(P 330) EFFICACY OF NON-CARDIOTOXIC ANTHRACYCLINES IN EWING SARCOMA

Objective: Dose escalation of anthracyclines is associated with improved cancer-free survival in Ewing sarcoma (EwS) patients but induces cardiotoxicity. Classic anthracyclines, such as doxorubicin (Doxo) are topoisomerase (topo) poisons that induce cell death via genotoxic damage. Recent discoveries showed that anthracyclines also kill cancer cells by damaging chromatin, and determined that the anthracyclines, Aclarubicin (Acla) and DiMethyl-Doxorubicin (DiMe-Doxo), selectively induce chromatin damage without DNA damage. Importantly, these agents did not induce cardiotoxicity in preclinical models. In the current study we evaluated the anti-tumor efficacy and safety of these agents in preclinical models and defined their impact on EwS cell chromatin landscapes.

Methods: Anti-tumor efficacy of Doxo, Acla, and DiMe-Doxo were tested in vitro in a panel of sarcoma cell lines in 2D monolayer and 3D tumor spheroids in collagen matrix. In vivo efficacy studies were performed in subcutaneous xenograft models in immune deficient mice. Non-tumor bearing C57BL/6 mice were exposed to a cumulative dose of 20mg/kg Doxo followed by an equivalent course of vehicle, Acla, or Doxo, and survival and toxicity was assessed for up to 12 weeks post-final dose. Cardiotoxicity was specifically evaluated by echocardiograms (echo) and histology. Cleavage Under Targets and Tagmentation (CUT&Tag)-seq was performed to profile H3K4me3, H3K27me3, H3K27ac, H3K4me1, and H3K9me3 marks in A673 cells before and after 48 hr exposure to 100nM Acla, DiMe-Doxo, and Doxo.

Results: Doxo, Acla, and DiMe-Doxo reproducibly induced apoptosis of established and early passage EwS cell lines and PDX-derived cells at nanomolar doses in vitro. Effects of non-cardiotoxic anthracyclines on cell viability were equivalent to Doxo in both 2D and 3D assays, but only cells treated with Doxo showed evidence of DNA damage. Mouse studies are ongoing and preliminary data suggests anti-EwS activity in vivo. To model the safety of delivering non-cardiotoxic anthracyclines to patients who have previously received Doxo, we exposed C57Bl/6 mice to serial dosing as above. Survival of mice that received a second course of Doxo was significantly shortened (p < 0.01) compared to mice that received either vehicle or Acla as their second treatment. Survival of Acla- and vehicle-treated mice was equivalent. In contrast to expectations, shortened survival of the Doxo-Doxo mice was not associated with diminished cardiac function or fibrosis as determined by echo or histology, respectively. Histone profiling of anthracycline-exposed A673 cells revealed drug-induced changes in chromatin landscapes. We identified eviction of H3K4me3 and H3K27ac following anthracycline treatment, suggesting preferential effects of anthracycline-induced chromatin damage at active promoters and enhancers in EwS cells. Nevertheless, despite a general loss of H3K4me3 peaks relative to control, specific gains of H3K4me3 at discrete loci were reproducibly identified with all three anthracyclines. Studies are ongoing to identify these regions and determine if they are mechanistically relevant for EwS cell killing.

Conclusion: Our studies demonstrate that non-cardiotoxic anthracyclines can effectively kill EwS cells in preclinical models and that they can be safely delivered after prior Doxo exposure. Further, chromatin profiling studies support the hypothesis that chromatin damaging effects of these agents be a primary mechanism of anti-cancer effect in these tumors. The therapeutic significance of this work is potentially profound as it suggests that these drugs could be safely administered to patients in a relapse setting in combination with other agents.