Apo-14´-Carotenoic Acid Is a Novel Endogenous and Bioactive Apo-Carotenoid

Carotenoids can be metabolized to various apo-carotenoids and retinoids. Apo-15´-carotenoic acid (retinoic acid, RA) is a potent activator of the retinoic acid receptor (RAR) in its all-trans- (ATRA) and 9-cis- (9CRA) forms. In this study we show firstly, that apo-14´-carotenoic acid (A14CA), besides retinoic acids, is present endogenously and with increased levels in the human organism after carrot juice supplementation rich in β-carotene. All-trans-A14C (ATA14CA) is just a moderate activator of RAR-transactivation in reporter cell lines but can potently activate retinoic acid response element (RARE)-mediated signalling in DR5/RARE-reporter mice and potently increase retinoid-reporter target gene expression in ATA14CA-supplemented mice and treated MM6 cells. Further metabolism to all-trans-13,14-dihydroretinoic acid (ATDHRA) may be the key for its potent effects on retinoid target gene activation in ATA14CA-treated MM6 cells and in liver of supplemented mice. We conclude that besides RAs, there are alternative ways to activate RAR-response pathways in the mammalian organism. ATA14CA alone and in combination with its metabolite ATDHRA may be an alternative pathway for potent RAR-mediated signalling.

Cooperative Effects of a Dnmt Inhibitor and All-Trans Retinoic Acid in an AML Cell Line Model Lacking PML-Rara

Introduction: All-trans retinoic acid (ATRA, RA) has powerful activity in acute promyelocytic leukemia (APL); its efficacy in non-APL acute myeloid leukemia (AML) is still unclear, but may be enhanced by epigenetic drugs such as azanucleoside DNMT inhibitors (Blagitko-Dorfs et al. PLoS ONE 2013). In a randomized phase II study (DECIDER trial, NCT00867672) the addition of RA to decitabine (DAC) in newly diagnosed non-fit older AML patients resulted in a clinically meaningful extension of survival. We hypothesize that the add-on of RA to DAC results in cooperative transcriptome changes (possibly associated with demethylation) which may, at least in part, explain these clinical results. Materials and Methods: U937 cells were treated in triplicates with daily pulses of 200 nM DAC, 1 µM RA was administered after 48 hours (hr). Cells were harvested after 72 and 120 hr of treatment. Proliferation and apoptosis were determined by acridine orange / propidium iodide staining. For the colony formation assay (CFA), 100 cells were seeded after 120 hr treatment in methylcellulose, colonies were counted after 21 days. Stranded cDNA libraries were sequenced on an Illumina HiSeq2000 with 60 million 125 bp paired-end reads. Read quality was checked with FastQC (per base Q-score >38), alignment to the GencodeCompV24 reference genome with RNAStar, generation of count matrices with ht-seq and differential expression testing with DeSeq2. Transcript changes were considered significant with an adjusted FDR p-value < 0.01. GO analysis was performed with Metascape. Methylomes were generated using Infinium Human Methylation 450K BeadChip arrays and differential methylation data was obtained using the RnBeads package for R (Assenov et al. Nature Methods 2014). Results: In suspension culture, RA alone had no effect on the proliferation of U937 cells, DAC alone reduced proliferation by 74 %, the DAC+RA combination by 81 % (with viability always ≥ 90 %). RA alone also had no effect on colony growth, DAC reduced colony formation by 85 %, and DAC+RA decreased colony numbers by 96 %. To determine transcriptional add-on effects of DAC+RA, transcriptomes of cells treated for 72 hr were analyzed by RNA-Seq. RA alone had a modest effect on transcription, whereas DAC alone induced / downregulated 2538 and 620 transcripts, DAC+RA induced / downregulated 2848 and 1173 transcripts, respectively (Table 1, Fig. 1). 29.6 % (1192 transcripts) of all DAC+RA-altered transcripts were uniquely regulated by the combination and not by DAC or RA alone. GO analysis of the DAC+ATRA alone upregulated genes (664 transcripts) revealed a significant enrichment for immune system and cellular decay terms, downregulated genes were enriched for transcription and cell cycle terms. Of the transcripts already up- or downregulated by DAC or RA alone, 55.5 % of were further enhanced by DAC+RA. Among those, the retinoic acid receptors RARA, RARB and the retinoic acid response element (RARE)-containing tumor suppressor HIC-1 (Hassan et al. Cell Reports 2017) were induced 8.6-, 8.3- and 179.8-fold, respectively, compared to 2.4- and 5.3-fold (RARA and RARB, DAC alone) and 26.5-fold (HIC-1, RA alone) (validated by qRT-PCR). Among the most regulated uniquely induced transcripts by DAC+RA, the RA-regulating cytochrome P450 member CYP26A1 (regulated by multiple RAREs) was upregulated 347.3-fold. In order to further investigate this add-on effect of RA on DAC-induced transcriptome changes, DNA methylation was analyzed (72 and 120 hr). As expected, DAC significantly reduced CpG methylation in gene bodies and promoters, without further demethylation upon RA add-on after 72 hr (after 120 hr, a trend towards further demethylation was noted with DAC+ATRA). Conclusion: In vitro, the combination of DAC with RA resulted in enhanced growth inhibition and reduced colony formation compared to DAC alone. Transcriptome analysis revealed a high number of uniquely regulated transcripts, and an enhancement of regulation by the combination treatment. As expected, this also included genes with RAREs. The enrichment for genes involved in immune response (e.g. multiple interferon-response genes) encourages the combination of DNMTi-based therapies with immunotherapeutic agents. Therefore, further transcriptome analyses of transposable elements, when reactivated, may trigger an interferon-mediated immune reaction, are currently ongoing. Disclosures Lübbert: TEVA: Other: Study drug; Celgene: Other: Travel Support; Cheplapharm: Other: Study drug; Janssen: Honoraria, Research Funding.

Caspase-8, RIPK1, and RIPK3 Coordinately Regulate Retinoic Acid-Induced Cell Differentiation and Necroptosis

Caspase-8, which is essential for death receptor-mediated apoptosis, inhibits necroptosis by suppressing the function of RIPK1 and RIPK3 to activate MLKL. We show that knockdown of caspase-8 expression in embryoid bodies derived from ES cells markedly enhances retinoic acid (RA)-induced cell differentiation and necroptosis, both of which are dependent on Ripkl and Ripk3. RA treatment obviously enhanced the expression of RA-specific genes having a retinoic acid response element (RARE) to induce cell differentiation, and induced marked expression of RIPK1, RIPK3 and MLKL to stimulate necroptosis. Caspase-8 knockdown induced RA receptor (RAR) to form a complex with RIPK1 and RIPK3 in the nucleus, and RAR interacting with RIPK1 and RIPK3 showed much stronger binding activity to RARE than RAR without RIPK1 or RIPK3. In Caspase-8-deficient mouse embryos, expression of RA-specific genes was obviously enhanced. Thus, caspase-8, RIPK1, and RIPK3 regulate RA-induced cell differentiation and necroptosis both in vitro and in vivo.

Pokemon decreases the transcriptional activity of RARα in the absence of ligand

Pokemon is a transcriptional repressor that belongs to the POZ and Krüppel (POK) protein family. In this study, we investigated the potential interaction between Pokemon and retinoic acid receptor alpha (RARα) and determined the role of Pokemon in regulation of RARα transcriptional activity in the absence of ligand. We found that Pokemon could directly interact with RARα. Moreover, we demonstrated that Pokemon could decrease the transcriptional activity of RARα in the absence of ligand. Furthermore, we showed that Pokemon could repress the transcriptional activity of RARα by increasing the recruitment of nuclear receptor co-repressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) to the retinoic acid response element (RARE) element. Taken together, these data suggest that Pokemon is a novel partner of RARα that acts as a co-repressor to regulate RARα transcriptional activity in the absence of ligand.

Retinoic acid triggers c-kit gene expression in spermatogonial stem cells through an enhanceosome constituted between transcription factor binding sites for retinoic acid response element (RARE), spleen focus forming virus proviral integration oncogene (SPFI1) (PU.1) and E26 transformation-specific (ETS)

Restricted availability of retinoic acid (RA) in the testicular milieu regulates transcriptional activity of c-kit (KIT, CD117), which aids in the determination of spermatogonial stem-cell differentiation. The effect of RA on c-kit has been reported previously, but its mode of genomic action remains unresolved. We studied the molecular machinery guiding RA responsiveness to the c-kit gene using spermatogonial stem-cell line C18–4 and primary spermatogonial cells. A novel retinoic acid response element (RARE) positioned at –989 nucleotides upstream of the transcription start site (TSS) was identified, providing a binding site for a dimeric RA receptor (i.e. retinoic acid receptor gamma (RARγ) and retinoic X receptor). RA treatment influenced c-kit promoter activity, along with endogenous c-kit expression in C18–4 cells. A comprehensive promoter deletion assay using the pGL3B reporter system characterised the region spanning –271 bp and –1011 bp upstream of the TSS, which function as minimal promoter and maximal promoter, respectively. In silico analysis predicted that the region –1011 to +58 bp comprised the distal enhancer RARE and activators such as spleen focus forming virus proviral integration oncogene (SPFI1) (PU.1), specificity protein 1 (SP1) and four E26 transformation-specific (ETS) tandem binding sites at the proximal region. Gel retardation and chromatin immunoprecipitation (ChIP) assays showed binding for RARγ, PU.1 and SP1 to the predicted consensus binding sequences, whereas GABPα occupied only two out of four ETS binding sites within the c-kit promoter region. We propose that for RA response, an enhanceosome is orchestrated through scaffolding of a CREB-binding protein (CBP)/p300 molecule between RARE and elements in the proximal promoter region, controlling germ-line expression of the c-kit gene. This study outlines the fundamental role played by RARγ, along with other non-RAR transcription factors (PU.1, SP1 and GABPα), in the regulation of c-kit expression in spermatogonial stem cells in response to RA.

The Retinoid Derivative Drug, Acitretin, Modulates CD38 Expression on MEC-1 Cells and Primary Chronic Lymphocytic Leukemia (CLL) Cells and Reduces Migration in Response to the Chemokine CXCL12

Background: CD38 is a cell surface receptor and ectoenzyme expressed in a variety of haematological malignancies. In CLL, CD38 expression is a marker of poor prognostic disease and is now attracting growing interest as a target molecule for anti CLL therapy. Retinoids modulate CD38 expression due to the presence of the Retinoic Acid Response Element (RARE) DNA sequence in the promotor region of the CD38 gene. In this study we demonstrate that the retinoic acid derivative, acitretin, upregulates the expression of CD38 on the MEC-1 cell line and on primary CLL cells from CD38 positive patients. We propose that, by doing so, Acitretin may render these cells more susceptible to the actions of anti CD38 monoclonal antibody drugs and thus have a potential role as an adjunct to such therapies. Methods: Primary CLL cells were freshly isolated from whole blood of CLL patients using the FICOLL density centrifugation technique. The CLL like cell line MEC-1 was obtained from DSMZ, Germany. MEC-1 cells and cells from three CD38 negative and five CD38 positive patients were treated with 10micM acitretin for 24, 48 and 72hrs. CD38 expression was measured by FACS analysis at baseline and at the different time points. Results are expressed as fold increase in Mean Fluorescence Intensity (MFI). Migration assays were performed using 2x10^6 cells per well incubated with 10microM acitretin overnight in 1%FBS. Cells were subsequently transferred to chamber inserts overlying 10%FBS media containing 200ng/ml CXCL12 in all wells, except negative controls. Cells were then allowed to migrate for 4hrs. Migration was calculated as percentage of cells in lower chamber to upper chamber. Results: Acitretin increased the expression of CD38 on MEC-1 cells by a mean of 7.4 fold and on cells from CD38 positive patients by a mean of 3.4 folds (n=5). Acitretin, however, did not modulate CD38 expression levels on primary CLL cells from CD38 negative patients (n=3). Migration of primary CLL cells from 11/14 patients toward the CXCL12 chemokine was reduced in response to Acitretin treatment as compared to untreated controls. Mean migration of treated cells was 12.3% versus 27.3% mean migration in untreated controls. Conclusion: Our findings re-affirm the potent effect of retinoid drugs as upregulators of CD38 expression and suggest that their use in CLL therapeutics in conjunction with anti CD38 monoclonal antibodies should be explored. We also propose that acitretin appears to exert a favourable blocking effect on CLL cell migration towards micro-environmental chemokines such as CXCL12, warranting further evaluation. Disclosures Murphy: Celgene: Honoraria.