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Seq-ing the Truth: Unraveling Normal and Malignant Myelopoiesis Employing Single-cell Technologies

December 5, 2020

“The real voyage of discovery consists, not in seeking new landscapes, but in having new eyes.” –La Prisonnière, Marcel Proust

My, what powerful eyes we now possess. As will be discussed by the heavyweights convening for today’s Scientific Program Joint Session, “Single Cell Analysis of Hematopoietic Development and Clonal Complexity of Malignant Hematopoiesis” (live Q&A Saturday, December 5, at 9:30 a.m. Pacific Time), the application of single-cell technologies enabled by advanced computational biology methods is disrupting the orthodox view of hematopoiesis as an orderly, hierarchal process and exposing striking heterogeneity within classically defined hematopoietic stem and progenitor cell (HSPC) populations. As aberrant cell fate decisions lie at the heart of disease, these insights are anything but esoteric.

Dr. Vijay Sankaran, a physician-scientist focused on pediatric red cell disorders and marrow failure, makes the compelling case that using single-cell genomics can provide insights into the pathways governing blood development that are translatable to the clinic using two vignettes. The first illustrates the utility of tracking somatic mutations in mitochondrial DNA (mtDNA) as a natural genetic barcode for lineage tracing in both normal and malignant hematopoiesis. Compared to the nuclear genome, mtDNA presents several advantages for clonal tracing at scale. At only 16.6 kb, the mitochondrial genome is more cost effective to sequence. Due to the combination of high copy numbers and more primitive DNA replication and repair systems, the somatic mutation rate is one to two logs higher. Finally, mtDNA is more accessible to transcription machinery, which makes employing assay for transposase-accessible chromatin using sequencing (ATAC-seq) and single-cell RNA sequencing (scRNA-seq) in parallel an attractive strategy. When these methods are performed in unison, they provide correlative information about chromatin state and gene expression. (At the single-cell level no less!) His second vignette will demonstrate how recognizing the cell-to-cell heterogeneity of gene expression within HSPC populations can deepen our knowledge of fundamental transcription circuits, which when perturbed, can result in rare bone marrow failure disorders.

Hematopoiesis occurs within anatomical bone marrow microenvironments whose diversity rivals that of the Galápagos Islands. Dr. Timm Schroeder, a leader of the Cell Systems Dynamics group, discusses how deep, dynamic imaging of the bone marrow can capture cell cycle–specific interactions between HSPCs and different marrow niches. “Quantitative multicolor 3-D imaging of very large tissue volumes is crucial to understand their organization in health and disease,” Dr. Schroeder said. “With our novel approaches for imaging complex bone marrow, we observed unexpected distributions of hematopoietic stem and progenitor cells and their cellular and molecular niches.” He also discusses how age-related remodeling of the marrow microenvironment promotes myeloid skewing, pointing to the microenvironment as an alternative therapeutic target in the treatment of high-risk clonal hematopoiesis (CH) and myeloid neoplasms.

Dr. Margaret Goodell, a doyenne of hematopoietic stem cell research and member of the National Academy of Medicine, will shift the viewpoint from the single-cell to the subclonal level. CH refers to HSPC clones often harboring specific, disruptive, and recurrent genetic variants in individuals without a demonstrable hematologic malignancy. Although CH confers a cumulative risk of subsequent hematologic malignancy, that risk is mutation- and context-specific. These clones tend to expand and thus become detectable with aging, providing mechanistic insight into the inextricable link between chronic inflammation and age-related diseases. Dr. Goodell shows how the timing and type of mutation likely have large influences on the evolution of CH and related downstream events. She shares emerging data on how the mutations that drive CH often occur much earlier than anticipated. “I am excited about work from our lab and others that shows that some mutations that appear in mid-life as CH can happen long before birth, adding literally months to the time that clones are expanding,” said Dr. Goodell. Using DNMT3A mutations as a prototype, she also discusses how functional profiling reveals important differences of prognostic and therapeutic relevance between mutant subtypes.

When Robert Hooke illustrated what he observed using the novel technology of light microscopy in the 1665 book Micrographia, it challenged what scientists thought they knew about the universe. Similarly, the development of novel molecular biomarkers and technologies (e.g., multicolor flow cytometry, real-time quantitative polymerase chain reaction (RT-qPCR), digital polymerase chain reaction, and next-generation sequencing)  allow us to detect measurable residual disease (MRD) with high sensitivity in a variety of hematologic malignancies. This in turn challenges us to refine response criteria beyond morphologic complete response (CR), for both clinical decision-making and assessing drug efficacy in clinical trials. Except for a few disease subsets driven by leukemogenic fusion transcripts that can be longitudinally tracked with RT-qPCR, as in chronic-phase chronic myeloid leukemia or acute promyelocytic leukemia, this has not until recently been the standard of care. Dr. Konstanze Döhner, founder of one of the leading reference laboratories for molecular diagnostics in acute myeloid leukemia (AML) and a member of the European LeukemiaNet MRD working party, provides a framework for understanding the movement toward CR MRD being the “ultimate” goal in leukemic therapy, as reflected in recent clinical guidelines1 and a U.S. Food and Drug Administration guidance document.2 She discusses suitable markers and timepoints for MRD assessment and proposes how molecular and/or multicolor flow cytometry MRD should be integrated into prospective clinical trials. Dr. Döhner highlights AML-specific considerations in MRD assessment, made particularly difficult given its molecular heterogeneity and the confounding impact of CH and germline mutations. Lastly, she addresses some of the barriers to widespread integration of MRD testing.

While studying the fundamentals of hematopoiesis was previously thought to be a “mature” field, Dr. Sankaran’s enthusiasm captures the renaissance afoot: “This is an exciting and important session,” he said. “The field is moving rapidly, and this session will discuss the enormous progress that is happening.”

Handy Abbreviations and Definitions
ATAC-seq Assay for Transposase-Accessible Chromatin using sequencing — a technique to assess chromatin accessibility and transcription factor occupancy that can be performed with even small amounts of genetic material. Works by leveraging the hyperactivity of a mutant Tn5 transposase
Barcodes Unique identifying genetic sequences, either manufactured or naturally occurring, incorporated into the DNA/RNA during sequencing studies used to attribute/track cell of origin.
Clone Cells that descend from a common ancestor. Does not automatically imply a malignant nature.
LIC Leukemia-initiating cell – a source of semantic confusion and debate. Similar terms include “leukemic stem cell,” “cell of origin,” and “pre-leukemic clone.” Hematopoietic stem cells or cells with stem cell like properties that are distinct from bulk leukemic cells that can engender therapeutic resistance and thus relapse.
Lineage A cell’s ancestral line
Multi-omics The integration of data sets from multiple “’omes” (genome, transcriptome, epigenome, proteome, etc.) to provide a multidimensional biological analysis, including at the single-cell level
scRNA-seq Single cell RNA sequencing — A variety of methods can be used to separate the bulk sample into individual cells (e.g., flow-activated cell sorting, microfluidics, etc.). The transcriptome of these cells is then sequenced with next-generation sequencing. Unique molecular barcodes can be incorporated during this sequencing process such that the transcripts produced can be traced back to the cells of origin.
  1. Döhner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129:424-447.
  2. Office of Communications, Division of Drug Information Center for Drug Evaluation and Research. Hematologic malignancies: Regulatory considerations for use of minimal residual disease in development of drug and biological products for treatment. Guidance for industry. U.S. Food and Drug Administration. January 2020. Last accessed Nov 25, 2020.
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