Ets-1 Is Essential for Connective Tissue Growth Factor (CTGF/CCN2) Induction by TGF-β1 in Osteoblasts

Ets-1 controls osteoblast differentiation and bone development; however, its downstream mechanism of action in osteoblasts remains largely undetermined. CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts; however, the molecular mechanisms that control CCN2 induction are poorly understood. In this study, we investigated the role of Ets-1 for CCN2 induction by TGF-β1 in primary osteoblasts.We demonstrated that Ets-1 is expressed and induced by TGF-β1 treatment in osteoblasts, and that Ets-1 over-expression induces CCN2 protein expression and promoter activity at a level similar to TGF-β1 treatment alone. Additionally, we found that simultaneous Ets-1 over-expression and TGF-β1 treatment synergize to enhance CCN2 induction, and that CCN2 induction by TGF-β1 treatment was impaired using Ets-1 siRNA, demonstrating the requirement of Ets-1 for CCN2 induction by TGF-β1. Site-directed mutagenesis of eight putative Ets-1 motifs (EBE) in the CCN2 promoter demonstrated that specific EBE sites are required for CCN2 induction, and that mutation of EBE sites in closer proximity to TRE or SBE (two sites previously shown to regulate CCN2 induction by TGF-β1) had a greater effect on CCN2 induction, suggesting potential synergetic interaction among these sites for CCN2 induction. In addition, mutation of EBE sites prevented protein complex binding, and this protein complex formation was also inhibited by addition of Ets-1 antibody or Smad 3 antibody, demonstrating that protein binding to EBE motifs as a result of TGF-β1 treatment require synergy between Ets-1 and Smad 3.This study demonstrates that Ets-1 is an essential downstream signaling component for CCN2 induction by TGF-β1 in osteoblasts, and that specific EBE sites in the CCN2 promoter are required for CCN2 promoter transactivation in osteoblasts

Ets-1 synergizes with TGF-β1 for CCN2 promoter induction in osteoblasts.

<p>(<b>A</b>) Osteoblasts were plated in 96 well tissue culture plates and transfected with either 0.4 µg of an empty vector control (−) or the Ets-1 expression construct (+). All samples were co-transfected with 0.4 µg of our previously described CCN2 promoter luciferase reporter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone.0035258-Arnott2" target="_blank">[22]</a> and 0.2 µg of a renilla luciferase expression vector as an internal control. The cells were serum starved for 24 hrs and then treated with TGF-β1 (5 ng/ml) (+) or mock treated (−) with TGF-β1 diluent for 24 hrs. Luciferase activity was then assessed and expressed as a ratio of firefly/renilla luciferase (+SEM, n = 6). A = p<0.05 compared to +TGF-β1 only or +Ets-1 only. (<b>B</b>) Osteoblasts were plated in 96 well tissue culture plates and transfected with either 100 nM of Ets-1 siRNA (Ets-1) or control siRNA (C) for 48 hrs. All samples were co-transfected with 0.4 µg of our previously described CCN2 promoter luciferase reporter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone.0035258-Arnott2" target="_blank">[22]</a> and 0.2 µg of a renilla luciferase expression vector as an internal control. The cells were serum starved for 24 hrs and then treated with 5 ng/ml of TGF-β1 (+) or mock treated (−) with TGF-β1 diluent for 24 hrs. Luciferase activity was then assessed and expressed as a ratio of firefly/renilla luciferase (+SEM, n = 6). Star symbol indicates p<0.05 compared to control siRNA.</p

EBE sites are required for CCN2 promoter activation by TGF-β1 in osteoblasts.

<p>Osteoblasts were plated in 96-well tissue culture plates and transfected with 0.4 µg of either EBE mutation construct 1–8, pGL3-Basic (negative control) or W787 (positive control) and all were co-transfected with 0.2 µg of a renilla luciferase expression vector (internal control) for 24 hrs. The cells were serum starved for 24 hrs and then treated with TGF-β1 (5 ng/ml) for 24 hrs. Luciferase activity was assessed, and expressed as a % of activity obtained using the full length W787 construct. (+SEM, n = 6). A = p<0.05 compared to W787.</p

Mutation of EBE sites prevents protein complex binding.

<p>(<b>A</b>) Electro-mobility shift assays (EMSA) probes were created that were homologues to the CCN2 promoter and contained either mutated or unmutated EBE sites (#5-8) as indicated. Each probe was dsDNA and 5′ biotinylated. (<b>B</b>) Electro-mobility shift assays (EMSA) from nuclear lysates were generated from osteoblasts that were treated with TGF-β1 (5 ng/ml) for 2 hrs. Nuclear protein binding to the wild type and mutated EBE sites in the CTGF promoter was assessed using 5 µg of nuclear lysates. The lane number above each well corresponds to the probe used for that reaction. The experiment was repeated four times with similar results.</p

Ets-1 binds to EBE sites in the CCN2 promoter in osteoblasts.

<p>Electro-mobility shift assays (EMSA) from nuclear lysates were generated from osteoblasts that were treated with TGF-β1 (5 ng/ml) for 2 hrs. (<b>A</b>) Nuclear protein binding to the wild type E-E-E (lanes 1–6) (this probe contains EBE # 6-8; for probe design see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone-0035258-g006" target="_blank">Figure 6</a>) probe was assessed using 5 µg of nuclear lysates for each reaction. In some reactions, Ets-1 antibody was added at increasing concentrations (1 ug of antibody in lane 4; Two micrograms of antibody in lane 5) to test for Ets-1/probe interaction. Control antibody (2 ug) was also used (lane 6; C). In some cases, probe only (lane 1) or a molar excess of unlabeled probe (lane 3) was also used to demonstrate specificity. (<b>B</b>) Nuclear protein binding to the wild type S-E-T (lanes 7–14) (this probe contains EBE#5; for probe design see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone-0035258-g006" target="_blank">Figure 6</a>) probe was assessed using 5 µg of nuclear lysates for each reaction. In some reactions, Ets-1 antibody was added at increasing concentrations (1 ug of antibody in lane 10; 2 ug of antibody in lane 11) to test for Ets-1 protein/probe interaction or Smad 3 antibody (1 ug of antibody in lane 12; 2 ug of antibody in lane 13) to test for Smad 3 protein/probe interaction. Control antibody (2 ug) was also used (lane 14). In some cases, probe only (lane 7) or a molar excess of unlabeled probe (lane 9) was also used to demonstrate specificity.</p

The Lysine-Specific Demethylase 1 (LSD1) Inhibitor Tranylcypromine (TCP) in Combination with ATRA Is Tolerable and Has Anti-Leukemic Activity in Adult Patients with Relapsed/Refractory AML and MDS

Abstract Introduction: Tranylcypromine (TCP) is an irreversible monoamine oxidase inhibitor, a potent antidepressant that has been in use since the 1960s. Additionally, TCP has been demonstrated to inhibit lysine-specific histone demethylase 1A (LSD1), which is highly expressed in AML (Lee 2006; Berglund 2008). Preclinical studies combining TCP and ATRA induced differentiation and impaired clonogenic survival in non-APL AML cell lines and primary patient samples. These findings were supported by mouse xenograft models (Schenk 2011). Based on this preclinical work, we pursued an investigator-initiated Phase 1 study of this combination at the University of Miami Sylvester Comprehensive Cancer Center (NCT02273102). Methods: A Phase 1 study was initiated to evaluate the safety, PK/PD, and preliminary clinical activity of TCP in combination with ATRA in patients (pts) with relapsed/refractory AML and high-grade MDS. The study followed a traditional 3+3 dose escalation design. Safety for all pts and efficacy for all evaluable pts to date are reported. All adverse events were recorded per NCI CTCAE v4.03. All pts received continuous daily dosing of both ATRA (45 mg/m2 in divided doses) and TCP (3 escalating dose levels: 10mg BID, 20mg BID and 30mg BID), with a 3-day lead-in of TCP only during cycle 1. Cycles were 21 days and pts were allowed to remain on study until progression or unacceptable toxicity. Results: At the time of data cutoff, 15 pts had received therapy with combination TCP/ATRA (8 AML and 7 MDS). Median age was 74 years, 40% were female and 67% white/27% Hispanic/7% black. Overall, the combination was well tolerated, with the majority of treatment emergent adverse effects (TEAEs) Grade 1 and 2. The most common TEAEs (all grades, ≥20%) included dry mouth (33%), dry skin (27%), febrile neutropenia (27%), dizziness (27%), fatigue (27%), headache (27%), rash (27%), increase in creatinine (27%); and vomiting, nausea, diarrhea, infection, urinary frequency and thrombocytopenia all with a frequency of 20%. The most common Grade 3/4 TEAEs included febrile neutropenia (27%), thrombocytopenia (20%), sepsis (13%), lung infection (13%) and anemia (13%). There was 1 DLT of dizziness at the TCP 20mg BID dose level (out of 8 pts) and 2 DLTs of generalized weakness and nausea/vomiting, respectively, at 30mg BID (out of 3 pts). All DLTs were grade 2, but persistent and poorly tolerated. Therefore, TCP 20mg BID was determined to be the MTD and selected as the RP2D. Best evaluable responses per modified IWG/ELN criteria included 5 pts with prolonged stable disease (all 3 months or more) (2 AML, 1 CMML, 2 MDS), 1 marrow CR (MDS) and 1 MLFS (AML). Three of the 4 MDS/CMML responders had hematologic improvement (HI) (2 HI-P and 1 HI-P and HI-E). One AML pt also recovered neutrophils (0.62 to 14.75) with a decrease in blasts but did not meet response criteria. The 2 pts with best response of marrow CR and MLFS continued on study for 7 and 10 months, respectively. Importantly, these 2 pts and a third pt who had prolonged SD (5 months) plus HI-P/HI-E were all taken off study for cumulative skin toxicity (not progression), and the marrow CR and MLFS pts are both still alive. Conclusions: TCP/ATRA combination therapy has demonstrated an acceptable safety profile in pts with R/R AML and MDS, and additionally has demonstrated clinical activity. TCP 20mg BID is the RP2D, and a phase 1 dose expansion at this dose level is ongoing. In responders, skin toxicity may be treatment duration-limiting due to continuous exposure to ATRA, and an intermittent ATRA schedule after cycle 4 may be pursued for the phase 2 study. Additional data will be presented at the meeting, including myeloid mutational analysis, RNA-seq and ATAC-seq, in order to delineate pre- and post-treatment molecular profiles and chromatin accessibility in these pts. Preliminary data (not shown) suggest that a baseline gene expression pattern may predict sensitivity or resistance to TCP/ATRA. Disclosures Watts: Takeda: Research Funding; Jazz Pharma: Consultancy, Speakers Bureau. Swords:AbbVie: Employment