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  • Although previous studies begin to recognize the potential c

    2018-10-24

    Although previous studies begin to recognize the potential complex genetic architecture of leukemia, unambiguous longitudinal detection of leukemic clones remains difficult to achieve. Ideally, clones should be prospectively defined by unique labeling of renin inhibitor before tumor initiation, and detection of different clones should be performed at high resolution. A recently described method, which would potentially accomplish this, is the marking of cells by the introduction of a unique, heritable mark that can be detected in its offspring experimentally (Bystrykh et al., 2012; Gerrits et al., 2010; Naik et al., 2014; Schepers et al., 2008). These methods rely on the viral integration of a random “barcode” sequence of fixed length in the genome of target cells. After transplantation of barcoded cells, their offspring can be traced by quantifying the abundance of unique barcodes using deep sequencing. Previously, we have shown that overexpression of the Polycomb PRC1 member Cbx7 in bone marrow cells causes leukemia (Klauke et al., 2013). While typically a single oncogene causes one specific tumor type, the epigenetic modifier CBX7 causes a wide spectrum of leukemias, including T-ALL, erythroid, and undifferentiated leukemias. Since only long-term hematopoietic stems cells (LT-HSCs), short-term HSCs (ST-HSCs), and multipotent progenitors (MPPs), but not lineage-restricted progenitors are responsive to Cbx7 overexpression (Klauke et al., 2013), the different types of leukemias are not likely to depend on the cell of origin in which Cbx7 is overexpressed. Rather, the phenotypic variation seems to be an inherent virtue of CBX7. In the present paper, we have generated a mouse model in which overexpression of Cbx7 serves as the initial leukemic “hit” and every pre-LSC is uniquely labeled by a barcode. We show how our approach allows for the identification of LSC-derived clones in the transplanted primary and secondary recipients. We prospectively describe clonal dynamics in mice that succumb to leukemia and highlight the complexity of clonal evolution.
    Results
    Discussion In early tumor models, malignant cells were thought to descend from a single common ancestor whose offspring sequentially acquired multiple mutations or other genetic abnormalities in time (Knudson, 1971; Nordling, 1953; Nowell, 1976). This implied that all cells in a full-blown tumor were genetically and functionally homogenous. However, emerging evidence suggests that leukemias (and other tumors for that matter) are comprised of a multitude of different subclones resulting in heterogeneity (Anderson et al., 2011; Landau et al., 2013; Magrangeas et al., 2013; Mullighan et al., 2008; Notta et al., 2011; Sanders and Valk, 2013; Schuh et al., 2012; Welch et al., 2012). This concept has thus far only been partially confirmed since previous experiments did not uniquely mark individual cells to allow prospective identification of leukemic clones. In the current study, we used a barcoding tool combined with overexpression of Cbx7, an epigenetic modifier that we recently found to regulate self-renewal of HSCs (Klauke et al., 2013), to discern patterns of clonality in highly variable leukemic subtypes. We delineate leukemic heterogeneity with high resolution and document the coexistence of (sometimes quiescent) LSCs with different leukemic properties in a single tumor. Additional genetic or epigenetic abnormalities that occur after the first leukemia-predisposing event (high Cbx7 expression in primitive cells) within a subset of cells from one leukemic clone (cells that carry the same barcode) are likely to be the driving force behind the leukemia dynamics that we observed in our study. Since CBX7 is an epigenetic modifier, it is tempting to speculate that overexpression of Cbx7 generates an altered chromatin structure that is susceptible to stochastic epigenetic or genetic secondary hits, which ultimately shape the disease phenotype. Identification of secondary genetic abnormalities would require additional assessment of the mutational status of subclones, which is beyond the scope of the present study.