Reeder heating driver#
Reeder heating software#

(D) HDAC6 was overexpressed (OE) in PANC-1 cells treated with pano in the presence or absence of 1 μM E64FC26 for 48 hours. Statistical significance was determined by Student’s t-test (n=12).

Reeder heating software#

The total area and the total number of ubiquitinated (Ub) aggregates per cell were calculated using IMARIS Microscopy Image Analysis Software (Bitplane). Cells were stained with ubiquitin-FITC and Hoechst 33342 (nucleus).

(C) Confocal microscopy images of PANC-1 cells treated with 1 μM E64FC26 alone or in combination with 50 nM pano or 100 nM romi for 16 hours. Western blots for ubiquitin (Ub), acetylated lysine (Ac-Lysine), and acetylated α-tubulin (Ac-Tubulin) are shown. (B) Indicated cell lines were treated with 1 μM E64FC26 alone or in combination with 50 nM pano or 100 nM romidepsin (romi) for 16 hours. Statistical significance was determined by Student’s t-test (n=6). A representative cell viability curve (left) and the change in EC 50 values are shown (right). Cells were treated with panobinostat (pano) in the presence or absence of 1 μM E64FC26 for 48 hours. (A) PDIA1 was stably knocked down in PANC-1 cells. Based on this equation, a score above 1-fold is indicative of synergy. EC 50s for each cell line were extrapolated from independent 8-dose cell viability curves. The “Pano Potentiation” score was calculated by dividing the EC 50 for pano in the absence of E64FC26 by the EC 50 for pano in the presence of E64FC26. (G) Waterfall plot showing the change in pano sensitivity in the presence of 1 μM E64FC26 and (H) 500 nM E64FC26 for the specified cell lines. EC 50 values were calculated from 3 individual dose curves. (E) EC 50 values of PANC-1 cells and (F) T98G cells treated with various epigenetic protein inhibitors alone or in combination with 1 μM E64FC26 (Pano: panobinostat, Vorino: vorinostat, Romi: romidepsin, Belino: belinostat, Rico: ricolinostat, Tuba: tubastatin, Entino: entinostat, 5-Aza: 5-azacytadine, Taz: tazemetostat). Curves that fall below the dotted line are indicative of synergy. The dotted line represents a combination index score of 1.0. Numbers inside the boxes represent cell viability (%). (C) Isobolograms of PANC-1 cells treated with E64FC26 in combination with panobinostat (top) or gemcitabine (bottom) and (D) isobolograms of T98G cells treated with E64FC26 in combination with panobinostat (top) or temozolomide (bottom). Thus, separation of the curves indicates synergistic drug interaction. Viability was normalized to the RLU values in the absence of the HDACi to account for cell death induced by E64FC26 alone. (B) Representative cell viability response curves of PANC-1 cells treated with panobinostat and T98G cells treated with vorinostat alone or in combination with 1 μM E64FC26 for 48 hours. Combination index scores less than 1.0 are indicative of synergistic potential. Cells were treated for 48 hours with 1 μM E64FC26 and the indicated drug. (A) Synergistic hits in PANC-1 (left) and T98G cells (right). ©2020 American Association for Cancer Research. SIGNIFICANCE: This study uses a first-in-class PDI inhibitor entering clinical development to enhance the effects of epigenetic drugs in some of the deadliest forms of cancer. Combined treatment with PDI and HDACi offers a dual therapeutic strategy in solid tumors and the opportunity to achieve previously unrealized activity of HDACi in oncology. In summary, this study presents a synergistic antitumor combination of PDI and HDAC inhibitors and demonstrates a mechanistic and tumor suppressive role of ATF3. The HSP40/HSP70 family genes DNAJB1 and HSPA6 were found to be critical ATF3-dependent genes that elicited the antitumor response after PDI and HDAC inhibition. These chromatin marks were associated with increased RNA polymerase II recruitment to the ATF3 promoter, a synergistic upregulation of ATF3, and a subsequent apoptotic response in cancer cells. ATF3 was highly induced by combined PDI and HDACi treatment as a result of increased acetylation of key histone lysine residues (acetylated histone 3 lysine 27 and histone 3 lysine 18) flanking the ATF3 promoter region.

Reeder heating driver#

RNA-sequencing screening coupled with gene silencing studies identified ATF3 as the driver of this antitumor synergy. In this study, we describe a new class of protein disulfide isomerase (PDI) inhibitors that significantly and synergistically enhance the antitumor activity of HDACi in glioblastoma and pancreatic cancer preclinical models. Histone deacetylase inhibitors (HDACi) are largely ineffective in the treatment of solid tumors.