ALBERT

Description: Self-renewal is a key feature of the cells that initiate acute myeloid leukemia. To identify the mechanisms involved in PML-RARA (PR)-driven self-renewal, we made use of Ctsg-PR mice, which have PR knocked into the UTR of the first exon of Ctsg. Ctsg-PR drives the expression of PR in myeloid progenitor cells and gives these cells the ability to serially replate in methylcellulose-based colony assays. Most Ctsg-PR mice develop acute promyelocytic leukemia (APL) with an average latency of ~300 days. To identify target genes regulated by Ctsg-PR, we performed single cell RNA-seq (scRNA-seq) on whole bone marrow from young, preleukemic Ctsg-PR mice or age-matched littermates. We identified 959 differentially expressed genes (DEGs) within myeloid progenitors (546 upregulated by PR, and 413 downregulated). Gata2 was identified as a DEG in this analysis, and we confirmed this phenotype with bulk RNA-seq of purified promyelocytes from young, preleukemic WT vs. Ctsg-PR mice. All APLs derived from Ctsg-PR mice also expressed high levels of Gata2. To identify the immediate-early target genes of PR, we transduced human CD34+ cord blood cells with MSCV-IRES-GFP retroviruses containing PR, a mutant PR with a RARA DNA binding mutation (C88A, known to abolish PR replating), or an empty vector. ScRNA-seq analysis of these cells after 7 days of ex vivo culture identified 1815 DEGs (1301 upregulated and 514 downregulated by PR) in GFP+ cells expressing PR, compared to GFP+ cells transduced with PRC88A or empty vector. Among the DEGs, Gata2 was upregulated 5-fold in the PR GFP+ cells. Identical short-term retroviral overexpression studies with mouse marrow revealed that PR expression caused an expansion of hematopoietic progenitor cells that overexpressed Gata2. Finally, although normal human promyelocytes do not express GATA2, virtually all primary human APL samples do. Combined, these studies strongly suggest that GATA2 is a target gene of PR, and may therefore play a role in the development of APL. Based on our expression data, we hypothesized that Gata2 inactivation would reduce PR-driven self-renewal. To test this hypothesis, we bred Ctsg-PR mice to Rosa26-Cas9 mice, which ubiquitously express Cas9. Marrow from the resulting Ctsg-PR x Cas9 mice was electroporated with guide RNAs (gRNAs) targeting the zinc finger 1 (ZF1) domain or exon 2 of Gata2, or control loci (Actb intron 5 or Rosa26 intron 1) and serially replated in methylcellulose with SCF, IL-3, and IL-6. CRISPR-Cas9 efficiently induced a wide array of indel mutations at all gRNA target sites. Two days after electroporation, the frequency of Gata2 alleles with indels at the target site ranged from 64% to 93% (n=4); hundreds of different Gata2 indels were generated in each experiment. To our surprise, the Gata2 targeted Ctsg-PR cells replated with dramatically higher efficiency than control locus targeted cells (Figure 1). The enhanced replating efficiency was dependent upon PR, since Gata2 targeted Rosa26-Cas9 bone marrow did not serially replate. Further, cells with Gata2 indels were positively selected for over time. For example, in one experiment, the frequency of a 12 bp deletion in Gata2 (that caused an in-frame deletion in ZF1) rose from an initial variant allele frequency (VAF) of 3% to 70% after 8 weeks of replating. A larger Gata2 deletion was co-selected at a similar frequency, and resulted in the deletion of Gata2 exon 4, which encodes 90% of ZF1. Virtually all of the positively selected cells contained Gata2 indels, demonstrating the competitive advantage of Gata2 loss-of-function mutations in this setting. Control gRNAs did not lead to significant changes in plating efficiency, nor were any indels selected for. Additionally, Ctsg-PR x Cas9 cells with Gata2 indels shifted from a neutrophilic phenotype (Gr1+ CD11b+) to a monocytic one (CD11b+ Gr1-) at late passages. To further investigate the role of Gata2 in Ctsg-PR induced APL, we sequenced the genomes of 16 mouse APLs and found that 2 samples had spontaneous mutations in Gata2 (R330L and N363fs, with VAFs of 74% and 39% respectively). In summary, these data provide evidence that PR positively regulates Gata2, and that Gata2 in turn promotes neutrophilic differentiation of committed, "reprogrammed" myelomonocytic progenitors. Surprisingly, Gata2 appears to contribute to the lineage fate and proliferative capacity of PR-expressing hematopoietic progenitor cells. Disclosures No relevant conflicts of interest to declare.

Description: Background: Expansion of one or more subclone occurs during progression from myelodysplastic syndrome (MDS) to secondary acute myeloid leukemia (sAML). Existing data suggest that acquired mutations in myeloid transcription factor (e.g., RUNX1, CEBPA, WT1) and signaling genes (e.g., receptor tyrosine kinases or RAS pathway genes) contribute to clonal evolution and the rising blast count that defines progression to sAML. While signaling gene (SG) mutations are typically acquired later in disease progression, our understanding of when transcription factor (TF) mutations occur, in what clone they occur (e.g. founding clone or subclone), and whether TF-mutated clones undergo further clonal evolution remains incomplete. This is largely due to the limited number of paired MDS and sAML samples analyzed, the limitation of current sequencing technology and the lack of serial samples, and incomplete characterization of tumor clonality. Methods: We banked paired MDS and sAML (plus skin) samples from 44 patients who progressed from MDS to sAML (median time to progression 306 days, range 21-3568). We sequenced sAML and skin samples for 285 recurrently mutated genes (RMGs) and genotyped the paired MDS sample in patients with TF and/or SG mutations. Twelve patients were selected for enhanced whole genome sequencing (eWGS) of MDS and sAML samples (plus skin) to characterize tumor clonality. Somatic mutations were validated using error-corrected sequencing and clones were identified in MDS and sAML samples using mutation variant allele frequencies (VAFs). We tracked clonal evolution by sequencing serial samples between MDS and sAML. Results: The frequency of both TF and SG mutations were elevated in the 44 sAML patients compared to our previously sequenced cohort of 150 independent de novo MDS patients (signaling: 36% vs. 15%, transcription factor: 30% vs. 11%, respectively, p

Description: Background. Acute Myeloid Leukemia (AML) is genetically and epigenetically heterogeneous. Most AML samples display clonal heterogeneity at presentation, which evolves with therapeutic interventions. To better understand the epigenetic consequences of clonal heterogeneity, we are using single-cell RNA-sequencing (scRNA-seq) to characterize expression heterogeneity in AML. To date, scRNA-seq has had limited utility in applications where it is essential to link transcriptional heterogeneity to genetic variation, because it has been difficult to identify specific mutations in individual cells using scRNA-seq data alone. To address this limitation, we developed an approach to use scRNA-seq data to identify expressed mutations in individual AML cells, and link these variants to the expression heterogeneity in the same samples. Methods. We generated duplicate cDNA libraries for each of 5 cryopreserved bone marrow samples from adult patients with de novo AML, using the 10x Genomics Chromium Single Cell 5' Gene Expression workflow for Single Cell RNA Sequencing. Single cell libraries were sequenced to yield a median of 20,474 cells per sample, and 192,427 reads per cell. Transcript alignment, counting, and inter-library normalization were performed using the Cell Ranger pipeline (10x Genomics). The Seurat R package was used for further normalization, filtering, principal component analysis, clustering, and t-SNE visualization. A nearest-neighbor algorithm was developed to assign each cell in the data set to the most transcriptionally similar hematopoietic lineage. For each case, we performed whole genome sequencing (WGS) to identify germline and somatic variants, and define clonal architecture. We then developed bioinformatic methods to determine which cells harbor these mutations, assign those cells to mutationally-defined subclones, and link mutations to defined expression clusters. Results. WGS identified 25-56 coding mutations per sample; we were able to identify 22%-46% of these mutations in at least one cell in the scRNA-seq data, including point mutations (e.g. DNMT3A, U2AF1, TP53, IDH1, IDH2, SRSF2, CEBPA, and others) and indels (e.g. FLT3-ITD, NPMc). Although the libraries were 5' biased, expressed mutations could be identified at long distances from the 5' end of transcripts; for example, an expressed DNMT3AR882H mutation (2.646 Kb from the initiating codon) was easily detected (Fig 1c). The frequency of detected mutations in the single-cell data varied widely (range: 1-1564 cells; median: 11 cells), and as expected, depended heavily on the expression level of the gene, and the size of the clone containing the mutation. Regardless, a median of 1378 cells (6.7%) had at least one identifiable mutation in the 5 samples. Using these data, we were able to 1) distinguish AML cells from normal cells in bone marrow samples (Fig 1a/b), 2) identify major subclones within the AML samples (Fig 1c/d), and 3) identify mutation-specific and subclone-specific expression profiles. In 2 samples with mutationally-defined subclones (one with a CEBPAR142fs mutation, and the other with a GATA2R361C mutation), subclone-specific gene expression profiles were clearly detected in the scRNA-seq data, and could be directly associated with cells containing the mutant transcription factors. In the case with the subclonal GATA2R361C mutation, cells with that mutation were restricted to a subset of expression clusters (Fig 1d). In this subset, we identified an expression signature that is supported by pre-existing knowledge of the GATA2/SPI1 transcriptional regulatory circuit. In addition, we observed that expression heterogeneity frequently occurs independent of mutations defined by specific subclones. For instance, the GATA2R361C subclone contained additional heterogeneity (5 independent expression clusters) that could not be accounted for by mutations (Fig 1a/d). Moreover, the other 3 cases exhibited extensive expression heterogeneity within the AML cells that was not explained by genetically defined subclones. In sum, scRNA-seq data, when adapted to detect mutations, has dramatically improved our understanding of the expression heterogeneity of AML, which arises from two main sources: 1) cell-type composition of the sample, and 2) expression variation among the AML cells themselves (caused by both mutation-associated and mutation-independent factors). Disclosures Williams: 10x Genomics: Employment, Equity Ownership. Fiddes:10x Genomics: Employment, Equity Ownership. Church:10x Genomics: Employment, Equity Ownership.

Description: Myelodysplastic syndromes (MDS) are the most common myeloid malignancies among the elderly. Patients present with bone marrow (BM) failure manifested by low peripheral blood (PB) counts and are at increased risk of developing acute myeloid leukemia. Mutations of U2AF1, a gene that encodes a spliceosome protein, are identified in 11% of MDS patients. The two most common U2AF1 mutants, S34F and Q157P, alter the splicing of two distinct sets of pre-mRNA targets in vitro and each co-occur with unique gene mutations in MDS patients, suggesting these mutants may affect MDS pathogenesis differently. In mice, U2AF1S34F expression leads to altered splicing, reduced B-cell counts, and features of MDS. Similar studies have not been performed for U2AF1Q157P. To study the impact of U2AF1Q157P expression on splicing and hematopoiesis in vivo, we created a doxycycline (DOX)-inducible ("Tet-On") transgenic mouse that expresses mutant U2AF1Q157P and is isogenic to our previously reported U2AF1S34F and U2AF1WT transgenic mice. First, we confirmed DOX-inducible expression of the U2AF1Q157P transgene in BM by RT-PCR-seq. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P, we performed non-competitive BM transplants into lethally irradiated congenic recipient mice. Donor BM from U2AF1WT or U2AF1S34F mice was also transplanted for comparison. Six weeks after transplant, mice were maintained on DOX chow to induce U2AF1 transgene (U2AF1WT, U2AF1S34F, or U2AF1Q157P) expression (n = 10 mice per genotype). After six weeks on DOX, there were no significant changes in PB counts for U2AF1Q157P mice compared to U2AF1WT controls. In contrast, white blood cell (WBC) and B-cell counts were significantly reduced in U2AF1S34F mice, as reported previously. Assessment of the BM revealed increased numbers (per five leg bones) of hematopoietic stem and progenitor cells (LSK [Lin− Sca-1+ c-kit+] and LK [Lin− Sca-1− c-kit+]) in U2AF1S34F mice (1.33×105 LSK and 7.13×105 LK cells) compared to U2AF1WT (1.04×105 LSK and 5.69×105 LK cells; p 〈 0.05 for LSK and LK), as reported previously. In contrast, there was no change in LSK cells (1.03×105, p = 0.9668) and a non-significant increase in LK cells (6.84×105, p = 0.0547) in U2AF1Q157P mice compared to U2AF1WT. Both U2AF1S34F and U2AF1Q157P mice shared a significant increase in the number of common myeloid progenitors (CMP) compared to U2AF1WT (2.43×105 and 2.39×105 vs. 1.66×105 cells; p 〈 0.001 and p 〈 0.01, respectively), although CFU-C interrogated by methylcellulose assay were significantly increased only for U2AF1S34F mice. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P on stem cell function, we mixed equal numbers of whole BM test cells (CD45.2+; U2AF1Q157P or U2AF1WT) with congenic control wild-type BM competitor cells (CD45.1+/CD45.2+) and transplanted them into lethally irradiated congenic recipient mice (CD45.1+/CD45.2+ ; n = 6 per genotype). As in non-competitive transplants, DOX chow was administered six weeks after transplant. After six weeks on DOX, we observed a relative multi-lineage competitive disadvantage by analysis of peripheral blood chimerism (%CD45.2+ WBC) for U2AF1Q157P test compared to U2AF1WT test cells (49.5% vs. 71.7%, respectively, p 〈 0.001). In addition, stem and progenitor cells were all significantly reduced in the BM of U2AF1Q157P competitive transplant mice compared to U2AF1WT after 18 weeks of DOX (LSK, 36.1% vs. 92.2%, respectively, p 〈 0.001; LK, 53.1% vs. 92.0%, p 〈 0.001). Lastly, using a Nanostring array, we identified consensus 3' splice sites of cassette exons that were increased or decreased in RNA from c-kit enriched mutant (U2AF1S34F or U2AF1Q157P) BM cells relative to U2AF1WT (FDR 〈 0.1). As expected, we observed altered consensus 3' splice sites at the −3 position (for U2AF1S34F) and +1 position (for U2AF1Q157P) of differentially spliced exons, indicating altered but different pre-mRNA splicing induced by either U2AF1 mutant. In aggregate, hematopoietic expression of U2AF1Q157P causes a multi-lineage competitive disadvantage of BM stem cells and expanded myeloid progenitors in the non-competitive transplant setting, like U2AF1S34F. However, PB counts and lineage distribution are not affected, indicating that the two common U2AF1 mutants, Q157P and S34F, are associated with different hematopoietic phenotypes and alterations to splicing, and may have different roles in MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

Description: Background: Previous studies indicate that mutations in signaling (e.g., receptor tyrosine kinases and RAS pathway members) and transcription factor genes are more common in secondary acute myeloid leukemia (sAML) than myelodysplastic syndrome (MDS), suggesting a role in disease progression. However, our understanding of the timing and order of mutation acquisition in these genes remains incomplete without analyzing paired MDS and sAML samples from the same patient. Defining the role of signaling gene mutations during progression should provide biologic insight into clonal evolution and help define prognostic markers for MDS progression. Methods: We banked paired MDS and sAML (and matched skin) samples from 44 patients (median time to progression: 306 days, range 21-3568). We sequenced 44 sAML (+ skin) samples for 285 recurrently mutated genes (RMGs) and 12 samples were selected for enhanced whole genome sequencing (eWGS, genome with deep exome coverage) of MDS and sAML samples (+ skin) to determine clonal hierarchy. Somatic mutations in these 12 samples were validated with high coverage error-corrected sequencing, and clonality was defined in MDS and sAML samples using mutation variant allele frequencies (VAFs). Additionally, error-corrected sequencing for all sAML RMG mutations, plus 40 additional genes, was performed on 43 of the MDS samples. Single cell DNA sequencing (scDNAseq, Mission Bio) was performed on 6 samples. Results: We identified 32 signaling gene mutations in 15 of the 44 sAML samples, with only 11 of 32 mutations (34%) detected in the initial, paired MDS sample (limit of detection;