Abstract A016: Selective delivery of fibroblast activation protein conjugated dual phosphoinositide 3-kinase–AKT Kinase-mTOR inhibitor associated with decreased tumor proliferation and on-target toxicity in high-grade soft-tissue sarcomas

Abstract

Abstract Soft-tissue sarcomas (STS) are a group of lethal mesenchymal tumors that are a major cause of cancer related morbidity for those under the age of 20.PI3K inhibitors have been found to be effective against subset of STS but are associated with significant toxicities limiting their use.We have thought to test a selective fibroblast activation protein (FAP) targeted delivery of PI3K/AKT/mTOR inhibitor omipalisib to STS in pre-clinical setting as FAP is overwhelmingly expressed agressive soft tissue malignancies. Experimental Design: Patient derived fibrosarcomas (aSMA-,FAP+,pAKT+) were isolated by flow cytometry.FAP(-)HT1080 fibrosarcoma cell lines were used as a negative control whereas methylcoanthrene induced mouse fibrosarcoma transfected with human FAP cDNA were used as positive control.Cell expression of FAP,AKT,mTOR was compared using SDS Page,IHC,and flow cytometry.FAP ligand coupled to omipalisib(FAPL-PI3Ki)was then evaluated in-vitro and in small animal models. Results: Cell lines with known FAP expression demonstrated significant uptake of FAPL conjugated NIR fluorochrome.Wild type cells(hFAP-) showed no ligand binding both in flow cytometry and fluorescent microscopy(p<0.05).Escalating doses of FAP-PI3Ki suppressed AKT/mTOR activity in HT-1080 hFAP cells with 10 nM being the smallest effective dose.In hFAP+cell lines 10 nM of FAP-PI3Ki inhibited the AKT/mTOR signaling but inhibition was not observed in hFAP- cell lines demonstrating the need for FAP presence for FAPL-PI3Ki activity(p<0.05).Pan non-targeted PI3Ki inhibited the AKT/mTOR pathway in both hFAP+ and hFAP-cell lines.Densitometric analysis demonstrated that in hFAP+ cells,FAP-PI3Ki at above 10 nM suppresses the AKT/mTOR activity with ratio of pAKT/AKT and pmTOR/mTOR decreasing 11 and 4-fold respectively(p<0.05).FAP-PI3Ki activity significantly reduced when hFAP+ cells were competitively inhibited with free FAPL(4.6x increase in AKT activity,p<0.05).hFAP+ cells treated with FAP-PI3Ki had significant decrease in cell migration.In small animal models,SCID Mice injected with 25 umol/kg of either FAP-PI3Ki and non-targeted PI3Ki showed marked elevation of serum insulin levels in the non-targeted PI3Ki group (0.91 ng/mL vs 0.58 ng/mL,p<0.05).Xenograft tumor digest demonstrated mice treated with FAP-PI3Ki had Akt/mTOR pathway downregulated only in hFAP+ cell lines and this was evident in mice which received 10 nM or higher dosing(pAkt/Akt ratio of 1.1 WT mice vs 0.55 in hFAP+ mice,p<0.05).There was near complete suppression of the mTOR expression in the hFAP+ treatment group (pmTOR/mTOR 1.05 vs 0.087,p<0.05).There was no treatment related mortality in FAPL-PI3Ki group as compared to non-targeted PI3Ki delivery (p<0.05). Conclusion: Our study demonstrated that FAP conjugated modified omipalisib binds FAP expressing soft-tissue sarcomas both in-vitro and in xenograft models in concordance with FAP receptor density.FAP-PI3Ki induces PI3K/AkT/mTOR pathway inhibition similar to non-targeted PI3K inhibitors without significant systemic toxicities observed in pan nontargeted PI3K inhibitors. Citation Format: Feredun Azari, Gregory T. Kennedy, Ashley Chang, Bilal Nadeem, Azra Din, Isvita Marfatia, Steven Albelda, Philip Low, Madduri Srinivasarao, Ramesh Mukkamala, Neil T. Sullivan, Evgeniy Eruslanov, Sunil Singhal. Selective delivery of fibroblast activation protein conjugated dual phosphoinositide 3-kinase–AKT Kinase-mTOR inhibitor associated with decreased tumor proliferation and on-target toxicity in high-grade soft-tissue sarcomas [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A016.