Peter J. Klauck

Rational Combination of a MEK Inhibitor, Selumetinib, and the Wnt/Calcium Pathway Modulator, Cyclosporin A, in Preclinical Models of Colorectal Cancer

Purpose: The mitogen-activated protein kinase (MAPK) pathway is a crucial regulator of cell proliferation, survival, and resistance to apoptosis. MEK inhibitors are being explored as a treatment option for patients with KRAS-mutant colorectal cancer who are not candidates for EGFR-directed therapies. Initial clinical results of MEK inhibitors have yielded limited single-agent activity in colorectal cancer, indicating that rational combination strategies are needed. Experimental Design: In this study, we conducted unbiased gene set enrichment analysis and synthetic lethality screens with selumetinib, which identified the noncanonical Wnt/Ca++ signaling pathway as a potential mediator of resistance to the MEK1/2 inhibitor selumetinib. To test this, we used shRNA constructs against relevant WNT receptors and ligands resulting in increased responsiveness to selumetinib in colorectal cancer cell lines. Further, we evaluated the rational combination of selumetinib and WNT pathway modulators and showed synergistic antiproliferative effects in in vitro and in vivo models of colorectal cancer. Results: Importantly, this combination not only showed tumor growth inhibition but also tumor regression in the more clinically relevant patient-derived tumor explant (PDTX) models of colorectal cancer. In mechanistic studies, we observed a trend toward increased markers of apoptosis in response to the combination of MEK and WntCa++ inhibitors, which may explain the observed synergistic antitumor effects. Conclusions: These results strengthen the hypothesis that targeting both the MEK and Wnt pathways may be a clinically effective rational combination strategy for patients with metastatic colorectal cancer. Clin Cancer Res; 19(15); 4149–62. ©2013 AACR.

Dual pharmacological targeting of the MAP kinase and PI3K/mTOR pathway in preclinical models of colorectal cancer.

Background The activation of the MAPK and PI3K/AKT/mTOR pathways is implicated in the majority of cancers. Activating mutations in both of these pathways has been described in colorectal cancer (CRC), thus indicating their potential as therapeutic targets. This study evaluated the combination of a PI3K/mTOR inhibitor (PF-04691502/PF-502) in combination with a MEK inhibitor (PD-0325901/PD-901) in CRC cell lines and patient-derived CRC tumor xenograft models (PDTX). Materials and Methods The anti-proliferative effects of PF-502 and PD-901 were assessed as single agents and in combination against a panel of CRC cell lines with various molecular backgrounds. Synergy was evaluated using the Bliss Additivity method. In selected cell lines, we investigated the combination effects on downstream effectors by immunoblotting. The combination was then evaluated in several fully genetically annotated CRC PDTX models. Results The in vitro experiments demonstrated a wide range of IC50 values for both agents against a cell line panel. The combination of PF-502 and PD-901 demonstrated synergistic anti-proliferative activity with Bliss values in the additive range. As expected, p-AKT and p-ERK were downregulated by PF-502 and PD-901, respectively. In PDTX models, following a 30-day exposure to PF-502, PD-901 or the combination, the combination demonstrated enhanced reduction in tumor growth as compared to either single agent regardless of KRAS or PI3K mutational status. Conclusions The combination of a PI3K/mTOR and a MEK inhibitor demonstrated enhanced anti-proliferative effects against CRC cell lines and PDTX models.

Antitumor Activity of the MEK Inhibitor TAK-733 Against Melanoma Cell Lines and Patient-Derived Tumor Explants

The goal of this study was to investigate the activity of the selective MEK1/2 inhibitor TAK-733 in both melanoma cell lines and patient-derived melanoma xenograft models. In vitro cell proliferation assays using the sulforhodamine B assay were conducted to determine TAK-733 potency and melanoma responsiveness. In vivo murine modeling with eleven patient-derived melanoma explants evaluated daily dosing of TAK-733 at 25 or 10 mg/kg. Immunoblotting was performed to evaluate on-target activity and downstream inhibition by TAK-733 in both in vitro and in vivo studies. TAK-733 demonstrated broad activity in most melanoma cell lines with relative resistance observed at IC50 > 0.1 μmol/L in vitro. TAK-733 also exhibited activity in 10 out of 11 patient-derived explants with tumor growth inhibition ranging from 0% to 100% (P < 0.001–0.03). Interestingly, BRAFV600E and NRAS mutational status did not correlate with responsiveness to TAK-733. Pharmacodynamically, pERK was suppressed in sensitive cell lines and tumor explants, confirming TAK-733–mediated inhibition of MEK1/2, although the demonstration of similar effects in the relatively resistant cell lines and tumor explants suggests that escape pathways are contributing to melanoma survival and proliferation. These data demonstrate that TAK-733 exhibits robust tumor growth inhibition and regression against human melanoma cell lines and patient-derived xenograft models, suggesting that further clinical development in melanoma is of scientific interest. Particularly interesting is the activity in BRAF wild-type models, where current approved therapy such as vemurafenib has been reported not to be active. Mol Cancer Ther; 14(2); 317–25. ©2014 AACR.

An integrated bioinformatics analysis to dissect kinase dependency in triple negative breast cancer

BackgroundTriple-Negative Breast Cancer (TNBC) is an aggressive disease with a poor prognosis. Clinically, TNBC patients have limited treatment options besides chemotherapy. The goal of this study was to determine the kinase dependency in TNBC cell lines and to predict compounds that could inhibit these kinases using integrative bioinformatics analysis.ResultsWe integrated publicly available gene expression data, high-throughput pharmacological profiling data, and quantitative in vitro kinase binding data to determine the kinase dependency in 12 TNBC cell lines. We employed Kinase Addiction Ranker (KAR), a novel bioinformatics approach, which integrated these data sources to dissect kinase dependency in TNBC cell lines. We then used the kinase dependency predicted by KAR for each TNBC cell line to query K-Map for compounds targeting these kinases. Wevalidated our predictions using published and new experimental data.ConclusionsIn summary, we implemented an integrative bioinformatics analysis that determines kinase dependency in TNBC. Our analysis revealed candidate kinases as potential targets in TNBC for further pharmacological and biological studies.

Antitumor activity of the aurora a selective kinase inhibitor, alisertib, against preclinical models of colorectal cancer

Background The Aurora kinases are a family of serine/threonine kinases comprised of Aurora A, B, and C which execute critical steps in mitotic and meiotic progression. Alisertib (MLN8237) is an investigational Aurora A selective inhibitor that has demonstrated activity against a wide variety of tumor types in vitro and in vivo, including CRC. Results CRC cell lines demonstrated varying sensitivity to alisertib with IC50 values ranging from 0.06 to > 5 umol/L. Following exposure to alisertib we observed a decrease in pAurora A, B and C in four CRC cell lines. We also observed an increase in p53 and p21 in a sensitive p53 wildtype cell line in contrast to the p53 mutant cell line or the resistant cell lines. The addition of alisertib to standard CRC treatments demonstrated improvement over single agent arms; however, the benefit was largely less than additive, but not antagonistic. Methods Forty-seven CRC cell lines were exposed to alisertib and IC50s were calculated. Twenty-one PDX models were treated with alisertib and the Tumor Growth Inhibition Index was assessed. Additionally, 5 KRAS wildtype and mutant PDX models were treated with alisertib as single agent or in combination with cetuximab or irinotecan, respectively. Conclusion Alisertib demonstrated anti-proliferative effects against CRC cell lines and PDX models. Our data suggest that the addition of alisertib to standard therapies in colorectal cancer if pursued clinically, will require further investigation of patient selection strategies and these combinations may facilitate future clinical studies.

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Antitumor activity of a potent MEK inhibitor, TAK-733, against colorectal cancer cell lines and patient derived xenografts

Background CRC is a significant cause of cancer mortality, and new therapies are needed for patients with advanced disease. TAK-733 is a highly potent and selective investigational novel MEK allosteric site inhibitor. Materials and Methods In a preclinical study of TAK-733, a panel of CRC cell lines were exposed to varying concentrations of the agent for 72 hours followed by a sulforhodamine B assay. Twenty patient-derived colorectal cancer xenografts were then treated with TAK-733 in vivo. Tumor growth inhibition index (TGII) was assessed to evaluate the sensitivity of the CRC explants to TAK-733 while linear regression was utilized to investigate the predictive effects of genotype on the TGII of explants. Results Fifty-four CRC cell lines were exposed to TAK-733, while 42 cell lines were deemed sensitive across a broad range of mutations. Eighty-two percent of the cell lines within the sensitive subset were BRAF or KRAS/NRAS mutant, whereas 80% of the cell lines within the sensitive subset were PIK3CA WT. Twenty patient-derived human tumor CRC explants were then treated with TAK-733. In total, 15 primary human tumor explants were found to be sensitive to TAK-733 (TGII ≤ 20%), including 9 primary human tumor explants that exhibited tumor regression (TGII > 100%). Explants with a BRAF/KRAS/NRAS mutant and PIK3CA wild-type genotype demonstrated increased sensitivity to TAK-733 with a median TGII of −6%. MEK-response gene signatures also correlated with responsiveness to TAK-733 in KRAS-mutant CRC. Conclusions The MEK inhibitor TAK-733 demonstrated robust antitumor activity against CRC cell lines and patient-derived tumor explants. While the preclinical activity observed in this study was considerable, single-agent efficacy in the clinic has been limited in CRC, supporting the use of these models in an iterative manner to elucidate resistance mechanisms that can guide rational combination strategies.

Combined inhibition of MEK and Aurora A kinase in KRAS/PIK3CA double-mutant colorectal cancer models

Aurora A kinase and MEK inhibitors induce different, and potentially complementary, effects on the cell cycle of malignant cells, suggesting a rational basis for utilizing these agents in combination. In this work, the combination of an Aurora A kinase and MEK inhibitor was evaluated in pre-clinical colorectal cancer models, with a focus on identifying a subpopulation in which it might be most effective. Increased synergistic activity of the drug combination was identified in colorectal cancer cell lines with concomitant KRAS and PIK3CA mutations. Anti-proliferative effects were observed upon treatment of these double-mutant cell lines with the drug combination, and tumor growth inhibition was observed in double-mutant human tumor xenografts, though effects were variable within this subset. Additional evaluation suggests that degree of G2/M delay and p53 mutation status affect apoptotic activity induced by combination therapy with an Aurora A kinase and MEK inhibitor in KRAS and PIK3CA mutant colorectal cancer. Overall, in vitro and in vivo testing was unable to identify a subset of colorectal cancer that was consistently responsive to the combination of a MEK and Aurora A kinase inhibitor.

Abstract IA20: Challenges, opportunities, and lessons learned in the bench-to-bedside translation of xenopatient studies

Progress in oncology drug development has been hampered by a lack of preclinical models that reliably predict clinical activity of novel compounds in cancer patients. This is thought to be largely due to the inability of cell culture and cell line xenograft models to faithfully recapitulate the complex genetic and histologic heterogeneity of tumors. Patient-derived xenograft (PDX) models, the topic of this meeting, have been shown to be biologically stable when passaged in mice in terms of global gene expression patterns, mutational status, drug responsiveness, and tumor architecture, thus providing an opportunity for more efficient and effective preclinical drug development. Potential applications include initial drug activity screening, biomarker development, assessment of combination strategies, and more recently, testing of immunotherapy strategies in humanized PDX models. With escalating concerns over heterogeneity between primary and metastatic sites and within the tumor itself, PDX models may also provide a platform for studying the evolution of heterogeneity, particularly within the context of drug resistance mechanisms. Despite all of the theoretical advantages of these models and potential novel applications, academic labs, which operate on a smaller scale than industry, must continue to refine and assess the opportunities and limitations of PDX models in order to ensure the greatest bench-to-bedside translation of novel therapies. Our group has been working with PDX models over the last seven years and has developed a large and robust bank of PDX models of which the majority has undergone full genomic annotation. In this session on practical applications of PDX models, specific examples will be presented that represent distinct scenarios of preclinical development within the context of the opportunities, challenges, and lessons learned in utilizing these models in xenopatient trials. There is no doubt that PDX models represent a more clinically relevant platform for oncology drug development, but it will be important to recognize their strengths and weaknesses in order to fully exploit their potential in the drug development process. This is particularly important for academic labs where partnerships with industry are valuable and resources may be limited. Citation Format: S. Gail Eckhardt, Todd Pitts, Aik Choon Tan, Stacey Bagby, John Arcaroli, Anna Capasso, Kit Wong, Peter Klauck, Wells Messersmith, S Lindsey Davis, Christopher Lieu, Stephen Leong, Jennifer Diamond, John Tentler. Challenges, opportunities, and lessons learned in the bench-to-bedside translation of xenopatient studies. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr IA20.