Dual CTLA-4 and PD-1 checkpoint blockade using CS1002 and CS1003 (nofazinlimab) in patients with advanced solid tumors: A first-in-human, dose-escalation, and dose-expansion study.

BACKGROUND: This study investigated the safety and efficacy of an anti-CTLA-4 monoclonal antibody (CS1002) as monotherapy and in combination with an anti-PD-1 monoclonal antibody (CS1003) in patients with advanced/metastatic solid tumors. METHODS: The phase 1 study involved phase 1a monotherapy dose-escalation (part 1) and phase 1b combination therapy dose escalation (part 2) and expansion (part 3). Various dosing schedules of CS1002 (0.3, 1, or 3 mg/kg every 3 weeks, or 3 mg/kg every 9 weeks) were evaluated with 200 mg CS1003 every 3 weeks in part 3. RESULTS: Parts 1, 2, and 3 included a total of 13, 18, and 61 patients, respectively. No dose-limiting toxicities or maximum tolerated doses were observed. Treatment-related adverse events (TRAEs) were reported in 30.8%, 83.3%, and 75.0% of patients in parts 1, 2, and 3, respectively. Grade ≥3 TRAEs were experienced by 15.4%, 50.0%, and 18.3% of patients in each part. Of 61 patients evaluable for efficacy, 23 (37.7%) achieved objective responses in multiple tumor types. Higher objective response rates were observed with conventional and high-dose CS1002 regimens (1 mg/kg every 3 weeks or 3 mg/kg every 9 weeks) compared to low-dose CS1002 (0.3 mg/kg every 3 weeks) in microsatellite instability-high/mismatch repair-deficient tumors, melanoma, and hepatocellular carcinoma (50.0% vs. 58.8%, 14.3% vs. 42.9%, and 0% vs. 16.7%). CONCLUSION: CS1002, as monotherapy, and in combination with CS1003, had a manageable safety profile across a broad dosing range. Promising antitumor activities were observed in patients with immune oncology (IO)-naive and IO-refractory tumors across CS1002 dose levels when combined with CS1003, supporting further evaluation of this treatment combination for solid tumors. PLAIN LANGUAGE SUMMARY: CS1002 is a human immunoglobulin (Ig) G1 monoclonal antibody that blocks the interaction of CTLA-4 with its ligands and increases T-cell activation/proliferation. CS1003, now named nofazinlimab, is a humanized, recombinant IgG4 monoclonal antibody that blocks the interaction between human PD-1 and its ligands. In this original article, we determined the safety profile of CS1002 as monotherapy and in combination with CS1003. Furthermore, we explored the antitumor activity of the combination in anti-programmed cell death protein (ligand)-1 (PD-[L]1)-naive microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) pan tumors, and anti-PD-(L)1-refractory melanoma and hepatocellular carcinoma (HCC). CS1002 in combination with CS1003 had manageable safety profile across a broad dosing range and showed promising antitumor activities across CS1002 dose levels when combined with CS1003. This supports further assessment of CS1002 in combination with CS1003 for the treatment of solid tumors.

Clinical pharmacokinetics and pharmacodynamics of ivosidenib in Chinese patients with relapsed or refractory IDH1-mutated acute myeloid leukemia.

PURPOSE: This study aimed to assess the pharmacokinetics (PK) and pharmacodynamics (PD) of ivosidenib in Chinese patients with relapsed or refractory acute myeloid leukemia (R/R AML) carrying the mIDH1 mutation. METHODS: A bridging study (NCT04176393) was conducted involving 29 Chinese patients who received a daily dose of ivosidenib 500 mg in 28-day cycles. Plasma concentrations of ivosidenib and D-2-hydroxyglutarate (2-HG) were measured before and after treatment. Non-compartmental analysis (NCA) was employed to evaluate the PK, and an established population pharmacokinetic (popPK) model developed from non-Chinese patients was externally validated. RESULTS: The findings revealed comparable PD effects of ivosidenib in Chinese patients with mIDH1 R/R AML. After adjusting for concomitant drug effects, PK characteristics were similar between Chinese and non-Chinese patients. Furthermore, the popPK model offered additional insights into the possible causes of the apparent ethnic difference in PK exposure. CONCLUSION: The study indicates that ivosidenib can be used effectively in Chinese patients, and the observed ethnic differences in PK exposure can be explained by concomitant drug effects. The popPK model contributes to a better understanding and optimization of personalized dosing in Chinese patients with mIDH1 R/R AML.

Tumor growth inhibition-overall survival modeling in non-small cell lung cancer: A case study from GEMSTONE-302.

Overall survival is vital for approving new anticancer drugs but is often impractical for early-phase studies. The tumor growth inhibition-overall survival (TGI-OS) model could bridge the gap between early- and late-stage development. This study aimed to identify an appropriate TGI-OS model for patients with non-small cell lung cancer from the GEMSTONE-302 study of sugemalimab. We used three TGI models to delineate tumor trajectories and investigated three OS model for linking TGI metric to OS. All three TGI models accurately captured tumor profiles at the individual level. The published atezolizumab-based TGI-OS model predicted survival time satisfactorily through simulation-based evaluation, whereas the other published model built from multi-treatment underestimated OS. Our study-specific TGI-OS model identified time-to-growth as the most significant metric with the number of metastatic sites and neutrophil-to-lymphocyte ratio at baseline as covariates and exhibited robust OS predictability. Our findings demonstrated the effectiveness of the TGI-OS models in predicting phase III outcomes, which underpins their value as a powerful tool for antitumor drug development.

A first-in-human phase 1 study of nofazinlimab, an anti-PD-1 antibody, in advanced solid tumors and in combination with regorafenib in metastatic colorectal cancer.

BACKGROUND: We assessed nofazinlimab, an anti-PD-1 antibody, in solid tumors and combined with regorafenib in metastatic colorectal cancer (mCRC). METHODS: This phase 1 study comprised nofazinlimab dose escalation (phase 1a) and expansion (phase 1b), and regorafenib dose escalation (80 or 120 mg QD, days 1-21 of 28-day cycles) combined with 300-mg nofazinlimab Q4W (part 2a) to determine safety, efficacy, and RP2D. RESULTS: In phase 1a (N = 21), no dose-limiting toxicity occurred from 1 to 10 mg/kg Q3W, with 200 mg Q3W determined as the monotherapy RP2D. In phase 1b (N = 87), 400-mg Q6W and 200-mg Q3W regimens were found comparable. In part 2a (N = 14), both regimens were deemed plausible RP2Ds. Fatigue was the most frequent treatment-emergent adverse event (AE) in this study. Any-grade and grade 3/4 nofazinlimab-related AEs were 71.4% and 14.3%, 56.3% and 5.7%, and 57.1% and 21.4% in phases 1a, 1b, and part 2a, respectively. ORRs were 14.3% and 25.3% in phases 1a and 1b, respectively. In part 2a, no patients had radiological responses. CONCLUSIONS: Nofazinlimab monotherapy was well tolerated and demonstrated preliminary anti-tumor activity in multiple tumor types. Regorafenib plus nofazinlimab had a manageable safety profile but was not associated with any response in mCRC. CLINICAL TRIAL REGISTR ATION: Clinicaltrials.gov (NCT03475251).

Can a propensity score matching method be applied to assessing efficacy from single-arm proof-of-concept trials in oncology?

As a result of the escalating number of new cancer treatments being developed and competition among pharmaceutical companies, decisions regarding how to proceed with phase III trials are frequently based on findings from either single-arm phase I expansion cohorts or phase II studies that compare the efficacy of the study drug to a standard-of-care benchmark derived from historical data. However, even when eligibility criteria are matched, differences in the distribution of baseline patient features may influence the outcome of single-arm trials in real-world scenarios. Therefore, novel methods are needed to enhance the accuracy of efficacy prediction from current cohorts relative to historical data. In this study, we demonstrated the feasibility of using the propensity score matching (PSM) method to improve decision making by matching relevant baseline features between current and historical cohorts. According to our findings, utilizing the PSM method may provide a less biased means of comparing outcomes between current and historical cohorts relative to a naïve approach, which relies solely on differences in average outcomes between the cohorts.

STimulator of INterferon Genes Agonism Accelerates Antitumor Activity in Poorly Immunogenic Tumors.

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile.

Oncology phase II proof-of-concept studies with multiple targets: Randomized controlled trial or single arm?

For oncology drug development, phase II proof-of-concept studies have played a key role in determining whether or not to advance to a confirmatory phase III trial. With the increasing number of immunotherapies, efficient design strategies are crucial in moving successful drugs quickly to market. Our research examines drug development decision making under the framework of maximizing resource investment, characterized by benefit cost ratios (BCRs). In general, benefit represents the likelihood that a drug is successful, and cost is characterized by the risk adjusted total sample size of the phases II and III studies. Phase III studies often include a futility interim analysis; this sequential component can also be incorporated into BCRs. Under this framework, multiple scenarios can be considered. For example, for a given drug and cancer indication, BCRs can yield insights into whether to use a randomized control trial or a single-arm study. Importantly, any uncertainty in historical control estimates that are used to benchmark single-arm studies can be explicitly incorporated into BCRs. More complex scenarios, such as restricted resources or multiple potential cancer indications, can also be examined. Overall, BCR analyses indicate that single-arm trials are favored for proof-of-concept trials when there is low uncertainty in historical control data and smaller phase III sample sizes. Otherwise, especially if the most likely to succeed tumor indication can be identified, randomized controlled trials may be a better option. While the findings are consistent with intuition, we provide a more objective approach.

Characterization of murine CEACAM1 in vivo reveals low expression on CD8+ T cells and no tumor growth modulating activity by anti-CEACAM1 mAb CC1.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been reported to mediate both tumorigenic and anti-tumor effects in vivo. Blockade of the CEACAM1 signaling pathway has recently been implicated as a novel mechanism for cancer immunotherapy. CC1, a mouse anti-CEACAM1 monoclonal antibody (mAb), has been widely used as a pharmacological tool in preclinical studies to inform on CEACAM1 pathway biology although limited data are available on its CEACAM1 blocking characteristics or pharmacodynamic-pharmacokinetic profiles. We sought to investigate CEACAM1 expression on mouse tumor and immune cells, characterize CC1 mAb binding, and evaluate CC1 in syngeneic mouse oncology models as a monotherapy and in combination with an anti-PD-1 mAb. CEACAM1 expression was observed at high levels on neutrophils, NK cells and myeloid-derived suppressor cells (MDSCs), while the expression on tumor-infiltrating CD8+ T cells was low. Unexpectedly, rather than blocking, CC1 facilitated binding of soluble CEACAM1 to CEACAM1 expressing cells. No anti-tumor effects were observed in CT26, MBT2 or A20 models when tested up to 30 mg/kg dose, a dose that was estimated to achieve >90% target engagement in vivo. Taken together, tumor infiltrating CD8+ T cells express low levels of CEACAM1 and CC1 Ab mediates no or minimal anti-tumor effects in vivo, as a monotherapy or in combination with anti-PD-1 treatment.

Predictive Gene Signatures Determine Tumor Sensitivity to MDM2 Inhibition.

Early clinical trials using murine double minute 2 (MDM2) inhibitors demonstrated proof-of-concept of p53-induced apoptosis by MDM2 inhibition in cancer cells; however, not all wild-type TP53 tumors are sensitive to MDM2 inhibition. Therefore, more potent inhibitors and biomarkers predictive of tumor sensitivity are needed. The novel MDM2 inhibitor DS-3032b is 10-fold more potent than the first-generation inhibitor nutlin-3a. TP53 mutations were predictive of resistance to DS-3032b, and allele frequencies of TP53 mutations were negatively correlated with sensitivity to DS-3032b. However, sensitivity to DS-3032b of TP53 wild-type tumors varied greatly. We thus used two methods to create predictive gene signatures. First, by comparing sensitivity to MDM2 inhibition with basal mRNA expression profiles in 240 cancer cell lines, a 175-gene signature was defined and validated in patient-derived tumor xenograft models and ex vivo human acute myeloid leukemia (AML) cells. Second, an AML-specific 1,532-gene signature was defined by performing random forest analysis with cross-validation using gene expression profiles of 41 primary AML samples. The combination of TP53 mutation status with the two gene signatures provided the best positive predictive values (81% and 82%, compared with 62% for TP53 mutation status alone). In addition, the top-ranked 50 genes selected from the AML-specific 1,532-gene signature conserved high predictive performance, suggesting that a more feasible size of gene signature can be generated through this method for clinical implementation. Our model is being tested in ongoing clinical trials of MDM2 inhibitors.Significance: This study demonstrates that gene expression profiling combined with TP53 mutational status predicts antitumor effects of MDM2 inhibitors in vitro and in vivoCancer Res; 78(10); 2721-31. ©2018 AACR.

A 2-in-1 adaptive phase 2/3 design for expedited oncology drug development.

We propose an adaptive design that allows us to expand an ongoing Phase 2 trial into a Phase 3 trial to expedite a drug development program with fewer patients. Rather than the usual practice of increasing sample size with a less positive interim outcome, here we propose maintaining sample size with such a result and wait for fully mature data. The final Phase 2 data may be negative, may warrant a larger Phase 3 trial, or, in the extreme, could provide a definitively positive outcome. If the interim outcome is more positive, the trial continues to an originally planned larger sample size for a definitive Phase 3 evaluation. All patients from the study are used for inference regardless of the interim expansion decision. We show that no penalty needs to be paid in order to control the overall Type I error of the study, under a mild assumption that is expected to generally hold in practice. The proposed design may be considered an alternative approach to sample size adjustment for ongoing trials. As such, the use of an intermediate endpoint for adaptive decision is a unique feature of the design. A hypothetical example is provided for illustration purpose.

Phase I Imaging and Pharmacodynamic Trial of CS-1008 in Patients With Metastatic Colorectal Cancer.

PURPOSE: CS-1008 (tigatuzumab) is a humanized, monoclonal immunoglobulin G1 (IgG1) agonistic antibody to human death receptor 5. The purpose of this study was to investigate the impact of CS-1008 dose on the biodistribution, quantitative tumor uptake, and antitumor response in patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: Patients with mCRC who had received at least one course of chemotherapy were assigned to one of five dosage cohorts and infused with a weekly dose of CS-1008. Day 1 and day 36 doses were trace-labeled with indium-111 ((111)In), followed by whole-body planar and regional single-photon emission computed tomography (SPECT) imaging at several time points over the course of 10 days. RESULTS: Nineteen patients were enrolled. (111)In-CS-1008 uptake in tumor was observed in only 12 patients (63%). (111)In-CS-1008 uptake and pharmacokinetics were not affected by dose or repeated drug administration. (111)In-CS-1008 biodistribution showed gradual blood-pool clearance and no abnormal uptake in normal tissue. No anti-CS-1008 antibody development was detected. One patient achieved partial response (3.7 months duration), eight patients had stable disease, and 10 patients had progressive disease. Clinical benefit rate (stable disease + partial response) in patients with (111)In-CS-1008 uptake in tumor was 58% versus 28% in patients with no uptake. An analysis of individual lesions showed that lesions with antibody uptake were one third as likely to progress as those without antibody uptake (P = .07). Death-receptor-5 expression in archived tumor samples did not correlate with (111)In-CS-1008 uptake (P = .5) or tumor response (P = .6). CONCLUSION: Death-receptor-5 imaging with (111)In-CS-1008 reveals interpatient and intrapatient heterogeneity of uptake in tumor, is not dose dependent, and is predictive of clinical benefit in the treatment of patients who have mCRC.

A phase I clinical trial of FOLFIRI in combination with the pan-cyclin-dependent kinase (CDK) inhibitor flavopiridol.

BACKGROUND: The cyclin-dependent kinase inhibitor flavopiridol increases irinotecan- and fluorouracil-induced apoptosis. We conducted a phase I trial of FOLFIRI + flavopiridol in patients with advanced solid tumors. DESIGN: FOLFIRI + flavopiridol were administered every 2 weeks. Based on sequence-dependent inhibition, flavopiridol was given 3 h after irinotecan but before 5-FU. Two maximum tolerated doses were determined, one with flavopiridol administered over 1 h, and one with flavopiridol split as a 30-min bolus followed by a 4-h infusion. RESULTS: A total of 74 patients were enrolled and 63 were evaluable. The MTD with FOLFIRI was flavopiridol 80 mg/m(2) over 1 h or 35 mg/m(2) bolus + 35 mg/m(2) over 4 h. Dose-limiting toxicities were diarrhea, fatigue, neutropenia, and neuropathy. Clinical activity included 2 partial responses in small bowel cancer and bladder cancer and 1 complete response in mucosal melanoma. Stable disease was seen in 22 patients. Pharmacokinetic studies showed increasing C(max) with increasing flavopiridol dose. Clinical benefit was correlated with the presence of wild-type p53. Of 25 patients with colorectal cancer, 11 had as best response SD for >3 m (median 6 m, range 4.2-15.4 m), despite failing ≥1 irinotecan-containing regimen. CONCLUSIONS: Treatment with flavopiridol and FOLFIRI is a safe and effective regimen. Concentrations of flavopiridol that enhance the effects of FOLFIRI can be achieved. Clinical activity is encouraging and includes prolonged stable disease in patients with irinotecan-refractory colorectal cancer.

Chk1 inhibition after replicative stress activates a double strand break response mediated by ATM and DNA-dependent protein kinase.

Checkpoint kinase 1 (Chk1) regulates cell cycle checkpoints and DNA damage repair in response to genotoxic stress. Inhibition of Chk1 is an emerging strategy for potentiating the cytotoxicity of chemotherapeutic drugs. Here, we demonstrate that AZD7762, an ATP -competitive Chk1/2 inhibitor induces gammaH2AX in gemcitabine-treated cells by altering both dynamics and stability of replication forks, allowing the firing of suppressed replication origins as measured by DNA fiber combing and causing a dramatic increase in DNA breaks as measured by comet assay. Furthermore, we identify ATM and DNA-PK, rather than ATR, as the kinases mediating gammaH2AX induction, suggesting AZD7762 converts stalled forks into double strand breaks (DSBs). Consistent with DSB formation upon fork collapse, cells deficient in DSB repair by lack of BRCA2, XRCC3 or DNA-PK were selectively more sensitive to combined AZD7762 and gemcitabine. Checkpoint abrogation by AZD7762 also caused premature mitosis in gemcitabine-treated cells arrested in G(1)/early S-phase. Prevention of premature mitotic entry via Cdk1 siRNA knockdown suppressed apoptosis. These results demonstrate that chemosensitization of gemcitabine by Chk1 inhibition results from at least three cellular events, namely, activation of origin firing, destabilization of stalled replication forks and entry of cells with damaged DNA into lethal mitosis. Additionally, the current study indicates that the combination of Chk1 inhibitor and gemcitabine may be particularly effective in targeting tumors with specific DNA repair defects.

A phase II study of flavopiridol (Alvocidib) in combination with docetaxel in refractory, metastatic pancreatic cancer.

BACKGROUND/AIMS: Pancreatic adenocarcinoma (PC) harbors frequent alterations in p16, resulting in cell cycle dysregulation. A phase I study of docetaxel and flavopiridol, a pan-cyclin-dependent kinase inhibitor, demonstrated encouraging clinical activity in PC. This phase II study was designed to further define the efficacy and toxicity of this regimen in patients with previously treated PC. METHODS: Patients with gemcitabine-refractory, metastatic PC were treated with docetaxel 35 mg/m(2) followed by flavopiridol 80 mg/m(2) on days 1, 8, and 15 of a 28-day cycle. Tumor measurements were performed every two cycles. A Simon two-stage design was used to evaluate the primary endpoint of response. RESULTS: Ten patients were enrolled, and 9 were evaluable for response. No objective responses were observed; however, 3 patients (33%) achieved transient stable disease, with one of these patients achieving a 20% reduction in tumor size. Median survival was 4.2 months, with no patients alive at the time of analysis. Adverse events were significant, with 7 patients (78%) requiring >or=1 dose reduction for transaminitis (11%), grade 4 neutropenia (33%), grade 3 fatigue (44%), and grade 3 diarrhea (22%). CONCLUSIONS: The combination of flavopiridol and docetaxel has minimal activity and significant toxicity in this patient population. These results reflect the challenges of treating patients with PC in a second-line setting where the risk/benefit equation is tightly balanced.

Aurora B kinase regulates the postmitotic endoreduplication checkpoint via phosphorylation of the retinoblastoma protein at serine 780.

The phenotypic change characteristic of Aurora B inhibition is the induction of polyploidy. Utilizing specific siRNA duplexes and a selective small molecule inhibitor (AZD1152) to inhibit Aurora B activity in tumor cells, we sought to elucidate the mechanism by which Aurora B inhibition results in polyploidy. Cells treated with AZD1152 progressed through mitosis with misaligned chromosomes and exited without cytokinesis and subsequently underwent endoreduplication of DNA despite activation of a p53-dependent pseudo G1 checkpoint. Concomitant with polyploid cell formation, we observed the appearance of Rb hypophosphorylation, an event that occurred independently of cyclin-dependent kinase inhibition. We went on to discover that Aurora B directly phosphorylates Rb at serine 780 both in vitro and in vivo. This novel interaction plays a critical role in regulating the postmitotic checkpoint to prevent endoreduplication after an aberrant mitosis. Thus, we propose for the first time that Aurora B determines cellular fate after an aberrant mitosis by directly regulating the Rb tumor suppressor protein.

A phase I study of gemcitabine given via intrahepatic pump for primary or metastatic hepatic malignancies.

PURPOSE: To determine the maximum tolerated dose and duration of hepatic arterial infusion (HAI) gemcitabine in patients with unresectable hepatic metastases from colorectal cancer or primary hepatic malignancies. METHODS: Patients received weekly gemcitabine via the side-port of an implantable HAI pump for 3 weeks in a 28-day cycle. During the dose escalation phase, increasing doses of HAI gemcitabine (800, 1,000, 1,200, and 1,500 mg/m(2)) were given at a fixed dose-rate of 10 mg/(m(2) min). This was followed by the infusion duration escalation (IDE) phase, in which HAI gemcitabine at 1,000 mg/m(2) was given over increasing lengths of time (200, 300, and 400 min). To estimate hepatic drug extraction, the pharmacokinetics of HAI gemcitabine was compared with those of intravenous gemcitabine given at the same dose-rate to the same patient in the IDE phase. RESULTS: Twenty-eight of 30 patients were evaluable. HAI gemcitabine was well tolerated up to 1,500 mg/m(2) given at 10 mg/(m(2) min) and up to 1,000 mg/m(2) infused over 400 min. There were no protocol-defined dose-limiting toxicities. One patient with cholangiocarcinoma had a partial response. Hepatic extraction of gemcitabine seems highly variable among patients and does not correlate with the length of HAI infusion. CONCLUSIONS: Hepatic arterial infusion of gemcitabine given at doses higher or longer than the recommended systemic dose of 1,000 mg/m(2) over 30 min is well tolerated. For future studies, we recommend an infusion of 1,500 mg/m(2) at a fixed dose-rate of 10 mg/(m(2) min).

90-kDa heat shock protein inhibition abrogates the topoisomerase I poison-induced G2/M checkpoint in p53-null tumor cells by depleting Chk1 and Wee1.

The G(2)/M cell cycle checkpoint is regulated by a multitude of signaling pathways after genotoxic stress. Herein, we report that treatment with the 90-kDa heat shock protein (Hsp90) molecular chaperone inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) selectively abrogates the G(2)/M checkpoint induced by 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, in p53-null compared with p53-intact HCT116 colon cancer cells. The basis for this selectivity can be explained in part by the lack of p21 induction in p53-null cells. In accord with published results, we could show that treatment with 17AAG resulted in depletion of Chk1, a known Hsp90 client protein. In addition, we observed a time- and dose-dependent decrease in Wee1 kinase level, a negative regulator of mitosis, after 17AAG treatment in gastrointestinal cancer cells. Depletion of Wee1 protein preceded mitotic entry induced by 17AAG, and this decrease could be partially rescued by cotreatment with a proteasome inhibitor. Coimmunoprecipitation experiments showed that Hsp90 and Wee1 interacted in whole cells, and 17AAG treatment decreased the degradative half-life of Wee1, indicating that Wee1 is another Hsp90 client in mammalian cells. Knockdown of Chk1 and Wee1 by short interfering RNA each resulted in abrogation of the G(2)/M checkpoint induced by SN-38. The combination of SN-38 and 17AAG was shown to be synergistic in p53-null but not in parental HCT116 cells by median effect/combination index analysis. Taken together, 17AAG specifically inhibits the G(2)/M checkpoint in p53-defective cells by down-regulation of two critical checkpoint kinases, Chk1 and Wee1.

A phase 1 dose-escalation study of irinotecan in combination with 17-allylamino-17-demethoxygeldanamycin in patients with solid tumors.

PURPOSE: Both heat shock protein 90 (Hsp90) and checkpoint kinase 1 (Chk1) have emerged as novel therapeutic targets. We conducted a phase I study of irinotecan and the Hsp90 inhibitor 17AAG, which can also down-regulate Chk1, in patients with solid tumors. EXPERIMENTAL DESIGN: During the dose escalation phase, patients received i.v. irinotecan followed by 17AAG once weekly for 2 weeks in a 21-day cycle. At the maximum tolerated dose (MTD), additional patients were enrolled to undergo pre- and post-17AAG tumor biopsies for pharmacodynamic evaluation. The pharmacokinetics of irinotecan, 17AAG, and their metabolites were characterized. Tumor p53 status as determined by immunohistochemistry was correlated with antitumor activity. RESULTS: Twenty-seven patients with a variety of solid tumors were enrolled. Four patients developed dose-limiting toxicity at dose level 4 (100 mg/m(2) irinotecan and 375 mg/m(2) 17AAG) including nausea, vomiting, diarrhea, and pulmonary embolism. The pharmacokinetics of 17AAG and its metabolite were not significantly affected by the coadministration of irinotecan, and vice versa. There was no partial response, although tumor shrinkage was observed in six patients. Five of 10 patients with p53-mutant tumor had stable disease as the best response compared with 2 of 6 patients with p53-wildtype tumor (P = 0.63). Evidence for Hsp90 inhibition by 17AAG, resulting in phospho-Chk1 loss, abrogation of the G(2)-M cell cycle checkpoint, and cell death could be shown in tumor biopsy samples obtained at the MTD. CONCLUSIONS: The combination of irinotecan and 17AAG can be given to patients with acceptable toxicity. The recommended phase II dose of the combination is 100 mg/m(2) irinotecan and 300 mg/m(2) 17AAG.