BET bromodomain inhibitors synergize with ATR inhibitors in melanoma

Abstract

Metastatic malignant melanoma continues to be a challenging disease despite clinical translation of the comprehensive understanding of driver mutations and how melanoma cells evade immune attack. In Myc-driven lymphoma, efficacy of epigenetic inhibitors of the bromodomain and extra-terminal domain (BET) family of bromodomain proteins can be enhanced by combination therapy with inhibitors of the DNA damage response kinase ATR. Whether this combination is active in solid malignancies like melanoma, and how it relates to immune therapy, has not previously investigated. To test efficacy and molecular consequences of combination therapies cultured melanoma cells were used. To assess tumor responses to therapies in vivo we use patient-derived xenografts and B6 mice transplanted with B16F10 melanoma cells. Concomitant inhibition of BET proteins and ATR of cultured melanoma cells resulted in similar effects as recently shown in lymphoma, such as induction of apoptosis and p62, implicated in autophagy, senescence-associated secretory pathway and ER stress. In vivo, apoptosis and suppression of subcutaneous growth of patient-derived melanoma and B16F10 cells were observed. Our data suggest that ATRI/BETI combination therapies are effective in melanoma.

Figure 1

ATRIs synergize with BETi to kill melanoma cells and induce SASP/ER stress. (a and b) A375 cells (BRAF^V600E) and MeWo cells (NF1^−/−) were treated with vehicle (0.1% DMSO), 10 μM VE821 (VE; AXON Medchem, Groningen, The Netherlands), 1 μM JQ1 (Cayman Chemicals, Ann Arbor, MI, USA), 10 μM RVX2135 (RVX) or indicated combinations. The experiments were repeated twice in biological triplicates. Cells were imaged in a light microscope (a) or counted in a hemocytometer (b). (c) A375 and MeWo cells were cultured in the presence of vehicle (DMSO), 10 μM of RVX2135 (RVX) and/or the ATRI VE821 (VE; 10 μM) for 48 h, and were assayed for viability with CellTiter Glo. Value to achieve synergy is shown with a dotted line. (d) A375 cells treated with vehicle, 10 μM VE821, 10 μM RVX2135 or both VE821/RVX2135 were analyzed by qRT-PCR for indicated genes. (e) A375 cells treated as described above and analyzed by western blot analysis using indicated antibodies

Figure 2

ATRIs synergize with BETi to kill melanoma cells. (a) The melanoma PDX model M121218 was initiated by thawing a stock of cryopreserved melanoma tumor cells, and injecting the cells subcutaneously into the flank of 10 immunocompromised NOD/SCID/IL2Rγ mice (Taconic). Tumor sizes were measured bi-weekly using an caliper. When the tumors reached 75–100 mm³ 5 mice each were randomized to receive either oral and i.p. vehicle, or oral RVX2135 at 75 mg/kg b.i.d. and i.p. injection of AZ20 (MedChemExpress, Princeton, NJ, USA) at 50 mg/kg q.d. for 5 days a week. (b) Four hours after the last dose, tumors were excised and weighed. (c) A blood sample was drawn from the saphenous vein of all mice before treatment and after 3 weeks of treatment. Plasma was isolated and used to determine the level of the melanoma marker S100B using an ELISA kit from Abcam (Elisa kit from Abnova, Taipei City, Taiwan). (d) Single cells were derived by trypsinization of excised tumors from vehicle-treated or combination-treated mice. The cells were lysed and their nuclei were labeled with 7-AAD. Sub-G1 content (apoptosis) was measured by flow cytometry. (e) Tumor pieces from M121218 PDXs treated with vehicle or the RVX2135/AZ20 combination treatment were subjected to western blot analysis

Figure 3

Combined ATR and BET inhibition reduces growth of patient-derived melanoma tumorgrafts in mice. (a) Growth of three melanoma PDX models, originally developed from a biopsies of patients’ metastases, transplanted subcutaneously onto the flank of NOG mice (n=4 per treatment group). When the tumors reached 75–100 mm³ they were randomized to receive either oral vehicle or 75 mg/kg RVX2135 b.i.d. and an intraperitoneal injection of vehicle or 50 mg/kg AZ20 q.d. five times per week. After the first week of treatment PDX model M120903 exhibited sings of treatment-induced distress so the dose of AZ20 was reduced to 25 mg/kg q.d. for all mice of all PDX models subsequently treated to avoid complications. (b) Four hours after the last dose, tumors were excised and weighed from the indicated PDX models. The tumor texture of M120903 was very loose preventing accurate weighing. (c) Tumor pieces from M130116 PDXs treated with the vehicle or the RVX2135/AZ20 combination treatment were subjected to western blot analysis

Figure 4

ATRI/BETI combination treatment kills B16 melanoma cells in vitro and in vivo. (a) B16F10-luciferase cells were cultured in RPMI-1640 emented with 10% fetal bovine serum and antibiotics. They were treated with vehicle (0.1% DMSO), 10 μM VE821 (VE), 10 μM RVX2135 (RVX) or indicated combinations. The experiments were repeated twice in biological triplicates. Cells were imaged in a light microscope (a) or counted in a hemocytometer (b). (c) B16F10-luciferase cells were cultured in the presence of vehicle (DMSO), 10 μM of RVX2135 (RVX) and/or the ATRI VE821 (VE; 10 μM) for 48 h and were assayed for viability by adding luciferin (Perkin-Elmer) to a final concentration of 100 μg/ml. Value to achieve synergy is shown with a dotted line. (d) B16F10-luciferase cells were cultured in the presence of vehicle (DMSO), 0.5 μM of iBET762 and/or 2 μM of the ATRI AZ20 for 48 h and were assayed for viability by adding luciferin (Perkin-Elmer) to a final concentration of 100 μg/ml. Value to achieve synergy is shown with a dotted line. (e) Eight 6- to 8-week-old C57BL/6 Albino mice were transplanted subcutaneously with 10⁵ B16F10-luciferase cells. Seven days after transplantation, mice were imaged in an IVIS Lumina III XR machine. After imaging, mice were treated with oral and i.p. vehicle, or oral RVX2135 at 75 mg/kg b.i.d. and i.p. injection of AZ20 at 25 mg/kg for 4 days, followed by imaging again. Shown is the fold change in luciferase signal during treatment (n=4 mice per treatment group)