In vivo interrogation of regulatory genomes reveals extensive quasi-insufficiency in cancer evolution.

In contrast to mono- or biallelic loss of tumor-suppressor function, effects of discrete gene dysregulations, as caused by non-coding (epi)genome alterations, are poorly understood. Here, by perturbing the regulatory genome in mice, we uncover pervasive roles of subtle gene expression variation in cancer evolution. Genome-wide screens characterizing 1,450 tumors revealed that such quasi-insufficiency is extensive across entities and displays diverse context dependencies, such as distinct cell-of-origin associations in T-ALL subtypes. We compile catalogs of non-coding regions linked to quasi-insufficiency, show their enrichment with human cancer risk variants, and provide functional insights by engineering regulatory alterations in mice. As such, kilo-/megabase deletions in a Bcl11b-linked non-coding region triggered aggressive malignancies, with allele-specific tumor spectra reflecting gradual gene dysregulations through modular and cell-type-specific enhancer activities. Our study constitutes a first survey toward a systems-level understanding of quasi-insufficiency in cancer and gives multifaceted insights into tumor evolution and the tissue-specific effects of non-coding mutations.

Arginine Methyltransferase PRMT7 Deregulates Expression of RUNX1 Target Genes in T-Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with no well-established prognostic biomarkers. We examined the expression of protein arginine methyltransferases across hematological malignancies and discovered high levels of PRMT7 mRNA in T-ALL, particularly in the mature subtypes of T-ALL. The genetic deletion of PRMT7 by CRISPR-Cas9 reduced the colony formation of T-ALL cells and changed arginine monomethylation patterns in protein complexes associated with the RNA and DNA processing and the T-ALL pathogenesis. Among them was RUNX1, whose target gene expression was consequently deregulated. These results suggest that PRMT7 plays an active role in the pathogenesis of T-ALL.

Therapeutic targeting of LCK tyrosine kinase and mTOR signaling in T-cell acute lymphoblastic leukemia.

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.

Expression of BCL6 in paediatric B-cell acute lymphoblastic leukaemia and association with prognosis.

B-cell lineage acute lymphoblastic leukaemia (B-ALL) is the most common paediatric malignancy. Transcription factor B-cell lymphoma 6 (BCL6) is essential to germinal centre formation and antibody affinity maturation and plays a major role in mature B-cell malignancies. More recently, it was shown to act as a critical downstream regulator in pre-BCR+ B-ALL. We investigated the expression of the BCL6 protein in a population-based cohort of paediatric B-ALL cases and detected moderate to strong positivity through immunohistochemistry in 7% of cases (8/117); however, only two of eight BCL6 cases (25%) co-expressed the ZAP70 protein. In light of these data, the subtype with active pre-BCR signalling constitutes a rare entity in paediatric B-ALL. In three independent larger cohorts with gene expression data, high BCL6 mRNA levels were associated with the TCF3-PBX1, Ph-like, NUTM1, MEF2D and PAX5-alt subgroups and the 'metagene' signature for pre-BCR-associated genes. However, higher-than-median BCL6 mRNA level alone was associated with favourable event free survival in the Nordic paediatric cohort, indicating that using BCL6 as a diagnostic marker requires careful design, and evaluation of protein level is needed alongside the genetic or transcriptomic data.

Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities.

BACKGROUND: Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. METHODS: We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. RESULTS: We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. CONCLUSIONS: Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia.

SIX6 is a TAL1-regulated transcription factor in T-ALL and associated with inferior outcome.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological malignancy driven by abnormal activity of transcription factors. Here we report an aberrant expression of the developmental transcription factor SIX6 in the TAL1-subtype of T-ALL. Our results demonstrate that the binding of TAL1 and GATA3 transcription factors into an upstream enhancer element directly regulates SIX6 expression. High expression of SIX6 was associated with inferior event-free survival within three independent patient cohorts. At a functional level, CRISPR-Cas9-mediated knockout of the SIX6 gene in TAL1 positive Jurkat cells induced changes in genes associated with the mTOR-, K-RAS-, and TNFα-related molecular signatures but did not impair cell proliferation or viability. There was also no acceleration of T-ALL development within a Myc driven zebrafish tumor model in vivo. Taken together, our results show that SIX6 belongs to the TAL1 regulatory gene network in T-ALL but is alone insufficient to influence the development or maintenance of T-ALL.

Clinicopathological features and prognostic value of SOX11 in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia is marked by aberrant transcriptional features that alter cell differentiation, self-renewal, and proliferative features. We sought to identify the transcription factors exhibiting altered and subtype-specific expression patterns in B-ALL and report here that SOX11, a developmental and neuronal transcription factor, is aberrantly expressed in the ETV6-RUNX1 and TCF3-PBX1 subtypes of acute B-cell leukemias. We show that a high expression of SOX11 leads to alterations of gene expression that are typically associated with cell adhesion, migration, and differentiation. A high expression is associated with DNA hypomethylation at the SOX11 locus and a favorable outcome. The results indicate that SOX11 expression marks a group of patients with good outcomes and thereby prompts further study of its use as a biomarker.

Overexpression of PTP4A3 in ETV6-RUNX1 acute lymphoblastic leukemia.

Cell signalling, which is often derailed in cancer, is a network of multiple interconnected pathways with numerous feedback mechanisms. Dynamics of cell signalling is intimately regulated by addition and removal of phosphate groups by kinases and phosphatases. We examined expression of members of the PTP4A family of phosphatases across acute leukemias. While expression of PTP4A1 and PTP4A2 remained relatively unchanged across diseases, PTP4A3 showed marked overexpression in ETV6-RUNX1 and BCR-ABL1 subtypes of precursor B cell acute lymphoblastic leukemia. We show that PTP4A3 is regulated by the ETV6-RUNX1 fusion, but noticed no marked impact on cell viability either after PTP4A3 silencing or treatment with a PTP4A3 inhibitor. Regulation of PTP4A3 expression is altered in specific subgroups of acute leukemias and this is likely brought about by expression of the aberrant fusion genes.

Genome-wide repression of eRNA and target gene loci by the ETV6-RUNX1 fusion in acute leukemia.

Approximately 20%-25% of childhood acute lymphoblastic leukemias carry the ETV6-RUNX1 (E/R) fusion gene, a fusion of two central hematopoietic transcription factors, ETV6 (TEL) and RUNX1 (AML1). Despite its prevalence, the exact genomic targets of E/R have remained elusive. We evaluated gene loci and enhancers targeted by E/R genome-wide in precursor B acute leukemia cells using global run-on sequencing (GRO-seq). We show that expression of the E/R fusion leads to widespread repression of RUNX1 motif-containing enhancers at its target gene loci. Moreover, multiple super-enhancers from the CD19+/CD20+-lineage were repressed, implicating a role in impediment of lineage commitment. In effect, the expression of several genes involved in B cell signaling and adhesion was down-regulated, and the repression depended on the wild-type DNA-binding Runt domain of RUNX1. We also identified a number of E/R-regulated annotated and de novo noncoding genes. The results provide a comprehensive genome-wide mapping between E/R-regulated key regulatory elements and genes in precursor B cell leukemia that disrupt normal B lymphopoiesis.

Transcription-coupled genetic instability marks acute lymphoblastic leukemia structural variation hotspots.

Progression of malignancy to overt disease requires multiple genetic hits. Activation-induced deaminase (AID) can drive lymphomagenesis by generating off-target DNA breaks at loci that harbor highly active enhancers and display convergent transcription. The first active transcriptional profiles from acute lymphoblastic leukemia (ALL) patients acquired here reveal striking similarity at structural variation (SV) sites. Specific transcriptional features, namely convergent transcription and Pol2 stalling, were detected at breakpoints. The overlap was most prominent at SV with recognition motifs for the recombination activating genes (RAG). We present signal feature analysis to detect vulnerable regions and quantified from human cells how convergent transcription contributes to R-loop generation and RNA polymerase stalling. Wide stalling regions were characterized by high DNAse hypersensitivity and unusually broad H3K4me3 signal. Based on 1382 pre-B-ALL patients, the ETV6-RUNX1 fusion positive patients had over ten-fold elevation in RAG1 while high expression of AID marked pre-B-ALL lacking common cytogenetic changes.