Dec 14, 2021

Dec. 14, 2021 Australian researchers have developed a new single-cell expressed barcoding strategy termed SPLINTR (Single-cell Profiling and LINeage TRacing), to investigate the key basic nongenetic transcriptional processes underlying malignant clonal fitness in mouse models of leukemia. In an accompanying editorial, researchers from the New York Genome Center described those processes as "heritable but nongenetic," and wrote that the findings "point to the need for a more holistic view of cancer evolution. This would help to define how genetic and nongenetic mechanisms jointly contribute to cellular phenotype, which is the substrate for selection." The study's findings may guide future cancer therapeutic strategies by casting new light on clonal fitness, which "describes the property of a cancer clone to contribute substantially to the overall tumor burden," said study leader Mark Dawson, head of the Cancer Biology and Therapeutic Program at Peter MacCallum Cancer Centre in Melbourne. To date, "most cancer research has been done at a population level, whereby thousands of cancer clones are homogenized for analysis as a single sample, but here we have investigated cancer cell behavior at a single clone resolution." Notably, the multicenter study's research team discovered that cancer stem cells (CSCs) do not contribute equally to the overall tumour burden. Rather, the malignant clonal yield was shown to be an intrinsic heritable property that dictates adaptive processes and sensitivity to chemotherapy. "To the best of our knowledge, this is the first time it has been experimentally proven that all CSCs do not contribute equally to the overall tumor burden," said Dawson. This is a clinically significant finding, "because that intrinsic clonal output shapes response to therapy," he told BioWorld Science. All cancers emerge following clonal selection and expansion. Although the evolutionary principles due to genetic intratumor heterogeneity (ITH) are becoming increasingly clear, little is known about nongenetic mechanisms underlying ITH and cancer clonal fitness. Acute myeloid leukemia (AML) provides a rare example whereby a single genetic abnormality initiates and drives an aggressive human malignancy. These mutationally inert and genetically stable cancers are ideal models for studying how nongenetic processes contribute to clonal dominance. In their new study reported in the December 9, 2021, edition of Nature, the authors used SPLINTR to investigate the influence of transcriptional programs on future cell fate at the single-cell level. To enable concurrent and/or sequential tracking of multiple cell populations, they also constructed three high-diversity libraries, each with a distinct barcode structure coupled to different light-emitting fluorochrome markers. "The three libraries comprised different barcode structures, allowing us to follow genetically diverse clones in the same assay and to follow clones serially by re-barcoding them prior to serial re-transplantation in mice," explained Dawson. SPLINTR barcodes are readily captured by single-cell RNA sequencing (scRNA-seq) and are identified at comparable frequencies to matched DNA barcode sequencing. Using SPLINTR, the researchers traced isogenic clones in three clinically relevant mouse models of AML. They found that malignant clonal dominance is a cell-intrinsic and heritable property facilitated by repression of antigen presentation and increased expression of the gene secretory leukocyte peptidase inhibitor (Slpi). The research team then genetically validated Slpi as being a new AML regulator, "by knocking out Slpi and showing that this reduced cancer cell fitness," said Dawson. This is an important discovery, "as it means that these clonal properties dictate the behavior of the cancer cell." Moreover, increased transcriptional heterogeneity was found to enable clonal fitness in diverse tissues and immune microenvironments, and within the context of clonal competition between genetically distinct clones. "We assessed clonal fitness in the spleen and bone marrow," noted Dawson. "Cancer cells have genetic as well as nongenetic heterogeneity, so we assessed how genetically diverse clones compete with each other." Similar to hematopoietic stem cells, leukemia stem cells (LSCs) were revealed to display heritable clone-intrinsic properties of high and low clonal output that contributed to the overall tumor mass. Importantly, the researchers further demonstrated that sensitivity to chemotherapy was dictated by LSC clonal output, or "the percentage of the overall tumor burden derived from a single clone," noted Dawson. Moreover, high and low output clones were shown to adapt differently to therapeutic pressure and emerge from minimal residual disease with increased LSC expression, meaning that there is not just one avenue to resistance. Taken together, these findings provide basic insights into nongenetic transcriptional processes underlying malignant clonal fitness and may guide future therapies. "Unless we understand the cell intrinsic properties of clonal fitness, we won't be able to design better therapies to counteract them," Dawson said. Looking forward, said the clinical hematologist, "we now need to study other cancers, particularly those with greater genetic diversity, in order to better understand how different mutations influence cancer cell behavior."