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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.

Supplemental Figure 5. Selective BPTF Bromdomain Inhibitor AU1 Recapitulates Effects from BPTF KD in Human MDA-MB-231 and Mouse 66cl4 and 4T07 Cell Lines.

Supplemental Figure 2. NURF Selectively Suppresses Toxicity to Topoisomerase 2 Poisons and Improves their DNA Damaging Effects.

Supplemental Figure 4. Selective BPTF Bromdomain Inhibitor AU1 Recapitulates Effects from BPTF KD in 4T1 Cells and Tumors.

Supplemental Figure 3. Validation of Autophagy by Microscopy and ATG5 KD in 4T1 Cells.

Early insults associated with cardiac transplantation increase the immunogenicity of donor microvascular endothelial cells (ECs), which interact with recipient alloreactive memory T cells and promote responses leading to allograft rejection. Thus, modulating EC immunogenicity could potentially alter T cell responses. Recent studies have shown modulating mitochondrial fusion/fission alters immune cell phenotype. Here, we assess whether modulating mitochondrial fusion/fission reduces EC immunogenicity and alters EC-T cell interactions. By knocking down DRP1, a mitochondrial fission protein, or by using the small molecules M1, a fusion promoter, and Mdivi1, a fission inhibitor, we demonstrate that promoting mitochondrial fusion reduced EC immunogenicity to allogeneic CD8+ T cells, shown by decreased T cell cytotoxic proteins, decreased EC VCAM-1, MHC-I expression, and increased PD-L1 expression. Co-cultured T cells also displayed decreased memory frequencies and Ki-67 proliferative index. For in vivo significance, we used a novel murine brain-dead donor transplant model. Balb/c hearts pretreated with M1/Mdivi1 after brain-death induction were heterotopically transplanted into C57BL/6 recipients. We demonstrate that, in line with our in vitro studies, M1/Mdivi1 pretreatment protected cardiac allografts from injury, decreased infiltrating T cell production of cytotoxic proteins, and prolonged allograft survival. Collectively, our data show promoting mitochondrial fusion in donor ECs mitigates recipient T cell responses and leads to significantly improved cardiac transplant survival.

Chronic obstructive pulmonary disease-associated chronic inflammation has been shown to lead to an autoimmune phenotype characterized in part by the presence of lung autoreactive antibodies. We hypothesized that ischemia-reperfusion injury (IRI) liberates epitopes that would facilitate preexisting autoantibody binding, thereby exacerbating lung injury after transplant. We induced emphysema in C57BL/6 mice through 6 months of cigarette smoke (CS) exposure. Mice with CS exposure had significantly elevated serum autoantibodies compared with non-smoke-exposed age-matched (NS) mice. To determine the impact of a full preexisting autoantibody repertoire on IRI, we transplanted BALB/c donor lungs into NS or CS recipients and analyzed grafts 48 hours after transplant. CS recipients had significantly increased lung injury and immune cell infiltration after transplant. Immunofluorescence staining revealed increased IgM, IgG, and C3d deposition in CS recipients. To exclude confounding alloreactivity and confirm the role of preexisting autoantibodies in IRI, syngeneic Rag1-/- (recombination-activating protein 1-knockout) transplants were performed in which recipients were reconstituted with pooled serum from CS or NS mice. Serum from CS-exposed mice significantly increased IRI compared with control mice, with trends in antibody and C3d deposition similar to those seen in allografts. These data demonstrate that pretransplant CS exposure is associated with increased IgM/IgG autoantibodies, which, upon transplant, bind to the donor lung, activate complement, and exacerbate post-transplant IRI.

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.