Identifying non-genetic determinants of malignant clonal fitness at single cell resolution [chemo scRNAseq]

All cancers emerge following a period of clonal selection and subsequent clonal expansion. Whilst the evolutionary principles imparted by genetic intra-tumour heterogeneity (ITH) are becoming increasingly clear, little is known about the non-genetic mechanisms that contribute to ITH and malignant clonal fitness. Using SPLINTR, a synthetic expressed barcoding strategy, in three clinically relevant mouse models of acute myeloid leukemia (AML) we find that malignant clonal dominance is a stable and heritable property that is facilitated by the repression of antigen presentation and the increased expression of Slpi, a leukocyte protease inhibitor that has not previously been characterised in AML. Increased transcriptional heterogeneity is a consistent feature enabling clonal fitness in diverse tissue / immune microenvironments and in the context of clonal competition between genetically distinct clones within a uniform microenvironment. Compared to extramedullary sites, leukemia initiating capacity is most enriched in malignant cells resident within the bone marrow microenvironment and leukemia stem cells (LSC), like normal haematopoietic stem cells, display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. Finally, we demonstrate that clonal output does not dictate sensitivity to chemotherapy and both high and low output LSC clones retain marked cellular plasticity enabling them to survive potent therapeutic challenge and persist as minimal residual disease. Together these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness which may inform future therapeutic strategies. Single-cell RNA-seq of MLL-AF9 leukemia cells with SPLINTR barcodes transplanted into Ptprca mice

所有恶性肿瘤均起源于一段时期的克隆选择及后续克隆扩增。尽管由遗传型肿瘤内异质性（genetic intra-tumour heterogeneity, ITH）所驱动的进化规律已逐渐明晰，但目前对于促成ITH与恶性克隆适应性的非遗传机制仍鲜有研究。本研究采用合成表达条形码技术SPLINTR，在三种临床相关的急性髓系白血病（acute myeloid leukemia, AML）小鼠模型中开展研究，结果显示：恶性克隆优势是一种稳定且可遗传的特性，其形成可通过抑制抗原呈递过程，以及上调Slpi的表达来实现——Slpi是一种此前未在AML中被报道过的白细胞蛋白酶抑制剂。在多样化的组织/免疫微环境中，以及在均一微环境内遗传异质性克隆间的克隆竞争情境下，转录异质性升高均是促进克隆适应性的共性特征。相较于髓外部位，骨髓微环境中的恶性细胞富集了更强的白血病起始能力；且白血病干细胞（leukemia stem cells, LSC）与正常造血干细胞类似，表现出可遗传的克隆固有特性：其克隆产出率存在高低差异，该差异会影响整体肿瘤负荷。最后，本研究证实克隆产出率并非决定肿瘤细胞化疗敏感性的因素，高、低产出率的LSC克隆均保留了显著的细胞可塑性，使其能够在强效治疗打击下存活，并以微小残留病的形式持续存在。综上，本研究为支撑恶性克隆适应性的非遗传转录过程提供了基础性见解，或可为未来的治疗策略提供参考。本研究数据集为：携带SPLINTR条形码的MLL-AF9白血病细胞移植至Ptprca小鼠后的单细胞RNA测序数据。