Nature reveals non-genetic determinants of malignant clone fitness at single-cell resolution

Article | The emergence of all cancers in Sister Xian is bound to go through a period of clonal selection and subsequent clonal expansion
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Tumors are composed of subpopulations (subclones) of cells that can be differentiated on the basis of various features that affect their phenotype, including genetic alterations (eg, mutations, gene amplifications, gene deletions, or chromosomal translocations)
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In most cancers, intratumoral genetic heterogeneity has been demonstrated and can serve as the basis for clonal evolution
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The basic biological mechanisms underlying the evolution of cancer clones are similar to those in the evolution of asexually reproducing species: replication, heritable variation, genetic drift, selection, and environmental change
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Despite the increasingly clear evolutionary principles conferred by intratumoral genetic heterogeneity, little is currently known about the non-genetic mechanisms responsible for intratumoral heterogeneity and the fitness of malignant clones [1,2]
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Lineage tracing studies in the developmental context have shown that genetic properties inherent in cells can guide cell fate potential and clonal dynamics [3,4], however, the analysis of how non-genetic diversity guides clonal behavior in malignant settings remains to be established
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Recently, the team of Mark A.
Dawson from the Peter McCarran Cancer Center in Australia published an article titled Non-genetic determinants of malignant clonal fitness at single-cell resolution online in Nature, using single-cell analysis and lineage tracing (SPLINTR) Tracking of isogenic clones in three clinically relevant mouse models of acute myeloid leukemia revealed that malignant clonal dominance is a cell-inherent heritable property that can be induced by inhibiting antigen presentation and increasing the secretory leukocyte peptidase inhibitor gene ( Slpi) expression to promote
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Increased transcriptional heterogeneity is a feature that allows clones to adapt to different tissue and immune microenvironments, as well as clonal competition among genetically distinct clones
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This provides fundamental insights into the non-genetic transcriptional processes that underlie malignant clonal fitness and provides important information for future therapeutic strategies
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Acute leukemia is driven by an oncogenic fusion involving a translocation of the mixed lineage leukemia (KMT2A, also known as MLL1) gene, making it a rare example of a human cancer—a single gene abnormality responsible for this aggressive and lethal malignancy necessary and sufficient conditions
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It is conceivable that this mutationally inert and genetically stable cancer is an ideal model to study the non-genetic mechanisms that lead to clonal dominance
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To assess the impact of transcriptional programs on future cell fate, we developed an expression barcoding strategy called SPLINTR to construct three highly diverse libraries, each with a different barcode structure and associated with a different fluorophore.
Dye coupled
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Meanwhile, SPLINTR barcodes can be easily captured by 3′ single-cell RNA sequencing (scRNA-seq) methods and can be identified with comparable frequency to matching DNA barcode sequencing
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Because these barcodes exist primarily as single-cell copies, they can accurately describe clonal fate and gene expression in a time-resolved manner
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The researchers first transformed mouse hematopoietic stem cells (HSCs) and granulocyte-monocyte progenitor cells (GMP) with the MLL-AF9 oncogene (a fusion of KMT2A and MLL T3), then labeled them with different SPLINTR libraries, and cloned them.
When expanded and transplanted into mice, both cell-derived clones were found to cause disease in all mice, suggesting that clonal fitness is a cell-intrinsic property independent of the source cell
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At the same time, transforming a heterogeneous mix of HSCs and hematopoietic progenitor cells (HPCs) with the MLL-AF9 oncogene to mimic MLL leukemia also observed the same leukemia-initiating clones in multiple mice
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Notably, the leukemia-initiating clones that predominated in mice were not necessarily the clones that expanded the fastest in vitro prior to transplantation, suggesting that properties conferring clonal fitness in vivo go beyond proliferative potential and are comparable to those conferred in vitro.
The characteristics of adaptability are different
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The use of SPLINTR to examine transcriptional programs present in leukemia-initiating clones immediately prior to transplantation found that cells of origin and leukemia stem cell (LSC) characteristics are not necessarily linked and that cell-inherent clonal fitness cannot simply be attributed to a unified transcriptional state
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To understand how different secondary mutations affect clonal fitness, we generated three genetically distinct MLL-AF9 leukemias from wild-type or isogenic C57BL/6 mice, two of which harbor Flt3 (Flt3ITD) or Endogenous knock-in activating mutation of Kras (KrasG12D)
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To examine the properties of these different AML genotypes, each cell line was barcoded with a unique SPLINTR barcode library and transplanted into mice individually (Figure 1)
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Analysis of the data suggests that clonal dominance is a cell-intrinsic property of disease-causing clones regardless of oncogenic genotype
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Evaluation of the transcriptome of the progenitor of the pathogenic clone immediately prior to transplantation found that the dominant clones of all three genotypes exhibited marked downregulation of immune-related gene signaling signatures, particularly by antigen processing of major histocompatibility complex class I and presentation pathways, and that these transcriptional signatures also appear in fulminant disease in vivo, suggesting that heritable reproduction of transcriptional programs has inherent clonal advantage in all genotypes
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The researchers then transplanted the three genotypes of MLL-AF9 cells into mice in equal or skewed proportions (more accurately reflecting the malignant subclonal structure found in the clinic) in an attempt to clone between genetically diverse clones These principles of clonal dominance are studied in the context of competition
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The results showed that the transcripts of competition-specific and isogenic pathogenic clones clustered roughly in a similar location, but the former exhibited greater transcriptional heterogeneity, a feature associated with adaptive potential in the context of environmental stress
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Analysis of scRNA-seq data revealed significant differences in the transcriptional burst kinetics of expressed genes related to chromatin remodeling and mRNA processing among competition-specific clones
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The findings suggest that intrinsic clonal dominance is influenced by competition among genetically diverse clones, in which case transcriptional heterogeneity may contribute to the fitness of malignant clones
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To investigate the role of different immune and tissue microenvironments on clonal dominance, we transplanted identical SPLINTR-coded leukemia cells into either immunodeficient mice (NSG) or immunocompetent mice (Ptprca)
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The experimental results showed that leukemia-initiating clones shared a microenvironment in which all transcriptions were clustered together and exhibited characteristics of dominant clones, including an abundant LSC program and overexpression of Slpi
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Figure 1 All in vivo experiments in this study identified pre-existing cell-intrinsic transcriptional states enriched in cancer-initiating clones.
To test whether these transcriptional states were heritable, we isolated from bone marrow and spleen of diseased mice SPLINTR GFP-tagged MLL-AF9 KrasG12D cells were generated, re-encoded with different SPLINTR libraries, and then mixed in equal proportions and re-transplanted into NSG or Ptprca mice (Figure 2)
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Experimental results find that transcriptional/epigenetic signatures inherent in cancer-initiating cells are best preserved in the native microenvironment of cancer-originating cells, while confirming non-inherited inheritance of clonal fitness traits - despite in vitro and in vivo exposure In different microenvironments, some cells still stably inherit transcriptional states necessary for cancer initiation and growth
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At the same time, the researchers found that LSC clones could also be classified as high, medium, or low yielding, and that this cell-intrinsic behavior was highly consistent across recipient mice
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Using a sequential combinatorial SPLINTR barcoding strategy (Figure 2) to track clonal behavior, we found that high-yielding clones retained this property in subsequent transplantations, while low- and medium-yielding clones never transitioned to high-yielding clones
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The diseased mice were further treated with intensive chemotherapy similar to human AML treatment, and a series of minimal residual disease (MRD) samples were collected for a detailed temporal analysis of the same clonal population with/without treatment stress.
Three main patterns are shown: (1) high/mid-yielding clones whose clonal yields are only briefly reduced by treatment; (2) high/mid-yielding clones that alter clonal behavior to adopt a low-yielding state; Under these circumstances, only low-yielding clones will transition to a high-yielding state, resulting in the vast majority of MRD burden after treatment
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This suggests that LSC clone yield determines sensitivity to chemotherapy, and although high-yielding and low-yielding clones adapt differently to treatment stress, they appear synergistically in MRD with increased expression of the LSC program
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Figure 2 In summary, this study used SPLINTR to track thousands of malignant clones simultaneously in a time-resolved manner to identify principles of clonal fitness beyond the cancer genome
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demonstrated that clonal dominance, regardless of the cell of origin, is a cell-inherent heritable state that cannot be attributed to a unified transcriptional program, and instead, increased transcriptional diversity is a consistent feature of clonal fitness, especially in more challenging microenvironments
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It also demonstrated that cancer stem cells do not play the same role in the overall tumor burden, and that malignant clonal yield is a cell-inherent and heritable property that determines adaptive processes and sensitivity to chemotherapy
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This underscores the urgent need to address both genetic and non-genetic heterogeneity, while underscoring the ambition to personalize cancer medicine
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Original link: https://doi.
org/10.
1038/s41586-021-04206-7 Publisher: Eleven References 1.
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DiSNE movie visualization and assessment of clonal kinetics reveal multiple trajectories of dendritic cell development.
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