Therapeutic targeting of EP300/CBP by bromodomain inhibition in hematologic malignancies.

CCS1477 (inobrodib) is a potent, selective EP300/CBP bromodomain inhibitor which induces cell-cycle arrest and differentiation in hematologic malignancy model systems. In myeloid leukemia cells, it promotes rapid eviction of EP300/CBP from an enhancer subset marked by strong MYB occupancy and high H3K27 acetylation, with downregulation of the subordinate oncogenic network and redistribution to sites close to differentiation genes. In myeloma cells, CCS1477 induces eviction of EP300/CBP from FGFR3, the target of the common (4; 14) translocation, with redistribution away from IRF4-occupied sites to TCF3/E2A-occupied sites. In a subset of patients with relapsed or refractory disease, CCS1477 monotherapy induces differentiation responses in AML and objective responses in heavily pre-treated multiple myeloma. In vivo preclinical combination studies reveal synergistic responses to treatment with standard-of-care agents. Thus, CCS1477 exhibits encouraging preclinical and early-phase clinical activity by disrupting recruitment of EP300/CBP to enhancer networks occupied by critical transcription factors.

A TIAM1-TRIM28 complex mediates epigenetic silencing of protocadherins to promote migration of lung cancer cells.

Lung cancer is the leading cause of cancer deaths. Its high mortality is associated with high metastatic potential. Here, we show that the RAC1-selective guanine nucleotide exchange factor T cell invasion and metastasis-inducing protein 1 (TIAM1) promotes cell migration and invasion in the most common subtype of lung cancer, non-small-cell lung cancer (NSCLC), through an unexpected nuclear function. We show that TIAM1 interacts with TRIM28, a master regulator of gene expression, in the nucleus of NSCLC cells. We reveal that a TIAM1-TRIM28 complex promotes epithelial-to-mesenchymal transition, a phenotypic switch implicated in cell migration and invasion. This occurs through H3K9me3-induced silencing of protocadherins and by decreasing E-cadherin expression, thereby antagonizing cell-cell adhesion. Consistently, TIAM1 or TRIM28 depletion suppresses the migration of NSCLC cells, while migration is restored by the simultaneous depletion of protocadherins. Importantly, high nuclear TIAM1 in clinical specimens is associated with advanced-stage lung adenocarcinoma, decreased patient survival, and inversely correlates with E-cadherin expression.

Differentiation block in acute myeloid leukemia regulated by intronic sequences of FTO.

Iroquois transcription factor gene IRX3 is highly expressed in 20-30% of acute myeloid leukemia (AML) and contributes to the pathognomonic differentiation block. Intron 8 FTO sequences ∼220kB downstream of IRX3 exhibit histone acetylation, DNA methylation, and contacts with the IRX3 promoter, which correlate with IRX3 expression. Deletion of these intronic elements confirms a role in positively regulating IRX3. RNAseq revealed long non-coding (lnc) transcripts arising from this locus. FTO-lncAML knockdown (KD) induced differentiation of AML cells, loss of clonogenic activity, and reduced FTO intron 8:IRX3 promoter contacts. While both FTO-lncAML KD and IRX3 KD induced differentiation, FTO-lncAML but not IRX3 KD led to HOXA downregulation suggesting transcript activity in trans. FTO-lncAMLhigh AML samples expressed higher levels of HOXA and lower levels of differentiation genes. Thus, a regulatory module in FTO intron 8 consisting of clustered enhancer elements and a long non-coding RNA is active in human AML, impeding myeloid differentiation.

Identification of curaxin as a potential new therapeutic for JAK2 V617F mutant patients.

Myelofibrosis is a myeloproliferative neoplasm (MPN) which typically results in reduced length and quality of life due to systemic symptoms and blood count changes arising from fibrotic changes in the bone marrow. While the JAK2 inhibitor ruxolitinib provides some clinical benefit, there remains a substantial unmet need for novel targeted therapies to better modify the disease process or eradicate the cells at the heart of myelofibrosis pathology. Repurposing drugs bypasses many of the hurdles present in drug development, such as toxicity and pharmacodynamic profiling. To this end we undertook a re-analysis of our pre-existing proteomic data sets to identify perturbed biochemical pathways and their associated drugs/inhibitors to potentially target the cells driving myelofibrosis. This approach identified CBL0137 as a candidate for targeting Jak2 mutation-driven malignancies. CBL0137 is a drug derived from curaxin targeting the Facilitates Chromatin Transcription (FACT) complex. It is reported to trap the FACT complex on chromatin thereby activating p53 and inhibiting NF-kB activity. We therefore assessed the activity of CBL0137 in primary patient samples and murine models of Jak2-mutated MPN and found it preferentially targets CD34+ stem and progenitor cells from myelofibrosis patients by comparison with healthy control cells. Further we investigate its mechanism of action in primary haemopoietic progenitor cells and demonstrate its ability to reduce splenomegaly and reticulocyte number in a transgenic murine model of myeloproliferative neoplasms.

A three-gene leukaemic stem cell signature score is robustly prognostic in chronic myelomonocytic leukaemia.

Leukaemic stem cell (LSC) gene expression has recently been linked to prognosis in patients with acute myeloid leukaemia (17-gene LSC score, LSC-17) and myelodysplastic syndromes. Although chronic myelomonocytic leukaemia (CMML) is regarded as a stem cell disorder, the clinical and biological impact of LSCs on CMML patients remains elusive. Making use of multiple independent validation cohorts, we here describe a concise three-gene expression signature (LSC-3, derived from the LSC-17 score) as an independent and robust prognostic factor for leukaemia-free and overall survival in CMML. We propose that LSC-3 could be used to supplement existing risk stratification systems, to improve prognostic performance and guide management decisions.

HMG20B stabilizes association of LSD1 with GFI1 on chromatin to confer transcription repression and leukemia cell differentiation block.

Pharmacologic inhibition of LSD1 induces molecular and morphologic differentiation of blast cells in acute myeloid leukemia (AML) patients harboring MLL gene translocations. In addition to its demethylase activity, LSD1 has a critical scaffolding function at genomic sites occupied by the SNAG domain transcription repressor GFI1. Importantly, inhibitors block both enzymatic and scaffolding activities, in the latter case by disrupting the protein:protein interaction of GFI1 with LSD1. To explore the wider consequences of LSD1 inhibition on the LSD1 protein complex we applied mass spectrometry technologies. We discovered that the interaction of the HMG-box protein HMG20B with LSD1 was also disrupted by LSD1 inhibition. Downstream investigations revealed that HMG20B is co-located on chromatin with GFI1 and LSD1 genome-wide; the strongest HMG20B binding co-locates with the strongest GFI1 and LSD1 binding. Functional assays demonstrated that HMG20B depletion induces leukemia cell differentiation and further revealed that HMG20B is required for the transcription repressor activity of GFI1 through stabilizing LSD1 on chromatin at GFI1 binding sites. Interaction of HMG20B with LSD1 is through its coiled-coil domain. Thus, HMG20B is a critical component of the GFI1:LSD1 transcription repressor complex which contributes to leukemia cell differentiation block.

Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity.

BACKGROUND: Disease relapse remains common following treatment of acute myeloid leukemia (AML) and is due to chemoresistance of leukemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. We sought to characterise in vivo chemoresistant blasts by studying the transcriptional and genetic features of blasts from before and shortly after induction chemotherapy using paired samples from six patients with primary refractory AML. METHODS: Leukemic blasts were isolated by fluorescence-activated cell sorting. Fluorescence in situ hybridization (FISH), targeted genetic sequencing and detailed immunophenotypic analysis were used to confirm that sorted cells were leukemic. Sorted blasts were subjected to RNA sequencing. Lentiviral vectors expressing short hairpin RNAs were used to assess the effect of FOXM1 knockdown on colony forming capacity, proliferative capacity and apoptosis in cell lines, primary AML cells and CD34+ cells from healthy donors. RESULTS: Molecular genetic analysis revealed early clonal selection occurring after induction chemotherapy. Immunophenotypic characterisation found leukemia-associated immunophenotypes in all cases that persisted following treatment. Despite the genetic heterogeneity of the leukemias studied, transcriptional analysis found concerted changes in gene expression in resistant blasts. Remarkably, the gene expression signature suggested that post-chemotherapy blasts were more proliferative than those at presentation. Resistant blasts also appeared less differentiated and expressed leukemia stem cell (LSC) maintenance genes. However, the proportion of immunophenotypically defined LSCs appeared to decrease following treatment, with implications for the targeting of these cells on the basis of cell surface antigen expression. The refractory gene signature was highly enriched with targets of the transcription factor FOXM1. shRNA knockdown experiments demonstrated that the viability of primary AML cells, but not normal CD34+ cells, depended on FOXM1 expression. CONCLUSIONS: We found that chemorefractory blasts from leukemias with varied genetic backgrounds expressed a common transcriptional program. In contrast to the notion that LSC quiescence confers resistance to chemotherapy we find that refractory blasts are both actively proliferating and enriched with LSC maintenance genes. Using primary patient material from a relevant clinical context we also provide further support for the role of FOXM1 in chemotherapy resistance, proliferation and stem cell function in AML.

Enhancer recruitment of transcription repressors RUNX1 and TLE3 by mis-expressed FOXC1 blocks differentiation in acute myeloid leukemia.

Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in ∼30% of HOXAhigh acute myeloid leukemia (AML) cases to confer blocked monocyte/macrophage differentiation. Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect.

Targeted nanopore sequencing for the identification of ABCB1 promoter translocations in cancer.

BACKGROUND: Resistance to chemotherapy is the most common cause of treatment failure in acute myeloid leukemia (AML) and the drug efflux pump ABCB1 is a critical mediator. Recent studies have identified promoter translocations as common drivers of high ABCB1 expression in recurrent, chemotherapy-treated high-grade serous ovarian cancer (HGSC) and breast cancer. These fusions place ABCB1 under the control of a strong promoter while leaving its open reading frame intact. The mechanisms controlling high ABCB1 expression in AML are largely unknown. We therefore established an experimental system and analysis pipeline to determine whether promoter translocations account for high ABCB1 expression in cases of relapsed human AML. METHODS: The human AML cell line THP-1 was used to create a model of chemotherapy resistance in which ABCB1 expression was driven by a promoter fusion. The THP-1 model was used to establish a targeted nanopore long-read sequencing approach that was then applied to cases of ABCB1high HGSC and AML. H3K27Ac ChIP sequencing was used to assess the activity of native promoters in cases of ABCB1high AML. RESULTS: Prolonged in vitro daunorubicin exposure induced activating ABCB1 promoter translocations in human THP-1 AML cells, similar to those recently described in recurrent high-grade serous ovarian and breast cancers. Targeted nanopore sequencing proved an efficient method for identifying ABCB1 structural variants in THP-1 AML cells and HGSC; the promoter translocations identified in HGSC were both previously described and novel. In contrast, activating ABCB1 promoter translocations were not identified in ABCB1high AML; instead H3K27Ac ChIP sequencing demonstrated active native promoters in all cases studied. CONCLUSIONS: Despite frequent high level expression of ABCB1 in relapsed primary AML we found no evidence of ABCB1 translocations and instead confirmed high-level activity of native ABCB1 promoters, consistent with endogenous regulation.

EVI1 phosphorylation at S436 regulates interactions with CtBP1 and DNMT3A and promotes self-renewal.

The transcriptional regulator EVI1 has an essential role in early development and haematopoiesis. However, acute myeloid leukaemia (AML) driven by aberrantly high EVI1 expression has very poor prognosis. To investigate the effects of post-translational modifications on EVI1 function, we carried out a mass spectrometry (MS) analysis of EVI1 in AML and detected dynamic phosphorylation at serine 436 (S436). Wild-type EVI1 (EVI1-WT) with S436 available for phosphorylation, but not non-phosphorylatable EVI1-S436A, conferred haematopoietic progenitor cell self-renewal and was associated with significantly higher organised transcriptional patterns. In silico modelling of EVI1-S436 phosphorylation showed reduced affinity to CtBP1, and CtBP1 showed reduced interaction with EVI1-WT compared with EVI1-S436A. The motif harbouring S436 is a target of CDK2 and CDK3 kinases, which interacted with EVI1-WT. The methyltransferase DNMT3A bound preferentially to EVI1-WT compared with EVI1-S436A, and a hypomethylated cell population associated by EVI1-WT expression in murine haematopoietic progenitors is not maintained with EVI1-S436A. These data point to EVI1-S436 phosphorylation directing functional protein interactions for haematopoietic self-renewal. Targeting EVI1-S436 phosphorylation may be of therapeutic benefit when treating EVI1-driven leukaemia.

Pre-clinical activity of combined LSD1 and mTORC1 inhibition in MLL-translocated acute myeloid leukaemia.

The histone demethylase lysine-specific demethylase 1 (LSD1 or KDM1A) has emerged as a candidate therapeutic target in acute myeloid leukaemia (AML); tranylcypromine-derivative inhibitors induce loss of clonogenic activity and promote differentiation, in particular in the MLL-translocated molecular subtype of AML. In AML, the use of drugs in combination often delivers superior clinical activity. To identify genes and cellular pathways that collaborate with LSD1 to maintain the leukaemic phenotype, and which could be targeted by combination therapies, we performed a genome-wide CRISPR-Cas9 dropout screen. We identified multiple components of the amino acid sensing arm of mTORC1 signalling-RRAGA, MLST8, WDR24 and LAMTOR2-as cellular sensitizers to LSD1 inhibition. Knockdown of mTORC1 components, or mTORC1 pharmacologic inhibition, in combination with LSD1 inhibition enhanced differentiation in both cell line and primary cell settings, in vitro and in vivo, and substantially reduced the frequency of clonogenic primary human AML cells in a modelled minimal residual disease setting. Synergistic upregulation of a set of transcription factor genes associated with terminal monocytic lineage differentiation was observed. Thus, dual mTORC1 and LSD1 inhibition represents a candidate combination approach for enhanced differentiation in MLL-translocated AML which could be evaluated in early phase clinical trials.

Enhancer Activation by Pharmacologic Displacement of LSD1 from GFI1 Induces Differentiation in Acute Myeloid Leukemia.

Pharmacologic inhibition of LSD1 promotes blast cell differentiation in acute myeloid leukemia (AML) with MLL translocations. The assumption has been that differentiation is induced through blockade of LSD1's histone demethylase activity. However, we observed that rapid, extensive, drug-induced changes in transcription occurred without genome-wide accumulation of the histone modifications targeted for demethylation by LSD1 at sites of LSD1 binding and that a demethylase-defective mutant rescued LSD1 knockdown AML cells as efficiently as wild-type protein. Rather, LSD1 inhibitors disrupt the interaction of LSD1 and RCOR1 with the SNAG-domain transcription repressor GFI1, which is bound to a discrete set of enhancers located close to transcription factor genes that regulate myeloid differentiation. Physical separation of LSD1/RCOR1 from GFI1 is required for drug-induced differentiation. The consequent inactivation of GFI1 leads to increased enhancer histone acetylation within hours, which directly correlates with the upregulation of nearby subordinate genes.

Extraction and analysis of signatures from the Gene Expression Omnibus by the crowd.

Gene expression data are accumulating exponentially in public repositories. Reanalysis and integration of themed collections from these studies may provide new insights, but requires further human curation. Here we report a crowdsourcing project to annotate and reanalyse a large number of gene expression profiles from Gene Expression Omnibus (GEO). Through a massive open online course on Coursera, over 70 participants from over 25 countries identify and annotate 2,460 single-gene perturbation signatures, 839 disease versus normal signatures, and 906 drug perturbation signatures. All these signatures are unique and are manually validated for quality. Global analysis of these signatures confirms known associations and identifies novel associations between genes, diseases and drugs. The manually curated signatures are used as a training set to develop classifiers for extracting similar signatures from the entire GEO repository. We develop a web portal to serve these signatures for query, download and visualization.