A Potent and Selective Small-Molecule Degrader of STAT3 Achieves Complete Tumor Regression In Vivo

Abstract

Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. Here we report the discovery of SD-36, a small-molecule degrader of STAT3. SD-36 potently induces the degradation of STAT3 protein in vitro and in vivo and demonstrates high selectivity over other STAT members. Induced degradation of STAT3 results in a strong suppression of its transcription network in leukemia and lymphoma cells. SD-36 inhibits the growth of a subset of acute myeloid leukemia and anaplastic large-cell lymphoma cell lines by inducing cell-cycle arrest and/or apoptosis. SD-36 achieves complete and long-lasting tumor regression in multiple xenograft mouse models at well-tolerated dose schedules. Degradation of STAT3 protein, therefore, is a promising cancer therapeutic strategy.

Keywords: PROTAC; SH2 domain; STAT3; c-Myc; degrader; leukemia; lymphoma; selectivity; transcriptional factor; undruggable.

Figure 1.. Structure-guided Design of STAT3 SH2 Domain Inhibitors and PROTAC Degraders

(A) Design of STAT3 SH2 domain inhibitors. K_i values are the means of three independent experiments. (B) Chemical structures of PROTAC STAT3 degrader SD-36 and its inactive control SD-36Me. (C) Co-crystal structure of SD-36 (orange) with STAT3 (grey). SI-109 binds to STAT3 in nearly identical binding modes as SD-36. Key residues in STAT3 interacting with SD-36 are labeled and hydrogen bonds are depicted as red dashed lines. A mediating water molecule is shown as a sphere. The hydrophobic patch formed by I659, M660 and L666 is shown as surface representation. See also Figure S1.

Figure 2.. SD-36-mediated Cellular STAT3 Protein Degradation

(A) Cells were treated with SD-336, 10 μM of SI-109 or SD-36Me for 3 hr (MOLM-16) or 24 hr (SU-DHL-1) for immunoblotting. (B) MOLM-16 cells were treated with 0.25 μM of SD-36 for immunoblotting. (C) MOLM-16 cells stably transduced with vector control or CRBN shRNAs were treated with 1 μM of SD-36 for 3 hr for immunoblotting. (D) MDA-MB-231 cells transfected with the indicated STAT3-expressing vectors were treated with 1 μM of SD-36 for 16 hr for immunoblotting. (E) Human PBMCs were treated with SD-36 or 10 μM of SD-36Me for 24 hr for immunoblotting. (F) Cells were treated with 10 μM of SD-36 for 2.5 hr for multiplexed quantitative proteomics analysis. p value: Student’s t-test. (G) Cells were treated with 1 μM of the indicated compounds for 24 hr for immunoblotting. (H) Parental DLD-1 (WT) and DLD-1/STAT3^Y705F/Y705F (Y705F) cells were pre-incubated with or without 10 ng/ml of IL-6 for 15 min, then 1 μM of SD-36 for 24 hr for immunoblotting. See also Figure S2.

Figure 3.. Effect of SD-36 on the Transcriptional Activity of STAT3

(A) MOLM-16 cells were treated for 5 hr. Whole cell lysis was subjected to native electrophoresis followed by immunoblotting. (B) Cells were treated with 1 μM of SD-36 or 10 μM of SI-109 for 8 hr. Nuclear extracts were prepared for STAT3 DNA binding activity assay (mean ± SEM; n=3). (C) MOLM-16 cells expressing STAT3-driven firefly luciferase gene were treated for 24 hr for luciferase reporter assay. Representative concentration-response curves are shown. (D) MOLM-16 cells were treated with 1 μM of SD-36 for 8 hr for RNA-seq analysis. The horizontal dashed line marks the statistical significance (FDR ≤ 0.05) and the vertical lines mark the fold change (FC) (|Log₂FC| ≥1). Black arrows indicate representative genes validated by qRT-PCR. The numbers indicate genes that meet these criteria: blue, down-regulated; red, up-regulated. FDR: false discovery rate. (E) GSEA showed the STAT3 targets gene set (GSE21670) is down-regulated by SD-36 in MOLM-16 cells. NES: normalized enrichment score. (F) MOLM-16 cells were treated with 1 μM of SD-36 or SD-36Me for 8 hr for qRT-PCR. Data are the means of two independent experiments. See also Figure S3.

Figure 4.. Growth-inhibitory Activity of SD-36 in AML and ALCL Cell Lines

(A) The expression of the indicated proteins was profiled by immunoblotting. SD-36 sensitive cell lines (IC₅₀ ≤ 2 μM) are labelled in green. (B) The growth-inhibitory activities of SD-36 and SI-109 in leukemia and lymphoma cell lines were determined by a 4-day CellTiter Glo assay. IC₅₀ values are the means of three independent experiments. DLBCL: diffuse large B-cell lymphoma; HL: Hodgkin’s lymphoma; nHL: non-Hodgkin’s lymphoma. SD-36 sensitive cell lines (IC₅₀ ≤ 2 μM) are labelled in green. (C) Representative concentration-response curves of CellTiter Glo assay. Len: lenalidomide; SI+Len: SI-109 plus lenalidomide at 1:1 molar ratio. (D) Cells were treated with SD-36 (0.5 μM), SI-109 (1 μM), lenalidomide (Len, 1 μM) and SI-109 plus Len (1:1 molar ratio at 1 μM) for 24 hr for immunoblotting. (E) Cells were treated with 1 μM of SD-36 or SD-36Me for 48 hr for cell cycle analysis (mean ± SEM; n=3). (F) Cells treated for 48 hr for apoptosis analysis by Annexin V staining (mean ± SEM; n=3). p value: Student’s t test. (G) Cells were treated with 1 μM of SD-36Me or SD-36 for 24 hr for qRT-PCR. Data are means of two independent experiments. See also Figure S4 and Table S1.

Figure 5.. Anti-tumor Activity of SD-36 in Xenograft Tumor Models

(A) NOD/SCID mice bearing MOLM-16 tumors were treated i.v. as indicated. Tumor lysates were analyzed by immunoblotting. (B) MOLM-16 tumors were analyzed by immunoblotting. (C) NOD/SCID mice bearing MOLM-16 tumors were treated i.v. with 100 mg/kg of SD-36Me or SI-109. Tumor lysates were analyzed by immunoblotting. (D) NOD/SCID mice bearing MOLM-16 tumors were treated with 25 mg/kg of SD-36 i.v. for 6 hr. Tumor lysates were analyzed by multiplexed proteomics analysis. p value: Student’s t test. (E) SCID mice bearing MOLM-16 tumors were treated with vehicle control (veh) or 25 mg/kg of SD-36 i.v.. Total RNAs were isolated from tumor lysates for qRT-PCR. (F) Tumor-bearing SCID mice were treated at the indicated dosing schedules, and tumor volumes were measured every 2–3 days (mean ± SEM; n=6–8). qD: daily; qW: weekly. See also Figure S5.

Figure 6.. Toxicity of SD-36 in Immunocompetent Mice

(A) Female CD-1 mice were treated with SD-36 at 100 mg/kg i.v. Mouse tissues were analyzed by immunoblotting. (B) CD-1 mice were treated i.v. for 3 weeks. Mouse body weight was measured every 2–3 days (mean ± SEM; n=4–8). qD: daily. See also Tables S2–S5 and Figure S6.