Detection of chemotherapy-resistant patient-derived acute lymphoblastic leukemia clones in murine xenografts using cellular barcodes

Clonal heterogeneity fuels leukemia evolution, therapeutic resistance, and relapse. Upfront detection of therapy-res istant leukemia clones at diagnosis may allow adaptation of treatment and prevention of relapse, but this is hampered by a paucity of methods to identify and trace single leukemia-propagating cells and their clonal offspring. Here, we tested methods of cellular barcoding analysis, to trace the in vivo competitive dynamics of hundreds of patient-derived leukemia clones upon chemotherapy-mediated selective pressure. We transplanted Nod/Scid/Il2Rg(-/-) (NSG) mice with barcoded patient-derived or SupB15 acute lymphoblastic leukemia (ALL) cells and assessed clonal responses to dexamethasone, methotrexate, and vincristine, longitudinally and across nine anatomic locations. We illustrate that chemotherapy reduces clonal diversity in a drug-dependent manner. At end-stage disease, methotrexate-treated patientderived xenografts had significantly fewer clones compared with placebo-treated mice (100 +/- 10 vs. 160 +/- 15 clones, p = 0.0005), while clonal complexity in vincristineand dexamethasone-treated xenografts was unaffected (115 +/- 33 and 150 +/- 7 clones, p = NS). Using tools developed to assess differential gene expression, we determined whether these clonal patterns resulted from random clonal drift or selection. We identified 5 clones that were reproducibly enriched in methotrexate-treated patient-derived xenografts, suggestive of pre-existent resistance. Finally, we found that chemotherapymediated selection resulted in a more asymmetric distribution of leukemia clones across anatomic sites. We found that cellular barcoding is a powerful method to trace the clonal dynamics of human patient-derived leukemia cells in response to chemotherapy. In the future, integration of cellular barcoding with single-cell sequencing technology may allow in-depth characterization of therapy-resistant leukemia clones and identify novel targets to prevent relapse. (C) 2020 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc

Correction:Quantitative distribution of patient-derived leukemia clones in murine xenografts revealed by cellular barcodes

An amendment to this paper has been published and can be accessed via a link at the top of the paper.</p

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Clonal analysis is important for many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle, they generally provide a lowresolution, biased, and incomplete assessment of clonality. To overcome those limitations, we labeled retroviral vectors with random sequence tags or &quot;barcodes.&quot; On integration, each vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each cell to be tracked over time. By coupling the barcoding method to a sequencing-based detection system, we could identify major and minor clones in 2 distinct cell culture systems in vitro and in a long-term transplantation setting. In addition, we demonstrate how clonal analysis can be complemented with transgene expression and integration site analysis. This cellular barcoding tool permits a simple, sensitive assessment of clonality and holds great promise for future gene therapy protocols in humans, and any other applications when clonal tracking is important. (Blood. 2010;115(13):2610-2618

Genetical genomics: spotlight on QTL hotspots

No abstract available

Expression quantitative trait loci are highly sensitive to cellular differentiation state

Blood cell development from multipotent hematopoietic stem cells to specialized blood cells is accompanied by drastic changes in gene expression for which the triggers remain mostly unknown. Genetical genomics is an approach linking natural genetic variation to gene expression variation, thereby allowing the identification of genomic loci containing gene expression modulators (eQTLs). In this paper, we used a genetical genomics approach to analyze gene expression across four developmentally close blood cell types collected from a large number of genetically different but related mouse strains. We found that, while a significant number of eQTLs (365) had a consistent “static” regulatory effect on gene expression, an even larger number were found to be very sensitive to cell stage. As many as 1,283 eQTLs exhibited a “dynamic” behavior across cell types. By looking more closely at these dynamic eQTLs, we show that the sensitivity of eQTLs to cell stage is largely associated with gene expression changes in target genes. These results stress the importance of studying gene expression variation in well-defined cell populations. Only such studies will be able to reveal the important differences in gene regulation between different ce

Combining transcriptional profiling and genetic linkage analysis to uncover gene networks operating in hematopoietic stem cells and their progeny

Stem cells are unique in that they possess both the capacity to self-renew and thereby maintain their original pool as well as the capacity to differentiate into mature cells. In the past number of years, transcriptional profiling of enriched stem cell populations has been extensively performed in an attempt to identify a universal stem cell gene expression signature. While stem-cell-specific transcripts were identified in each case, this approach has thus far been insufficient to identify a universal group of core “stemness” genes ultimately responsible for self-renewal and multipotency. Similarly, in the hematopoietic system, comparisons of transcriptional profiles between different hematopoietic cell stages have had limited success in revealing core genes ultimately responsible for the initiation of differentiation and lineage specification. Here, we propose that the combined use of transcriptional profiling and genetic linkage analysis, an approach called “genetical genomics”, can be a valuable tool to assist in the identification of genes and gene networks that specify “stemness” and cell fate decisions. We review past studies of hematopoietic cells that utilized transcriptional profiling and/or genetic linkage analysis, and discuss several potential future applications of genetical genomics

A verification protocol for the probe sequences of Affymetrix genome arrays reveals high probe accuracy for studies in mouse, human and rat

BACKGROUND: The Affymetrix GeneChip technology uses multiple probes per gene to measure its expression level. Individual probe signals can vary widely, which hampers proper interpretation. This variation can be caused by probes that do not properly match their target gene or that match multiple genes. To determine the accuracy of Affymetrix arrays, we developed an extensive verification protocol, for mouse arrays incorporating the NCBI RefSeq, NCBI UniGene Unique, NIA Mouse Gene Index, and UCSC mouse genome databases. RESULTS: Applying this protocol to Affymetrix Mouse Genome arrays (the earlier U74Av2 and the newer 430 2.0 array), the number of sequence-verified probes with perfect matches was no less than 85% and 95%, respectively; and for 74% and 85% of the probe sets all probes were sequence verified. The latter percentages increased to 80% and 94% after discarding one or two unverifiable probes per probe set, and even further to 84% and 97% when, in addition, allowing for one or two mismatches between probe and target gene. Similar results were obtained for other mouse arrays, as well as for human and rat arrays. Based on these data, refined chip definition files for all arrays are provided online. Researchers can choose the version appropriate for their study to (re)analyze expression data. CONCLUSION: The accuracy of Affymetrix probe sequences is higher than previously reported, particularly on newer arrays. Yet, refined probe set definitions have clear effects on the detection of differentially expressed genes. We demonstrate that the interpretation of the results of Affymetrix arrays is improved when the new chip definition files are used

CBX7 Induces Self-Renewal of Human Normal and Malignant Hematopoietic Stem and Progenitor Cells by Canonical and Non-canonical Interactions

In this study, we demonstrate that, among all five CBX Polycomb proteins, only CBX7 possesses the ability to control self-renewal of human hematopoietic stem and progenitor cells (HSPCs). Xenotransplantation of CBX7-overexpressing HSPCs resulted in increased multi-lineage long-term engraftment and myelopoiesis. Gene expression and chromatin analyses revealed perturbations in genes involved in differentiation, DNA and chromatin maintenance, and cell cycle control. CBX7 is upregulated in acute myeloid leukemia (AML), and its genetic or pharmacological repression in AML cells inhibited proliferation and induced differentiation. Mass spectrometry analysis revealed several non-histone protein interactions between CBX7 and the H3K9 methyltransferases SETDB1, EHMT1, and EHMT2. These CBX7- binding proteins possess a trimethylated lysine peptide motif highly similar to the canonical CBX7 target H3K27me3. Depletion of SETDB1 in AML cells phenocopied repression of CBX7. We identify CBX7 as an important regulator of self-renewal and uncover non-canonical crosstalk between distinct pathways, revealing therapeutic opportunities for leukemia

Asymmetry in skeletal distribution of mouse hematopoietic stem cell clones and their equilibration by mobilizing cytokines

Hematopoietic stem cells (HSCs) are able to migrate through the blood stream and engraft bone marrow (BM) niches. These features are key factors for successful stem cell transplantations that are used in cancer patients and in gene therapy protocols. It is unknown to what extent transplanted HSCs distribute throughout different anatomical niches in the BM and whether this changes with age. Here we determine the degree of hematopoietic migration at a clonal level by transplanting individual young and aged mouse HSCs labeled with barcoded viral vector, followed by assessing the skeletal distribution of hundreds of HSC clones. We detected highly skewed representation of individual clones in different bones at least 11 mo after transplantation. Importantly, a single challenge with the clinically relevant mobilizing agent granulocyte colony-stimulating factor (G-CSF) caused rapid redistribution of HSCs across the skeletal compartments. Old and young HSC clones showed a similar level of migratory behavior. Clonal make- up of blood of secondary recipients recapitulates the barcode composition of HSCs in the bone of origin. These data demonstrate a previously unanticipated high skeletal disequilibrium of the clonal composition of HSC pool long- term after transplantation. Our findings have important implications for experimental and clinical and stem cell transplantation protocols