Analysis of synthetic cellular barcodes in the genome and transcriptome with BARtab and bartools.

Cellular barcoding is a lineage-tracing methodology that couples heritable synthetic barcodes to high-throughput sequencing, enabling the accurate tracing of cell lineages across a range of biological contexts. Recent studies have extended these methods by incorporating lineage information into single-cell or spatial transcriptomics readouts. Leveraging the rich biological information within these datasets requires dedicated computational tools for dataset pre-processing and analysis. Here, we present BARtab, a portable and scalable Nextflow pipeline, and bartools, an open-source R package, designed to provide an integrated end-to-end cellular barcoding analysis toolkit. BARtab and bartools contain methods to simplify the extraction, quality control, analysis, and visualization of lineage barcodes from population-level, single-cell, and spatial transcriptomics experiments. We showcase the utility of our integrated BARtab and bartools workflow via the analysis of exemplar bulk, single-cell, and spatial transcriptomics experiments containing cellular barcoding information.

Inhibition of the CtBP complex and FBXO11 enhances MHC class II expression and anti-cancer immune responses.

There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.

Non-genetic determinants of malignant clonal fitness at single-cell resolution.

All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.

Two new RHD alleles with deletions spanning multiple exons.

BACKGROUND: The most common large-deletion RHD allele (RHD*01N.01) includes the entire coding sequence, intervening regions and untranslated regions. The rest of large-deletion RHD alleles reported to-date consist of single-exon deletions, such as RHD*01N.67 which includes exon 1. MATERIALS AND METHODS: Samples from two donors with RhD-negative serology yielded unclear or inconclusive results when subject to confirmatory testing on RHD genotyping arrays. To determine their RHD genotypes, genomic DNA was analyzed with a combination of allele-specific PCR, long-range PCR, Sanger sequencing, and next-generation sequencing assays. RESULTS: Allele-specific PCR failed to detect products for RHD exons 1 to 3 in one sample and RHD exons 1 to 5 in the other. A quantitative next-generation sequencing assay confirmed deletion of exons 1 to 3 and 1 to 5 respectively, and detected the absence of an RHD gene in trans in both samples. Long-range PCR and Sanger sequencing enabled identification of the breakpoints for both alleles. Both deletions start within the 5' Rhesus box (upstream of the identity region for the 1-to-3 deletion, downstream of it for the 1-to-5 deletion), and end within introns. CONCLUSIONS: Resolution of unclear or inconclusive results from targeted genotyping arrays often leads to the discovery of new alleles. The 5' Rhesus box may be a hot spot for genetic recombination events, such as the large deletions described in this report.

RH genotyping by nonspecific quantitative next-generation sequencing.

BACKGROUND: Conventional sequencing uses gene-specific primers to determine the location of RH variants and permits a qualitative assessment of zygosity. Whole-genome and whole-exome sequencing determine the genetic location of variants and enable a quantitative assessment of zygosity. Nonspecific sequencing uses RH-consensus primers to detect variants and sequencing-read ratios to quantify their copy number. STUDY DESIGN AND METHODS: Two hundred seventy eight samples with diverse genotypes were analyzed by next-generation sequencing with RH- consensus primers. Custom-developed data analysis software was used to detect individual variants and infer the RH genotype. The method was evaluated for its quantitative nature, its ability to discriminate similar genotypes, its accuracy to detect variants, and its accuracy to assign them to RHD or RHCE. RESULTS: As a measure of balanced amplification of RHD and RHCE sequences, observed ratio medians deviate from expected ratios by 3% or less of the ratio range. As a measure of discriminatory power, contiguous RHCE / [RHD + RHCE] ratio averages are separated by 4 or more standard deviations of the mean. Variants are detected with a sensitivity and specificity greater than 99%, and variants at consensus positions are correctly assigned to RHD vs RHCE with a sensitivity greater than 72% and a specificity greater than 99%. The method is successful in the identification of genotypes with large conversion events and in the detection of copy number variation. CONCLUSION: Nonspecific sequencing of homologous gene sets combines detection and quantification of genetic variation in a single assay. Evidence is provided for the quantitative nature of the method, its sensitivity and specificity, and its ability to identify complex RH genotypes.

Epigenetic therapies in acute myeloid leukemia: where to from here?

A hallmark of acute myeloid leukemia (AML) is epigenetic dysregulation, which is initiated by recurrent translocations and/or mutations in transcription factors and chromatin regulators. This manifests as a block in myeloid differentiation and an increase in malignant self-renewal. These common features of AML have led to widespread optimism that epigenetic therapies would dramatically change the natural history of this disease. Although preclinical studies with these drugs fueled this optimism, results from early clinical trials have offered a more sobering message. Here, we provide an overview of epigenetic therapies that are currently approved by therapeutic regulatory authorities across the world and those undergoing early-phase clinical trials. We also discuss the conceptual and molecular factors that may explain some of the disparity between the bench and bedside, as well as emerging avenues for combining the current generation of epigenetic therapies with other classes of agents and the development of novel epigenetic therapies. With further research and development of this exciting class of drugs, we may finally be able to dramatically improve outcomes for patients afflicted with this aggressive and often incurable malignancy.

Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.

Effect on gene expression of three allelic variants in GATA motifs of ABO, RHD, and RHCE regulatory elements.

BACKGROUND: Only a few genetic variants have been reported in regulatory elements of blood group genes. Most of them affect GATA motifs, binding sites for the GATA-1 transcription factor. STUDY DESIGN AND METHODS: Samples from two patients and one donor with unusual or discrepant serology for ABO, RhD, and RhCE antigens were analyzed by DNA sequencing. Analyzed regions included the coding sequence and portions of regulatory elements. The effect of some variants on gene expression was evaluated in reporter gene assays. RESULTS: Three new alleles were identified. Their key variants are located in the ABO Intron 1 enhancer, the RHD proximal promoter, and the RHCE proximal promoter. IVS1 + 5859A was found in an African American patient with a group O forward type and a group B reverse type. 5'UTR-115C was the only RHD variant sequence found in a mixed-race black and Caucasian prenatal patient showing mixed-field agglutination with anti-D. 5'UTR-83T was found in several black donors and patients in the context of the genetically related RHCE*ceBI and RHCE*ceSM alleles. Reporter assays of promoter constructs including 5'UTR-115C or 5'UTR-83T showed a significant reduction in RH gene expression. CONCLUSION: Three new alleles in the ABO, RHD, and RHCE genes consist of single-nucleotide changes within GATA motifs, emphasizing the key role of GATA transcription factors in the expression of blood group genes.

Knockdown of core binding factorß alters sphingolipid metabolism.

Core binding factor (CBF) is a heterodimeric transcription factor containing one of three DNA-binding proteins of the Runt-related transcription factor family (RUNX1-3) and the non-DNA-binding protein, CBFß. RUNX1 and CBFß are the most common targets of chromosomal rearrangements in leukemia. CBF has been implicated in other cancer types; for example RUNX1 and RUNX2 are implicated in cancers of epithelial origin, including prostate, breast, and ovarian cancers. In these tumors, CBF is involved in maintaining the malignant phenotype and, when highly over-expressed, contributes to metastatic growth in bone. Herein, lentiviral delivery of CBFß-specific shRNAs was used to achieve a 95% reduction of CBFß in an ovarian cancer cell line. This drastic reduction in CBFß expression resulted in growth inhibition that was not associated with a cell cycle block or an increase in apoptosis. However, CBFß silencing resulted in increased autophagy and production of reactive oxygen species (ROS). Since sphingolipid and ceramide metabolism regulates non-apoptotic cell death, autophagy, and ROS production, fumonsin B1 (FB1), an inhibitor of ceramide synthase, was used to alter ceramide production in the CBFß-silenced cells. FB1 treatment inhibited the CBFß-dependent increase in autophagy and provided a modest increase in cell survival. To document alterations to sphingolipids in the CBFß-silenced cells, ceramide, and lactosylceramide levels were directly examined by mass spectrometry. Substantial increases in ceramide species and decreases in lactosylceramides were identified. Altogether, this report provides evidence that CBF transcriptional pathways control cellular survival, at least in part, through sphingolipid metabolism.

Association of core-binding factor ß with the malignant phenotype of prostate and ovarian cancer cells.

Core binding factor (CBF) is a transcription factor complex that plays roles in development, stem-cell homeostasis, and human disease. CBF is a heterodimer composed of one of three DNA-binding RUNX proteins plus the non-DNA-binding protein, CBFß. Recent studies have showed that the RUNX factors exhibit complex expression patterns in prostate, breast, and ovarian cancers, and CBF has been implicated in the control of cancer-related genes. However, the biologic roles of CBF in solid tumors have not been fully elucidated. To test whether CBF is required for the malignant phenotype of various epithelial cancers, we used lentiviral delivery of CBFß-specific shRNA to significantly decrease CBFß expression in two prostate cancer cell lines (PPC1 and PC-3) and the SKOV-3 ovarian cancer cell line. We found that knockdown of CBFß significantly inhibited anchorage independent growth of each cell line. Further, CBFß knockdown in PPC1 cells suppressed xenograft tumor growth compared to controls. Mice injected with SKOV-3 ovarian cancer cells knocked-down for CBFß exhibited a survival time similar to control mice. However, human cells recovered from the ascites fluid of these mice showed CBFß expression levels similar to those from mice injected with control SKOV-3 cells, suggesting that CBFß knockdown is incompatible with tumor cell growth. Gene expression profiling of CBFß knockdown cells revealed significant changes in expression in genes involved in various developmental and cell signaling pathways. These data collectively suggest that CBFß is required for malignancy in some human cancers.