Epigenetic control over the cell-intrinsic immune response antagonizes self-renewal in acute myeloid leukemia.

ABSTRACT: Epigenetic modulation of the cell-intrinsic immune response holds promise as a therapeutic approach for leukemia. However, current strategies designed for transcriptional activation of endogenous transposons and subsequent interferon type-I (IFN-I) response, show limited clinical efficacy. Histone lysine methylation is an epigenetic signature in IFN-I response associated with suppression of IFN-I and IFN-stimulated genes, suggesting histone demethylation as key mechanism of reactivation. In this study, we unveil the histone demethylase PHF8 as a direct initiator and regulator of cell-intrinsic immune response in acute myeloid leukemia (AML). Site-specific phosphorylation of PHF8 orchestrates epigenetic changes that upregulate cytosolic RNA sensors, particularly the TRIM25-RIG-I-IFIT5 axis, thereby triggering the cellular IFN-I response-differentiation-apoptosis network. This signaling cascade largely counteracts differentiation block and growth of human AML cells across various disease subtypes in vitro and in vivo. Through proteome analysis of over 200 primary AML bone marrow samples, we identify a distinct PHF8/IFN-I signature in half of the patient population, without significant associations with known clinically or genetically defined AML subgroups. This profile was absent in healthy CD34+ hematopoietic progenitor cells, suggesting therapeutic applicability in a large fraction of patients with AML. Pharmacological support of PHF8 phosphorylation significantly impairs the growth in samples from patients with primary AML. These findings provide novel opportunities for harnessing the cell-intrinsic immune response in the development of immunotherapeutic strategies against AML.

The neuropeptide receptor calcitonin receptor-like (CALCRL) is a potential therapeutic target in acute myeloid leukemia.

Calcitonin receptor-like (CALCRL) is a G-protein-coupled neuropeptide receptor involved in the regulation of blood pressure, angiogenesis, cell proliferation, and apoptosis, and is currently emerging as a novel target for the treatment of migraine. This study characterizes the role of CALCRL in acute myeloid leukemia (AML). We analyzed CALCRL expression in collectively more than 1500 well-characterized AML patients from five international cohorts (AMLCG, HOVON, TCGA, Leucegene, and UKM) and evaluated associations with survival. In the AMLCG analytic cohort, increasing transcript levels of CALCRL were associated with decreasing complete remission rates (71.5%, 53.7%, 49.6% for low, intermediate, high CALCRL expression), 5-year overall (43.1%, 26.2%, 7.1%), and event-free survival (29.9%, 15.8%, 4.7%) (all P < 0.001). CALCRL levels remained associated with all endpoints on multivariable regression analyses. The prognostic impact was confirmed in all validation sets. Genes highly expressed in CALCRLhigh AML were significantly enriched in leukemic stem cell signatures and CALCRL levels were positively linked to the engraftment capacity of primary patient samples in immunocompromised mice. CRISPR-Cas9-mediated knockout of CALCRL significantly impaired colony formation in human myeloid leukemia cell lines. Overall, our study demonstrates that CALCRL predicts outcome beyond existing risk factors and is a potential therapeutic target in AML.

RNA-Guided CRISPR-Cas9 System-Mediated Engineering of Acute Myeloid Leukemia Mutations.

Current acute myeloid leukemia (AML) disease models face severe limitations because most of them induce un-physiological gene expressions that do not represent conditions in AML patients and/or depend on external promoters for regulation of gene expression/repression. Furthermore, many AML models are based on reciprocal chromosomal translocations that only reflect the minority of AML patients, whereas more than 50% of patients have a normal karyotype. The majority of AML, however, is driven by somatic mutations. Thus, identification as well as a detailed molecular and functional characterization of the role of these driver mutations via improved AML models is required for better approaches toward novel targeted therapies. Using the IDH2 R140Q mutation as a model, we present a new effective methodology here using the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to reproduce or remove AML-associated mutations in or from human leukemic cells, respectively, via introduction of a DNA template at the endogenous gene locus via homologous recombination. Our technology represents a precise way for AML modeling to gain insights into AML development and progression and provides a basis for future therapeutic approaches.

p73 and IGF1R Regulate Emergence of Aggressive Cancer Stem-like Features via miR-885-5p Control.

Cancer stem-like cells (CSC) have been proposed to promote cancer progression by initiating tumor growth at distant sites, suggesting that stem-like cell features can support metastatic efficiency. Here, we demonstrate that oncogenic DNp73, a dominant-negative variant of the tumor-suppressor p73, confers cancer cells with enhanced stem-like properties. DNp73 overexpression in noninvasive melanoma and lung cancer cells increased anchorage-independent growth and elevated the expression of the pluripotency factors CD133, Nanog, and Oct4. Conversely, DNp73 depletion in metastatic cells downregulated stemness genes, attenuated sphere formation and reduced the tumor-initiating capability of spheroids in tumor xenograft models. Mechanistic investigations indicated that DNp73 acted by attenuating expression of miR-885-5p, a direct regulator of the IGF1 receptor (IGF1R) responsible for stemness marker expression. Modulating this pathway was sufficient to enhance chemosensitivity, overcoming DNp73-mediated drug resistance. Clinically, we established a correlation between low p73 function and high IGF1R/CD133/Nanog/Oct4 levels in melanoma specimens that associated with reduced patient survival. Our work shows how DNp73 promotes cancer stem-like features and provides a mechanistic rationale to target the DNp73-IGF1R cascade as a therapeutic strategy to eradicate CSC.

Epigenetic factor EPC1 is a master regulator of DNA damage response by interacting with E2F1 to silence death and activate metastasis-related gene signatures.

Transcription factor E2F1 is a key regulator of cell proliferation and apoptosis. Recently, it has been shown that aberrant E2F1 expression often detectable in advanced cancers contributes essentially to cancer cell propagation and characterizes the aggressive potential of a tumor. Conceptually, this requires a subset of malignant cells capable of evading apoptotic death through anticancer drugs. The molecular mechanism by which the pro-apoptotic activity of E2F1 is antagonized is widely unclear. Here we report a novel function for EPC1 (enhancer of polycomb homolog 1) in DNA damage protection. Depletion of EPC1 potentiates E2F1-mediated apoptosis in response to genotoxic treatment and abolishes tumor cell motility. We found that E2F1 directly binds to the EPC1 promoter and EPC1 vice versa physically interacts with bifunctional E2F1 to modulate its transcriptional activity in a target gene-specific manner. Remarkably, nuclear-colocalized EPC1 activates E2F1 to upregulate the expression of anti-apoptotic survival genes such as BCL-2 or Survivin/BIRC5 and inhibits death-inducing targets. The uncovered cooperativity between EPC1 and E2F1 triggers a metastasis-related gene signature in advanced cancers that predicts poor patient survival. These findings unveil a novel oncogenic function of EPC1 for inducing the switch into tumor progression-relevant gene expression that may help to set novel therapies.

Eradication of metastatic melanoma through cooperative expression of RNA-based HDAC1 inhibitor and p73 by oncolytic adenovirus.

Malignant melanoma is a highly aggressive cancer that retains functional p53 and p73, and drug unresponsiveness largely depends on defects in death pathways after epigenetic gene silencing in conjunction with an imbalanced p73/DNp73 ratio. We constructed oncolytic viruses armed with an inhibitor of deacetylation and/or p73 to specifically target metastatic cancer. Arming of the viruses is aimed at lifting epigenetic blockage and re-opening apoptotic programs in a staggered manner enabling both, efficient virus replication and balanced destruction of target cells through apoptosis. Our results showed that cooperative expression of shHDAC1 and p73 efficiently enhances apoptosis induction and autophagy of infected cells which reinforces progeny production. In vitro analyses revealed 100% cytotoxicity after infecting cells with OV.shHDAC1.p73 at a lower virus dose compared to control viruses. Intriguingly, OV.shHDAC1.p73 acts as a potent inhibitor of highly metastatic xenograft tumors in vivo. Tumor expansion was significantly reduced after intratumoral injection of 3 x 108 PFU of either OV.shHDAC1 or OV.p73 and, most important, complete regression could be achieved in 100 % of tumors treated with OV.shHDAC1.p73. Our results point out that the combination of high replication capacity and simultaneous restoration of cell death routes significantly enhance antitumor activity.

Association of RHAMM with E2F1 promotes tumour cell extravasation by transcriptional up-regulation of fibronectin.

Dissemination of cancer cells from primary to distant sites is a complex process; little is known about the genesis of metastatic changes during disease development. Here we show that the metastatic potential of E2F1-dependent circulating tumour cells (CTCs) relies on a novel function of the hyaluronan-mediated motility receptor RHAMM. E2F1 directly up-regulates RHAMM, which in turn acts as a co-activator of E2F1 to stimulate expression of the extracellular matrix protein fibronectin. Enhanced fibronectin secretion links E2F1/RHAMM transcriptional activity to integrin-ß1-FAK signalling associated with cytoskeletal remodelling and enhanced tumour cell motility. RHAMM depletion abolishes fibronectin expression and cell transmigration across the endothelial layer in E2F1-activated cells. In a xenograft model, knock-down of E2F1 or RHAMM in metastatic cells protects the liver parenchyma of mice against extravasation of CTCs, whereas the number of transmigrated cells increases in response to E2F1 induction. Expression data from clinical tissue samples reveals high E2F1 and RHAMM levels that closely correlate with malignant progression. These findings suggest a requirement for RHAMM in late-stage metastasis by a mechanism involving cooperative stimulation of fibronectin, with a resultant tumourigenic microenvironment important for enhanced extravasation and distant organ colonization. Therefore, stimulation of the E2F1-RHAMM axis in aggressive cancer cells is of high clinical significance. Targeting RHAMM may represent a promising approach to avoid E2F1-mediated metastatic dissemination.

DNp73 exerts function in metastasis initiation by disconnecting the inhibitory role of EPLIN on IGF1R-AKT/STAT3 signaling.

Dissemination of cancer cells from primary tumors is the key event in metastasis, but specific determinants are widely unknown. Here, we show that DNp73, an inhibitor of the p53 tumor suppressor family, drives migration and invasion of nonmetastatic melanoma cells. Knockdown of endogenous DNp73 reduces this behavior in highly metastatic cell lines. Tumor xenografts expressing DNp73 show a higher ability to invade and metastasize, while growth remains unaffected. DNp73 facilitates an EMT-like phenotype with loss of E-cadherin and Slug upregulation. We provide mechanistic insight toward regulation of LIMA1/EPLIN by p73/DNp73 and demonstrate a direct link between the DNp73-EPLIN axis and IGF1R-AKT/STAT3 activation. These findings establish initiation of the invasion-metastasis cascade via EPLIN-dependent IGF1R regulation as major activity of DNp73.

E2F1 promotes angiogenesis through the VEGF-C/VEGFR-3 axis in a feedback loop for cooperative induction of PDGF-B.

Angiogenesis is essential for primary tumor growth and metastatic dissemination. E2F1, frequently upregulated in advanced cancers, was recently shown to drive malignant progression. In an attempt to decipher the molecular events underlying this behavior, we demonstrate that the tumor cell-associated vascular endothelial growth factor-C/receptor-3 (VEGF-C/VEGFR-3) axis is controlled by E2F1. Activation or forced expression of E2F1 in cancer cells leads to the upregulation of VEGFR-3 and its ligand VEGF-C, whereas E2F1 depletion prevents their expression. E2F1-dependent receptor induction is crucial for tumor cells to enhance formation of capillary tubes and neovascularization in mice. We further provide evidence for a positive feedback loop between E2F1 and VEGFR-3 signaling to stimulate pro-angiogenic platelet-derived growth factor B (PDGF-B). E2F1 or VEGFR-3 knockdown results in reduced PDGF-B levels, while the coexpression synergistically upregulates promoter activity and endogenous protein expression of PDGF-B. Our findings delineate an as yet unrecognized function of E2F1 as enhancer of angiogenesis via regulation of VEGF-C/VEGFR-3 signaling in tumors to cooperatively activate PDGF-B expression. Targeting this pathway might be reasonable to complement standard anti-angiogenic treatment of cancers with deregulated E2F1.

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry.

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.

Predicting and preventing melanoma invasiveness: advances in clarifying E2F1 function.

Malignant melanoma of the skin is one of the most aggressive human cancers with increasing incidence, despite efforts to improve primary prevention. In particular, the prognosis of patients at late stages of the disease has not significantly improved in the last three decades, because systemic therapies have proven disappointing. Thus, metastatic melanoma continues to be a daunting clinical problem. The increasingly high rates of lethal outcome associated with advanced melanoma rely on the acquisition of invasiveness, early metastatic dissemination of tumor cells from their primary sites, and generation of chemoresistance as a consequence of alteration of key molecules involved in the regulation of cell survival. Thus far, extensive studies have been conducted to understand the molecular mechanisms that drive tumor progression, but the specific requirements underlying the aggressive behavior are still widely unknown. Understanding the determinants of this process is key to unveiling its dynamics, especially those that promote invasiveness, and may open new routes for the development of therapeutic strategies that control metastatic spread, and eventually the prevention of life-threatening metastases. Here, we review recent advances on molecular aspects, particularly of E2F1 transcription factor function, in the context of patient data, and discuss the implications for targeting melanoma cells when they begin to invade and metastasize.

E2F1 in melanoma progression and metastasis.

Metastases are responsible for cancer deaths, but the molecular alterations leading to tumor progression are unclear. Overexpression of the E2F1 transcription factor is common in high-grade tumors that are associated with poor patient survival. To investigate the association of enhanced E2F1 activity with aggressive phenotype, we performed a gene-specific silencing approach in a metastatic melanoma model. Knockdown of endogenous E2F1 via E2F1 small hairpin RNA (shRNA) expression increased E-cadherin expression of metastatic SK-Mel-147 melanoma cells and reduced their invasive potential but not their proliferative activity. Although growth rates of SK-Mel-147 and SK-Mel-103 xenograft tumors expressing E2F1 shRNA or control shRNA were similar, mice implanted with cells expressing E2F1 shRNA had a smaller area of metastases per lung than control mice (n = 3 mice per group; 5% vs 46%, difference = 41%, 95% confidence interval = 15% to 67%; P = .01; one-way analysis of variance). We identified epidermal growth factor receptor as a direct target of E2F1 and demonstrated that inhibition of receptor signaling abrogates E2F1-induced invasiveness, emphasizing the importance of the E2F1-epidermal growth factor receptor interaction as a driving force in melanoma progression that may serve as a paradigm for E2F1-induced metastasis in other human cancers.

Transcriptional repression of the prosurvival endoplasmic reticulum chaperone GRP78/BIP by E2F1.

The endoplasmic reticulum chaperone GRP78/BIP plays a central role in the prosurvival machinery, and its enhanced expression has been implicated in drug resistance, carcinogenesis, and metastasis. E2F1, as part of an antitumor safeguard mechanism, promotes apoptosis regardless of functional p53. Using cells that are defective in p53, we show that E2F1 represses GRP78/BIP at the transcriptional level, and this requires its DNA binding domain. Analysis of human GRP78/BIP promoter reporter constructs revealed that the region between -371 and -109 of the proximal promoter contains major E2F1-responsive elements. Toward understanding the underlying mechanism of this regulation, we performed chromatin immunoprecipitation and gel shift assays, demonstrating that E2F1 directly binds to GC-rich regions in the distal GC-box and endoplasmic reticulum stress response element -126 by interfering with the binding of positive regulatory proteins Sp1 and TFII-I of the ER stress response element-binding factor complex. We further show that TFII-I, which is required for optimal stress induction of GRP78/BIP, is suppressed by E2F1 on the protein level. Finally, our studies suggest a molecular link between the inhibition of GRP78/BIP and E2F1-mediated chemosensitization of tumor cells, underscoring its relevance for cancer treatment. Together, the data provide a new mechanism for the incompletely understood tumor suppressor function of E2F1.

Human leukocyte elastase counteracts matrix metalloproteinase-7 induced apoptosis resistance of tumor cells.

Matrix metalloproteinase-7 (MMP-7/Matrilysin) is a component of the tumor microenvironment associated with malignant progression. Its expression in tumors protects tumor cells from CD95-mediated apoptosis and the cytotoxic activity of tumor specific CD8(+) T cells. In the present study, we show that human leukocyte elastase (HLE) secreted by polymorphonuclear leukocytes cleaves MMP-7 resulting in loss of enzymatic activity. The anti-apoptotic effect of MMP-7 is reduced in the presence of HLE for CD95-, doxorubicin- and CTL-mediated apoptosis. Our data indicates that HLE may be a natural inactivator of MMP-7 which can counteract MMP-7-induced apoptosis resistance.

Cleavage of CD95 by matrix metalloproteinase-7 induces apoptosis resistance in tumour cells.

The ability of tumour cells to resist apoptosis-inducing signals by cytotoxic T cells may decide the success or failure of tumour elimination. An important effector of apoptosis is the CD95/CD95 ligand system (APO-1/Fas) that mediates perforin-independent cytotoxic T-cell killing of tumour cells. We propose a new strategy by which tumour cells can resist CD95-induced apoptosis. We identified matrix metalloproteinase-7, MMP-7 (Martilysin), as the first physiologically relevant protease that can specifically cleave CD95. MMP-7 is of unique importance because it is produced by the tumour cells themselves at early stages of tumour development. Microsequencing of the positions in CD95 cleaved by MMP-7 revealed two sites in the N-terminal extracellular domain of CD95, important for preligand assembly of CD95. MMP-7 cleavage of CD95 results in reduced CD95 surface expression and decreased CD95-mediated apoptosis sensitivity of tumour cells. Treatment of MMP-7-positive HT-29 tumour cells with MMP-7-antisense oligonucleotides led to an increase in CD95-mediated apoptosis sensitivity. Finally, specific cytotoxic T-cell killing was reduced in the presence of MMP-7. Thus, MMP-7 expression in tumour cells may contribute to an apoptosis-resistant phenotype, which ultimately promotes immune escape. This activity may account for the well-established role of MMP-7 in early tumour development.