Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer

Mammalian switch/sucrose non-fermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, a first-in-class, orally bioavailable proteolysis targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 (BRD4) and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.


Figures S1 to S5
Tables S1 to S2 Supplementary Methods

Synthesis of AU-24118
General Information: All chemicals and solvents were obtained from commercial suppliers and sed without further purification.Purification was performed using combi-flash Nextgen300.All reactions were monitored by TLC, using silica gel plates with fluorescence F254 and UV light visualization. 1 H NMR spectra was recorded on a Varian Mercury Plus at 400 MHz, and 13 C NMR spectra was recorded on a JEOL-ECZ-400S spectrometer at 100 MHz.Coupling constants (J) are expressed in hertz (Hz).Chemical shifts (δ) of NMR are reported in parts per million (ppm) units relative to internal control (TMS).Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad (br), or a combination thereof.The low resolution of ESI-MS was recorded on an Agilent-6120, and the highresolution mass (resolution-70000) for the compound was generated using Q-Exactive Plus orbitrap system (Thermo Scientific) using electrospray ionization (ESI).Melting point was recorded in Stuart instrument, model smp30.HPLC was recorded in Agilent 1260 infinity II with PDA detector; the column used was a KINETEX EVO C-18(150mm x 4.6mm, 5μ) using A: 0.01%TFA IN WATER B: ACN 100%; Method -T/%B: 0/5,1/5,6/100,8/100,10/5,12/5 method and Flow rate: 1.0 ml/min.FTIR spectrum was recorded using Spectrum Two™ instrument (Scanning range 4000 cm-1 to 450 cm-1, Pallet making -For solid sample with KBr).

Synthesis
To a stirred solution of 4-bromo-6-chloropyridazin-3-amine (8.3g, 39.84 mmol), 1-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidin-4-one (10.0g, 33.20 mmol) in 1,4dioxane (80 mL) and water (10 mL) was added K2CO3 (13.76g, 99.6 mmol) and degassed with nitrogen for 10 min.This was followed by adding Pd(dppf)2Cl2.DCM (2.71g, 3.32 mmol), and the reaction mixture was heated at 100 ºC in a sealed tube for 16 h.Once the reaction was completed (monitored by TLC), the reaction mixture was diluted with EtOAc.The combined organic layer was washed with water, brine, dried over anhydrous sodium sulphate, and concentrated under vacuum to get crude product which was purified by combi flash column chromatography using 70-90% EtOAc in hexane as eluent to afford the title compound white solid (5.0g, 50%).10 min.This was followed by adding Pd(dppf)2Cl2.DCM (2.96 g, 3.63 mmol), and the reaction mixture was heated for 4 h at 120 ºC in a sealed tube.Once the reaction was completed (monitored by TLC), the reaction mixture was diluted with EtOAc.The combined organic layer was washed with water, brine, dried over anhydrous sodium sulphate, and concentrated under vacuum to give the residue which was purified by combi flash column chromatography using 90 -100% EtOAc in hexane as eluent to afford the title compound off white solid (7.0g, 54%).The reaction was monitored by TLC.After completion of the reaction, the reaction mixture was extracted in DCM, and a saturated ammonium chloride wash was given to the organic layer followed by a saturated sodium chloride wash.The organic layer was dried over anhydrous sodium sulphate and then concentrated under reduced pressure, and the resultant residue was washed with diethyl ether and dried under vacuum to afford the title compound light yellow solid (0.310g, 28.25 %).LCMS: m/z 633.05 (M+H).

Fig
Fig. S4.(A) Volcano plot visualizing overall transcriptomic alterations as assessed by RNA-seq in 22Rv1-AUR-2 versus 22Rv1-WT cells.(B) Immunoblots of 22Rv1-wild type (WT) or 22Rv1 AUR-2 cells treated with 4h AU-15330 or AU-24118 with indicated concentration showing changes in the labeled targets.Vinculin was used as a loading control.This experiment was repeated independently twice.(C) Dose-response curves of 22Rv1-wild type (WT) or 22Rv1 AUR-2 cells treated with AU-15330 or AU-24118.Data are presented as mean +/− SD (n = 6) from one-of-three independent experiments.

Fig
Fig. S5.(A) Immunoblot illustrating levels of ABCB1 proteins in C4-2B xenografts after long-term (24 days) AU-15330 treatment.Vinculin is utilized as the loading control across immunoblots.CDX, cell line-derived xenograft.(B) qPCR of C4-2B xenografts treated with AU-15330 or vehicle for 5 days showing changes in RNA levels of ABCB1.Data shown are technical triplicates.T-tests were performed