(P 29) COMBINATION TREATMENT WITH TEMOZOLOMIDE AND THE DEATH RECEPTOR-5 AGONIST INBRX-109 EFFECTIVELY TARGETS SUCCINATE DEHYDROGENASE -DEFICIENT GASTROINTESTINAL STROMAL TUMOR

Objective: Succinate Dehydrogenase (SDH)-def Gastrointestinal Stromal Tumor (GIST) may present as indolent tumors or aggressive cancers that respond poorly to current FDA-approved therapies for GIST. They are caused by inherited mutations in the SDH (A/B/C/D) genes, or by epigenetic silencing of the SDHC promoter. Both causes result in loss of a functional SDH complex that abolishes the cell’s ability to convert succinate to fumarate. In turn, elevated succinate levels suppress the homologous recombination DNA Damage Repair (DDR) pathway required to resolve DNA double-strand breaks, thus rendering SDH-def tumor cells vulnerable to temozolomide (TMZ). Furthermore, we have shown that TMZ induces endoplasmic reticulum (ER) stress, which causes SDH-def GIST to upregulate DR5 (Death Receptor 5) expression. We now hypothesize that DR5 is a therapeutic vulnerability induced by TMZ, and that combination treatment with INBRX-109, a tetravalent DR5 agonist, will enhance the efficacy of TMZ monotherapy

Methods: We previously reported the development of the 3 human SDH-def cell models isolated from patients with SDH-def GIST (Yebra et al., CCR 2022). Using these models, we performed immunoblot analyses, single cell (sc) RNAseq, flow cytometry-based ER stress assays, and pharmacologic synergy analyses with 4 methods (Bliss, ZIP, Loewe, HSA). We also performed single nucleus (sn) RNAseq in 13 SDH-def GIST. Finally, we performed DR5 immunohistochemical (IHC) staining of resected SDH-def GIST treated with TMZ in a clinical trial (NCT03556384).

Results: Using the 3 cell models, immunoblotting revealed that TMZ treatment significantly increased expression of the transcription factor CHOP, which in turn increased DR5 expression. snRNAseq of 13 SDH-def GIST also showed that multiple TMZ-naïve tumor cell clusters had basal DR5 expression. In vitro, we utilized scRNAseq to confirm that SDHC mutant GIST cells treated with TMZ (600 μM, 7d) had increased DR5 expression versus untreated (control) cells in most cell clusters. Finally, IHC staining of 3 TMZ-treated SDH-def GIST showed high DR5 immunostaining, confirming our in vitro findings. We then treated the SDHA and SDHC mutant cell models with TMZ+INBRX-109 and performed cell viability assays. These showed that combination treatment is either additive (0-9.9) or synergistic (≥10) in targeting cells in vitro. To further understand the mechanism of cell killing, we performed flow cytometric quantitation analyses of polyubiquitination and tetraphenylethene maleimide (TMI), which independently confirmed that TMZ+INBRX-109 increased global ER stress in cells. Further analyses showed significantly increased levels of both mRNA (scRNAseq) and protein (immunoblot) expression of PERK (protein kinase R-like ER kinase) pathway components in ER stress signaling (i.e., p-elF2α, ATF4, CHOP, DR5) and apoptotic pathways (i.e., BAX, CASP3). Hence, treatment with TMZ+INBRX-109 activated two independent mechanisms that each led to tumor cell death, namely: 1) the DDR pathway; and 2) the PERK ER stress pathway leading to increased apoptosis. Finally, in an unbiased high-throughput drug screen using a chemical library of 5,034 compounds, we identified 4 compounds with IC50 < 100 nM in the 3 SDH-def GIST models that were ER stress inducers, further suggesting that this pathway may represent an effective target in SDH-def tumor cells.

Conclusion: Herein, we report that SDH-def tumors are sensitive to TMZ, which induces ER stress and upregulates DR5, a pro-apoptotic receptor that can be targeted by INBRX-109 (Figure 1). In turn, TMZ+INBRX-109 additively/synergistically reduces SDH-def GIST cell viability in vitro. These data provide the preclinical evidence for a Phase I clinical trial (3+3 dose escalation design with two dosing cohorts, NCT03715933) to study the safety/tolerability of TMZ+INBRX-109 in patients with aggressive SDH-def cancers, including GIST (Figure 2). As of the submission date, the first subject has been safely dosed and a second subject is in screening.