BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design

Abstract

Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.

Figure 1. Rational design and evaluation of a partial degrader of SMARCA2 and SMARCA4, PROTAC 1

a) 2D chemical structure of a SMARCA^BD ligand and crystal structure of this ligand in complex with SMARCA2^BD. The piperazine ring was selected as an exit vector for PROTAC linkage as it is directed into solvent away from the binding site. b) 2D chemical structure of PROTAC 1 c) Inverse ITC titrations of VCB into PROTAC 1 (left) and VCB into the preformed PROTAC 1–SMARCA2^BD complex (right), n = 2. PROTAC 1 binds VCB with higher affinity when in complex with SMARCA2^BD and is therefore cooperative, α = 4.8. d) Degradation of SMARCA2 and SMARCA4 in MV-4-11 cells following treatment with PROTAC 1, analysed via capillary electrophoresis (see online methods). For SMARCA2 and SMARCA4, maximal degradation is ~65 % and ~70%, and DC₅₀ is 300 nM and 250 nM, respectively. Data represent means from two biologically independent experiments, ± S.E.M.

Figure 2. Ternary co-crystal structure of SMARCA2^BD: PROTAC 1: VCB.

a) Overall co-crystal structure of VCB: PROTAC 1: SMARCA2^BD in ribbon representation with bound PROTAC shown in stick with magenta carbons. b) F_o-F_c omit map (green meshes) of PROTAC 1 prior to ligand modelling (complex 1, interface of chains A/B) contoured at 3.0 σ with a carve radius of 2.2 Å. c) Key electrostatic protein-protein interactions between R69 of VHL and C-terminal backbone carbonyl groups of the B helix of SMARCA2^BD d) Surface representation colored using normalized consensus hydrophobicity scale, red = hydrophobic, white = hydrophilic. Linker of PROTAC 1 seen to be collapsing on to a lipophilic face formed in part by Y98 of VHL.

Figure 3. Ternary co-crystal structure of SMARCA2^BD: PROTAC 2: VCB and biophysical data validate a rational design strategy

a) 2D chemical structures of PROTAC 2 and ACBI1 b) Overlays of ternary crystal structures of VCB: PROTAC 1 : SMARCA2^BD (orange, PROTAC carbons in magenta) and VCB: PROTAC 2 : SMARCA2^BD (2.35 Å, salmon, PROTAC carbons in green). Near identical ternary complexes are formed by both PROTACs; with the phenyl ring of PROTAC 2 in close proximity to Y98 of VHL. c) Overlays show that the constrained 1,4-disubstituted phenyl ring of PROTAC 2 (green carbons) accurately recapitulates the linker geometry observed previously for PROTAC 1 (magenta carbons). d) F_o-F_c omit map (green meshes) of PROTAC 2 prior to ligand modelling contoured at 3.0 σ with a carve radius of 2.2 Å. e) Fitted curves from Fluorescence Polarization competition assays measuring displacement of a VHL peptide by PROTACs in the presence or absence of SMARCA2^BD. ACBI1 forms more cooperative and stable complexes compared with PROTAC 1. Curves are a best fit of means from n = 3 biologically independent experiments, ± S.E.M. f) Fitted curves for TR-FRET assays measuring displacement of a biotinylated SMARCA2^BD probe by PROTAC alone, in complex with VCB or in complex with an R69A variant of VCB. A significant rightward shift when using VCBR69A vs VCB highlights the importance of this residue in ternary complex formation in solution. Curves are a best fit of means from n = 3 biologically independent experiments, ± S.D.

Figure 4. ACBI1 is a potent and selective degrader of SMARCA2, SMARCA4 and PBRM1.

a) Degradation of endogenous SMARCA2, SMARCA4 and PBRM1 in MV-4-11 cells treated for 18 h gave a DC₅₀ of 6, 11 and 32 nM respectively (left). Time course of degradation in MV-4-11 in the presence of 1 µM of ACBI1 or 1 µM cis-ACBI1 (right). Two independent experiments. b) Degradation of endogenous SMARCA2 in NCI-H1568 cells treated for 18 h gave a DC₅₀ of 3.3nM for SMARCA2 and 15.6 nM for PBRM1 Two independent experiments. c) Effects of ACBI1 (blue) and cis-ACBI1 (red) at 333 nM for 8 h on the proteome of MV-4-11 cells. Data plotted Log₂ of the fold change versus DMSO control against –Log₁₀ of the p value per protein from n = 3 independent experiments. All t-tests performed were two-tailed t-tests assuming equal variances d) SWI/SNF complexes were immuno-purified from MV-4-11 cell lysates following PROTAC treatment and abundance of subunits was determined by label free quantitation. Data plotted as fold change in abundance for PROTAC 2 vs cis-PROTAC 2 treatment against –Log₁₀ of the p value per protein from n = 3 biologically independent experiments. Subunits of the SWI/SNF complex are highlighted. All t-tests performed were two-tailed t-tests assuming equal variances.

Figure 5. Effects on the proliferation and apoptosis of cancer cells in the presence of ACBI1.

a) Cell viability of an AML cell line, MV-4-11 (left), a SMARCA4 deficient melanoma cell line, SK-MEL-5 (centre) and a SMARCA2 and SMARCA4 deficient NSCLC cell line, NCI-H1703 (right), after 7 days in the presence of ACB.I1. IC₅₀s for ACBI1 and cis-ACBI1 in MV-4-11 cells are 29 nM and 1.4 µM, and in SK-MEL-5 cells are 77 nM and > 10000 nM, respectively. Two independent experiments. b) Cell viability of a melanoma cell line, SK-MEL-5 after 3 days in the presence of dose titrations of ACBI1, the SMARCA^BD ligand or a dose titration of ACBI1 in the presence of 10 µM of SMARCA^BD ligand, representative of two biologically independent experiments. c) Real-time measurement of proliferation (left) and apoptosis (right) in SK-MEL-5 cells after treatment with doxorubicin (1 µM), ACBI1 (0.3 µM) and cis-ACBI1 (0.3 µM). Representative of two biologically independent experiments.