩增，但在个体白血病患者之间和内部存在异质性。除了 BAX 或 NOXA 亚克隆缺失的个别病例外，促凋亡基因的变化非常罕见。更突出的是普遍的 MCL1 基因上调。这是由 NF-κB（核因子 kappa B）激活的覆盖层驱动的，该激活在 VEN 治疗期间持续存在于循环细胞中。我们发现MCL1可能是NF-κB的直接转录靶标。停止使用 VEN 后，转向替代性促生存因子和 NF-κB 激活都会很大程度上消失。我们的研究揭示了 CLL 细胞逃避 VEN 诱导的细胞凋亡能力的可塑性程度。重要的是，这些发现指出了规避 VEN 耐药性的新方法，并为达到最大反应后停止 VEN 的策略提供了具体的生物学依据，而不是维持药物的长期选择压力。