Virtually all tumors are genetically heterogeneous, containing mutationally-defined subclonal cell populations that often have distinct phenotypes. Single-cell RNA-sequencing has revealed that a variety of tumors are also transcriptionally heterogeneous, but the relationship between expression heterogeneity and subclonal architecture is unclear. Here, we address this question in the context of Acute Myeloid Leukemia (AML) by integrating whole genome sequencing with single-cell RNA-sequencing (using the 10x Genomics Chromium Single Cell 5’ Gene Expression workflow). Applying this approach to five cryopreserved AML samples, we identify hundreds to thousands of cells containing tumor-specific mutations in each case, and use the results to distinguish AML cells (including normal-karyotype AML cells) from normal cells, identify expression signatures associated with subclonal mutations, and find cell surface markers that could be used to purify subclones for further study. This integrative approach for connecting genotype to phenotype is broadly applicable to any sample that is phenotypically and genetically heterogeneous.

Natural killer (NK) cells are an emerging cancer cellular therapy and potent mediators of antitumor immunity. Cytokine-induced memory-like (ML) NK cellular therapy is safe and induces remissions in patients with acute myeloid leukemia (AML). However, the dynamic changes in phenotype that occur after NK-cell transfer that affect patient outcomes remain unclear. Here, we report comprehensive multidimensional correlates from ML NK cell–treated patients with AML using mass cytometry. These data identify a unique in vivo differentiated ML NK–cell phenotype distinct from conventional NK cells. Moreover, the inhibitory receptor NKG2A is a dominant, transcriptionally induced checkpoint important for ML, but not conventional NK-cell responses to cancer. The frequency of CD8α+ donor NK cells is negatively associated with AML patient outcomes after ML NK therapy. Thus, elucidating the multidimensional dynamics of donor ML NK cells in vivo revealed critical factors important for clinical response, and new avenues to enhance NK-cell therapeutics. Significance: Mass cytometry reveals an in vivo memory-like NK-cell phenotype, where NKG2A is a dominant checkpoint, and CD8α is associated with treatment failure after ML NK–cell therapy. These findings identify multiple avenues for optimizing ML NK–cell immunotherapy for cancer and define mechanisms important for ML NK–cell function. This article is highlighted in the In This Issue feature, p. 1775

Bam-readcount is a utility for generating low-level information about sequencing data at specific nucleotide positions. Originally designed to help filter genomic mutation calls, the metrics it outputs are useful as input for variant detection tools and for resolving ambiguity between variant callers (Koboldt et al., 2013a;Kothen-Hill et al., 2018). In addition, it has found broad applicability in diverse fields including tumor evolution, single-cell genomics, climate change ecology, and tracking community spread of SARS-CoV-2 (

Mutations in KIT and TET2 are associated with myeloid malignancies. We show that loss of TET2-induced PI3K activation and -increased proliferation is rescued by targeting the p110α/δ subunits of PI3K. RNA-Seq revealed a hyperactive c-Myc signature in Tet2-/- cells, which is normalized by inhibiting PI3K signaling. Loss of TET2 impairs the maturation of myeloid lineage-derived mast cells by dysregulating the expression of Mitf and Cebpa, which is restored by low-dose ascorbic acid and 5-azacytidine. Utilizing a mouse model in which the loss of TET2 precedes the expression of oncogenic Kit, similar to the human disease, results in the development of a non-mast cell lineage neoplasm (AHNMD), which is responsive to PI3K inhibition. Thus, therapeutic approaches involving hypomethylating agents, ascorbic acid, and isoform-specific PI3K inhibitors are likely to be useful for treating patients with TET2 and KIT mutations.

Mutant U2AF1-induced alternative splicing of H2afy (macroH2A1) regulates B-lymphopoiesis in mice Graphical abstract Highlights d Mutant U2AF1(S34F) induces alternative splicing of H2AFY d H2afy À/À mice have defective B cell development similar to U2AF1(S34F) mice d The H2afy1.1 splice isoform, reduced by U2AF1(S34F), regulates B cell development d H2AFY occupies the Ebf1 promoter, a master regulator of B cell development

Nonsense-mediated RNA decay (NMD) is recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTC) for degradation, however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system to accurately measure NMD activity in individual cells. A genome-wide CRISPR-Cas9 knockout screen using this reporter system identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, cells with mutations in the spliceosome genes SF3B1 and U2AF1, which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Compared with wild-type (WT) cells, SF3B1- and U2AF1-mutant cells were more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage, and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition was rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, these findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel synthetic lethal strategy for the treatment of MDS and cancers with spliceosome mutations. Significance: This study has developed a novel NMD reporter system and identified a potential therapeutic approach of targeting the NMD pathway to treat cancer with spliceosome gene mutations.

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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.