Coordinated alterations in RNA splicing and epigenetic regulation drive leukaemogenesis.

Transcription and pre-mRNA splicing are key steps in the control of gene expression and mutations in genes regulating each of these processes are common in leukaemia1,2. Despite the frequent overlap of mutations affecting epigenetic regulation and splicing in leukaemia, how these processes influence one another to promote leukaemogenesis is not understood and, to our knowledge, there is no functional evidence that mutations in RNA splicing factors initiate leukaemia. Here, through analyses of transcriptomes from 982 patients with acute myeloid leukaemia, we identified frequent overlap of mutations in IDH2 and SRSF2 that together promote leukaemogenesis through coordinated effects on the epigenome and RNA splicing. Whereas mutations in either IDH2 or SRSF2 imparted distinct splicing changes, co-expression of mutant IDH2 altered the splicing effects of mutant SRSF2 and resulted in more profound splicing changes than either mutation alone. Consistent with this, co-expression of mutant IDH2 and SRSF2 resulted in lethal myelodysplasia with proliferative features in vivo and enhanced self-renewal in a manner not observed with either mutation alone. IDH2 and SRSF2 double-mutant cells exhibited aberrant splicing and reduced expression of INTS3, a member of the integrator complex3, concordant with increased stalling of RNA polymerase II (RNAPII). Aberrant INTS3 splicing contributed to leukaemogenesis in concert with mutant IDH2 and was dependent on mutant SRSF2 binding to cis elements in INTS3 mRNA and increased DNA methylation of INTS3. These data identify a pathogenic crosstalk between altered epigenetic state and splicing in a subset of leukaemias, provide functional evidence that mutations in splicing factors drive myeloid malignancy development, and identify spliceosomal changes as a mediator of IDH2-mutant leukaemogenesis.

Conserved role of FOXC1 in TNBC is parallel to FOXA1 in ER+ breast cancer.

Triple-negative breast cancer (TNBC) is characterized by lack of the estrogen (ER) receptor, progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2), and standard receptor-targeted therapies are ineffective. FOXC1, a transcription factor aberrantly overexpressed in many cancers, drives growth, metastasis, and stem-cell-like properties in TNBC. However, the molecular function of FOXC1 is unknown, partly due to heterogeneity of TNBC. Here, we show that although FOXC1 regulates many cancer hallmarks in TNBC, its function is varied in different cell lines, highlighted by the differential response to CDK4/6 inhibitors upon FOXC1 loss. Despite this functional heterogeneity, we show that FOXC1 regulates key oncogenes and tumor suppressors and identify a set of core FOXC1 peaks conserved across TNBC cell lines. We identify the ER-associated and drug-targetable nuclear receptor NR2F2 as a cofactor of FOXC1. Finally, we show that core FOXC1 targets in TNBC are regulated in parallel by the pioneer factor FOXA1 and the nuclear receptor NR2F2 in ER + breast cancer.

CRISPR-based large-scale modeling of loss-of-function mutations to investigate mechanisms of stress resistance in cancer.

Dissecting mechanisms driving subclone expansion in primary cancers has been challenging. Here, we present a protocol to systematically disrupt entire gene networks and assess the functional impact of this perturbation on cancer cell fitness. By combining arrayed CRISPR libraries and high-content microscopy, we describe steps to identify classes of genes whose inactivation promotes resistance to environmental challenges faced by cancer cells during tumor growth or upon therapy. A proof-of-principle interrogation of the epigenetic regulatory network is described. For complete details on the use and execution of this protocol, please refer to Loukas et al. (2022).1.

Selective advantage of epigenetically disrupted cancer cells via phenotypic inertia.

The evolution of established cancers is driven by selection of cells with enhanced fitness. Subclonal mutations in numerous epigenetic regulator genes are common across cancer types, yet their functional impact has been unclear. Here, we show that disruption of the epigenetic regulatory network increases the tolerance of cancer cells to unfavorable environments experienced within growing tumors by promoting the emergence of stress-resistant subpopulations. Disruption of epigenetic control does not promote selection of genetically defined subclones or favor a phenotypic switch in response to environmental changes. Instead, it prevents cells from mounting an efficient stress response via modulation of global transcriptional activity. This "transcriptional numbness" lowers the probability of cell death at early stages, increasing the chance of long-term adaptation at the population level. Our findings provide a mechanistic explanation for the widespread selection of subclonal epigenetic-related mutations in cancer and uncover phenotypic inertia as a cellular trait that drives subclone expansion.

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.

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.

Enhancer recruitment of a RUNX1, HDAC1 and TLE3 co-repressor complex by mis-expressed FOXC1 blocks differentiation in acute myeloid leukemia.

Tissue-inappropriate expression of FOXC1 (Forkhead Box C1) in acute myeloid leukemia confers a monocyte/macrophage lineage differentiation block. We discovered that FOXC1 interacts with RUNX1 (Runt-Related Transcription Factor 1) to stabilize a RUNX1, HDAC1 (Histone Deacetylase 1) and TLE3 (Transducin-like enhancer protein 3) repressor complex at enhancers controlling myeloid differentiation genes.

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.

A stress-responsive enhancer induces dynamic drug resistance in acute myeloid leukemia.

The drug efflux pump ABCB1 is a key driver of chemoresistance, and high expression predicts treatment failure in acute myeloid leukemia (AML). In this study, we identified and functionally validated the network of enhancers that controls expression of ABCB1. We show that exposure of leukemia cells to daunorubicin activated an integrated stress response-like transcriptional program to induce ABCB1 through remodeling and activation of an ATF4-bound, stress-responsive enhancer. Protracted stress primed enhancers for rapid increases in activity following re-exposure of cells to daunorubicin, providing an epigenetic memory of prior drug treatment. In primary human AML, exposure of fresh blast cells to daunorubicin activated the stress-responsive enhancer and led to dose-dependent induction of ABCB1. Dynamic induction of ABCB1 by diverse stressors, including chemotherapy, facilitated escape of leukemia cells from targeted third-generation ABCB1 inhibition, providing an explanation for the failure of ABCB1 inhibitors in clinical trials. Stress-induced upregulation of ABCB1 was mitigated by combined use of the pharmacologic inhibitors U0126 and ISRIB, which inhibit stress signaling and have potential for use as adjuvants to enhance the activity of ABCB1 inhibitors.

Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer caused by the deregulation of key T-cell developmental pathways, including Notch signaling. Aberrant Notch signaling in T-ALL occurs by NOTCH1 gain-of-function mutations and by NOTCH3 overexpression. Although NOTCH3 is assumed as a Notch1 target, machinery driving its transcription in T-ALL is undefined in leukemia subsets lacking Notch1 activation. Here, we found that the binding of the intracellular Notch3 domain, as well as of the activated Notch1 fragment, to the NOTCH3 gene locus led to the recruitment of the H3K27 modifiers JMJD3 and p300, and it was required to preserve transcriptional permissive/active H3K27 marks and to sustain NOTCH3 gene expression levels. Consistently, pharmacological inhibition of JMJD3 by GSKJ4 treatment or of p300 by A-485 decreased the levels of expression of NOTCH3, NOTCH1 and of the Notch target genes DELTEX1 and c-Myc and abrogated cell viability in both Notch1- and Notch3-dependent T-cell contexts. Notably, re-introduction of exogenous Notch1, Notch3 as well as c-Myc partially rescued cells from anti-growth effects induced by either treatment. Overall our findings indicate JMJD3 and p300 as general Notch1 and Notch3 signaling co-activators in T-ALL and suggest further investigation on the potential therapeutic anti-leukemic efficacy of their enzymatic inhibition in Notch/c-Myc axis-related cancers and diseases.

Revert the SIRT: Normalizing SIRT1 Activity in Myelodysplastic Stem Cells.

Myelodysplastic syndromes are hematologic malignancies with few treatment options and a propensity to transform to acute myeloid leukemia. In this issue of Cell Stem Cell, Sun et al. (2018) report that low SIRT1 levels in myelodysplastic stem cells contribute to aberrant self-renewal through enabling hyperacetylation and reduced activity of TET2.

Derepression of the Iroquois Homeodomain Transcription Factor Gene IRX3 Confers Differentiation Block in Acute Leukemia.

The Iroquois homeodomain transcription factor gene IRX3 is expressed in the developing nervous system, limb buds, and heart, and transcript levels specify obesity risk in humans. We now report a functional role for IRX3 in human acute leukemia. Although transcript levels are very low in normal human bone marrow cells, high IRX3 expression is found in ∼30% of patients with acute myeloid leukemia (AML), ∼50% with T-acute lymphoblastic leukemia, and ∼20% with B-acute lymphoblastic leukemia, frequently in association with high-level HOXA gene expression. Expression of IRX3 alone was sufficient to immortalize hematopoietic stem and progenitor cells (HSPCs) in myeloid culture and induce lymphoid leukemias in vivo. IRX3 knockdown induced terminal differentiation of AML cells. Combined IRX3 and Hoxa9 expression in murine HSPCs impeded normal T-progenitor differentiation in lymphoid culture and substantially enhanced the morphologic and phenotypic differentiation block of AML in myeloid leukemia transplantation experiments through suppression of a terminal myelomonocytic program. Likewise, in cases of primary human AML, high IRX3 expression is strongly associated with reduced myelomonocytic differentiation. Thus, tissue-inappropriate derepression of IRX3 contributes significantly to the block in differentiation, which is the pathognomonic feature of human acute leukemias.

Identification of a novel chalcone derivative that inhibits Notch signaling in T-cell acute lymphoblastic leukemia.

Notch signaling is considered a rational target in the therapy of several cancers, particularly those harbouring Notch gain of function mutations, including T-cell acute lymphoblastic leukemia (T-ALL). Although currently available Notch-blocking agents are showing anti-tumor activity in preclinical studies, they are not effective in all the patients and often cause severe side-effects, limiting their widespread therapeutic use. Here, by functional and biological analysis of the most representative molecules of an in house library of natural products, we have designed and synthetized the chalcone-derivative 8 possessing Notch inhibitory activity at low micro molar concentration in T-ALL cell lines. Structure-activity relationships were afforded for the chalcone scaffold. Short term treatments with compound 8 resulted in a dose-dependent decrease of Notch signaling activity, halted cell cycle progression and induced apoptosis, thus affecting leukemia cell growth. Taken together, our data indicate that 8 is a novel Notch inhibitor, candidate for further investigation and development as an additional therapeutic option against Notch-dependent cancers.