Autophagy-Dependent Sensitization of Triple-Negative Breast Cancer Models to Topoisomerase II Poisons by Inhibition of the Nucleosome Remodeling Factor

Abstract

Epigenetic regulators can modulate the effects of cancer therapeutics. To further these observations, we discovered that the bromodomain PHD finger transcription factor subunit (BPTF) of the nucleosome remodeling factor (NURF) promotes resistance to doxorubicin, etoposide, and paclitaxel in the 4T1 breast tumor cell line. BPTF functions in promoting resistance to doxorubicin and etoposide, but not paclitaxel, and may be selective to cancer cells, as a similar effect was not observed in embryonic stem cells. Sensitization to doxorubicin and etoposide with BPTF knockdown (KD) was associated with increased DNA damage, topoisomerase II (TOP2) crosslinking and autophagy; however, there was only a modest increase in apoptosis and no increase in senescence. Sensitization to doxorubicin was confirmed in vivo with the syngeneic 4T1 breast tumor model using both genetic and pharmacologic inhibition of BPTF. The effects of BPTF inhibition in vivo are autophagy dependent, based on genetic autophagy inhibition. Finally, treatment of 4T1, 66cl4, 4T07, MDA-MB-231, but not ER-positive 67NR and MCF7 breast cancer cells with the selective BPTF bromodomain inhibitor, AU1, recapitulates genetic BPTF inhibition, including in vitro sensitization to doxorubicin, increased TOP2-DNA crosslinks and DNA damage. Taken together, these studies demonstrate that BPTF provides resistance to the antitumor activity of TOP2 poisons, preventing the resolution of TOP2 crosslinking and associated autophagy. These studies suggest that BPTF can be targeted with small-molecule inhibitors to enhance the effectiveness of TOP2-targeted cancer chemotherapeutic drugs. Implications: These studies suggest NURF can be inhibited pharmacologically as a viable strategy to improve chemotherapy effectiveness.

New Design Rules for Developing Potent Cell-Active Inhibitors of the Nucleosome Remodeling Factor (NURF) via BPTF Bromodomain Inhibition

“…BPTF has been implicated in resistance to chemotherapeutics for treating hepatocellular carcinoma and BRAF inhibitors for melanoma therapy . We recently identified the BPTF suppression of topoisomerase 2 poisons, including doxorubicin and etoposide, whose cytotoxic activities were enhanced with BPTF knockdown or bromodomain inhibition with AU1, respectively . While the knockdown of BPTF in 4T1 mouse breast cancer cells does not exhibit toxicity on its own, the AU1 treatment exhibited toxicity at higher concentrations, which is consistent with an off-target effect.…”

“…16 We recently identified the BPTF suppression of topoisomerase 2 poisons, including doxorubicin and etoposide, whose cytotoxic activities were enhanced with BPTF knockdown or bromodomain inhibition with AU1, respectively. 31 While the knockdown of BPTF in 4T1 mouse breast cancer cells 44 does not exhibit toxicity on its own, 45 the AU1 treatment exhibited toxicity at higher concentrations, which is consistent with an off-target effect. We first tested several of our pyridazinones and found them to be well-tolerated by the 4T1 cells up to midmicromolar concentrations with the exception of BZ1, which started to exhibit some toxicity at 8 μM (% survival = 56% and 89% in two separate experiments) (Figures 6A and S12).…”

“…30 Most recently, AU1 showed an enhancement of anticancer activity when used in combination with the chemotherapeutic drug doxorubicin in 4T1 breast cancer models both in vitro and in vivo. 31 Mechanistic studies showed these processes to be autophagy-dependent, and AU1's effects on topo2-isomerase-DNA cross-links and DNA damage recapitulated the effects from BPTF knockdown experiments. However, the off-target kinase activity of AU1, its poor physicochemical properties, and low ligand efficiency posed significant challenges to inhibitor development and highlighted the need for new and more potent BPTF inhibitors.…”

“…AU1 has since been used in mouse mammary epithelial cells showing decreased proliferation, cell cycle arrest, and reduced c-Myc-DNA occupancy; however, off-target activity was identified in other cell lines . Most recently, AU1 showed an enhancement of anticancer activity when used in combination with the chemotherapeutic drug doxorubicin in 4T1 breast cancer models both in vitro and in vivo . Mechanistic studies showed these processes to be autophagy-dependent, and AU1’s effects on topo2-isomerase-DNA cross-links and DNA damage recapitulated the effects from BPTF knockdown experiments.…”

“…The high affinity, aqueous solubility, and physicochemical properties of these inhibitors enabled us to obtain several co-crystal structures with BPTF to rationalize our structure−activity relationship data and to identify an acidic triad as a targetable feature of the binding site for future inhibitor development. Finally, we use the 4T1 breast cancer cell chemotherapeutic synergy model previously validated for BPTF on-target engagement, 31 to show that our scaffolds are generally well-tolerated by cells and enhance doxorubicin cytotoxic effects to wild type breast cancer cells. They do not enhance cytotoxic effects in identical cells with BPTF knockdown, demonstrating specificity in their biological activity.…”