A Novel HSP90 Inhibitor-Drug Conjugate to SN38 Is Highly Effective in Small Cell Lung Cancer

Abstract

Purpose: Small cell lung cancer (SCLC) is a highly aggressive disease representing 12% to 13% of total lung cancers, with median survival of <2 years. No targeted therapies have proven effective in SCLC. Although most patients respond initially to cytotoxic chemotherapies, resistance rapidly emerges, response to second-line agents is limited, and dose-limiting toxicities (DLT) are a major issue. This study performs preclinical evaluation of a new compound, STA-8666, in SCLC.

Experimental design: To avoid DLT for useful cytotoxic agents, the recently developed drug STA-8666 combines a chemical moiety targeting active HSP90 (concentrated in tumors) fused via cleavable linker to SN38, the active metabolite of irinotecan. We compare potency and mechanism of action of STA-8666 and irinotecan in vitro and in vivo RESULTS: In two SCLC xenograft and patient-derived xenograft models, STA-8666 was tolerated without side effects up to 150 mg/kg. At this dose, STA-8666 controlled or eliminated established tumors whether used in a first-line setting or in tumors that had progressed following treatment on standard first- and second-line agents for SCLC. At 50 mg/kg, STA-8666 strongly enhanced the action of carboplatin. Pharmacokinetic profiling confirmed durable STA-8666 exposure in tumors compared with irinotecan. STA-8666 induced a more rapid, robust, and stable induction of cell-cycle arrest, expression of signaling proteins associated with DNA damage and cell-cycle checkpoints, and apoptosis in vitro and in vivo, in comparison with irinotecan.

Conclusions: Together, these results strongly support clinical development of STA-8666 for use in the first- or second-line setting for SCLC. Clin Cancer Res; 22(20); 5120-9. ©2016 AACR.

Figure 1. In vivo response to STA-8666 treatment of NCI-H69 xenografts

All graphs in A-D represent tumor volume (TV) fold changes in individual mice following dosing with indicated drugs. A. Mice treated with STA-8666 at 150 mg/kg, received 3 weekly doses, then were observed; reinitiation of dosing with STA-8666 at 150mg/kg occurred 1 week after observation of tumor recurrence in 3/6 mice. B. TV in mice, treated with vehicle. C. Tumor volumes in vehicle cohort (from B) after treatment was switched to STA-8666 at 150 mg/kg; for starting volume, “1” references TV indicated in B. D. TV in mice treated with irinotecan at 60 mg/kg administered in continuous dosing. E. Lines indicate average TV in drug treatment cohorts, presented as a ratio to initial TV. All drugs were administered continuously except for the STA-8666/carboplatin combination, where mice received 3 weekly doses, then were observed; dosing with STA-8666 at 150mg/kg occurred 1 week after observation of tumor recurrence in 5/11 of these mice (see F). For other drugs, lines terminate when treatment is switched to STA-8666 at 150mg/kg. F. Lines indicate average TV fold changes in treatment cohorts after treatment was switched to STA-8666 at 150 mg/kg. G. Table representing statistical significance of complete response (CR) at 4 weeks, for each treatment group.

Figure 2. In vivo response to STA-8666 treatment of PDX LX-36 xenografts

A,C-G. All graphs represent fold change in TV in individual mice following dosing with indicated drugs, before and after switch to STA-8666 150 mg/kg as indicated. Genotype data for LX-36 model is provided in Table S1. B. Table representing statistical significance of complete response (CR) at 4 weeks, for each treatment group

Figure 3. Pharmacokinetics and histopathological evaluation of STA-8666 treated SCLC tumors

A. Concentration of STA-8666 and SN-38 in plasma (pl) or tumors from NCI-H69 xenografts, 72 hours post-treatment. B. Quantification of necrotic area in STA-8666 (150mg/kg), irinotecan (60mg/kg) or vehicle-treated NCI-H69 xenografts, 72 hours after single injection (top) and 48 hours after second weekly injection (bottom) C. Representative images of H&E stained tumors obtained from indicated treatment groups 72 hours after a single injection (left) and 48 hours after second weekly injection (right). Scale bar, 3mm D. Quantification of tumor area in STA-8666 (150mg/kg) vs. irinotecan (60mg/kg) or vehicle-treated NCI-H69 xenografts, 72 hours after single dose of drugs (left) and 48 hours after second weekly injection (right). E. Quantification of immunohistochemical analysis of cleaved caspase 3, phosphor (π)-H2AX, HSP70, and HSP90 at times indicated. All graphs: *, p<0.05; **, p<0.01, ***, p<0.001, ****, p<0.0001.

Figure 4. STA-8666 mediated DNA damage and cell cycle arrest

A. Schematic representation of signaling pathway activated upon SN38-mediated DNA damage. Proteins shown in gray inhibit G2/M transition; indicated amino acid residues are phosphorylated during regulation of DDR response (H2AX, KAP1, CHK1) or G2/M cell cycle arrest (cyclin B1, CDK1). Hatched proteins promote G2/M transition. Sensors of DNA damage are shown in white. B-G. Western blot analysis of NCI-H69 xenograft tumors harvested at 24, 48 and 72 hours after single dose of vehicle, irinotecan (60 mg/kg), or STA-8666 (50 mg/kg and 150 mg/kg). Representative images (left) and quantification (right) for the following proteins: phospho and total KAP1 (B), phospho and total CHK1 (C), phospho and total CDK1 (D), phospho and total Cyclin B1 (E), total and cleaved PARP (F), HSP90 and HSP70 (G). All protein levels are normalized to GAPDH or vinculin loading control*, P < 0.05, **, P < 0.01, ***, P<0.001, ****, P<0.0001 compared to vehicle; ^#, P < 0.05, ^##, P <0.01, ^####, P < 0.0001 versus irinotecan.

Figure 5. G2/M cell cycle arrest and checkpoint response to STA-8666 in NCI-H69 and NCI-H157 cell lines

A. Kinome profiling of NCI-H69 xenograft tumors treated with STA-8666, irinotecan, or vehicle. Waterfall plot indicated kinases that were detected in 2/3 biological replicates as having an activity fold change >1.3 in at least one treatment condition. Analysis was performed 48 hr after single dose of STA-8666 (50 mg/kg) and irinotecan (60 mg/kg). B. Percentage of cells in G2/M phase of cell cycle after treatment with vehicle, 100 nM irinotecan and 100 nM STA-8666 in NCI-H69 (left) and NCI-H157 (right) cell lines. C. Phospho-H2AX induction in NCI-H69 (left) and NCI-H157 (right) cells treated with vehicle, 100 nM STA-8666 and 100 nM irinotecan. MFI (mean fluorescence intensity) calculated from FACS data is presented on arbitrary scale. D-H: Western blot analysis of NCI-H69 (left) and NCI-H157 (right) cell lysates reflecting checkpoint induction after STA-8666 treatment: phospho KAP1 (D), phospho CDK1 (E), cleaved PARP (F), HSP90 (G) and HSP70 (H). All protein levels are normalized to GAPDH or vinculin loading control. Data expressed as mean ± SEM are the average of 3 independent replications. *, P < 0.05, **, P < 0.01 compared to vehicle treated. Refer to Fig S4 for representative images.