Abstract C017: Signaling through Ref-1 and STAT3 in soft tissue sarcoma (MPNST) and the effects of perturbing this pathway on tumor cell survival and gene expression
Authors: Shah Fenil , Babb Olivia , Zhang Chi , Gampala Silpa , Zhang Emily , Rhodes Steven D , Tee Andrew R. , Calver Brian , Rad Ellie , Staedtke Verena , Pollok Karen E , Clapp D. Wade , Kelley Mark R. , Fishel Melissa L. 
Published: March 2020

Abstract
Malignant Peripheral Nerve Sheath Tumor (MPNST) is a rare soft tissue sarcoma that can arise from patients with NF1 (neurofibromatosis type 1). These patients are at a much greater risk of developing MPNST than the general population (10% vs. 0.01%, respectively). Existing chemotherapeutic and targeted agents have thus far not been successful in MPNST treatment, with a 5-year patient survival rate of just 35%-50%. Recent research implicates Signal Transducer and Activator of Transcription-3 (STAT3) and Hypoxia Inducible Factor 1 (HIF1) in driving MPNST. STAT3 is a transcription factor implicated in several cancers, and phosphorylated STAT3 (p-STAT3) expression indicates aggressive disease at disease onset in MPNST. STAT3 is activated upon phosphorylation by Janus Kinase (JAK), but its DNA binding and transcriptional activity is regulated by Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1 or Ref-1) redox function. Ref-1 is a multifunctional protein involved in repairing DNA damage via endonuclease activity and in redox regulation of various transcription factors including STAT3, HIF-1α, and NFκB. High expression levels of Ref-1 indicate decreased survival in several cancers. We characterized Ref-1 and p-STAT3 expression in several MPNST samples including: the IN Pediatric BioBank, and established MPNST cell lines, new patient-derived tumor cells, and genetically engineered mouse models. We observed strong Ref-1 and phospho-STAT3 staining in the majority of these samples. We examined the expression of p-STAT3 and Ref-1 in mice that have conditional ablation in the Schwann cells of Nf1-/-; Arf+/- and Nf1-/-; Arf-/- mice and found a significant increase in positivity of p-STAT3 and Ref-1 expression in the sections containing MPNST. It also appears that there may be differences in staining intensity based on Arf genotype as the Nf1-/-; Arf-/- have stronger staining than the Nf1-/-; Arf+/-. Knocking down Ref-1 or STAT3 impairs MPNST growth in vitro. Inhibiting the redox activity of Ref-1 using the redox-specific APE1 inhibitor, APX3330 (and its next generation analogs), or inhibiting STAT3 activity using Napabucasin also resulted in reduced in vitro growth. Investigation into the apoptotic pathways that are activated following treatment demonstrate activation of caspase 3/7. Several biomarkers downstream of Ref-1 and STAT3 were downregulated following Ref-1 redox or STAT3 inhibition. We also performed RNA-sequencing to identify differentially expressed genes (DEGs) following Ref-1 or STAT3 knockdown and implicate new targets associated with clinical response in MPNST. In vivo experiments performed on mice implanted with ST88-14 cells using either APX2009 (APX3330 analog) or Napabucasin both resulted in significantly reduced tumor growth and additional combination studies are underway. Obtaining these results with clinically-tested Ref-1 and STAT3 inhibitors make the translation of this work highly plausible for pediatric patients with MPNST in the future. We will use both PDX and genetically engineered models of MPNST to test and validate these novel targets.
Citation Format: Fenil Shah, Olivia Babb, Chi Zhang, Silpa Gampala, Emily Zhang, Steven D Rhodes, Andrew R. Tee, Brian Calver, Ellie Rad, Verena Staedtke, Karen E Pollok, D. Wade Clapp, Mark R. Kelley, Melissa L. Fishel. Signaling through Ref-1 and STAT3 in soft tissue sarcoma (MPNST) and the effects of perturbing this pathway on tumor cell survival and gene expression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C017. doi:10.1158/1535-7163.TARG-19-C017