OTX015, a novel BET inhibitor is a promising anticancer agent for multiple myeloma Maria Todaro1, Michela Boi1, Valentina Vurchio1, Elisabetta Ercole1, Rodolfo Machiorlatti1, Katia Messana1, Indira Landra1, Susanna Urigu1, Sabrina Aliberti1, Eugenia Riveiro2, Francesco Bertoni3, Giorgio Inghirami1 Pathology and Center for Experimental Research and Medical Studies (CeRMS), University of Turin, Turin, Italy; 2 Oncology Therapeutic Development, Clichy, France; 3 Lymphoma and Genomics Research Program, IOR Institute of Oncology Research, Bellinzona, Switzerland. 1600000 E 6 5 48H 60 80 50 % of cells RPMI U266 60 KMS27 JJN3 RLU (luminescence) 1200000 Ctct 40 250nM OTX250 OTX500 500nM 30 40 24h Ct 24h 500 nM 1000000 48h Ct 48h 500 nM 800000 72h Ct 72h 500 nM 600000 CTRL 1400000 100 4 72H KMS27 96H Brd4 Ct 3 250nM 500nM Brd2 2 20 400000 20 C-Myc 10 1 200000 0 NT 3 nM 25 nM 74 nM 222 nM 666 nM 2uM 0 6uM G1 S G2 Death G1 KMS27 -20 S G2 Death G1 S RPMI8226 G2 Act Death 0 JJn3 KMS27 MYC BRD2 BRD3 BRD4 NUC CAD ODC The effect on c-MYC mRNA (D) and protein levels (E) after different time exposure to OTX015 at a assay and IC50s were calculated (A). Cell lines were treated with 250 and 500 nM OTX015 and cell cycle analysis was performed after 72h of drug exposure. Both range of concentrations (250-500nM) was evaluated in KMS27 cells, showing c-MYC doses induced a consistent G1 cell cycle arrest and in some cell lines an increase of death rate was observed (B). Anti-proliferative activity was evaluated by ATP-Lite downregulation at both mRNA and protein levels. mRNA levels of downstream c-MYC target genes luminescent assay after 24h, 48h and 72h of OTX015 (500nM) exposure. Proliferation block is observed already after 48h of treatment but it is more consistent at 72h (C). (NUC, CAD, ODC) and BRD2/3/4 were also evaluated (D,E). 24h 12h F 48h We compared OTX015 activity (250 and 500 nM) in RPMI 8226, with the “bona fide” BET inhibitor, JQ1. OTX015 treatment (250nm) 6h WO 1h 2h 4h 6h 8h 24h JQ1 250nM Toxicity Test downregulation on c-MYC levels 35 was seen with both inhibitors (F). CT Harvest, RNA and real-time 100mg/kg OTX015, bid, po Similar dose- and time- dependent OTX015 treatment (250nm) 30 weight 30’ 50mg/kg OTX015, bid, po MYC expression was evaluated. JQ1 500nM OTX015 500nM CT OTX015 250nM JQ1 500nM JQ1 500nM OTX015 500nM CT OTX015 250nM JQ1 250nM JQ1 500nM OTX015 500nM CT CT OTX015 250nM and 48h of drug exposure and c- JJN3 Water, po H RNA was extracted after 12h, 24h G 0 RPMI 8266 A panel of six MM cell lines was treated with a range of OTX015 concentrations (from 3 nM to 6 µM) for 72 h and antiproliferative activity was evaluated with the MTT Material and Methods Contact: Maria Todaro, Department of Pathology and CeRMS, University of Turin, Italy. [email protected]. KMS27 D 1800000 CTRL 70 RPMI8266 Human cell lines derived from MM (KMS27, RPMI 8226, JJN3) were treated with increasing doses of OTX015 (OncoEthix SA, Switzerland). Cell proliferation was evaluated by ATPlite and MTT assays over time, and cell cycle analysis by FACScan flow cytometer. RNA was extracted using TRIzol and reverse-transcribed with the Superscript First-Strand Synthesis System kit. RT-qPCR was performed using SYBR Green Master Mix on a Bio-RAD Real-Time PCR System. For Western blot analysis, lysates were fractionated on 8% polyacrilamide gels and membranes incubated with commercial antibodies. In vivo toxicity study was performed with three doses(25,50 and 100mg/kg) in NSG mice. MM1S cells (1.106 cells each flanks) were inoculated in NSG mice. Treatment was started 72h-post inoculation with vehicle (water) or OTX015 (50-100 mg/kg bid, po, 5 days on-2 days off) for 4 weeks. Tumor growth was measured over time. ATP Lite C 80 OTX 500 120 CTRL OTX015 treatment, 72h B 140 OTX 500 OTX015 IC50 A % viable cells The activity of bromodomain and extra-terminal (BET) protein inhibitors has recently been demonstrated in several human hematologic malignancy models, including multiple myeloma (MM). Exposure of cancer cells lines to BET inhibitors results in a significant downregulation of c-MYC expression, and suppression of transcriptional programs associated with proliferation and survival. OTX015 is a new selective orally bioavailable inhibitor of the BET family proteins currently in clinical development. We evaluated its activity in a panel of human MM cell lines for whom a known overactivaction of the c-MYC pathway has been described. Moreover, OTX015 activity was also assessed in an in vivo MM model. RESULTS OTX 500 BACKGROUND fold change (respect to ct) 1 Department of RPMI8226 6h WO 30’ 1h 3298 25 3299 3300 20 3301 3302 15 2h 4h 6h 8h 24h 3303 10 0 Harvest, RNA and real-time 2 4 7 9 11 14 16 18 21 days of treatments with OTX015 100mg/kg 23 25 NSG mice were inoculated with 1.106 MM1S cells in each flanks and treated with different schedules starting day 3 from the injection during 30 days. The effect of 50 mg/kg or 100 mg/kg OTX015 (twice daily; po) for up to 4 weeks in NSG mice inoculated was evaluated in terms of tumor volume (H). Insert. The effect of OTX015 on c-MYC mRNA down-regulation after shorter drug exposures was evaluated. Cells were treated with OTX015 for 6h after which the culture medium was replaced with fresh medium without the drug (washout). C-MYC levels were downregulated following 6h treatment and NSG mice were treated with OTX015 (25, 50, or 100 mg/kg daily doses/orally once a day) and toxicity was assessed testing the total body mass (mouse body weight). No decrease of animal weight was observed after 30 days of treatment for any of the tested doses, suggesting that OTX015 has minimal toxicity (data are presented for mice treated with 100 mg/kg) recovery of c-Myc levels was time and cell line dependent (G). CONCLUSIONS The new bromodomain inhibitor, OTX015, shows antiproliferative activity in a panel of MM cell lines and induced cell cycle arrest in a dose- and time-dependent manner concomitant to c-MYC downregulation. OTX015 is active in vitro for up to 72h in almost all cell lines, including after drug wash-out. In vivo experiments show tumor volume decrease after drug exposure. Our preclinical results suggest that OTX015 may also be active in MM patients, currently a phase I clinical study (NCT01713582; AACR2014 oral presentation CT231) is recruiting patients affected by hematologic cancer. Further preclinical evaluation of its activity in combination with standard therapy regimens for MM patients is ongoing.
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