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.

Reptin drives tumour progression and resistance to chemotherapy in nonsmall cell lung cancer.

While targeted nonsmall cell lung cancer (NSCLC) therapies have improved the outcome of defined disease subtypes, prognosis for most patients remains poor. We found the AAA+ ATPase Reptin to be highly expressed in the vast majority of 278 NSCLC tumour samples. Thus, the objective of the study was to assess the role of Reptin in NSCLC.Survival analyses of 1145 NSCLC patients revealed that high RNA expression levels of Reptin are associated with adverse outcome. Knockdown of Reptin in human NSCLC cells impaired growth ex vivo and eliminated engraftment in a xenograft model. Reptin directly interacted with histone deacetylase 1 (HDAC1) as the critical mechanism driving NSCLC tumour progression. Pharmacological disruption of the Reptin/HDAC1 complex resulted in a substantial decrease in NSCLC cell proliferation and induced significant sensitisation to cisplatin.Our results identify Reptin as a novel independent prognostic factor and as a key regulator mediating proliferation and clonal growth of human NSCLC cells ex vivo and in vivo We unveil a Reptin/HDAC1 protein complex whose pharmacological disruption sensitises NSCLC cells to cisplatin, suggesting this approach for application in clinical trials.

High expression of transcription factor POU2F1 confers improved survival on smokers with lung adenocarcinoma: a retrospective study of two cohorts.

Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide and its most important risk factor is tobacco smoking. While smoking is associated with inferior outcome in NSCLC patients, smoking also correlates with a higher tumor mutational burden. In contrast to adenocarcinomas (ADC) of non-smokers, that frequently harbor targetable gain-of-function mutations, NSCLC smokers largely present with non-targetable loss-of-function mutations of genes associated with DNA-damage repair. The transcription factor Pit-1, Oct1/2, Unc-86 (POU) domain class 2 transcription factor 1 (POU2F1) is a widely expressed bipotential stabilizer of repressed and inducible transcriptional states and frequently deregulated in cancer. Methods: Via immunohistochemistry, we evaluated POU2F1 protein expression on a tissue micro array of 217 operable stage I-III NSCLC patients. Findings were reproduced in a gene expression database of 1144 NSCLC patients, filtered for POU2F1 mRNA expression. After retroviral overexpression of POU2F1 in A549 cells, we evaluated for clonogenic growth and proliferation. Additionally, CRISPR-Cas9 mediated POU2F1 knockdown in A549 cells was likewise analyzed. Results: High protein expression of POU2F1 in 217 NSCLC patients resulted in improved outcome of smokers with ADC [hazard ratio (HR) 0.30 (0.09-0.99), P=0.035]. Moreover, gene expression analysis confirmed favorable outcome of high POU2F1 mRNA expression in smokers with ADC [HR 0.41 (0.24-0.69), P<0.001]. Other than that, retrovirally induced overexpression of POU2F1 in A549 cells significantly reduced both, clonogenic growth as well as proliferation of NSCLC cells, whereas CRISPR-Cas9 mediated knockdown of the protein did not have any impact. Conclusions: Our data suggest that high expression of POU2F1 mediates a less aggressive cancer phenotype in smokers with ADC NSCLC. Pharmacological induction of genes and signaling pathways controlled by POU2F1 may provide novel avenues for future targeted NSCLC therapies in smokers.

Calcitonin receptor-like (CALCRL) is a marker of stemness and an independent predictor of outcome in pediatric AML.

We have recently identified the G protein-coupled neuropeptide receptor calcitonin receptor-like (CALCRL) as an independent prognostic biomarker and a therapeutic target in more than 1500 adult patients with acute myeloid leukemia (AML). Here, we confirmed CALCRL expression as a prognostic factor in a cohort of 284 pediatric patients with AML. High CALCRL expression was independently associated with event-free survival (hazard ratio [HR], 1.87; 95% confidence interval [CI], 1.36-2.57; P = .0001), overall survival (HR, 1.55; 95% CI, 1.06-2.27; P = .025), and cumulative incidence of relapse (HR, 2.10; 95% CI, 1.49-1.96; P < .0001) when adjusting for age, white blood cell count, and genetic risk. Despite its association with leukemia stem cell signatures, CALCRL expression remained associated with all end points when compared with the 17-gene leukemic stem cell score. The strong association of CALCRL expression with the risk of relapse also in the pediatric population supports its role as novel age-independent master regulator of relapse-initiating, drug-tolerant AML cells in humans.

C/EBPß is a MYB- and p300-cooperating pro-leukemogenic factor and promising drug target in acute myeloid leukemia.

Transcription factor MYB has recently emerged as a promising drug target for the treatment of acute myeloid leukemia (AML). Here, we have characterized a group of natural sesquiterpene lactones (STLs), previously shown to suppress MYB activity, for their potential to decrease AML cell proliferation. Unlike what was initially thought, these compounds inhibit MYB indirectly via its cooperation partner C/EBPß. C/EBPß-inhibitory STLs affect the expression of a large number of MYB-regulated genes, suggesting that the cooperation of MYB and C/EBPß broadly shapes the transcriptional program of AML cells. We show that expression of GFI1, a direct MYB target gene, is controlled cooperatively by MYB, C/EBPß, and co-activator p300, and is down-regulated by C/EBPß-inhibitory STLs, exemplifying that they target the activity of composite MYB-C/EBPß-p300 transcriptional modules. Ectopic expression of GFI1, a zinc-finger protein that is required for the maintenance of hematopoietic stem and progenitor cells, partially abrogated STL-induced myelomonocytic differentiation, implicating GFI1 as a relevant target of C/EBPß-inhibitory STLs. Overall, our data identify C/EBPß as a pro-leukemogenic factor in AML and suggest that targeting of C/EBPß may have therapeutic potential against AML.

Multiple translational isoforms give functional specificity to serum- and glucocorticoid-induced kinase 1.

Serum- and glucocorticoid-induced kinase 1 is a ubiquitous kinase that regulates diverse processes such as ion transport and cell survival. We report that a single SGK1 mRNA produces isoforms with different N-termini owing to alternative translation initiation. The long isoforms, 49 and 47 kDa, are the most abundant, localize to the ER membrane, exhibit rapid turnover, their expression is decreased by ER stress, activate the epithelial sodium channel (ENaC) and translocate FoxO3a transcriptional factors from the nucleus to the cytoplasm. The short isoforms, 45 and 42 kDa, localize to the cytoplasm and nucleus, exhibit long half-life and phosphorylate glycogen synthase kinase-3beta. The data indicate that activation of Sgk1 in different cellular compartments is key to providing functional specificity to Sgk1 signaling pathways. We conclude that the distinct properties and functional specialization of Sgk1 given by the N-terminus confer versatility of function while maintaining the same core kinase domain.

Focal adhesion kinase confers pro-migratory and antiapoptotic properties and is a potential therapeutic target in Ewing sarcoma.

Oncogenesis of Ewing sarcoma (EwS), the second most common malignant bone tumor of childhood and adolescence, is dependent on the expression of chimeric EWSR1-ETS fusion oncogenes, most often EWSR1-FLI1 (E/F). E/F expression leads to dysregulation of focal adhesions (FAs) enhancing the migratory capacity of EwS cells. Here, we show that, in EwS cell lines and tissue samples, focal adhesion kinase (FAK) is expressed and phosphorylated at Y397 in an E/F-dependent way involving Ezrin. Employing different EwS cell lines as in vitro models, we found that key malignant properties of E/F are mediated via substrate-independent autophosphorylation of FAK on Y397. This phosphorylation results in enhanced FA formation, Rho-dependent cell migration, and impaired caspase-3-mediated apoptosis in vitro. Conversely, treatment with the FAK inhibitor 15 (1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) enhanced caspase-mediated apoptosis and EwS cell migration, independent from the respective EWSR1-ETS fusion type, mimicking an anoikis-like phenotype and paralleling the effects of FAK siRNA knockdown. Our findings were confirmed in vivo using an avian chorioallantoic membrane model and provide a first rationale for the therapeutic use of FAK inhibitors to impair metastatic dissemination of EwS.

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.

AAA+ ATPases Reptin and Pontin as potential diagnostic and prognostic biomarkers in salivary gland cancer - a short report.

PURPOSE: Salivary gland cancer (SGC) is a rare and heterogeneous disease with significant differences in recurrence and metastasis characteristics. As yet, little is known about the mechanisms underlying the initiation and/or progression of these diverse tumors. In recent years, the AAA+ ATPase family members Pontin (RuvBL1, Tip49a) and Reptin (RuvBL2, Tip49b) have been implicated in various processes, including transcription regulation, chromatin remodeling and DNA damage repair, that are frequently deregulated in cancer. The aim of this study was to assess the clinical and functional significance of Reptin and Pontin expression in SGC. METHODS: Immunohistochemical staining of Pontin, Reptin, ß-catenin, Cyclin D1, TP53 and MIB-1 was performed on a collection of 94 SGC tumor samples comprising 13 different histological subtypes using tissue microarrays. RESULTS: We found that Reptin and Pontin were expressed in the majority of SGC samples across all histological subtypes. Patients with a high Reptin expression showed a significantly inferior 5-year overall survival rate compared to patients with a low Reptin expression (47.7% versus 78.3%; p = 0.033), whereas no such difference was observed for Pontin. A high Reptin expression strongly correlated with a high expression of the proliferation marker MIB-1 (p = 0.003), the cell cycle regulator Cyclin D1 (p = 0.006), accumulation of TP53 as a surrogate p53 mutation marker (p = 0.042) and cytoplasmic ß-catenin expression (p = 0.002). Increased Pontin expression was found to significantly correlate with both cytoplasmic and nuclear ß-catenin expression (p = 0.037 and p = 0.018, respectively), which is indicative for its oncogenic function. CONCLUSIONS: Our results suggest a role of Reptin and Pontin in SGC tumor progression and/or patient survival. Therefore, SGC patients exhibiting a high Reptin expression may benefit from more aggressive therapeutic regimens. Future studies should clarify whether such patients may be considered for more radical surgery, extended adjuvant therapy and/or targeted therapy.

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.

hIscA: a protein implicated in the biogenesis of iron-sulfur clusters.

This article describes the gene called hIscA, its transcription product and protein (hIscA) and its putative function. We screened a human brain cDNA expression library with serum from a patient suffering from the autoimmune Sjögren's syndrome (S5:823/94). One cDNA of 1.6-kbp clone was isolated. This clone contains the entire coding sequence for a protein unknown in human. IscA is ubiquitously expressed and expression levels vary among tissues. The 15.5-kDa predicted protein contains a structural domain named HESB, is located in the mitochondria and is implicated in the biogenesis of iron-sulfur clusters. Since this unknown protein is related to IscA-like protein, we suggest as name for this protein hIscA. The recombinant protein is recognized by a rabbit polyclonal antiserum generated against the carboxyl extreme of the Saccharomyces cerevisiae orthologue Isa1. In this article, we demonstrate the functional homology between hIscA and Isa1 proteins using Isa1 null mutant S. cerevisiae transformed with hIscA in a yeast functional complementation test. We also describe the rat homologue to this gene.

MYST2 acetyltransferase expression and Histone H4 Lysine acetylation are suppressed in AML.

Chromatin-modifying enzymes are frequently altered in acute myeloid leukemia (AML). In the current study, we identified MYST2, a core histone acetyltransferase, to be suppressed in blast cells from AML patients compared with nonmalignant hematopoietic progenitor cells. Functionally, loss of MYST2 accelerated leukemic growth and colony formation, while forced expression of MYST2 induced H4K5 acetylation (H4K5Ac) and suppressed hematopoietic progenitor cell growth. Consistently, global H4K5Ac levels were frequently decreased in AML blasts. Low levels of H4K5Ac were most prominent in patients with complex karyotype AML and were associated with inferior overall survival in univariate but not multivariate analysis. ChIP-seq experiments in primary AML patients' blasts revealed widespread H4K5Ac deregulation, most prominent at gene promoters. Taken together, MYST2 is a repressed growth suppressor in AML mediating reduced acetylation of histone 4 at residue 5 and is associated with inferior AML patient survival.

The histone demethylase PHF8 governs retinoic acid response in acute promyelocytic leukemia.

While all-trans retinoic acid (ATRA) treatment in acute promyelocytic leukemia (APL) has been the paradigm of targeted therapy for oncogenic transcription factors, the underlying mechanisms remain largely unknown, and a significant number of patients still relapse and become ATRA resistant. We identified the histone demethylase PHF8 as a coactivator that is specifically recruited by RARα fusions to activate expression of their downstream targets upon ATRA treatment. Forced expression of PHF8 resensitizes ATRA-resistant APL cells, whereas its downregulation confers resistance. ATRA sensitivity depends on the enzymatic activity and phosphorylation status of PHF8, which can be pharmacologically manipulated to resurrect ATRA sensitivity to resistant cells. These findings provide important molecular insights into ATRA response and a promising avenue for overcoming ATRA resistance.

Autoantigenic nuclear proteins of a clinically atypical renal vasculitis.

BACKGROUND: Systemic vasculitides constitute a heterogeneous group of diseases of autoimmunological origin characterized by inflammation of blood vessels and antibodies that react against autoantigens in a process that ultimately affects blood vessel walls. An important number of these patients present kidney disease. An endeavour of this area of research is the identification of autoantigens involved in these diseases. Accordingly, we used serum from a patient suffering from a microscopic polyangiitis, P-ANCA positive, manifesting a clinically atypical renal necrotizing glomerulonephritis and interstitial nephropathy for the identification of autoantigens; we also determined the prevalence of corresponding autoantibodies in other vasculitides, diabetic microangiopathy and in general population. METHODS: The patient's serum was used as a probe for the immunoscreening method SEREX to screen a human brain cDNA expression library. RESULTS: Four positive clones were isolated and sequenced. Clones Jos002 code for protein HDAC5, Jos014 for TFC4, Jos107 for RTF1, and Jos313 for POLDIP3 polymerase. The four proteins are of nuclear localization. None of them had been reported as autoantigen. Recombinant proteins were synthesised and checked as antigens by western blot with different sera from controls and patients affected with other vasculitides and diabetic microangiopathy as well. Only the serum from the patient origin of this study recognized all recombinant proteins. CONCLUSION: We identify four nuclear proteins, HDAC5, TFC4, RTF1 and POLDIP3 polymerase as new autoantigens that could be used as markers in the diagnosis of subfamilies in immune diseases, although we cannot determine the role of these proteins in the aetiopathogenic process.

An amphipathic helix targets serum and glucocorticoid-induced kinase 1 to the endoplasmic reticulum-associated ubiquitin-conjugation machinery.

Serum- and glucocorticoid-induced kinase 1 (Sgk1) regulates many ion channels and transporters in epithelial cells and promotes cell survival under stress conditions. In this study we demonstrate that Sgk1 is a short-lived protein regulated by the endoplasmic reticulum (ER)-associated degradation system and subcellular localization to the ER. We identified a hydrophobic motif (residues 18-30) as the signal for ER localization and rapid degradation by the ubiquitin (Ub)/proteasome pathway in both yeast and mammalian cells. Deletion or reduction of hydrophobicity of the motif redistributes Sgk1 to the cytosol and nucleus and markedly increases its half-life. We determined that the Ub-conjugating UBC6 and UBC7 and the Ub ligase HRD1 are the ER-associated Ub enzymes that mediate degradation of Sgk1; thus, Sgk1 has been identified as a cytosolic substrate for mammalian HRD1. Compartmentalization of Sgk1 controls the functional and spatial specificities of Sgk1-mediated signaling pathways, whereas rapid protein turnover provides a means to rapidly adjust Sgk1 abundance in response to different hormonal and external stimuli that increase Sgk1 gene transcription.

Functional specificity of Sgk1 and Akt1 on ENaC activity.

Reabsorption of sodium by the epithelial sodium channel (ENaC) is essential for maintaining the volume of the extracellular compartment and blood pressure. The function of ENaC is regulated primarily by aldosterone, antidiuretic hormone [arginine vasopressin (AVP)], and insulin, but the molecular mechanisms that increase channel activity are still poorly understood. It has been proposed that the related serine/threonine kinases serum- and glucocorticoid-induced kinase (Sgk1) and protein kinase B (Akt) mediate activation of ENaC. Here, we addressed the question of whether there is functional specificity of these kinases for the activation of ENaC in epithelial cells of the distal renal tubule. We demonstrate that Akt does not increase ENaC function under basal conditions or after stimulation with aldosterone, insulin, or AVP. In contrast, under the same experimental conditions, Sgk1 increases ENaC activity by 10-fold. The effect of Sgk1 is additive to that of aldosterone, whereas, in the presence of active Sgk1, cells do not further respond to insulin or AVP. We conclude that, in cells expressing both kinases, modulation of ENaC activity is mediated by Sgk1 but not by Akt1.