Retinoic Acid Receptor Alpha Research Articles

Superenhancer Analysis Defines Novel Epigenomic Subtypes of Non-APL AML, Including an RARα Dependency Targetable by SY-1425, a Potent and Selective RARα Agonist.

We characterized the enhancer landscape of 66 patients with acute myeloid leukemia (AML), identifying 6 novel subgroups and their associated regulatory loci. These subgroups are defined by their superenhancer (SE) maps, orthogonal to somatic mutations, and are associated with distinct leukemic cell states. Examination of transcriptional drivers for these epigenomic subtypes uncovers a subset of patients with a particularly strong SE at the retinoic acid receptor alpha (RARA) gene locus. The presence of a RARA SE and concomitant high levels of RARA mRNA predisposes cell lines and ex vivo models to exquisite sensitivity to a selective agonist of RARα, SY-1425 (tamibarotene). Furthermore, only AML patient-derived xenograft (PDX) models with high RARA mRNA were found to respond to SY-1425. Mechanistically, we show that the response to SY-1425 in RARA-high AML cells is similar to that of acute promyelocytic leukemia treated with retinoids, characterized by the induction of known retinoic acid response genes, increased differentiation, and loss of proliferation.Significance: We use the SE landscape of primary human AML to elucidate transcriptional circuitry and identify novel cancer vulnerabilities. A subset of patients were found to have an SE at RARA, which is predictive for response to SY-1425, a potent and selective RARα agonist, in preclinical models, forming the rationale for its clinical investigation in biomarker-selected patients. Cancer Discov; 7(10); 1136-53. ©2017 AACR.See related commentary by Wang and Aifantis, p. 1065.This article is highlighted in the In This Issue feature, p. 1047.

The short isoform of PML‐RARα activates the NRF2/HO‐1 pathway through a direct interaction with NRF2

The NF-E2 p45-related factor 2 (NRF2)-Kelch-like ECH-associated protein 1 signaling pathway plays an important role in cytoprotection. In acute promyelocytic leukemia, fusion of the promyelocytic leukemia protein (PML) with retinoic acid receptor alpha (RARα) results in an oncogene, PML-RARα (PR). Although previous studies have shown that both RARα and PML inhibit NRF2 activity, how PR regulates NRF2 has not been reported. Here, we discovered that PR-S, the short isoform of PR, potentiates NRF2 activity in a tert-butylhydroquinone (tBHQ) concentration-dependent manner. Furthermore, PR-S colocalized with NRF2 in HeLa and HEK293T cells. The association of PR-S and NRF2 is mediated by the DNA-binding domains of RARα and the Neh7 domain of NRF2. Our results define a novel function of PR-S as a NRF2-transcriptional co-activator.

Abstract 1511: AML patient clustering by super-enhancers reveals an RARA associated transcription factor signaling partner

Abstract Prior studies have shown that the RARA gene is associated with a super-enhancer (SE) and has upregulated mRNA expression in a subset of AML patients. Furthermore, this has been found to confer increased sensitivity to SY-1425, a potent and selective RARα agonist. We sought to better characterize the cell state and transcription factor circuitry in these RARA-high AML cells. Clustering of 62 primary AML patient samples based on their genome wide SE maps identified six discrete clusters. RARA-high patients partitioned principally into cluster 2, and to a lesser extent 1, suggesting that RARA upregulation is associated with a specific transcription factor (TF) network and cell state. To start unraveling the TF circuitry in the RARA-high cluster, we investigated which other TFs were SE associated with clusters 1 and 2. In particular, interferon regulatory factor 8 (IRF8) was found to be strongly associated with clusters 1 and 2 by SE and mRNA expression, similar to RARA. Moreover, the expression of both genes is correlated in primary patient samples. IRF8 is involved in interferon signaling and previous studies have shown crosstalk between interferon and retinoic acid signaling. Furthermore, aberrant IRF8 pathway signaling is implicated in AML and CML pathogenesis. The tight clustering of RARA and IRF8 in patient subgroups defined by genome wide enhancer maps suggests RARα and IRF8 may form an integrated transcriptional circuit. Indeed, treatment with SY-1425 was found to strongly induce interferon-like gene expression changes in AML cells with high RARA or IRF8 levels, including the tumor suppressive IFN responsive gene IRF1. While RARA-high AML cell line models have been previously shown to respond to SY-1425, we found that models with high IRF8 expression and low levels of RARA were also found to respond to SY-1425. Such IRF8-high, RARA-low AML cell lines showed activation of similar transcriptional pathways as RARA-high cell lines in response to SY-1425 based on GSEA. IRF8-high AML also had comparable low nM EC50 anti-proliferative effects following SY-1425 treatment. In addition, SY-1425 was found to elicit differentiation in both RARA-high and IRF8-high AML cell lines based on flow cytometry. While RARA and IRF8 expression appear correlated, this data suggests that IRF8 levels may predict for sensitivity to SY-1425 in addition to RARA levels, particularly in cases of AML with high IRF8 expression but low RARA levels. Insights derived from enhancer analysis, transcriptional profiling and differentiation response in preclinical models support the recently initiated Phase 2 trial of SY-1425 (NCT02807558) in which we are evaluating the SE based patient selection strategies and gene circuitry derived pharmacodynamics clinical measurements, including differentiation markers, in patients with AML and MDS. Citation Format: Michael R. McKeown, Matthew L. Eaton, Chris Fiore, Emily Lee, Katie Austgen, Darren Smith, M. Ryan Corces, Ravindra Majeti, Christian C. Fritz. AML patient clustering by super-enhancers reveals an RARA associated transcription factor signaling partner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1511. doi:10.1158/1538-7445.AM2017-1511

Blue light potentiates neurogenesis induced by retinoic acid-loaded responsive nanoparticles.

Controlling the differentiation of stem cells would support the development of promising brain regenerative therapies. Blue light transiently increased reactive oxygen species, resulting in neuronal differentiation and increased retinoic acid receptor (RARα) levels. Additionally, the same blue light stimulation was capable of triggering the release of RA from light-responsive nanoparticles (LR-NP). The synergy between blue light and LR-NP led to amplified neurogenesis, while offering a temporal and spatial control of RA release. In this sense, our approach relying on the modulation of endogenous stem cells for the generation of new neurons may support the development of novel clinical therapies.

Diverse Regulation of Vitamin D Receptor Gene Expression by 1,25-Dihydroxyvitamin D and ATRA in Murine and Human Blood Cells at Early Stages of Their Differentiation

Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of VDR transcription, and unliganded RARα acts as a transcriptional repressor to VDR gene in acute myeloid leukemia (AML) cells. This is why we decided to examine the effects of the combination of 1,25D and all-trans-retinoic acid (ATRA) on VDR gene expression in normal human and murine blood cells at various steps of their development. We tested the expression of VDR and regulation of this gene in response to 1,25D or ATRA, as well as transcriptional activities of nuclear receptors VDR and RARs in human and murine blood cells. We discovered that regulation of VDR expression in humans is different from in mice. In human blood cells at early stages of their differentiation ATRA, but not 1,25D, upregulates the expression of VDR. In contrast, in murine blood cells 1,25D, but not ATRA, upregulates the expression of VDR. VDR and RAR receptors are present and transcriptionally active in blood cells of both species, especially at early steps of blood development.

Characterization of the differential coregulator binding signatures of the Retinoic Acid Receptor subtypes upon (ant)agonist action

Retinoic Acid Receptor alpha (RARα/NR1B1), Retinoic Acid Receptor beta (RARβ/NR1B2) and Retinoic Acid Receptor gamma (RARγ/NR1B3) are transcription factors regulating gene expression in response to retinoids. Within the RAR genomic pathways, binding of RARs to coregulators is a key intermediate regulatory phase. However, ligand-dependent interactions between the wide variety of coregulators that may be present in a cell and the different RAR subtypes are largely unknown. The aim of this study is to characterize the coregulator binding profiles of RARs in the presence of the pan-agonist all-trans-Retinoic Acid (AtRA); the subtype-selective agonists Am80 (RARα), CD2314 (RARβ) and BMS961 (RARγ); and the antagonist Ro415253. To this end, we used a microarray assay for coregulator-nuclear receptor interactions to assess RAR binding to 154 motifs belonging to >60 coregulators. The results revealed a high number of ligand-dependent RAR-coregulator interactions among all RAR variants, including many binding events not yet described in literature. Next, this work confirmed a greater ligand-independent activity of RARβ compared to the other RAR subtypes based on both higher basal and lower ligand-driven coregulator binding. Further, several coregulator motifs showed selective binding to a specific RAR subtype. Next, this work showed that subtype-selective agonists can be successfully discriminated by using coregulator binding assays. Finally this study demonstrated the possible applications of a coregulator binding assay as a tool to discriminate between agonistic/antagonistic actions of ligands. The RAR-coregulator interactions found will be of use to direct further studies to better understand the mechanisms driving the eventual actions of retinoids.

The change in retinoic acid receptor signaling induced by prenatal marginal vitamin A deficiency and its effects on learning and memory

This study investigated the prenatal marginal vitamin A deficiency (mVAD)-related impairment in learning and memory and the interactions between RARα, Src and NR1. Learning and memory were assessed in adult rats that were exposed to prenatal mVAD with Morris water maze. The average escape time was longer in mVAD rats, and they passed the hidden platform fewer times during the memory retention test than normal vitamin A intake (VAN) rats. The mRNA and protein levels of RARα, Src and NR1 in mVAD rats were significantly lower than those in VAN rats. RARα and Src, but not NR1, were in the same protein complex. RA treatment-induced increase in RARα, Src and NR1 expressions in mVAD neurons was much lower than that in VAN neurons. Overexpression of RARα gene in VAN neurons induced an increase in RARα, Src and NR1 expressions, while silencing of RARα gene induced a decrease in expressions of RARα and Src, but not that of of NR1. In mVAD neurons, however, overexpression of RARα did not induce an increase in NR1 expression, while silencing of RARα gene had no effect on Src and NR1 expressions. Furthermore, inhibition of Src was associated with a decrease in NR1 expression but not that of RARα. Prenatal mVAD was associated with impaired learning and memory in adult rats. It is possible that mVAD-related decrease in RARα led to a decrease in Src expression, which in turn down-regulated NR1 expression and Ca2+ influx and eventually caused learning and memory deficits.

A biomarker-directed phase 2 trial of SY-1425, a selective retinoic acid receptor alpha agonist, in adult patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).

TPS7071 Background: SY-1425 (tamibarotene) is an orally available, synthetic retinoid approved in Japan for the treatment of relapsed/refractory (R/R) APL. SY-1425 is a more potent and selective retinoic acid receptor alpha (RARα) agonist with improved pharmacologic properties compared to all-trans retinoic acid (ATRA) including increased half-life and lack of metabolism by CYP26A1 resulting in extended relative exposures. SY-1425 binding to RARα relieves pathogenic repression of myeloid differentiation. Super-enhancers associated with RARA and upregulation of RARA expression correlate with increased sensitivity to SY-1425 in vitro and predict for response to SY-1425 with induced differentiation and reduced proliferation in RARA-high PDX AML models, but not in RARA-low models. SY-1425 also induces the RARα target gene DHRS3 in RARA-high AML cell lines. This study is designed to demonstrate pharmacodynamic (PD) and clinical effects of SY-1425 in non-APL AML and MDS patients (pts) positive for the RARA super-enhancer associated biomarker or exploratory RARA pathway biomarker, IRF8. Methods: This study is enrolling pts with R/R AML, R/R higher-risk MDS, newly-diagnosed AML ≥60 yrs unlikely to respond to or tolerate standard therapy, and transfusion dependent lower-risk MDS pts without del 5q who are unlikely to respond to or have failed ESAs. Pts must be biomarker positive based on centralized testing of tumor cells from blood. All pts receive SY-1425 at 6 mg/m2/day PO with continuous twice daily dosing. Primary objectives are to characterize the activity of SY-1425 by ORR in AML and higher-risk MDS pts or transfusion independence in lower-risk MDS pts. Secondary objectives include event-free and relapse-free survival, duration of response, overall survival, hematologic improvement and safety. PD evaluation includes induction of DHRS3 and expression of myeloid differentiation markers. Target enrollment is 80 pts. This trial opened in September 2016. Through a protocol amendment, SY-1425 treatment in combination with azacitidine will also be evaluated. ClinicalTrials.gov identifier: NCT02807558.

Xanthophylls increased HDLC level and nuclear factor PPARγ, RXRγ and RARα expression in hens and chicks.

This study was designed to investigate effects of xanthophylls on serum lipid profile (triglyceride, TG; cholesterol, CHO; high-density lipoprotein cholesterol, HDLC; and low-density lipoprotein cholesterol, LDLC) and nuclear factor (peroxisome proliferator-activated receptor gamma, PPARγ; PPAR gamma coactivator 1 alpha, PGC1α; retinoid X receptor gamma, RXRγ; and retinoic acid receptor alpha, RARα) gene expression of breeding hens and chicks. In experiment 1, 432 hens were divided into three groups and fed diets supplemented with 0 (as control group), 20 or 40mg/kg xanthophylls. Blood was sampled at 7, 14, 21, 28 and 35 days of trial. Liver, duodenum, jejunum and ileum were sampled at 35 days of trial. Results showed that serum HDLC level of hens was increased after dietary 40mg/kg xanthophyll addition for 21, 28 and 35 days, while serum TG, CHO and LDLC were not affected. Xanthophyll addition also increased PPARγ expression in jejunum, RXRγ expression in duodenum and jejunum, and RARα expression in liver and duodenum. Experiment 2 was a 2×2 factorial design. Male chicks hatched from 0 or 40mg/kg xanthophyll diet of hens were fed diet containing either 0 or 40mg/kg xanthophylls. Liver, duodenum, jejunum and ileum were sampled at 0, 7, 14 and 21 days after hatching. Blood samples were also collected at 21 days. Results showed that in ovo xanthophylls elevated PPARγ in duodenum and jejunum, and RXRγ and RARα in liver of chicks mainly within 1week after hatching, while dietary xanthophylls increased serum HDLC level and PPARγ and RXRγ in liver from 2weeks onwards. In conclusion, our research suggested xanthophylls can regulate serum lipid profile and nuclear factor expression in hens and chicks.

Gut Microbiota Species Can Provoke both Inflammatory and Tolerogenic Immune Responses in Human Dendritic Cells Mediated by Retinoic Acid Receptor Alpha Ligation.

Dendritic cells are considered as the main coordinators of both mucosal and systemic immune responses, thus playing a determining role in shaping the outcome of effector cell responses. However, it is still uncovered how primary human monocyte-derived DC (moDC) populations drive the polarization of helper T (Th) cells in the presence of commensal bacteria harboring unique immunomodulatory properties. Furthermore, the individual members of the gut microbiota have the potential to modulate the outcome of immune responses and shape the immunogenicity of differentiating moDCs via the activation of retinoic acid receptor alpha (RARα). Here, we report that moDCs are able to mediate robust Th1 and Th17 responses upon stimulation by Escherichia coli Schaedler or Morganella morganii, while the probiotic Bacillus subtilis strain limits this effect. Moreover, physiological concentrations of all-trans retinoic acid (ATRA) are able to re-program the differentiation of moDCs resulting in altered gene expression profiles of the master transcription factors RARα and interferon regulatory factor 4, and concomitantly regulate the cell surface expression levels of CD1 proteins and also the mucosa-associated CD103 integrin to different directions. It was also demonstrated that the ATRA-conditioned moDCs exhibited enhanced pro-inflammatory cytokine secretion while reduced their co-stimulatory and antigen-presenting capacity thus reducing Th1 and presenting undetectable Th17 type responses against the tested microbiota strains. Importantly, these regulatory circuits could be prevented by the selective inhibition of RARα functionality. These results altogether demonstrate that selected commensal bacterial strains are able to drive strong effector immune responses by moDCs, while in the presence of ATRA, they support the development of both tolerogenic and inflammatory moDC in a RARα-dependent manner.

Acute promyelocytic leukemia with a cryptic insertion of RARA into PML on chromosome 15 due to uniparental isodisomy: A case report.

Acute promyelocytic leukemia is a myeloid disorder that is characterized by the specific t(15;17) variant in ~98% of cases. The typical hypergranular and microgranular or hypogranular types exist, and are frequently associated with disseminated intravascular coagulopathy. Rare cases of promyelocytic leukemia-retinoic acid receptor α (PML-RARA) fusion without the reciprocal RARA-PML have been reported in cytogenetically normal samples. Conversely, fluorescence in situ hybridization (FISH) analysis has revealed a cryptic insertion of the RARA gene into the PML gene on chromosome 15. The current study reports a unique case with a normal karyotype and molecular evidence of the PML-RARA short isoform 3-fusion transcript, with FISH analysis revealing two fusion signals on the two copies of chromosome 15, but absence of the reciprocal on the two copies of chromosome 17. This finding raised the hypothesis of chromosome 15 uniparental isodysomy as consequence of normal chromosome 15 loss and duplication of the rearranged chromosome, as supported by polymorphic loci molecular analysis. The clinical, cytogenetic and molecular characterization of this case are presented and discussed in the present study.

Synthesis of short retinoidal amides related to fenretinide: antioxidant activities and differentiation-inducing ability.

By a scaffold shortening strategy, a small series of retinoidal amides fenretinide (4-HPR) analogs have been synthesized from α, β-ionones and tested for their antiproliferative and differentiating activities, and antioxidant effect. The antiproliferative activity and triggering of apoptosis of our short retinoids were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 4'-6-diamidino-2-phenylindole staining and microscope evaluation after 3- or 6-day exposure, while their differentiating activity was established by the analysis of the expression of the CD11b marker of differentiation in treated HL60 target cells and by the superoxide production assayed colorimetrically by the nitro blue tetrazolium-reducing activity assay. Finally, the antioxidant activity was determined by the 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt radical cation decolourisation assay utilizing the antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) as reference (Trolox equivalent antioxidant capacity, or TEAC). Docking analysis was performed to study the binding features to the Retinoic Acid Receptor alpha (RARα). While no pharmacologically relevant antiproliferative activity was evidenced, some of our short retinoids showed a differentiating and antioxidant activity similar to that of 4-HPR. In particular, compound 2b6 displayed a scavenging activity two times more efficient than 4-HPR itself. Finally, the docking analysis showed that these short retinoids, like 4-HPR, bind to the RARα protein with good fitness scores. Our data could pave the way for the design of new potent and less toxic antioxidant and differentiating compounds related to 4-HPR.

Retinoic acid receptor alpha drives cell cycle progression and is associated with increased sensitivity to retinoids in T-cell lymphoma.

Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with generally poor outcomes following standard therapy. Few candidate therapeutic targets have been identified to date. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to retinoids. While retinoids have been used to treat some cutaneous T-cell lymphomas (CTCLs), their mechanism of action and the role of RARA in CTCL and other mature T-cell lymphomas remain poorly understood. After identifying a PTCL with a RARAR394Q mutation, we sought to characterize the role of RARA in T-cell lymphoma cells. Overexpressing wild-type RARA or RARAR394Q significantly increased cell growth in RARAlow cell lines, while RARA knockdown induced G1 arrest and decreased expression of cyclin-dependent kinases CDK2/4/6 in RARAhigh cells. The retinoids, AM80 (tamibarotene) and all-trans retinoic acid, caused dose-dependent growth inhibition, G1 arrest, and CDK2/4/6 down-regulation. Genes down-regulated in transcriptome data were enriched for cell cycle and G1-S transition. Finally, RARA overexpression augmented chemosensitivity to retinoids. In conclusion, RARA drives cyclin-dependent kinase expression, G1-S transition, and cell growth in T-cell lymphoma. Synthetic retinoids inhibit these functions in a dose-dependent fashion and are most effective in cells with high RARA expression, indicating RARA may represent a therapeutic target in some PTCLs.

Abstract P6-11-18: A novel subgroup of estrogen receptor positive breast cancer may benefit from super-enhancer guided patient selection for retinoic acid receptor α agonist treatment

Abstract Endocrine-resistance remains a major challenge for treatment of breast cancer. Multiple mechanisms for endocrine resistance have been proposed, including altered expression of ER co-regulators such as Retinoic Acid Receptor Alpha (RARα). Furthermore, crosstalk between estradiol and RA signaling is known and upregulation of RARα has been observed in tamoxifen resistance. We propose a novel treatment paradigm for a newly-defined subset of HR+ patients based on our discovery of a super-enhancer (SE) associated with the RARA locus. SEs are large, highly active chromatin regions that pinpoint cancer vulnerabilities. The RARA SE-identified vulnerability can be targeted using the potent, selective, and metabolically stable RARα agonist SY-1425 (tamibarotene). SY-1425 is approved in Japan to treat Acute Promyelocytic Leukemia, has a well-established efficacy and safety profile, and may enhance response to hormonal therapy (HT) in this newly-defined subset of HR+ patients potentially delaying the need for alternate treatment. Tumor samples from 42 breast cancer patients were analyzed across a range of molecular subtypes. We identified an SE linked to the RARA gene in 54.5% of the hormone positive patient samples. RARA SEs predicted sensitivity to SY-1425 in 12 breast cancer cell lines confirming their functional role, and showed a correlation with RARA gene expression. A panel of 37 breast cancer cell lines was tested for SY-1425 anti-proliferative activity and gene expression levels, and identified RARA as the single best predictor of response. Proliferation of RARA-high cells was inhibited by SY-1425 with low nanomolar EC50s. Transcriptional profiling was performed on 4 HR+ and 3 HER2+/HR- breast cancer cell lines and analyzed by GSEA to examine the molecular response to SY-1425. Signatures for growth including E2F, MYC, DNA replication, and cell cycle were significantly downregulated while retinol metabolism and luminal signaling were upregulated. Estrogen signaling was also significantly altered by SY-1425, supporting known crosstalk between RARα and ER. Consistent with differentiation, CYP26A1 and VE-Cadherin were induced and Actin and Ki67 were diminished at relevant concentrations of SY-1425 and could serve as pharmacodynamic markers of response. To test responses to SY-1425 in vivo, two cell line-derived models and two patient-derived breast cancer models (one RARA-high, and one RARA-low each) were treated with SY-1425. SY-1425 inhibited tumor growth in the RARA-high models, but not the RARA-low models (43% versus 0% TGI). Consistent with the observed changes in transcription, SY-1425 in combination with tamoxifen synergistically inhibited proliferation of RARA-high breast cancer cell lines. Although a few clinical studies have investigated the use of ATRA in HR+ breast cancer without success, our results suggest that patient selection based on the RARA SE may predict which HR+ breast cancer patients could derive benefit by adding an RARα agonist to HT. The potential to prolong or increase the clinical effect of anti-estrogen therapy with SY-1425, which has improved potency, selectivity, and PK stability versus ATRA, would be an attractive strategy to explore. Citation Format: McKeown MR, Fiore C, Lee E, Eaton ML, Orlando D, Guenther MG, Collins C, Chen MW, Fritz CC, di Tomaso E. A novel subgroup of estrogen receptor positive breast cancer may benefit from super-enhancer guided patient selection for retinoic acid receptor α agonist treatment [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-18.

The effect of microRNA-329 on proliferation and differentiation of human osteosarcoma U-2OS cells by regulating E2F1

Objective To explore the effect of microRNA (miRNA, miR)-329 on the proliferation and differentiation of human osteosarcoma U-2OS cells by regulating E2F1. Methods The U-2OS and human osteoblast line hFOB1.19 cells were cultured. The U-2OS cells were randomly divided into three groups. In blank control group (blank), the cells were transfected with only reagent liposome. In negative control group (negative control, NC), the cells were transfected with irrelated sequence. In miR-329 group, the cells were transfected with miR-329. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-329 mRNA and E2F1 mRNA in the osteosarcoma cells and human osteoblasts. Cell counting kit-8 (CCK-8) was used to detecte the proliferation in three groups of the osteosarcoma cells at 48, 72, and 96 h. The expression levels of E2F1, related transcription factor-2 (Runx2), osteopontin (OPN), and retinoic acid alpha receptor (RARα) proteins in three groups of the osteosarcoma cells were detected by Western blotting after the cells were transfected at 48 h. Results In human osteosarcoma U-2OS cells and human osteoblasts, the expression levels of miR-329 and E2F1 mRNA respectively were 0.736±0.013, 1.215±0.026 and 1.329±0.015, 0.894±0.017. The expression level of miR-329 mRNA in the osteosarcoma cells was lower than that in the osteoblasts (P=0.008). The expression level of E2F1 mRNA in the osteosarcoma cells was higher than that in the osteoblasts (P=0.003). After transfection, the proliferation level of osteosarcoma cells in the blank group, NC group and miR-329 group at 48, 72 and 96 h was respectively 2.635±0.214, 2.584±0.107 and 1.319±0.135; 2.912±0.152, 2.826±0.165 and 1.136±0.214; and 3.109±0.143, 3.095±0.129 and 1.153±0.172. In miR-329 group, the proliferation level of the osteosarcoma cells was lower than that in the blank control group and negative control group, and the inhibitory effect increased with time, with statistically significant differences (Fintragroup=4.351, P=0.036; Ftime=2.366, P=0.043). In the miR-329 group, the expression of E2F1 protein was lower than that in the blank group and negative control group, and the expression of Runx2, OPN and RARα proteins was higher than that in the blank group and negative control group, with the differences being statistically significant (Fintragroup=9.037, P=0.025; Fintergroup=7.779, P=0.028). Conclusion The miR-329 can inhibit the proliferation of U-2OS cells by down-regulatiing the E2F1 expression, and promote osteogenic differentiation of the cells. Key words: MicroRNA-329; E2F1; Osteosarcoma; Proliferation; Differentiation

Radiolabeling and initial biological evaluation of [18F]KBM-1 for imaging RAR-α receptors in neuroblastoma

Retinoic acid receptor alpha (RAR-α) plays a significant role in a number of diseases, including neuroblastoma. Children diagnosed with high-risk neuroblastoma are treated13-cis-retinoic acid, which reduces risk of cancer recurrence. Neuroblastoma cell death is mediated via RAR-α, and expression of RAR-α is upregulated after treatment. A molecular imaging probe that binds RAR-α will help clinicians to diagnose and stratify risk for patients with neuroblastoma, who could benefit from retinoid-based therapy. In this study, we report the radiolabeling, and initial in vivo evaluation of [18F]KBM-1, a novel RAR-α agonist. The radiochemical synthesis of [18F]KBM-1 was carried out through KHF2 assisted substitution of [18F]− from aryl-substituted pinacolatoesters-based retinoid precursor. In vitro cell uptake assay in human neuroblastoma cell line showed that the uptake of [18F]KBM-1 was significantly inhibited by all three blocking agents (KBM-1, ATRA, BD4) at all the selected incubation times. Standard biodistribution in mice bearing neuroblastoma tumors demonstrated increased tumor uptake from 5min to 60min post radiotracer injection and the uptake ratios for target to non-target (tumor: muscle) increased 2.2-fold to 3.7-fold from 30min to 60min post injection. Tumor uptake in subset of 30min blocking group was 1.7-fold lower than unblocked. These results demonstrate the potential utility of [18F]KBM-1 as a RAR-α imaging agent.

Retinoic Acid Receptor Alpha Expression Drives Cell-Cycle Progression and Is Associated with Increased Sensitivity to Retinoids in Peripheral T-Cell Lymphoma

Background: Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with marked clinical, pathological, and molecular heterogeneity. Outcomes following standard therapy generally are poor; however, few candidate therapeutic targets have been identified for precision medicine approaches. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to natural or synthetic retinoids. Retinoids have been used successfully to treat acute promyelocytic leukemia and some cutaneous T-cell lymphomas (CTCLs). However, the function of RARA and the action of retinoids in PTCL have not been defined.Methods:Based on identification of a PTCL patient with a non-synonymous point mutation, RARA R394Q, identified in the Mayo Clinic Center for Individualized Medicine, we sought to characterize the role of RARA in PTCL cells. To investigate the role of wild-type and mutant RARA, we constructed expression vectors containing either wild-type RARA or RARA R394Q coding sequences, and also used siRNAs targeting RARA to study the role of native RARA expression. Cell lines derived from post-thymic T-cell malignancies were used for in vitro studies, including HuT78 and Mac-1 (both derived from circulating tumor cells from CTCL patients) and Karpas 299 (from an ALK-positive anaplastic large cell lymphoma). Following RARA overexpression or knockdown, we measured cell growth, cell cycle regulation, and sensitivity to synthetic retinoids. In addition, RNA sequencing and pathway analysis were performed to profile the transcriptomic response to retinoids in malignant T cells.Results:In two RARAlow cell lines, Karpas 299 and HuT78, overexpression of wild-type RARA or RARA R394Q significantly increased cell growth (p<0.001), with a greater increase observed from mutant versus wild-type RARA in Karpas 299 (136% of control versus 122%; p=0.04). Accordingly, knockdown of wild-type RARA in the RARAhigh cell line, Mac-1, resulted in a 22% inhibition of cell growth (p=0.0002). This inhibition specifically was associated with G1 cell cycle arrest (120% of control; p=0.004) and decreased protein expression of the G1-S-associated cyclin-dependent kinases, CDK2, CDK4, and CDK6. These kinases were up-regulated by overexpression of RARA in RARAlow HuT78 cells. The relatively RARA-specific retinoid, AM80 (tamibarotene), and the less specific retinoid, all-trans retinoic acid (ATRA), resulted in RARA protein degradation, cell growth inhibition that was both dose-dependent and proportional to baseline RARA expression, G1 arrest, and CDK protein up-regulation. Gene-set enrichment analysis (GSEA) of transcriptome data confirmed that genes down-regulated by AM80 were highly enriched for regulators of cell cycle and particularly G1-S transition. Finally, overexpressing RARA in RARAlow Karpas 299 and HuT78 cell lines significantly increased the ability of AM80 to inhibit CDK2/4/6 expression and cell growth (16% to 23% greater growth inhibition than control; p<0.05).Conclusions:RARA drives cyclin-dependent kinase expression and G1-S transition in malignant T cells, and promotes cell growth. These functions may be enhanced by specific RARA gene mutations. Synthetic retinoids inhibit these functions in a dose-dependent fashion, and are most effective in cells with high RARA expression. These data suggest RARA as a candidate therapeutic target in some PTCL patients. DisclosuresNowakowski:Celgene: Research Funding; Morphosys: Research Funding; Bayer: Consultancy, Research Funding.

Dissecting the Autophagy Tumor Suppressor Pathway Network in Acute Promyelocytic Leukemia Therapy

Acute promyelocytic leukemia (APL) is a rare disease that is successfully treated with targeted therapy in the clinic using all-transretinoic acid (ATRA) in combination with chemotherapy or arsenic trioxide (ATO). APL is invariably characterized by a translocation involving the retinoic acid receptor alpha (RARA) and genes encoding proteins with self-aggregation motifs. The translocation t(15;17) leads to the expression of the oncofusion protein PML-RARA that results in a differentiation arrest, an increase of self-renewal potential and an anti-apoptotic phenotype. Unfortunately, resistance to ATRA and ATO treatment are described in patients due to mutations in the RARA or PML moiety, respectively. Therefore, a better understanding of APL therapy induced molecular responses will allow to address treatment resistances. We and others described that current APL therapies induce autophagy. Autophagy is a proteolytic self-degradation process characterized by the formation of double-membraned vesicles, so called autophagosomes, which engulf cytoplasmic contents such as protein aggregates or defective organelles. In APL therapy autophagy is among others involved in PML-RARA degradation. We also found that key autophagy-related proteins such as WIPI1, DRAM1, ATG7 and ATG5 are essential for APL cells differentiation. A comprehensive study of APL therapy induced autophagy is needed to design meaningful combination therapies with autophagy modulating drugs.We previously found significantly lower Death-Associated Protein Kinase 2 (DAPK2) expression levels in acute myeloid leukemia (AML) patients with particular low levels in APL (Humbert et al, J Leukoc Biol 2014). DAPK2 is a positive mediator of autophagy and cell death in different cellular systems. To test if DAPK2 is also involved in autophagy and cell death responses to APL therapy, we first determined DAPK2 levels upon ATRA or ATO treatment. Both treatments significantly induced DAPK2 expression in NB4 and HT93 APL cell lines model paralleled by autophagy and cell death induction. Next, we used an immunoprecipitation screening approach to identify DAPK2 binding partners during APL therapy. We found that the known tumor suppressor p73 as well as the autophagy-related (ATG) protein ATG5 and a short form of Beclin1 that is involved in apoptosis co-precipitate with DAPK2. Then, we knocked down DAPK2 in NB4 cells via shRNAs and determined autophagic and cell death responses upon ATRA and ATO treatment. Autophagic flux was determined by endogenous Light Chain 3 (LC3)B-II puncta formation and levels by immunofluorescence microscopy and by western blot in presence or absence of the lysosomal inhibitor, Bafilomycin A1. Silencing of DAPK2 led to decreased autophagic activity upon ATRA treatment. In addition, DAPK2 depletion in NB4 cells caused a decrease in ATG5-ATG12 complex formation in both APL therapies as well as an accumulation of full-length Beclin1. Surprisingly, inhibiting DAPK2 expression did not significantly impair autophagic activity upon ATO treatment but attenuated ATO-induced apoptosis (p<0.05). Since ATRA induces a Beclin-1 independent, non-canonical, autophagy, we propose that DAPK2 regulates autophagy via ATG5-ATG12 complex formation and apoptosis via Beclin1 cleavage regulation. Moreover, knocking down p73 resulted in autophagy and apoptosis inhibition during ATRA and ATO treatment, potentially due to its downstream targets DRAM1, ATG5 and CDKN1A. Furthermore, we showed that DAPK2 stabilizes p73 protein levels within the nucleus and that silencing DAPK2 led to a significant reduction of the p73 transcriptional target gene, CDKN1A (p<0.05). Additionally, DAPK2 transcription is directly regulated by p73 thereby creating a positive feedback loop during APL treatment. Together, we identified novel protein-protein interaction networks involved in ATRA and ATO induced autophagy and cell death responses.In conclusion, we provide strong evidence that DAPK2 is at the nexus of apoptosis (DAPK2-Beclin1 short) and autophagy (DAPK2-ATG5) pathways in response to APL therapies. Thus, we identified an autophagy-related network involved in APL therapy responses that represents a promising new target in therapeutic strategies to treat APL. DisclosuresNo relevant conflicts of interest to declare.

SY1425 (tamibarotene) Induces Profound Transcriptional Changes in AML Tumors with High Retinoic Acid Receptor Alpha

Retinoic acid receptor alpha (RARα) regulates myeloid differentiation and proliferation through the regulation of specific sets of genes. When unbound by a ligand, RARα is a repressive transcription factor while in its ligand-bound state it functions as a transcriptional activator. Previously, blast cells from a subset of individuals with non-APL AML were found to have a super-enhancer (SE), as revealed by H3K27 acetyl ChIP-Seq, associated with the RARA locus (hereafter called RARA-high), suggesting that tumor cell proliferation may have a dependency on RARA that can be exploited for therapeutic benefit. SEs are exceptionally large, highly active chromatin regions that are densely occupied by transcription factors and have been implicated in oncogene expression. Indeed, RARA-high non-APL AML cell lines showed >1000-fold increased sensitivity compared to RARA-low cells to the potent and selective RARα agonist SY-1425 (tamibarotene) as well as efficacy in non-APL AML patient derived xenograft models with a dependency on RARA.Since RARα is a transcription factor and the direct target of SY-1425, we investigated the change SY-1425 had on the transcriptional program of non-APL AML cell lines and the mechanism underlying those changes. Expression profiling on a panel of AML cell lines revealed that RARA-high AML cell lines had profound transcriptional changes in response to SY-1425, with 437 genes significantly changed, while RARA-low cell lines did not show significant gene expression changes. Gene set enrichment analysis (GSEA) of three RARA-high AML cell lines revealed that the genes upregulated by SY-1425 in the RARA-high cells are associated with immune signaling, interferon induction, protein secretion, and pathways associated with complement, MHC and integrin functions, all pathways indicative of more differentiated blood cells. Signatures downregulated by SY-1425 include MYC target genes. These findings are consistent with SY-1425 increasing the expression of genes involved in differentiation and decreasing those involved in proliferation. Genome-wide ChIP-Seq analysis revealed an increase in H3K27 acetylation at loci found to have strong RARα peaks as well as increased expression of those genes upon treatment with SY-1425. Together, these data support a model in which RARα binding nucleates functional enhancers in response to SY-1425 thereby upregulating proximal target gene expression and promotion of differentiation.The gene expression and epigenomic responses of RARA-high AML cell lines to SY-1425 were found to be similar to the responses of an APL cell line (NB-4) to retinoids or SY-1425. Gene sets identified in response to either retinoid treatment or genetic perturbation, such as forced expression or RAR-fusions or knockdown, matched the gene sets identified in RARA-high AML cell lines. Furthermore, the quantitative response of both NB-4 and RARA-high AML cell lines to SY-1425 was found to be similar. Across the genome, RARα binding was highly conserved between NB-4 and RARA-high AML cell lines with less overlap with the RARA-low cell lines. For example, the transcriptional and H3K27 acetylation alterations at the known PML-RARα target gene TGM2 following retinoid treatment was similar in NB-4 and the RARA- high cell lines. This locus also had a strong RARα binding site that is conserved among the cell lines and co-localized with a strong H3K27 acetylation peak. Consistent with the pattern of occupancy of RARα on the genome, the transcriptional response of the RARA enhancer-high cell lines to SY-1425 treatment was similar to the response of APL ex-vivo patient samples to retinoic acid treatment. These data support a model of a common biological response to retinoids between cells with the RARA-PML translocation in APL and cells with the RARA SE in AML. The mechanistic studies described here support the therapeutic potential of SY-1425 in myeloid leukemia patients who have a SE associated with RARA. A biomarker directed clinical trial of SY-1425, a potent and selective RARα agonist, in a subset of AML and MDS patients with an altered RARA locus (clinicaltrials.gov, NCT02807558) is supported by these data. DisclosuresFiore:Syros Pharmaceuticals: Employment, Equity Ownership. McKeown:Syros Pharmaceuticals: Employment, Equity Ownership. Lee:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership.

Pml Nuclear Body Disruption Cooperates in APL Pathogenesis, Impacting DNA Damage Repair Pathways

Acute promyelocytic leukemia (APL) is driven by the oncogene PML-RARA which is generated by fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes, and which strongly interferes with downstream signalling and the architecture of multiprotein structures known as PML nuclear bodies (NBs). NB disruption is a diagnostic hallmark of APL, yet the significance of this phenomenon to disease pathogenesis and treatment response remains poorly understood. The majority of APL patients can now be cured with combination therapy with arsenic trioxide (ATO) and ATRA (All Trans-Retinoic Acid), which synergize promoting re-formation of disrupted Pml NBs. To date, the importance of NB disruption has only been studied in vitro. To address this, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues (C62A/C65A) in the RING domain of Pml. Homozygous PmlC62A/C65A mice are viable, and developmentally normal. At a cellular level, Pml NB disruption was confirmed and treatment with ATO was associated with defective Pml SUMOylation and degradation. A key feature of APL fusion proteins is the capacity to homodimerise (mediated by the fusion partner e.g. PML), which is not a feature of wild-type RARα. This forced homodimerisation of RARα has been shown to be critical for APL pathogenesis. We investigated whether Pml NB disruption could cooperate in vivo with forced RARα homodimerisation (mediated artificially by linking RARα to the dimerisation domain of the NFκB p50 subunit). While no leukemias arose in PmlC62A/C65A mice, p50-RARα mice expressing PmlC62A/C65A presented a doubling in the rate of leukemia development (p<0.0001) compared to PmlWT-p50-RARα, leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency period to onset of leukemia was significantly reduced in the context of NB disruption (p=0.008). ATRA treatment significantly improved the survival of mice transplanted with PmlWT-p50-RARα or Pml-RARα leukemic blasts, but not with PmlC62A/C65A-p50-RARα. These data reveal not only the key role of PML-RARα expression-induced NB disruption in APL development, but also the importance of re-formation of NBs for an effective response to differentiating drug.While formation of the PML-RARA fusion is considered an initiating event in APL pathogenesis, it is insufficient for the full leukemic phenotype. Exome sequencing studies have consistently identified presence of cooperating mutations. Since Pml and Pml NB have established roles in DNA repair and in the maintenance of genomic stability, we speculated that loss of NB integrity could affect these functions. Whole exome sequencing revealed a pattern of higher genomic instability in PmlC62A/C65A-p50-RARα leukemia as compared to PmlWT-p50-RARα, with detection of mutations found in human APL, including Kras, Ptpn11 and Usp9y. Using DNA repair reporter assays, we demonstrated that DNA repair via both non-homologous end joining (NHEJ; p=0.01) and homologous recombination (HR; p=0.006) pathways was less efficient in PmlC62A/C65A primary cells than in PmlWT cells. Importantly, using a PML-RARα-inducible cell line, comparable defects in the NHEJ and HR pathways, which were PML-RARα dependent, were identified. These data were also supported by an increase in sister-chromatid exchange (p<0.0001) and chromosome abnormality (p=0.0002) in the context of PmlC62A/C65A versus PmlWT. Interestingly, the kinetic of repair of ionising radiation (IR)-induced DNA double-strand breaks, assessed by analysis of γH2AX foci formation and clearance, was not affected. None of the DNA repair players analysed (e.g. Blm, Rad51 and 53BP1) failed to form foci in response to IR. However, their basal levels of foci were significantly greater in the presence of PmlC62A/C65A (p<0.04; quantified using Amnis ImageStreamX Mk II imaging flow cytometer). Additionally, we found that Rad51 foci showed a defect in localisation post-IR when PmlC62A/C65A was expressed, with impairment of Rad51 co-localisation and interaction with γH2AX. Altogether, our data therefore highlight the significant contribution of Pml NB to the effectiveness of DNA damage repair processes, and the manner in which their disruption mediated by the PML-RARα oncoprotein can assist APL pathogenesis. DisclosuresHills:TEVA: Honoraria. Grimwade:TEVA: Research Funding.