Wednesday, April 2, 2025
4/2/2025
Mechanistic studies of hirsutinolides and azetidine-based small molecule inhibitors of STAT3 - PROJECT SUMMARY/ABSTRACT Triple-negative breast cancer (TNBC) is generally treated with chemotherapy, which only achieves incomplete response. Relapsed disease has no new treatment options and the long-term outcomes are poor. The central premise of our proposal is that the TNBC’s survival dependency on high glucose-6-phosphate dehydrogenase (G6PD) and thioredoxin reductase 1 (TrxR1) functions, elevated NADPH and GSH levels, and aberrantly-active signal transducer and activator of transcription (STAT)3, which are adaptation mechanisms, makes them therapeutically vulnerable to two chemotypes: R001, R002, and H182, H333. R001/R002 (natural products) moderated the high G6PD, TrxR1, and STAT3 functions, thereby decreased GSH levels, and induced gene expression changes, while H182/H333 (azetidine-based) potently and selectively inhibited STAT3 activity and gene expression in TNBC cells. The collective inhibition of the high G6PD, TrxR1, STAT3 and GSH functions and gene expression changes led to reactive oxygen species (ROS) build-up in TNBC cells, DNA damage, cell cycle arrests, cell death, and tumor growth inhibition in vivo of TNBC models. Though indeed, G6PD, TrxR1 or STAT3 knockdown alone led to ROS build up in TNBC cells, and RNAseq profiling showed STAT3 inhibition alone led to enrichment for the genes in the DNA damage, ER stress response, UPR, and senescence pathways in H182-treated TNBC cells, we anticipate differences between the chemotypes’ activities, which we seek to define in order to better tailor these compounds for TNBC treatment. Normal cells, which have low background G6PD, TrxR1, NADPH, GSH, and STAT3 functions are unaffected. We hypothesize that the moderation of the redox/metabolic events and gene regulation through the targeting of G6PD, TrxR1, and STAT3 by R001, R002, H182 and H333 inhibits TNBC growth. We further hypothesize that the compounds’ effects on the metabolic, redox and STAT3 pathways in non-tumor cells in the TME contribute to the antitumor response. We will define the differences between the chemotypes. 1. Investigate the interactions of STAT3, G6PD, and TrxR1 with the compounds in vitro to define their inhibition modes; 2. Study how the specific targeting of STAT3 by the azetidines and the modulation of G6PD, TrxR1 and STAT3 functions by natural products differ in the downstream changes in glycolysis and pentose phosphate pathway (PPP) that feed into NADPH and GSH levels and in gene expression in vitro and in vivo; 3. Investigate ROS induction and the associated stress response, DNA damage, cell cycle changes and cell death, their molecular regulators, including oxidoreductases in TNBC cells and tumor tissues following treatment; and 4. Investigate the antitumor responses to the inhibitors in vivo in CDX, PDX, 4T1/Balb-c, and E0771/BL6 models. We will gain new, indept insights into TNBC’s dependence on dysregulated G6PD, TrxR1 NADPH, GSH and STAT3 functions and the preferential effects of inhibitors. X-ray crystallographic studies will give atomic level details into the direct interactions. The successful development of R001, R002 or H182, H333 and their combination therapy with ICI will provide a novel effective treatment for TNBC.
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