566 GNS396 and analogues are potent new small molecules to target and kill chemotherapy-resistant subpopulation cells in acute myeloid leukemia

Background: Standard combination chemotherapy for advanced SCLC has not changed substantially in the last two decades. The challenge when identifying novel SCLC therapies is the prevalence of multiple inactivating mutations in tumor suppressor genes (TP53, PTEN, RB1) and gain-of-function oncogenic mutations (PI3K3CA, MYC family). BET bromodomain inhibitors such as OTX015 (OncoEthix SA) which binds to human bromodomain proteins BRD2/3/4, modulate the transcription of target genes via epigenetic mechanisms. OTX015 shows preclinical activity in hematologic and solid tumors and is currently in clinical development. Here we characterized the effects of OTX015 on a series of SCLC cell lines. Material and Methods: OTX015 growth inhibition 50% (GI50) values were determined with the MTT assay after 72 h exposure in four established SCLC cell lines (H82, H69, DMS79, DMS114). Protein levels were analyzed by Western Blot using commercial antibodies and RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System at baseline, then 4 and 24 h post-treatment. For cell cycle analysis, cells were stained with propidium iodide and analyzed for DNA content with a FACScan flow cytometer. Results: DMS114 cells showed dose-dependent sensitivity to OTX015 [GI50 = 120 (84–172) nM], while H82, H69, DMS79 cells were resistant [GI50 >6 mM], although they overexpressed CMYC and/or NMYC proteins. While all four SCLC cell lines are KRAS wild-type and TP53 mutated, OTX015-resistant SCLC cell lines harbor a homozygous mutation in RB1, while DMS144 cells express RB1 wild-type. All cell lines exhibited similar basal mRNA levels of BRD2/3/4, CMYC, NMYC, HEXIM, BCL-2 and p21. Following OTX015 treatment at 500 nM, CMYC and NMYC mRNA levels were unchanged, while mRNA levels of HEXIM and genes coding for histones (HIST1H2BK, HIST2H2BJ) were upregulated in all four cell lines. In DMS114 cells, OTX015 caused cell cycle arrest in G1 in a timedependent manner, which may be explained by an upregulation at the protein level of the cell cycle inhibitor p27. Conclusion: Our findings suggest that the MYC family does not mediate OTX015 antitumor effects in SCLC cell lines. The presence of functional RB1 protein, controlling cell progression at G1, may explain the cytostatic effects of OTX015. Further studies investigating the therapeutic potential of OTX015 in SCLC are ongoing.