A novel HSP90 inhibitor-drug conjugate to SN38 is highly effective in small cell lung cancer (SCLC)

Abstract

Purpose

Small cell lung cancer (SCLC) is a highly aggressive disease representing 12-13% of total lung cancers, with median survival <2 years. No targeted therapies have proven effective in SCLC. While 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 2 SCLC xenograft and patient-derived xenograft (PDX) 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 to 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 to irinotecan.

Conclusions

Together, these results strongly support clinical development of STA-8666 for use in the first or second line setting for SCLC.

Introduction

Of the one in four deaths occurring annually due to cancer in the US, approximately 16% (160,000 patients) are due to lung cancer (1). Small-cell lung cancer (SCLC) represents ~12-14% of the total lung cancer population, affecting over 30,000 patients annually, and has a particularly poor prognosis (2). Post-diagnosis median survival is 15-20 months for patients diagnosed with limited stage disease, and 9-12 months for patients with extensive stage (ES) disease. In contrast to other tumor types, such as non-small cell lung cancer (NSCLC) (3), SCLC typically is not characterized by kinase-activating mutations and other changes in targetable signaling pathways; rather, characteristic features include inactivation of the tumor suppressors TP53 and RB1, and changes limiting Notch and altering TP73 function (4, 5). Compatible with this profile, targeted therapies have not proven effective to date in SCLC. Standard front line therapies include a combination of a platinum agent and etoposide or irinotecan/topotecan (6), and tumors typically recur rapidly and are resistant to second line therapies. One critical barrier to effective treatment of SCLC tumors with cytotoxic agents has been the inability to concentrate these drugs in the tumor at sufficient levels to achieve therapeutic benefit without simultaneously inducing untenable degrees of toxicity in normal tissues. While a great deal of effort has been devoted to developing strategies to concentrate cytotoxic agents within tumors (7), so far these approaches have met with equivocal success.

STA-8666 (8) is a recently described tripartite molecule designed to address the issue of targeted delivery of cytotoxic agents to the tumor by using heat shock protein 90 (HSP90) as a tumor targeting agent. As a result of stresses existing within tumors and the tumor microenvironment, HSP90 is both highly overexpressed and in an activated configuration in tumors relative to normal tissue (9, 10). STA-8666 is comprised of an HSP90-targeting moiety fused via a cleavable carbamate linker to the cytotoxic compound SN-38 (the active metabolite of irinotecan (11)), which inhibits topoisomerase I to induce double and single strand DNA breaks. As with irinotecan, carboxylesterase (CES) activity in normal tissues (primarily liver) and tumors cleaves the linker region, releasing SN-38 over an extended period (8). Hence, STA-8666 has the potential for greater antitumor activity than other SN-38 delivery vehicles because of its ability to be concentrated in tumor tissue, where intratumoral cleavage provides high, selective SN-38 exposure. Such an approach has theoretical advantages versus other targeting strategies, such as antibody-drug conjugates, in that low molecular weight (880 Da) STA-8666 does not require a cell surface antigen for binding and active endocytosis, while the abundance of HSP90 in tumor cells obviates the requirement for a cytotoxic component active at extremely low concentrations.

The first study of STA-8666 reported exceptional biological activity of this compound against xenograft models of pancreatic and breast cancer, and demonstrated activity was dependent on the ability of this compound to interact with HSP90 (8). In this study, we therefore evaluated STA-8666 in SCLC xenografts, benchmarking against standard first and second line therapies, determining whether tumors resistant to such therapies were responsive to STA-8666, and performing parallel mechanistic analysis. The extraordinary potency of STA-8666 in SCLC patient-derived xenograft (PDX) and cell line models, coupled with the identification of response biomarkers, provides significant preclinical support for the development of this agent towards clinical trials for SCLC.

Material and Methods

Cell lines and xenograft analysis

The NCI-H69 human SCLC cell line (12), obtained from the American Type Culture Collection (ATCC), and the NCI-H157 (a gift of J. Kurie) have been described. Cell lines were sent for authentication to IDEXX Bioresearch (Westbrook, ME). Short tandem repeat (STR) analysis using the Promega CELL ID™ System (8 STR markers + amelogenin) was performed and verified that the genetic profile of the sample matches the known profile of the cell line. The samples were confirmed to be of human origin and no mammalian interspecies contamination was detected.

The LX-36 (also known as CTG-0199 and BML-4) PDX model was derived at Fox Chase Cancer Center (see Supp. Methods), and was used at passage 4. The genotype of mutations relevant to common mutations in SCLC was determined by exome sequencing performed by Ambry Genetics (Aliso Viejo, CA).

For xenograft analysis of the NCI-H69 and SCLC1 cell lines and 10 × 10⁶ cells were introduced into the flank of 6-8 week old, female C-B17.SCID mice. For the LX-36 model, tumors were minced on ice to 1-2 mm fragments, mixed 1:1 in RPMI/Matrigel Basement Membrane Matrix (#354234; BD Biosciences; Bedford, Massachusetts, USA), and implanted subcutaneously in both flanks of 25 C-B17.SCID mice. Mice were monitored twice a week. Tumor volumes (V) were calculated by caliper measurement of the width (W), length (L) and thickness (T) of tumors using the following formula: V = 0.5236 x (L x W x T). Mice with tumor size in the range of 90 to 250 mm³ (~day 24, and 60% of total mice; average size 150 mm³) were selected and randomized into treatment groups for treatment with vehicle, STA-8666, irinotecan, carboplatin, etoposide, topotecan, or ganetespib (details in Supp. Methods). Mice with tumors >1500 mm³, or experiencing 10% weight loss or with appearance of distress were switched from original treatment to 150 mg/kg STA-8666, as detailed in Results. All mice were euthanized by C0₂ inhalation at 120 days after commencement of dosing, or in cases of distress, or >10% weight loss. Tumors were excised and divided for formalin fixation and paraffin embedding (FFPE), or flash frozen and stored at −80°C.

The NCI-H69 cell line was used for assessment of pharmacokinetics (PK), and intratumoral response following transient dosing. Xenografts were established as described above, except tumors were allowed to grow to 1000 mm³ before commencing dosing. For signaling, tumors reached 600 mm³ mice were injected with vehicle, STA-8666, or irinotecan, then euthanized at times indicated after dosing as noted in Results, with tumor tissue collected, divided, and processed by FFPE or flash freezing. For kinome analysis, tumor tissues were weighed and homogenized in a 3x volume of PBS containing 40 mg/ml NaF, extracted by protein precipitation, and analyzed using a LC-MS/MS with a Waters SymmetryShield RP18 column (5μm, 2.1×100mm).

Bioanalysis

Tumor xenografts and plasma samples were collected and snap frozen 72 hours after a single injection of 150 mg/kg STA-8666, or 60 mg/kg irinotecan and sent for further analysis to Synta Pharmaceuticals. Plasma and tumor samples were received frozen in dry ice and stored at −80°C. Prior to analysis, tumor samples were weighed and homogenized in 3x volumes of phosphate buffered saline containing 40 mg/mL sodium fluoride, an esterase inhibitor. Homogenized tissue and plasma samples were extracted by protein precipitation with 0.5/95.5 (v/v) formic acid/acetonitrile containing stable isotope stable isotope labeled internal standards for STA-8666, STA-12-8663, and SN-38. An Agilent 1100 high pressure liquid chromatography (HPLC) pump was coupled to a Waters SymmetryShield RP18 column (100 × 2.1 mm, 5 μm). The analytes were eluted at a flow rate of 0.5 mL/min using a gradient mobile phase composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The eluent was introduced via electrospray ionization directly into an AB Sciex API 4000 triple quadrupole mass spectrometer operated in the positive ion mode. Total run time was 5 minutes per sample.

Immunohistopathology

Pathology specimens were stained with hematoxylin and eosin (H&E) (Sigma-Aldrich, St. Louis, MO). Tumor sections were immunostained with antibodies to cleaved caspase 3 (Cell Signaling Technology), γH2AX (EMD Millipore, Billerica, MA), HSP70 (Novus Biologicals, Littleton, CO), HSP90 (Cell Signaling Technology, Danvers, MA), and Ki-67 (DAKO, Carpinteria, CA). All analyses were performed by a board-certified pathologist (K.Q.C.) blind to sample identity. Quantification of signal in immunostained slides was performed using an Aperio ScanScope CS scanner (Aperio) and Vectra Automated Quantitative Pathology Imaging System (Perkin Elmer) (see Supp Methods).

Western Blot Analysis

To analyze the expression levels of individual proteins tumor tissue and cells were lysed in T-PER lysis buffer (ThermoScientific, Waltham, MA) and CelLytic MT Cell Lysis Reagent (Sigma-Aldrich, St. Louis, MO). Protein concentrations of the resulting lysates were measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). Western Blotting was performed using standard procedures, and blots developed by chemiluminescence using Luminata Western HRP substrates (Classico, Crescendo and Forte, EMD Millipore) and Immun-Star AP Substrate (Bio-Rad Laboratories).

Primary antibodies were used in 1:1000 dilution (if not indicated differently) and included: anti-PARP (#9542), anti-phospho CHK1 Ser345 (#2348), anti-CHK1 (#2360), anti-phospho Cyclin B1 Ser133 (#4133), anti-Cyclin B1 (#4135), anti-phospho-CDK1 Tyr15 (#9111) (all from Cell Signaling, Denvers, MA); anti-HSP70 (monoclonal, mouse, #ADI-SPA-820, Enzo, Farmingdale, NY) or anti-HSP90α (#ADI-SPS-771, Enzo, Farmingdale, NY and #4877, Cell Signaling, Denvers, MA); anti-CDK1 (#sc-54, Santa Cruz, Dallas, TX); anti-KAP1 Ser824 (ab70369), anti-KAP1 (ab56587), anti-GAPDH (ab9482 and ab8245, 1:10,000) (from Abcam, Cambridge, UK); anti-vinculin (mouse, monoclonal hVIN-1, #V9131, Sigma-Aldrich, St. Louis, MO). Secondary anti-mouse and anti-rabbit HRP-conjugated antibodies (GE Healthcare, Little Chalfont, UK) were used at a dilution of 1:10,000 and secondary anti-mouse and anti-rabbit AP-conjugated antibodies (Jackson Immunoresearch Labs, West Grove, PA) were used at a dilution of 1:5,000. Quantification of signals on Western blots was done using the NIH ImageJ Imaging and Processing Analysis Software (13) with signaling intensity normalized to loading control. IRDye800CW Goat anti-Mouse and IRDye 680 RD Goat anti-Rabbit secondary antibodies were used at 1:20,000 dilution (LI-COR, Lincoln, NE). Membranes were imaged and quantified in separate channels (700 and 800 nm) in the Odyssey Infrared Imaging System using Odyssey V3.0 software (LI-COR, Lincoln, NE).

Multiplexed inhibitor beads/mass spectrometry (MIBs/MS)

Flash frozen tumors collected from mice treated for 48 h with either vehicle control, 150 mg/kg and 50 mg/kg HDC or 60 mg/kg irinotecan (n=3/group) were randomized into three experimental replicate groups consisting of a single tumor from each of the three treatment conditions for processing and subsequent quantitative LC/MS analysis. MIB preparation and chromatography was performed as described in (14), and detailed in Supp Methods.

Cell cycle analysis & cytometric assessment of pH2AX

H69 and H157 cells treated for 24hr, 48hrs and 72 hr with 100nM of STA-8666 or irinotecan were collected, pelleted by centrifugation at 1500 rpm for 4 min and suspended in 0.5 ml phosphate-buffered saline (PBS). Cells were subsequently fixed and stained for phosphorylated (γ) H2AX as described in (15). Cells were incubated overnight at 4°C in anti-γH2AX primary antibodies (1:200) (Mouse Monoclonal, Millipore Upstate, Billerica, MA) and 1 hour at RT with FITC-tagged secondary antibodies (1:500) (Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate, Thermo Scientific, Waltham, MA). Cells were pelleted and resuspended in 300 μL of propidium iodide solution (PI/RNase Staining Buffer, BD Pharmingen, San Diego, CA). Following incubation at RT for 30 min, cell cycle distribution and γH2AX expression were analyzed using FACS (Becton Dickinson, UK).

Statistical Analysis

For Figures 1G and 2B, we used Fisher's exact tests for comparison of CR rates between groups. For the other figures, we generally used Wilcoxon Rank-sum tests for pairwise comparisons, unless otherwise noted. Analyses were performed by using STATA versions 12 and 13 and Prism 6 (GraphPad Software, San Diego, CA).

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.

Results

High dose STA-8666 or low dose STA-8666 in combination with carboplatin eliminates or controls H69 SCLC xenograft tumors

We first examined the effectiveness of weekly STA-8666 in comparison to vehicle or irinotecan in controlling xenograft growth for the NCI-H69 (12) SCLC cell line in SCID mice (Figs 1, S1). For this, we explored a dose range below the previously identified maximum tolerated dose (MTD) of 200 mg/kg (16) to establish efficacy in the context of a clear therapeutic window. For mice dosed with STA-8666 at 150 mg/kg, tumors regressed below detectability after 3 initial doses (Fig 1A). After the third dose, observations continued without treatment. No recurrence was observed for 3/6 mice in STA 150 mg/kg group over 3 months; recurrent tumors in 3/6 mice remained sensitive to re-dosing and were eliminated with 3 further weekly doses (Fig 1A). Importantly, no weight loss or sign of distress was seen in mice receiving 150 mg/kg STA-8666 or irinotecan at 60 mg/kg (Fig S1A).

By contrast, tumors grew very rapidly in mice dosed with vehicle, approaching volumes that required humane euthanasia within 3 weeks (Fig 1B,). However, crossing these animals onto STA-8666 treatment led to rapid, complete responses (Fig 1C). By comparison, continuous weekly treatment of animals with the SN-38 prodrug irinotecan at 60 mg/kg, representing a concentration equimolar for the SN-38 moiety of STA-8666 at 100 mg/kg, also resulted in stable disease (Figs 1D, S1A). Dosing with irinotecan at 100 mg/kg, a level equimolar for SN-38 from STA-8666 at 150 mg/kg, exceeded the MTD for this compound, based on initial safety studies.

Dosing with STA-8666 at lower levels (100, comparable to irinotecan 60 mg/kg; and 50 mg/kg) was less effective than dosing STA-8666 at 150 mg/kg, with 100 mg/kg causing transient complete regression of tumors and 50 mg/kg controlling tumor growth for up to 3 months (Figs 1E, S1B,C). However, these dose levels required continuous weekly administration of STA-8666 to maintain stable disease. In additional benchmarking experiments to compare to compounds currently used for SCLC, topotecan (12.5 mg/kg weekly) and etoposide (8 mg/kg, 3 days/week) used at concentrations at MTD induced limited tumor control in the first 2 weeks of dosing, but each caused significant side effects, including rapid weight loss, forcing discontinuation of treatment (Fig 1E, S1D,E). Similar to a recent report (17), inhibition of HSP90 with ganetespib only partially inhibited the growth of SCLC xenografts, and mice treated with this agent gradually lost weight as tumors progressed (Fig 1E S1F). Carboplatin (30 mg/kg) caused significant weight loss and only modestly slowed tumor growth even with continuous dosing, with 4/5 mice showing early resistance (Figs 1E S1G). However, combination of 30 mg/kg carboplatin with STA-8666 at 50 mg/kg was highly effective in controlling tumor growth, as after three doses 11/11 tumors shrunk below detection limits, and the combination was well tolerated (Fig 1E, S1H). This response was durable for at least 120 days in 6/11 mice.

Mice previously treated with vehicle, 50 mg/kg STA-8666, carboplatin, topotecan, etoposide, or 30 mg/kg carboplatin+50 mg/kg STA-8666 that had progressed, had stable disease for 6-8 weeks, or were unable to tolerate treatment, were then administered 3 doses of STA-8666 at 150 mg/kg (Fig 1F and S1). Importantly, in all cases except prior dosing with STA-8666 at 50 mg/kg, this resulted in rapid and sustained elimination of tumors, cessation of negative side effects, and regaining of lost weight (Fig S1). In sum, statistical analysis of results comparing efficacy of all treatments by 3 weeks of dosing showed much higher efficacy of STA-8666 at 150 and 100 mg/kg, and STA-8666 at 50 mg/kg combined with carboplatin at 30 mg/kg, versus all other treatments (Fig 1G).

Efficacy of STA-8666 in an SCLC patient-derived xenograft (PDX)

As an independent model of SCLC, we used the patient-derived xenograft LX-36 (Table S1) and compared STA-8666 (150 mg/kg and 75 mg/kg) to vehicle, irinotecan (60 mg/kg), and the combination of etoposide (4 mg/kg days 1, 2, 3) plus carboplatin (30 mg/kg) (Figs 2, S2). As with the NCI-H69 model, three doses of 150 mg/kg STA-8666 eliminated tumors in 5/5 animals and these responses remained durable until at least 80 days from the start of treatment in 4/5 animals. In the single animal in which a tumor recurred it was eliminated with another 3 week cycle of treatment (Fig 2A), resulting in a statistically significant outcome from vehicle treated animals (Fig 2B). Vehicle treated animals progressed rapidly (Fig 2C), but substitution of treatment with STA-8666 2-3 days prior to ethically required euthanasia durably eliminated large tumors, as in NCI-H69 xenografts (Fig 2D). Over a month of continuous weekly dosing, 75 mg/kg STA-8666 caused tumor regression or stable disease; subsequent substitution of 150 mg/kg caused transient regression, but 2 of 3 tumors ultimately grew uncontrollably (Fig 2E). Neither irinotecan at its MTD nor etoposide/carboplatin effectively controlled tumor growth in this model: however, administration of STA-8666 at 150 mg/kg was again well tolerated, and effective in reducing or eliminating detectable tumors in mice unsuccessfully treated with the other compounds (Figs 2F-G).

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

Concentration and biological activity of STA-8666 in xenograft tumors

The high efficacy and low toxicity of STA-8666 in tumor xenografts is compatible with higher retention of the SN-38 moiety in the tumor. We analyzed retention of compounds in plasma versus tumors of mice with established H69 xenografts treated 72 hours previously with either 150 mg/kg STA-8666, or 60 mg/kg irinotecan. The prodrug irinotecan and its metabolite, SN-38, were below quantifiable limits (BQL) in irinotecan-treated animals, suggesting rapid clearance. STA-8666 was concentrated 46-fold in tumor (7.5 nmol/g tissue) versus plasma (163 nM), while its cleaved SN-38 metabolite was concentrated 10-fold (0.56 nmol/g tumor, versus 0.05 nM in plasma) (Fig 3A). The high tumor concentration of STA-8666 in mice treated with STA-8666 suggested that at this time point, much of the compound was uncleaved, leaving a residual pool for continual SN-38 release. Notably, the levels of SN-38 released from STA-8666 were ~4-fold lower than intact STA-8666, suggesting that SN-38 is actively being cleared from tumor cells, as inferred for irinotecan.

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.

We performed quantitative histopathological analysis of tissues harvested from mice 72 hours after a single dose of 150 mg/kg STA-8666, 60 mg/kg irinotecan, or vehicle (equivalent to the time point for pharmacokinetic analysis), or 48 hours after a second weekly dose of compound, a time at which STA-8666-treated tumors have begun to significantly shrink (Figs 3B-E and S3). At both time points, the necrotic area of STA-8666-treated tumors exceeded that of irinotecan- and vehicle-treated tumors (59% versus 41% and 27% at the first time point; and 44% versus 29% or 39% at the second). By the second time point, STA-8666 had significantly reduced total tumor area versus vehicle or irinotecan (36 versus 64 mm² for vehicle (P=0.007) or 55 mm² for irinotecan (P=0.00013) (Figs 3B-D, S3). In addition, tumor size reduction was more homogeneous with STA-8666 than irinotecan (p=0.003 at the first time point, and p=0.054 at the later time point). By the early time points, both irinotecan and STA-8666 modestly elevated expression of the DNA damage marker phosphorylated (p) H2AX and the apoptotic marker, cleaved caspase 3. By the later time point, both pH2AX and cleaved caspase 3 were strongly elevated by STA-8666 relative to vehicle (5-fold and 13-fold) or irinotecan (2.3-fold and 10.5-fold) in nonnecrotic areas of the tumor (Figs 3E, S3B).

Levels of HSP90 were generally consistent between all treatment groups at both time points, with a slight reduction in expression associated with high treatment levels of STA-8666 at the early time point. No statistically significant change in HSP70 expression was observed, reflecting the biologically insignificant HSP90-inhibitory activity of STA8666 (8). Interestingly, analysis of the small number of tumors treated with lower doses of STA-8666 that developed resistance to this compound (data in Figs S1B,C) showed a similar profile of HSP70 and HSP90 expression to transiently treated tumors, except that 2 of 3 resistant tumors had barely detectable levels of pH2AX (Fig S4), potentially suggesting greater capacity to efflux the compound. Together, these results indicate that the primary biological activity of STA-8666 is through prolonged SN-38 exposure provided by the selective retention of STA-8666 in the tumor, and superior to that of irinotecan.

Distinct kinetics of DNA damage and cell cycle checkpoint response induced by STA-8666

Cellular exposure to SN-38 is characteristically associated with DNA damage and G2/M checkpoints and previously reported as maximal at 24-48 hours following cell exposure to SN-38 (18, 19) (Fig 4A). We hypothesized the targeting activity of STA-8666 and associated greater retention of SN-38 in tumors would result in a stronger and sustained imposition of DNA damage, and stimulus to arrest, than would irinotecan. Supporting this idea, tumors harvested from mice 24, 48, or 72 hours after STA-8666 treatment showed a more rapid, robust, and sustained induction of phS⁸²⁴-KAP1 (Fig 4B), a product of damage-associated ATM phosphorylation, compared to irinotecan (20). Similar results were observed with the checkpoint-associated phS³⁴⁵-CHK1 protein (Fig 4C). STA-8666 caused marked elevation of phY¹⁵-CDK1 (Fig 4D) and downregulation of cyclin B1 (CCNB1) and PLK1-phosphorylated phS¹³³-CCNB1 (Fig 4E) (21), compatible with imposition of a strong G2/M checkpoint arrest by STA-8666, with this peaking at 24-48 hours, as previously described (18, 19), and more marked for 150 mg/kg STA-8666 than observed with irinotecan. Cleavage of PARP1 was strongly increased in all drug-treated tumors, with this indicator of apoptosis peaking at 72 hours, and much higher in tumors treated with STA-8666 than with irinotecan (21-fold for 150 mg/kg STA-8666) (Fig 4F). Aside from a transient minor elevation of HSP70, expression of HSP90 and HSP70 was not affected by either drug treatment (Fig 4G).

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.

To further support this analysis, we used a recently described multiplexed inhibitor beads (MIBs, (14, 22)) technique coupled with mass spectrometry to profile the intratumoral activity of 148 kinases 48 hours after dosing 3 mice bearing NCI-H69 tumors with irinotecan (60 mg/kg), STA-8666 (50 mg/kg), or vehicle (Fig 5A). STA-8666 durably induced a number of kinases associated with onset of G2/M arrest and DNA damage checkpoints, while irinotecan treatment did not; rather, irinotecan-treated tumors showed modest depression of many kinases relative to vehicle treatment, potentially due to the weaker, more transient levels of drug in the tumor. In sum, these data suggested a more robust activity of STA-8666 in inducing G2/M arrest, potentially because of the more durable concentration and retention of the compound. To further test this idea, we compared the ability of STA-8666 and irinotecan treatment to induce cell cycle arrest in vitro (Fig 5B). At isomolar concentrations of 100 nM, STA-8666 induced a more significant accumulation of cells in G2/M than did irinotecan in both NCI-H69 and NCI-H157 lung cancer cells, with differences appearing 24-48 hours after addition of drug. Paralleling these results, flow cytometry analysis of treated cells indicated STA-8666 also more effectively induced γH2AX at similar time points (Fig 5C). Based on Western analysis, STA-8666 also induced phS⁸²⁴-KAP1 and phY¹⁵-CDK1, at earlier time points and to a greater degree than irinotecan, in a dose-responsive manner (Figs. 5D, E, S5 A, B). Subsequently, PARP cleavage was elevated, with the greatest effect seen with 100 nM STA-8666 (Figs 5F, S5). HSP90 and HSP70 levels were unaffected (Figs 5G,H, S5).

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.

Discussion

Our data for the first time demonstrates potential clinical value for application of STA-8666 in the setting of SCLC. Our results differentiate STA-8666 from irinotecan in both the intensity of the DNA damage and cell cycle checkpoint response as well as the induction of apoptosis and necrosis in human tumors. These results are compatible with pharmacokinetic results indicating delivery of high levels of SN-38 directly to target tumor tissue and superior retention over time, which is important in tumors with intrinsic resistance to therapeutics, such as SCLC (23). Systemic treatment for SCLC has not significantly improved for decades (2), while 5-year survival remains at <7% overall; for the 60-70% of patients diagnosed with extensive stage (ES) SCLC, 2-year survival is <5% (24). Factors contributing to these abysmal figures include the failure of early detection approaches to effectively identify rapidly dividing SCLC tumors before progression to the ES stage, and the characteristic mutational profile of SCLC, which has differed from many other tumor types in failing to suggest strategies for application of targeted therapeutic agents (2).

In this context, an HSP90-targeted strategy is potentially particularly valuable, as the rapid proliferation rate of the tumors enforces dependence on HSP90 expression. Indeed, the prior treatment of SCLC with other cytotoxic agents or radiation may contribute to the vulnerability to STA-8666, as such treatments would increase cellular dependence on heat shock and other stress response systems (25). The initial report of STA-8666 demonstrated that while an HSP90-binding capacity was important for functional activity, HSP90 inhibition per se was insufficient to limit tumor growth (8); although we cannot rule out a minor contribution of HSP90-inhibitory activity in the observed tumor control, the data in this study confirms that the HSP90 inhibitor ganetespib was ineffective in controlling SCLC xenograft growth and we did not observe elevation of the heat shock response.

In this study, we also found that prior treatment with a number of agents currently used for front line therapy for SCLC did not result in resistance to STA-8666; rather, rapid tumor shrinkage was observed after substitution of treatment with 150 mg/kg STA-8666, and responses were typically durable. In addition, the combination of low dose (50 mg/kg) STA-8666 with carboplatin was extremely effective. In this work, the only prior treatment that led to resistance to 150 mg/kg of STA-8666 was the same compound used at lower doses. This is compatible with the interpretation that these lower doses are not sufficient for complete eradication of tumors in the in vivo microenvironment, in spite of the large concentration of STA-8666 and SN-38 relative to plasma. Given the dose-limiting toxicity of untargeted SN-38, it has previously been impossible to perform analysis of curative doses for SCLC, which our studies suggest must likely fall in the range of 0.6 nM/g tumor tissue. For mice previously treated with high doses of STA-8666, responses were either cure or extending through the 4 months of observation, or tumor recurrence, followed by subsequent remission. Subsequent experiments prior to clinical development of STA-8666 should address long-term consequences of relapse, whether tumors remain susceptible to cycles of redosing, and what mechanisms contribute to resistance to this compound.

Over the past several decades, advances in cancer treatment have divided tumors into classes for which very significant gains have been made, yielding regimens that are curative or suitable for chronic maintenance, versus other classes for which treatments remain largely ineffective. Taken in sum, these results strongly support the further development of STA-8666 for clinical evaluation in limited SCLC or ES-SCLC, in either the first or second line setting.

Translational Statement.

There are few effective therapies for SCLC, which remains one of the most lethal cancers. STA-8666 represents a first-in-class member of a new treatment platform that shows striking efficacy in controlling or eliminating SCLC tumors, and offers a new therapeutic direction for clinical management.