ERG and c-MYC regulate a critical gene network in BCR::ABL1-driven B cell acute lymphoblastic leukemia.

Philadelphia chromosome-positive B cell acute lymphoblastic leukemia (B-ALL), characterized by the BCR::ABL1 fusion gene, remains a poor prognosis cancer needing new therapeutic approaches. Transcriptomic profiling identified up-regulation of oncogenic transcription factors ERG and c-MYC in BCR::ABL1 B-ALL with ERG and c-MYC required for BCR::ABL1 B-ALL in murine and human models. Profiling of ERG- and c-MYC-dependent gene expression and analysis of ChIP-seq data established ERG and c-MYC coordinate a regulatory network in BCR::ABL1 B-ALL that controls expression of genes involved in several biological processes. Prominent was control of ribosome biogenesis, including expression of RNA polymerase I (POL I) subunits, the importance of which was validated by inhibition of BCR::ABL1 cells by POL I inhibitors, including CX-5461, that prevents promoter recruitment and transcription initiation by POL I. Our results reveal an essential ERG- and c-MYC-dependent transcriptional network involved in regulation of metabolic and ribosome biogenesis pathways in BCR::ABL1 B-ALL, from which previously unidentified vulnerabilities and therapeutic targets may emerge.

IKAROS and AIOLOS directly regulate AP-1 transcriptional complexes and are essential for NK cell development.

Ikaros transcription factors are essential for adaptive lymphocyte function, yet their role in innate lymphopoiesis is unknown. Using conditional genetic inactivation, we show that Ikzf1/Ikaros is essential for normal natural killer (NK) cell lymphopoiesis and IKZF1 directly represses Cish, a negative regulator of interleukin-15 receptor resulting in impaired interleukin-15 receptor signaling. Both Bcl2l11 and BIM levels, and intrinsic apoptosis were increased in Ikzf1-null NK cells, which in part accounts for NK lymphopenia as both were restored to normal levels when Ikzf1 and Bcl2l11 were co-deleted. Ikzf1-null NK cells presented extensive transcriptional alterations with reduced AP-1 transcriptional complex expression and increased expression of Ikzf2/Helios and Ikzf3/Aiolos. IKZF1 and IKZF3 directly bound AP-1 family members and deletion of both Ikzf1 and Ikzf3 in NK cells resulted in further reductions in Jun/Fos expression and complete loss of peripheral NK cells. Collectively, we show that Ikaros family members are important regulators of apoptosis, cytokine responsiveness and AP-1 transcriptional activity.

Dividing out quantification uncertainty allows efficient assessment of differential transcript expression with edgeR.

Differential expression analysis of RNA-seq is one of the most commonly performed bioinformatics analyses. Transcript-level quantifications are inherently more uncertain than gene-level read counts because of ambiguous assignment of sequence reads to transcripts. While sequence reads can usually be assigned unambiguously to a gene, reads are very often compatible with multiple transcripts for that gene, particularly for genes with many isoforms. Software tools designed for gene-level differential expression do not perform optimally on transcript counts because the read-to-transcript ambiguity (RTA) disrupts the mean-variance relationship normally observed for gene level RNA-seq data and interferes with the efficiency of the empirical Bayes dispersion estimation procedures. The pseudoaligners kallisto and Salmon provide bootstrap samples from which quantification uncertainty can be assessed. We show that the overdispersion arising from RTA can be elegantly estimated by fitting a quasi-Poisson model to the bootstrap counts for each transcript. The technical overdispersion arising from RTA can then be divided out of the transcript counts, leading to scaled counts that can be input for analysis by established gene-level software tools with full statistical efficiency. Comprehensive simulations and test data show that an edgeR analysis of the scaled counts is more powerful and efficient than previous differential transcript expression pipelines while providing correct control of the false discovery rate. Simulations explore a wide range of scenarios including the effects of paired vs single-end reads, different read lengths and different numbers of replicates.

Population-level integration of single-cell datasets enables multi-scale analysis across samples.

The increasing generation of population-level single-cell atlases has the potential to link sample metadata with cellular data. Constructing such references requires integration of heterogeneous cohorts with varying metadata. Here we present single-cell population level integration (scPoli), an open-world learner that incorporates generative models to learn sample and cell representations for data integration, label transfer and reference mapping. We applied scPoli on population-level atlases of lung and peripheral blood mononuclear cells, the latter consisting of 7.8 million cells across 2,375 samples. We demonstrate that scPoli can explain sample-level biological and technical variations using sample embeddings revealing genes associated with batch effects and biological effects. scPoli is further applicable to single-cell sequencing assay for transposase-accessible chromatin and cross-species datasets, offering insights into chromatin accessibility and comparative genomics. We envision scPoli becoming an important tool for population-level single-cell data integration facilitating atlas use but also interpretation by means of multi-scale analyses.

MsImpute: Estimation of Missing Peptide Intensity Data in Label-Free Quantitative Mass Spectrometry.

Mass spectrometry (MS) enables high-throughput identification and quantification of proteins in complex biological samples and can provide insights into the global function of biological systems. Label-free quantification is cost-effective and suitable for the analysis of human samples. Despite rapid developments in label-free data acquisition workflows, the number of proteins quantified across samples can be limited by technical and biological variability. This variation can result in missing values which can in turn challenge downstream data analysis tasks. General purpose or gene expression-specific imputation algorithms are widely used to improve data completeness. Here, we propose an imputation algorithm designated for label-free MS data that is aware of the type of missingness affecting data. On published datasets acquired by data-dependent and data-independent acquisition workflows with variable degrees of biological complexity, we demonstrate that the proposed missing value estimation procedure by barycenter computation competes closely with the state-of-the-art imputation algorithms in differential abundance tasks while outperforming them in the accuracy of variance estimates of the peptide abundance measurements, and better controls the false discovery rate in label-free MS experiments. The barycenter estimation procedure is implemented in the msImpute software package and is available from the Bioconductor repository.

Biologically informed deep learning to query gene programs in single-cell atlases.

The increasing availability of large-scale single-cell atlases has enabled the detailed description of cell states. In parallel, advances in deep learning allow rapid analysis of newly generated query datasets by mapping them into reference atlases. However, existing data transformations learned to map query data are not easily explainable using biologically known concepts such as genes or pathways. Here we propose expiMap, a biologically informed deep-learning architecture that enables single-cell reference mapping. ExpiMap learns to map cells into biologically understandable components representing known 'gene programs'. The activity of each cell for a gene program is learned while simultaneously refining them and learning de novo programs. We show that expiMap compares favourably to existing methods while bringing an additional layer of interpretability to integrative single-cell analysis. Furthermore, we demonstrate its applicability to analyse single-cell perturbation responses in different tissues and species and resolve responses of patients who have coronavirus disease 2019 to different treatments across cell types.

MicroRNA-21 is immunosuppressive and pro-metastatic via separate mechanisms.

MiR-21 was identified as a gene whose expression correlated with the extent of metastasis of murine mammary tumours. Since miR-21 is recognised as being associated with poor prognosis in cancer, we investigated its contribution to mammary tumour growth and metastasis in tumours with capacity for spontaneous metastasis. Unexpectedly, we found that suppression of miR-21 activity in highly metastatic tumours resulted in regression of primary tumour growth in immunocompetent mice but did not impede growth in immunocompromised mice. Analysis of the immune infiltrate of the primary tumours at the time when the tumours started to regress revealed an influx of both CD4+ and CD8+ activated T cells and a reduction in PD-L1+ infiltrating monocytes, providing an explanation for the observed tumour regression. Loss of anti-tumour immune suppression caused by decreased miR-21 activity was confirmed by transcriptomic analysis of primary tumours. This analysis also revealed reduced expression of genes associated with cell cycle progression upon loss of miR-21 activity. A second activity of miR-21 was the promotion of metastasis as shown by the loss of metastatic capacity of miR-21 knockdown tumours established in immunocompromised mice, despite no impact on primary tumour growth. A proteomic analysis of tumour cells with altered miR-21 activity revealed deregulation of proteins known to be associated with tumour progression. The development of therapies targeting miR-21, possibly via targeted delivery to tumour cells, could be an effective therapy to combat primary tumour growth and suppress the development of metastatic disease.

TGFß and CIS Inhibition Overcomes NK-cell Suppression to Restore Antitumor Immunity.

Antibodies targeting "immune checkpoints" have revolutionized cancer therapy by reactivating tumor-resident cytotoxic lymphocytes, primarily CD8+ T cells. Interest in targeting analogous pathways in other cytotoxic lymphocytes is growing. Natural killer (NK) cells are key to cancer immunosurveillance by eradicating metastases and driving solid tumor inflammation. NK-cell antitumor function is dependent on the cytokine IL15. Ablation of the IL15 signaling inhibitor CIS (Cish) enhances NK-cell antitumor immunity by increasing NK-cell metabolism and persistence within the tumor microenvironment (TME). The TME has also been shown to impair NK-cell fitness via the production of immunosuppressive transforming growth factor ß (TGFß), a suppression which occurs even in the presence of high IL15 signaling. Here, we identified an unexpected interaction between CIS and the TGFß signaling pathway in NK cells. Independently, Cish- and Tgfbr2-deficient NK cells are both hyperresponsive to IL15 and hyporesponsive to TGFß, with dramatically enhanced antitumor immunity. Remarkably, when both these immunosuppressive genes are simultaneously deleted in NK cells, mice are largely resistant to tumor development, suggesting that combining suppression of these two pathways might represent a novel therapeutic strategy to enhance innate anticancer immunity.

Loss of TAF8 causes TFIID dysfunction and p53-mediated apoptotic neuronal cell death.

Mutations in genes encoding general transcription factors cause neurological disorders. Despite clinical prominence, the consequences of defects in the basal transcription machinery during brain development are unclear. We found that loss of the TATA-box binding protein-associated factor TAF8, a component of the general transcription factor TFIID, in the developing central nervous system affected the expression of many, but notably not all genes. Taf8 deletion caused apoptosis, unexpectedly restricted to forebrain regions. Nuclear levels of the transcription factor p53 were elevated in the absence of TAF8, as were the mRNAs of the pro-apoptotic p53 target genes Noxa, Puma and Bax. The cell death in Taf8 forebrain regions was completely rescued by additional loss of p53, but Taf8 and p53 brains failed to initiate a neuronal expression program. Taf8 deletion caused aberrant transcription of promoter regions and splicing anomalies. We propose that TAF8 supports the directionality of transcription and co-transcriptional splicing, and that failure of these processes causes p53-induced apoptosis of neuronal cells in the developing mouse embryo.

SFPQ-ABL1 and BCR-ABL1 use different signaling networks to drive B-cell acute lymphoblastic leukemia.

Philadelphia-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype of B-cell ALL characterized by a gene expression profile resembling Philadelphia chromosome-positive ALL (Ph+ ALL) in the absence of BCR-ABL1. Tyrosine kinase-activating fusions, some involving ABL1, are recurrent drivers of Ph-like ALL and are targetable with tyrosine kinase inhibitors (TKIs). We identified a rare instance of SFPQ-ABL1 in a child with Ph-like ALL. SFPQ-ABL1 expressed in cytokine-dependent cell lines was sufficient to transform cells and these cells were sensitive to ABL1-targeting TKIs. In contrast to BCR-ABL1, SFPQ-ABL1 localized to the nuclear compartment and was a weaker driver of cellular proliferation. Phosphoproteomics analysis showed upregulation of cell cycle, DNA replication, and spliceosome pathways, and downregulation of signal transduction pathways, including ErbB, NF-κB, vascular endothelial growth factor (VEGF), and MAPK signaling in SFPQ-ABL1-expressing cells compared with BCR-ABL1-expressing cells. SFPQ-ABL1 expression did not activate phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling and was associated with phosphorylation of G2/M cell cycle proteins. SFPQ-ABL1 was sensitive to navitoclax and S-63845 and promotes cell survival by maintaining expression of Mcl-1 and Bcl-xL. SFPQ-ABL1 has functionally distinct mechanisms by which it drives ALL, including subcellular localization, proliferative capacity, and activation of cellular pathways. These findings highlight the role that fusion partners have in mediating the function of ABL1 fusions.

Metastasis-Entrained Eosinophils Enhance Lymphocyte-Mediated Antitumor Immunity.

The recognition of the immune system as a key component of the tumor microenvironment (TME) led to promising therapeutics. Because such therapies benefit only subsets of patients, understanding the activities of immune cells in the TME is required. Eosinophils are an integral part of the TME especially in mucosal tumors. Nonetheless, their role in the TME and the environmental cues that direct their activities are largely unknown. We report that breast cancer lung metastases are characterized by resident and recruited eosinophils. Eosinophil recruitment to the metastatic sites in the lung was regulated by G protein-coupled receptor signaling but independent of CCR3. Functionally, eosinophils promoted lymphocyte-mediated antitumor immunity. Transcriptome and proteomic analyses identified the TME rather than intrinsic differences between eosinophil subsets as a key instructing factor directing antitumorigenic eosinophil activities. Specifically, TNFα/IFNγ-activated eosinophils facilitated CD4+ and CD8+ T-cell infiltration and promoted antitumor immunity. Collectively, we identify a mechanism by which the TME trains eosinophils to adopt antitumorigenic properties, which may lead to the development of eosinophil-targeted therapeutics. SIGNIFICANCE: These findings demonstrate antitumor activities of eosinophils in the metastatic tumor microenvironment, suggesting that harnessing eosinophil activity may be a viable clinical strategy in patients with cancer.

SOX9 Defines Distinct Populations of Cells in SHH Medulloblastoma but Is Not Required for Math1-Driven Tumor Formation.

Medulloblastoma is the most common malignant pediatric brain tumor and there is an urgent need for molecularly targeted and subgroup-specific therapies. The stem cell factor SOX9, has been proposed as a potential therapeutic target for the treatment of Sonic Hedgehog medulloblastoma (SHH-MB) subgroup tumors, given its role as a downstream target of Hedgehog signaling and in functionally promoting SHH-MB metastasis and treatment resistance. However, the functional requirement for SOX9 in the genesis of medulloblastoma remains to be determined. Here we report a previously undocumented level of SOX9 expression exclusively in proliferating granule cell precursors (GCP) of the postnatal mouse cerebellum, which function as the medulloblastoma-initiating cells of SHH-MBs. Wild-type GCPs express comparatively lower levels of SOX9 than neural stem cells and mature astroglia and SOX9low GCP-like tumor cells constitute the bulk of both infant (Math1Cre:Ptch1lox/lox ) and adult (Ptch1LacZ/+ ) SHH-MB mouse models. Human medulloblastoma single-cell RNA data analyses reveal three distinct SOX9 populations present in SHH-MB and noticeably absent in other medulloblastoma subgroups: SOX9 + MATH1 + (GCP), SOX9 + GFAP + (astrocytes) and SOX9 + MATH1 + GFAP + (potential tumor-derived astrocytes). To functionally address whether SOX9 is required as a downstream effector of Hedgehog signaling in medulloblastoma tumor cells, we ablated Sox9 using a Math1Cre model system. Surprisingly, targeted ablation of Sox9 in GCPs (Math1Cre:Sox9lox/lox ) revealed no overt phenotype and loss of Sox9 in SHH-MB (Math1Cre:Ptch1lox/lox;Sox9lox/lox ) does not affect tumor formation. IMPLICATIONS: Despite preclinical data indicating SOX9 plays a key role in SHH-MB biology, our data argue against SOX9 as a viable therapeutic target.

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Elp2 mutations perturb the epitranscriptome and lead to a complex neurodevelopmental phenotype.

Intellectual disability (ID) and autism spectrum disorder (ASD) are the most common neurodevelopmental disorders and are characterized by substantial impairment in intellectual and adaptive functioning, with their genetic and molecular basis remaining largely unknown. Here, we identify biallelic variants in the gene encoding one of the Elongator complex subunits, ELP2, in patients with ID and ASD. Modelling the variants in mice recapitulates the patient features, with brain imaging and tractography analysis revealing microcephaly, loss of white matter tract integrity and an aberrant functional connectome. We show that the Elp2 mutations negatively impact the activity of the complex and its function in translation via tRNA modification. Further, we elucidate that the mutations perturb protein homeostasis leading to impaired neurogenesis, myelin loss and neurodegeneration. Collectively, our data demonstrate an unexpected role for tRNA modification in the pathogenesis of monogenic ID and ASD and define Elp2 as a key regulator of brain development.

Type 2 Innate Lymphoid Cells Protect against Colorectal Cancer Progression and Predict Improved Patient Survival.

Chronic inflammation of the gastrointestinal (GI) tract contributes to colorectal cancer (CRC) progression. While the role of adaptive T cells in CRC is now well established, the role of innate immune cells, specifically innate lymphoid cells (ILCs), is not well understood. To define the role of ILCs in CRC we employed complementary heterotopic and chemically-induced CRC mouse models. We discovered that ILCs were abundant in CRC tumours and contributed to anti-tumour immunity. We focused on ILC2 and showed that ILC2-deficient mice developed a higher tumour burden compared with littermate wild-type controls. We generated an ILC2 gene signature and using machine learning models revealed that CRC patients with a high intratumor ILC2 gene signature had a favourable clinical prognosis. Collectively, our results highlight a critical role for ILC2 in CRC, suggesting a potential new avenue to improve clinical outcomes through ILC2-agonist based therapeutic approaches.

Hhex Directly Represses BIM-Dependent Apoptosis to Promote NK Cell Development and Maintenance.

Hhex encodes a homeobox transcriptional regulator important for embryonic development and hematopoiesis. Hhex is highly expressed in NK cells, and its germline deletion results in significant defects in lymphoid development, including NK cells. To determine if Hhex is intrinsically required throughout NK cell development or for NK cell function, we generate mice that specifically lack Hhex in NK cells. NK cell frequency is dramatically reduced, while NK cell differentiation, IL-15 responsiveness, and function at the cellular level remain largely normal in the absence of Hhex. Increased IL-15 availability fails to fully reverse NK lymphopenia following conditional Hhex deletion, suggesting that Hhex regulates developmental pathways extrinsic to those dependent on IL-15. Gene expression and functional genetic approaches reveal that Hhex regulates NK cell survival by directly binding Bcl2l11 (Bim) and repressing expression of this key apoptotic mediator. These data implicate Hhex as a transcriptional regulator of NK cell homeostasis and immunity.

Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia.

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.

Cotargeting BCL-2 and MCL-1 in high-risk B-ALL.

Improving survival outcomes in adult B-cell acute lymphoblastic leukemia (B-ALL) remains a clinical challenge. Relapsed disease has a poor prognosis despite the use of tyrosine kinase inhibitors (TKIs) for Philadelphia chromosome positive (Ph+ ALL) cases and immunotherapeutic approaches, including blinatumomab and chimeric antigen receptor T cells. Targeting aberrant cell survival pathways with selective small molecule BH3-mimetic inhibitors of BCL-2 (venetoclax, S55746), BCL-XL (A1331852), or MCL1 (S63845) is an emerging therapeutic option. We report that combined targeting of BCL-2 and MCL1 is synergistic in B-ALL in vitro. The combination demonstrated greater efficacy than standard chemotherapeutics and TKIs in primary samples from adult B-ALL with Ph+ ALL, Ph-like ALL, and other B-ALL. Moreover, combined BCL-2 or MCL1 inhibition with dasatinib showed potent killing in primary Ph+ B-ALL cases, but the BH3-mimetic combination appeared superior in vitro in a variety of Ph-like ALL samples. In PDX models, combined BCL-2 and MCL1 targeting eradicated ALL from Ph- and Ph+ B-ALL cases, although fatal tumor lysis was observed in some instances of high tumor burden. We conclude that a dual BH3-mimetic approach is highly effective in diverse models of high-risk human B-ALL and warrants assessment in clinical trials that incorporate tumor lysis precautions.

An Erg-driven transcriptional program controls B cell lymphopoiesis.

B lymphoid development is initiated by the differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating mature B cells. This highly regulated process generates clonal immunological diversity via recombination of immunoglobulin V, D and J gene segments. While several transcription factors that control B cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for early B lymphoid differentiation. Erg initiates a transcriptional network involving the B cell lineage defining genes, Ebf1 and Pax5, which directly promotes expression of key genes involved in V(D)J recombination and formation of the B cell receptor. Complementation of Erg deficiency with a productively rearranged immunoglobulin gene rescued B lineage development, demonstrating that Erg is an essential and stage-specific regulator of the gene regulatory network controlling B lymphopoiesis.

Snail induces epithelial cell extrusion by regulating RhoA contractile signalling and cell-matrix adhesion.

Cell extrusion is a morphogenetic process that is implicated in epithelial homeostasis and elicited by stimuli ranging from apoptosis to oncogenic transformation. To explore whether the morphogenetic transcription factor Snail (SNAI1) induces extrusion, we inducibly expressed a stabilized Snail6SA transgene in confluent MCF-7 monolayers. When expressed in small clusters (less than three cells) within otherwise wild-type confluent monolayers, Snail6SA expression induced apical cell extrusion. In contrast, larger clusters or homogenous cultures of Snail6SA cells did not show enhanced apical extrusion, but eventually displayed sporadic basal delamination. Transcriptomic profiling revealed that Snail6SA did not substantively alter the balance of epithelial and mesenchymal genes. However, we identified a transcriptional network that led to upregulated RhoA signalling and cortical contractility in cells expressing Snail6SA Enhanced contractility was necessary, but not sufficient, to drive extrusion, suggesting that Snail collaborates with other factors. Indeed, we found that the transcriptional downregulation of cell-matrix adhesion cooperates with contractility to mediate basal delamination. This provides a pathway for Snail to influence epithelial morphogenesis independently of classic epithelial-to-mesenchymal transition.