First-in-human trial of a STAT3 decoy oligonucleotide in head and neck tumors: implications for cancer therapy

Abstract

Despite evidence implicating transcription factors, including STAT3, in oncogenesis, these proteins have been regarded as "undruggable." We developed a decoy targeting STAT3 and conducted a phase 0 trial. Expression levels of STAT3 target genes were decreased in head and neck cancers following injection with the STAT3 decoy compared with tumors receiving saline control. Decoys have not been amenable to systemic administration due to instability. To overcome this barrier, we linked the oligonucleotide strands using hexaethylene glycol spacers. This cyclic STAT3 decoy bound with high affinity to STAT3 protein, reduced cellular viability, and suppressed STAT3 target gene expression in cancer cells. Intravenous injection of the cyclic STAT3 decoy inhibited xenograft growth and downregulated STAT3 target genes in the tumors. These results provide the first demonstration of a successful strategy to inhibit tumor STAT3 signaling via systemic administration of a selective STAT3 inhibitor, thereby paving the way for broad clinical development.

Significance: This is the fi rst study of a STAT3-selective inhibitor in humans and the fi rst evidence that a transcription factor decoy can be modifi ed to enable systemic delivery. These findings have therapeutic implications beyond STAT3 to other “undruggable” targets in human cancers.

Trial registration: ClinicalTrials.gov NCT00696176.

Figures

Figure 1

Intratumoral administration of a STAT3 decoy oligonucleotide abrogates target gene expression in HNSCC patients. (A) Schema of phase 0 trial. HNSCC tumors were biopsied and injected with a single dose of a STAT3 decoy oligonucleotide (or saline) followed by tumor resection and analysis of target gene expression in the paired tumor samples. (B) Downmodulation of STAT3 target genes in representative HNSCC tumors injected with STAT3 decoy as shown by western blot analyses. Whole tumor lysates were prepared from 4 HNSCC tumors pre- and post-injection with the STAT3 decoy enrolled on the first dose tier. Proteins (40 ug) were resolved on a 12.5% SDS/PAGE gel and subjected to immunoblotting with anti-Bcl-XL and cyclin D1 antibody. β-actin was used as a loading control. (C) Representative images of IHC staining for cyclin D1 protein expression in pre- and post-STAT3 decoy oligonucleotide or saline injections from 6 patients. (D) Cumulative quantitative determination of cyclin D1 expression in all 30 HNSCC patient tumors showed a significant decrease in expression in tumors injected with STAT3 decoy oligonucleotide compared to saline injection (p=0.0431). (E) Representative images of IHC staining for Bcl-XL protein expression in pre- and post-STAT3 decoy oligonucleotide or saline injections from 6 HNSCC patients. (F) Cumulative quantitative determination of Bcl-XL expression in all 30 HNSCC patient tumors demonstrated decreased expression in STAT3 decoy injected patient tumors compared to the patient tumors injected with saline (p=0.0634).

Figure 2

Structure of STAT3 decoys. The parental STAT3 decoy was modified by a 4-nucleotide hairpin (DN4), or a hexa-ethyleneglycol linkage (DS18) to generate unimolecular structures. Two hexa-ethyleneglycol linkages were used to generate the completely circularized cyclic STAT3 decoy.

Figure 3

The modified STAT3 decoys exhibit enhanced stabilities in mouse serum. (A) Parental STAT3 decoy, DN4, DS18, and cyclic STAT3 decoy were incubated for varying lengths of time in mouse serum, electrophoresed on 15% TBE + 7 M Urea gels (polyacrylamide), and stained with SYBR-Gold, as described in Materials and Methods. Undigested indicates decoy in the absence of serum. (B) Densitometric analyses were performed on the parental STAT3 decoy, DN4, DS18, and cyclic STAT3 decoy samples shown in Panel A, and results were expressed relative to the corresponding undigested forms.

Figure 4

Modified STAT3 decoys bind to phosphorylated STAT3 protein with similar affinity as the parental STAT3 decoy. Quantitative assessment of the binding of parental STAT3 decoy, DN4, DS18 and cyclic STAT3 decoy to recombinant pSTAT3 protein by Surface Plasmon Resonance. Binding to pSTAT3 protein immobilized on a carboxymethylated dextran matrix (CM5) chip was determined at six different concentrations of the parental, DN4, DS18, and cyclic STAT3 decoys (0.313 µM, 0.625 µM, 1.25 µM, 2.5 µM, 5.0 µM, 10.0 µM).

Figure 5

Systemic delivery of cyclic STAT3 decoy suppresses HNSCC xenograft tumor growth and expression of STAT3 target genes in the tumors. (A) Mean tumor volume by day of treatment and treatment groups. UM-SCC1 cells (3×106 cells) were inoculated subcutaneously in the right flank of athymic nude mice. Following the development of palpable tumors, mice were randomized, then given daily IV injections of cyclic STAT3 decoy or the corresponding cyclic mutant STAT3 decoy as a control (5 mg/kg/day; 10 mice/group). Tumor volume measurements were obtained 3 times/week and measured to day 19. A linear model was fit to daily tumor volumes with a non - linear day effect described by a 3 knot restricted cubic spline. The test of interaction between day and group was significant (p < 0.0001) indicating differential growth curves with decreased tumor growth for STAT3 decoy compared to mutant control. Solid lines are predicted means according to the linear model; dotted lines indicated 95% confidence bounds for daily mean values. (B) At the end of 19 days of treatment, tumors were harvested, and whole cell lysates were prepared and subjected to immunoblotting for cyclin D1 and Bcl-XL. β-actin was used to assess protein loading. The bar graph is a quantitative representation of the cyclin D1/β-actin (p=0.0015) and Bcl-XL/β-actin (p=0.0021) ratios in tumors from mice treated systemically with cyclic STAT3 decoy versus the cyclic mutant control decoy.