Feature selection followed by a residuals-based normalization simplifies and improves single-cell gene expression analysis.

Normalization is a critical step in the computational analysis of single-cell RNA-sequencing (scRNA-seq) counts data. The objective is to reduce systematic biases introduced by technical sources that can obscure underlying biological differences. This is typically accomplished by re-scaling the observed counts to reduce the differences in total counts between the cells and then transforming the scaled counts to stabilize the variances. In the standard scRNA-seq workflow, this is followed by feature selection to identify genes that capture most of the biologically meaningful variation across the cells. Here, we propose a simple feature selection method and show that we can perform feature selection before normalization. We also propose a novel residuals-based normalization method that includes a monotonic non-linear transformation to ensure effective variance stabilization of the residuals. We demonstrate significant improvements in downstream clustering analyses through the application of our feature selection and normalization methods to truth-known biological as well as simulated counts data sets. Based on these results, we make the case for a revised scRNA-seq analysis workflow wherein feature selection precedes and in fact informs our residuals-based normalization. This novel workflow has been implemented in an R package called Piccolo.

Therapeutic targeting of ACLY in T-ALL in vivo.

T-cell Acute Lymphoblastic Leukemia (T-ALL) is a hematological malignancy in need of novel therapeutic approaches. Here, we identify the ATP-citrate lyase ACLY as a novel therapeutic target in T-ALL. Our results show that ACLY is overexpressed in T-ALL, and its expression correlates with NOTCH1 activity. To test the effects of ACLY in leukemia progression and the response to NOTCH1 inhibition, we developed an isogenic model of NOTCH1-induced Acly conditional knockout leukemia. Importantly, we observed intrinsic antileukemic effects upon loss of ACLY, which further synergized with NOTCH1 inhibition in vivo . Gene expression profiling analyses showed that the transcriptional signature of ACLY loss very significantly correlates with the signature of NOTCH1 inhibition in vivo , with significantly downregulated pathways related to oxidative phosphorylation, electron transport chain, ribosomal biogenesis and nucleosome biology. Consistently, metabolomic profiling upon ACLY loss revealed a metabolic crisis with accumulation of nucleotide intermediates and reduced levels of several amino acids. Overall, our results identify a link between NOTCH1 and ACLY and unveil ACLY as a novel promising target for T-ALL treatment.

A Therapeutically Targetable NOTCH1-SIRT1-KAT7 Axis in T-cell Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a NOTCH1-driven disease in need of novel therapies. Here, we identify a NOTCH1-SIRT1-KAT7 link as a therapeutic vulnerability in T-ALL, in which the histone deacetylase SIRT1 is overexpressed downstream of a NOTCH1-bound enhancer. SIRT1 loss impaired leukemia generation, whereas SIRT1 overexpression accelerated leukemia and conferred resistance to NOTCH1 inhibition in a deacetylase-dependent manner. Moreover, pharmacologic or genetic inhibition of SIRT1 resulted in significant antileukemic effects. Global acetyl proteomics upon SIRT1 loss uncovered hyperacetylation of KAT7 and BRD1, subunits of a histone acetyltransferase complex targeting H4K12. Metabolic and gene-expression profiling revealed metabolic changes together with a transcriptional signature resembling KAT7 deletion. Consistently, SIRT1 loss resulted in reduced H4K12ac, and overexpression of a nonacetylatable KAT7-mutant partly rescued SIRT1 loss-induced proliferation defects. Overall, our results uncover therapeutic targets in T-ALL and reveal a circular feedback mechanism balancing deacetylase/acetyltransferase activation with potentially broad relevance in cancer. SIGNIFICANCE: We identify a T-ALL axis whereby NOTCH1 activates SIRT1 through an enhancer region, and SIRT1 deacetylates and activates KAT7. Targeting SIRT1 shows antileukemic effects, partly mediated by KAT7 inactivation. Our results reveal T-ALL therapeutic targets and uncover a rheostat mechanism between deacetylase/acetyltransferase activities with potentially broader cancer relevance. This article is highlighted in the In This Issue feature, p. 1.

Error-prone repair of stalled replication forks drives mutagenesis and loss of heterozygosity in haploinsufficient BRCA1 cells.

Germline mutations in the BRCA genes are associated with a higher risk of carcinogenesis, which is linked to an increased mutation rate and loss of the second unaffected BRCA allele (loss of heterozygosity, LOH). However, the mechanisms triggering mutagenesis are not clearly understood. The BRCA genes contain high numbers of repetitive DNA sequences. We detected replication forks stalling, DNA breaks, and deletions at these sites in haploinsufficient BRCA cells, thus identifying the BRCA genes as fragile sites. Next, we found that stalled forks are repaired by error-prone pathways, such as microhomology-mediated break-induced replication (MMBIR) in haploinsufficient BRCA1 breast epithelial cells. We detected MMBIR mutations in BRCA1 tumor cells and noticed deletions-insertions (>50 bp) at the BRCA1 genes in BRCA1 patients. Altogether, these results suggest that under stress, error-prone repair of stalled forks is upregulated and induces mutations, including complex genomic rearrangements at the BRCA genes (LOH), in haploinsufficient BRCA1 cells.

Super-enhancer hypermutation alters oncogene expression in B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common B cell non-Hodgkin lymphoma and remains incurable in around 40% of patients. Efforts to sequence the coding genome identified several genes and pathways that are altered in this disease, including potential therapeutic targets1-5. However, the non-coding genome of DLBCL remains largely unexplored. Here we show that active super-enhancers are highly and specifically hypermutated in 92% of samples from individuals with DLBCL, display signatures of activation-induced cytidine deaminase activity, and are linked to genes that encode B cell developmental regulators and oncogenes. As evidence of oncogenic relevance, we show that the hypermutated super-enhancers linked to the BCL6, BCL2 and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (targeting BCL6) and the steroid receptor NR3C1 (targeting BCL2 and CXCR4). Genetic correction of selected mutations restored repressor DNA binding, downregulated target gene expression and led to the counter-selection of cells containing corrected alleles, indicating an oncogenic dependency on the super-enhancer mutations. This pervasive super-enhancer mutational mechanism reveals a major set of genetic lesions deregulating gene expression, which expands the involvement of known oncogenes in DLBCL pathogenesis and identifies new deregulated gene targets of therapeutic relevance.

Mutational signatures reveal ternary relationships between homologous recombination repair, APOBEC, and mismatch repair in gynecological cancers.

BACKGROUND: Revealing the impacts of endogenous and exogenous mutagenesis processes is essential for understanding the etiology of somatic genomic alterations and designing precise prognostication and treatment strategies for cancer. DNA repair deficiency is one of the main sources of endogenous mutagenesis and is increasingly recognized as a target for cancer therapeutics. The role and prevalence of mechanisms that underly different forms of DNA repair deficiencies and their interactions remain to be elucidated in gynecological malignancies. METHODS: We analyzed 1231 exomes and 268 whole-genomes from three major gynecological malignancies including uterine corpus endometrial carcinoma (UCEC) as well as ovarian and cervical cancers. We also analyzed data from 134 related cell lines. We extracted and compared de novo and refitted mutational signature profiles using complementary and confirmatory approaches and performed interaction analysis to detect co-occurring and mutually exclusive signatures. RESULTS: We found an inverse relationship between homologous recombination deficiency (HRd) and mismatch repair deficiency (MMRd). Moreover, APOBEC co-occurred with HRd but was mutually exclusive with MMRd. UCEC tumors were dominated by MMRd, yet a subset of them manifested the HRd and APOBEC signatures. Conversely, ovarian tumors were dominated by HRd, while a subset represented MMRd and APOBEC. In contrast to both, cervical tumors were dominated by APOBEC with a small subsets showing the POLE, HRd, and MMRd signatures. Although the type, prevalence, and heterogeneity of mutational signatures varied across the tumor types, the patterns of co-occurrence and exclusivity were consistently observed in all. Notably, mutational signatures in gynecological tumor cell lines reflected those detected in primary tumors. CONCLUSIONS: Taken together, these analyses indicate that application of mutation signature analysis not only advances our understanding of mutational processes and their interactions, but also it has the potential to stratify patients that could benefit from treatments available for tumors harboring distinct mutational signatures and to improve clinical decision-making for gynecological malignancies.

Cilium Expression Score Predicts Glioma Survival.

The accurate classification, prognostication, and treatment of gliomas has been hindered by an existing cellular, genomic, and transcriptomic heterogeneity within individual tumors and their microenvironments. Traditional clustering is limited in its ability to distinguish heterogeneity in gliomas because the clusters are required to be exclusive and exhaustive. In contrast, biclustering can identify groups of co-regulated genes with respect to a subset of samples and vice versa. In this study, we analyzed 1,798 normal and tumor brain samples using an unsupervised biclustering approach. We identified co-regulated gene expression profiles that were linked to proximally located brain regions and detected upregulated genes in subsets of gliomas, associated with their histologic grade and clinical outcome. In particular, we present a cilium-associated signature that when upregulated in tumors is predictive of poor survival. We also introduce a risk score based on expression of 12 cilium-associated genes which is reproducibly informative of survival independent of other prognostic biomarkers. These results highlight the role of cilia in development and progression of gliomas and suggest potential therapeutic vulnerabilities for these highly aggressive tumors.

Germline Testing Data Validate Inferences of Mutational Status for Variants Detected From Tumor-Only Sequencing.

Pathogenic germline variants (PGVs) in cancer susceptibility genes are usually identified through germline testing of DNA from blood or saliva: their detection can affect treatment options and potential risk-reduction strategies for patient relatives. PGV can also be identified in tumor sequencing assays, which, when performed without patient-matched normal specimens, render determination of variants' germline or somatic origin critical. METHODS: Tumor-only sequencing data from 1,608 patients were retrospectively analyzed to infer germline versus somatic status of variants using an information-theoretic, gene-independent approach. Loss of heterozygosity was also determined. Predicted mutational models were compared with clinical germline testing results. Statistical measures were computed to evaluate performance. RESULTS: Tumor-only sequencing detected 3,988 variants across 70 cancer susceptibility genes for which germline testing data were available. We imputed germline versus somatic status for > 75% of all detected variants, with a sensitivity of 65%, specificity of 88%, and overall accuracy of 86% for pathogenic variants. False omission rate was 3%, signifying minimal error in misclassifying true PGV. A higher portion of PGV in known hereditary tumor suppressors were found to be retained with loss of heterozygosity in the tumor specimens (72%) compared with variants of uncertain significance (58%). CONCLUSION: Analyzing tumor-only data in the context of specimens' tumor cell content allows precise, systematic exclusion of somatic variants and suggests a balance between type 1 and 2 errors for identification of patients with candidate PGV for standard germline testing. Although technical or systematic errors in measuring variant allele frequency could result in incorrect inference, misestimation of specimen purity could result in inferring somatic variants as germline in somatically mutated tumor suppressor genes. A user-friendly bioinformatics application facilitates objective analysis of tumor-only data in clinical settings.

JAK/STAT of all trades: linking inflammation with cancer development, tumor progression and therapy resistance.

Inflammation is at the forefront of carcinogenesis, tumor progression and resistance to therapy. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling axis is a central pathway that mediates the cellular response to inflammation and contributes to carcinogenesis. The JAK/STAT pathway coordinates intercellular communication between tumor cells and their immune microenvironment, and JAK/STAT activation leads to the expression of a variety of proteins involved in cell proliferation, cell survival, stemness, self-renewal, evasion of immunosurveillance mechanisms and overall tumor progression. Activation of JAK/STAT signaling also mediates resistance to radiation therapy or cytotoxic agents and modulates tumor cell responses to molecularly targeted and immune modulating drugs. Despite extensive research focused on understanding its signaling mechanisms and downstream phenotypic and functional consequences in hematological disorders, the importance of JAK/STAT signaling in solid tumor initiation and progression has been underappreciated. We highlight the role of chronic inflammation in cancer, the epidemiological evidence for contribution of JAK/STAT to carcinogenesis, the current cancer prevention measures involving JAK/STAT inhibition and the impact of JAK/STAT signaling activity on cancer development, progression and treatment resistance. We also discuss recent therapeutic advances in targeting key factors within the JAK/STAT pathway with single agents and the use of these agents in combination with other targeted therapies and immune checkpoint inhibitors.

A novel and highly effective mitochondrial uncoupling drug in T-cell leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy. Despite recent advances in treatments with intensified chemotherapy regimens, relapse rates and associated morbidities remain high. In this context, metabolic dependencies have emerged as a druggable opportunity for the treatment of leukemia. Here, we tested the antileukemic effects of MB1-47, a newly developed mitochondrial uncoupling compound. MB1-47 treatment in T-ALL cells robustly inhibited cell proliferation via both cytostatic and cytotoxic effects as a result of compromised mitochondrial energy and metabolite depletion, which severely impaired nucleotide biosynthesis. Mechanistically, acute treatment with MB1-47 in primary leukemias promoted adenosine monophosphate-activated serine/threonine protein kinase (AMPK) activation and downregulation of mammalian target of rapamycin (mTOR) signaling, stalling anabolic pathways that support leukemic cell survival. Indeed, MB1-47 treatment in mice harboring either murine NOTCH1-induced primary leukemias or human T-ALL patient-derived xenografts (PDXs) led to potent antileukemic effects with a significant extension in survival without overlapping toxicities. Overall, our findings demonstrate a critical role for mitochondrial oxidative phosphorylation in T-ALL and uncover MB1-47-driven mitochondrial uncoupling as a novel therapeutic strategy for the treatment of this disease.

A Tumor Suppressor Enhancer of PTEN in T-cell development and leukemia.

Long-range oncogenic enhancers play an important role in cancer. Yet, whether similar regulation of tumor suppressor genes is relevant remains unclear. Loss of expression of PTEN is associated with the pathogenesis of various cancers, including T-cell leukemia (T-ALL). Here, we identify a highly conserved distal enhancer (PE) that interacts with the PTEN promoter in multiple hematopoietic populations, including T-cells, and acts as a hub of relevant transcription factors in T-ALL. Consistently, loss of PE leads to reduced PTEN levels in T-ALL cells. Moreover, PE-null mice show reduced Pten levels in thymocytes and accelerated development of NOTCH1-induced T-ALL. Furthermore, secondary loss of PE in established leukemias leads to accelerated progression and a gene expression signature driven by Pten loss. Finally, we uncovered recurrent deletions encompassing PE in T-ALL, which are associated with decreased PTEN levels. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in cancer.

Spleen plays a major role in DLL4-driven acute T-cell lymphoblastic leukemia.

The Notch pathway is highly active in almost all patients with T-cell acute lymphoblastic leukemia (T-ALL), but the implication of Notch ligands in T-ALL remains underexplored. Methods: We used a genetic mouse model of Notch ligand delta like 4 (DLL4)-driven T-ALL and performed thymectomies and splenectomies in those animals. We also used several patient-derived T-ALL (PDTALL) models, including one with DLL4 expression on the membrane and we treated PDTALL cells in vitro and in vivo with demcizumab, a blocking antibody against human DLL4 currently being tested in clinical trials in patients with solid cancer. Results: We show that surgical removal of the spleen abrogated T-ALL development in our preclinical DLL4-driven T-ALL mouse model. Mechanistically, we found that the spleen, and not the thymus, promoted the accumulation of circulating CD4+CD8+ T cells before T-ALL onset, suggesting that DLL4-driven T-ALL derives from these cells. Then, we identified a small subset of T-ALL patients showing higher levels of DLL4 expression. Moreover, in mice xenografted with a DLL4-positive PDTALL model, treatment with demcizumab had the same therapeutic effect as global Notch pathway inhibition using the potent γ-secretase inhibitor dibenzazepine. This result demonstrates that, in this PDTALL model, Notch pathway activity depends on DLL4 signaling, thus validating our preclinical mouse model. Conclusion: DLL4 expression in human leukemic cells can be a source of Notch activity in T-ALL, and the spleen plays a major role in a genetic mouse model of DLL4-driven T-ALL.

Gene expression of adipokines and adipokine receptors in the tumor microenvironment: associations of lower expression with more aggressive breast tumor features.

PURPOSE: Limited epidemiologic data are available on the expression of adipokines leptin (LEP) and adiponectin (ADIPOQ) and adipokine receptors (LEPR, ADIPOR1, ADIPOR2) in the breast tumor microenvironment (TME). The associations of gene expression of these biomarkers with tumor clinicopathology are not well understood. METHODS: NanoString multiplexed assays were used to assess the gene expression levels of LEP, LEPR, ADIPOQ, ADIPOR1, and ADIPOR2 within tumor tissues among 162 Black and 55 White women with newly diagnosed breast cancer. Multivariate mixed effects models were used to estimate associations of gene expression with breast tumor clinicopathology (overall and separately among Blacks). RESULTS: Black race was associated with lower gene expression of LEPR (P = 0.002) and ADIPOR1 (P = 0.01). Lower LEP, LEPR, and ADIPOQ gene expression were associated with higher tumor grade (P = 0.0007, P < 0.0001, and P < 0.0001, respectively) and larger tumor size (P < 0.0001, P = 0.0005, and P < 0.0001, respectively). Lower ADIPOQ expression was associated with ER- status (P = 0.0005), and HER2-enriched (HER2-E; P = 0.0003) and triple-negative (TN; P = 0.002) subtypes. Lower ADIPOR2 expression was associated with Ki67+ status (P = 0.0002), ER- status (P < 0.0001), PR- status (P < 0.0001), and TN subtype (P = 0.0002). Associations of lower adipokine and adipokine receptor gene expression with ER-, HER2-E, and TN subtypes were confirmed using data from The Cancer Genome Atlas (P-values < 0.005). CONCLUSION: These findings suggest that lower expression of ADIPOQ, ADIPOR2, LEP, and LEPR in the breast TME might be indicators of more aggressive breast cancer phenotypes. Validation of these findings are warranted to elucidate the role of the adipokines and adipokine receptors in long-term breast cancer prognosis.

Pan-Cancer Analysis of BRCA1 and BRCA2 Genomic Alterations and Their Association With Genomic Instability as Measured by Genome-Wide Loss of Heterozygosity.

PURPOSE: BRCA1 or BRCA2 loss of function results in homologous recombination deficiency (HRD), which is targetable by poly (ADP-ribose) polymerase (PARP) inhibitors and other DNA-damaging agents. In cancers associated with germline BRCA1/2 alterations (BRCA1/2-associated cancers: breast, ovarian, pancreatic, prostate), BRCA1/2 alterations result in HRD and are biomarkers for PARP inhibitor use. In other (non-BRCA1/2-associated) cancer types, the association between BRCA1/2 alteration and HRD is less clear. METHODS: A total of 234,154 tumor samples were sequenced by hybrid capture-based comprehensive genomic profiling. Somatic, germline, and zygosity status was determined computationally. BRCA1/2 alterations were classified as predicted germline/somatic and biallelic/monoallelic. Genome-wide loss of heterozygosity (gLOH) was evaluated as a marker of HRD. RESULTS: BRCA1/2 alterations were observed at a 4.7% frequency. BRCA1/2 mutations were predicted germline in 57.4% of BRCA1/2-associated and 37.2% of non-BRCA1/2-associated cancers. The fraction of BRCA1/2-altered cases that were biallelic was 68.7%, with a higher biallelic fraction in BRCA1/2-associated (89.9%) versus non-BRCA1/2-associated cancers (43.6%). Differences in tissue distribution of biallelic BRCA1 versus BRCA2 alterations were noted, including a higher rate of biallelic BRCA2 alteration in prostate cancer. Biallelic BRCA1/2 alteration was observed at a 3.2% frequency (BRCA1/2-associated cancers, 8.9%; non-BRCA1/2-associated cancers, 1.3%) and > 1% frequency in at least 13 cancer types. Across cancer types, biallelic BRCA1/2 alteration was associated with increased gLOH versus monoallelic or wild-type BRCA1/2; predicted germline or somatic mutations were both associated with elevated gLOH. CONCLUSION: Biallelic BRCA1/2 alterations were associated with elevated gLOH in diverse cancer types, including those not traditionally associated with BRCA1/2 cancer syndromes. Biomarker development for PARP inhibitors should integrate methods to distinguish biallelic from monoallelic BRCA1/2 status, and biallelic BRCA1/2 alteration should be broadly evaluated across cancer types as a biomarker for underlying HRD and PARP inhibitor sensitivity.

Tissue- and development-stage-specific mRNA and heterogeneous CNV signatures of human ribosomal proteins in normal and cancer samples.

We give results from a detailed analysis of human Ribosomal Protein (RP) levels in normal and cancer samples and cell lines from large mRNA, copy number variation and ribosome profiling datasets. After normalizing total RP mRNA levels per sample, we find highly consistent tissue specific RP mRNA signatures in normal and tumor samples. Multiple RP mRNA-subtypes exist in several cancers, with significant survival and genomic differences. Some RP mRNA variations among subtypes correlate with copy number loss of RP genes. In kidney cancer, RP subtypes map to molecular subtypes related to cell-of-origin. Pan-cancer analysis of TCGA data showed widespread single/double copy loss of RP genes, without significantly affecting survival. In several cancer cell lines, CRISPR-Cas9 knockout of RP genes did not affect cell viability. Matched RP ribosome profiling and mRNA data in humans and rodents stratified by tissue and development stage and were strongly correlated, showing that RP translation rates were proportional to mRNA levels. In a small dataset of human adult and fetal tissues, RP protein levels showed development stage and tissue specific heterogeneity of RP levels. Our results suggest that heterogeneous RP levels play a significant functional role in cellular physiology, in both normal and disease states.

Subclonal NT5C2 mutations are associated with poor outcomes after relapse of pediatric acute lymphoblastic leukemia.

Activating mutations in cytosolic 5'-nucleotidase II (NT5C2) are considered to drive relapse formation in acute lymphoblastic leukemia (ALL) by conferring purine analog resistance. To examine the clinical effects of NT5C2 mutations in relapsed ALL, we analyzed NT5C2 in 455 relapsed B-cell precursor ALL patients treated within the ALL-REZ BFM 2002 relapse trial using sequencing and sensitive allele-specific real-time polymerase chain reaction. We detected 110 NT5C2 mutations in 75 (16.5%) of 455 B-cell precursor ALL relapses. Two-thirds of relapses harbored subclonal mutations and only one-third harbored clonal mutations. Event-free survival after relapse was inferior in patients with relapses with clonal and subclonal NT5C2 mutations compared with those without (19% and 25% vs 53%, P < .001). However, subclonal, but not clonal, NT5C2 mutations were associated with reduced event-free survival in multivariable analysis (hazard ratio, 1.89; 95% confidence interval, 1.28-2.69; P = .001) and with an increased rate of nonresponse to relapse treatment (subclonal 32%, clonal 12%, wild type 9%, P < .001). Nevertheless, 27 (82%) of 33 subclonal NT5C2 mutations became undetectable at the time of nonresponse or second relapse, and in 10 (71%) of 14 patients subclonal NT5C2 mutations were undetectable already after relapse induction treatment. These results show that subclonal NT5C2 mutations define relapses associated with high risk of treatment failure in patients and at the same time emphasize that their role in outcome is complex and goes beyond mutant NT5C2 acting as a targetable driver during relapse progression. Sensitive, prospective identification of NT5C2 mutations is warranted to improve the understanding and treatment of this aggressive ALL relapse subtype.

All-FIT: allele-frequency-based imputation of tumor purity from high-depth sequencing data.

SUMMARY: Clinical sequencing aims to identify somatic mutations in cancer cells for accurate diagnosis and treatment. However, most widely used clinical assays lack patient-matched control DNA and additional analysis is needed to distinguish somatic and unfiltered germline variants. Such computational analyses require accurate assessment of tumor cell content in individual specimens. Histological estimates often do not corroborate with results from computational methods that are primarily designed for normal-tumor matched data and can be confounded by genomic heterogeneity and presence of sub-clonal mutations. Allele-frequency-based imputation of tumor (All-FIT) is an iterative weighted least square method to estimate specimen tumor purity based on the allele frequencies of variants detected in high-depth, targeted, clinical sequencing data. Using simulated and clinical data, we demonstrate All-FIT's accuracy and improved performance against leading computational approaches, highlighting the importance of interpreting purity estimates based on expected biology of tumors. AVAILABILITY AND IMPLEMENTATION: Freely available at http://software.khiabanian-lab.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.