Abstract 2009: Elucidating the mechanistic effect of targeting Ref-1 redox function on MPNST survival signaling using patient-derived xenolines
Authors: Gampala Silpa , Babb Olivia , Ganesh Nikkitha Umesh , Rhodes Steven D. , Saadatzadeh Reza M. , Pollard Kai , Pratilas Christine , Yeh Jing-Ruey Joanna , Pollok Karen E. , Clapp Wade D. , Kelley Mark R. , Zhang Chi , Fishel Melissa L. 
Published: June 2022

Malignant Peripheral Nerve Sheath Tumor (MPNST) is a rare soft tissue sarcoma common in patients with NF1 (neurofibromatosis type 1). MPNSTs respond poorly to most chemotherapeutics due to molecular heterogeneity and altered signal transduction pathways. Ref-1 and STAT3 are highly expressed in MPNST patient samples offering druggable pathways. Inhibition of one singular protein, like Ref-1 to block the activity of many important transcription factors (TFs), STAT3, HIF1a, and NFkB is key to improving success in MPNST therapy. Inhibition of both Ref-1 and STAT3 in MPNST lines resulted in decreased proliferation, wound healing, tumor signaling, and deactivation of MPNST survival genes. Further, knockdown of Ref-1 or STAT3 resulted in a concordant decrease in NFkB activity. Ref-1 redox inhibitor, APX3330 that completed Phase I clinical trial (NCT03375086), potently inhibited in vitro growth of a panel of MPNST cells. We have also been developing new more potent analogs of APX3330 for inhibition of Ref-1 redox function and potent cell killing in our panel of MPNST cells. Several of these analogs significantly and potently reduced NFkB and HIF1a activity at concentrations where cell killing was minimal, pointing toward an on-target effect. Based on the role of Ref-1 in transcriptional regulation of MPNST, RNA sequencing after knockdown of Ref-1 was used to determine mechanistic effects on MPNST gene expression. We have identified 443 genes up-regulated and 758 genes down-regulated in two MPNST cell lines with siRef-1. The pathways enriched by the commonly up-regulated genes included RNA polymerase, P53 downstream, glycerophospholipid, and other lipid metabolism pathways; the pathways enriched by the commonly down-regulated genes included cell cycle, adaptive immune response, and VEGF signaling pathways. From this data, we also found that OXPHOS (Oxidative Phosphorylation) pathway genes (like NDUFS2, SURF1, COX15) were down with siRef-1 along with others like AURKA, RNASEH2A, CDC20, GINS4, TIMELESS that were identified in our previous publication to be MPNST survival genes. Based on our published observations that Ref-1 inhibition dramatically affects metabolic pathways, we used OXPHOS deficient and proficient osteosarcoma cells and confirmed the impact of Ref-1 redox activity on metabolism. Furthermore, if we combine Ref-1 inhibition with a-ketoglutarate (aKG) and target the tumor cells’ dependence on aspartate biosynthesis, the tumor cell death was dramatic (p &lt; 0.0001). Two new xenolines were established from patient PDXs and are being validated for growth inhibition and downregulation of MPNST survival genes with Ref-1 knockdown and redox inhibition using APX analogs both in vitro and in vivo. Successful derailing of MPNST survival pathways by targeting Ref-1 redox function is our aim to treat this rare but deadly cancer.

Citation Format: Silpa Gampala, Olivia Babb, Nikkitha Umesh Ganesh, Steven D. Rhodes, Reza M. Saadatzadeh, Kai Pollard, Christine Pratilas, Jing-Ruey Joanna Yeh, Karen E. Pollok, Wade D. Clapp, Mark R. Kelley, Chi Zhang, Melissa L. Fishel. Elucidating the mechanistic effect of targeting Ref-1 redox function on MPNST survival signaling using patient-derived xenolines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2009.