Patient-derived Tumor Explants Research Articles

Abstract 4148: Novel ERX-11 and CDK4/6 inhibitor combination therapy for treating therapy resistant breast cancer

Abstract BACKGROUND: The majority of the breast cancer is estrogen receptor alpha (ESR1) positive. While tamoxifen and letrozole therapies are effective, therapy resistance is common. Importantly, both therapy-sensitive and therapy-resistant tumors retain ESR1 signaling, via interaction with critical oncogenic coregulator proteins. Further, resistant tumors commonly acquire cyclin D1:CDK4/6 signaling via multiple mechanisms, cyclin D1 can independently activate ESR1 and thus contribute to estrogen independence of ESR+ tumor. Currently, CDK4/6 inhibitors in clinical trials for treating breast cancer, however, considering complex signaling interplay of estrogen and CDK axis, combination therapy of CDK inhibitor with other potent ESR1 targeted agents may have better utility and may prevent development of resistance to the CDK4/6 inhibitors. We recently developed a small organic molecule, ESR1 coregulator binding inhibitor ERX-11 (EtiraRx-11). The objective of this study is to test the utility of novel combination therapy of ERX-11 with CDK4/6 inhibitor palbociclib in treating therapy resistant cancer. METHODS: We have utilized multiple therapy sensitive and therapy-resistant models with various genetic back grounds. We tested efficacy using both acquired resistance and engineered models that express ESR1 mutations or oncogenes. Efficacy of combination therapy was tested using established in vitro assays including, MTT, colony formation, apoptosis, and cell cycle progression. Mechanistic studies were conducted using reporter gene assays, gene expression and signaling alterations. Xenograft studies were used to determine the in vivo efficacy of the combination therapy. RESULTS: ERX-11 effectively blocked ESR1-mediated oncogenic signaling and has potent anti-proliferative activity against therapy-sensitive and therapy-resistant breast cancer cells. Mechansistic studies showed that ERX-11 blocks the interaction between a subset of coregulators with both native and mutant forms of ESR1. ERX-11 showed potent activity in both preclinical xenograft models and patient-derived breast tumor explant models. Co-treatment of ERX-11 with palbociclib synergistically reduced cell viability and induced apoptosis of therapy sensitive and resistant breast cancer model cells. Importantly, combination therapy of ERX-11 and the palbociclib synergistically reduced the growth and induced apoptosis of tamoxifen and letrozole resistant xenograft tumors compared to either drug alone. Mechanistic studies showed combination therapy significantly altered E2F1 and ESR1 signaling pathways and promoted apoptosis. CONCLUSIONS: Collectively our studies have discovered a novel combinational treatment with ERX-11 and palbociclib for patients with therapy-sensitive and therapy-resistant breast cancers. Citation Format: Suryavathi Viswanadhapalli, Gangadhara Reddy Sareddy, Shi-Hong Ma, Tae-Kyung Lee, Rajeshwar Rao Tekmal, Jung-Mo Ahn, Ganesh Raj, Ratna K. Vadlamudi. Novel ERX-11 and CDK4/6 inhibitor combination therapy for treating therapy resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4148. doi:10.1158/1538-7445.AM2017-4148

Abstract 1246: Development of a novel KRAS-targeting agent: systematic validation using in silico, in solution, cell models, PDX and transgenic mouse models

Abstract Aberrant KRAS signaling plays an important role in the pathogenesis of malignancies including pancreatic (PDAC), colon and lung cancer. Because therapies targeting either downstream or associated factors of KRAS pathway have shown dismal results in clinics, there is a need to identify effective agents targeting active KRAS (GTP-KRAS) protein. In this study, we screened a chemical library for their efficacy to inhibit KRAS activity using in silico methods. Based on their binding energy, we selected a candidate agent (LP1) that exhibited high potential of binding to KRAS protein at a region where KRAS-activator protein “SOS1” binds to, and GDP-GTP exchange takes place. We next investigated if LP1 exhibits the efficacy of KRAS binding in biological solution using recombinant proteins and employing drug development techniques i.e., florescence (FL)-based competition, Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) assays. The FL-based study show that LP1 inhibits the binding of SOS1 protein and GTP molecules to KRAS protein. The ITC and SPR analysis showed that LP1 binds to the KRAS protein (at 10 μM). We next determined the KRAS-inhibitory potential of LP1 in activated-KRAS cells representing premalignant and malignant models of PDAC, colon and lung cancer. Dissociation study involving incubation of KRAS/SOS1 proteins showed the LP1 inhibits the binding of SOS1 to KRAS. Notably, LP1 therapy significantly decreased the (i) KRAS-GTP protein levels (ii) growth and (iii) proliferation of activated-KRAS single cell and 3D Organoid cultures. We next tested KRAS-inhibitory efficacy of LP1 in PDAC patient-derived (PD) tumor explants and found that LP1 therapy (10 days) significantly decreased growth of, and KRAS-GTP levels in PD explants. Based on the in silico, in solution, cell model and PD-explant data, we determined the efficacy of LP1 as a KRAS inhibitor in in vivo conditions. We first performed the pharmacokinetic study of LP1 in mice and observed that LP1 is physiologically available in the blood (peak of 15-20 μM) after oral and intraperitoneal administrations and detectable up to 24h post-administration. We next tested LP1 therapy in tumor xenograft models and show that LP1 therapy significantly reduces the tumorigenicity of KRAS-activated PDAC and colon cell-derived tumors implanted in athymic mice. We next tested efficacy of LP1 administration on the developmental phases of PDAC disease (from normal to high grade neoplasia/early stage carcinoma) and show that LP1 feeding for six months caused a significant inhibition of PanIN development in KPCG12D transgenic mouse model. Notably the analysis of tumors xenograft models and pancreatic tissues of KPCG12D mice receiving LP1 therapy exhibited reduced KRAS-GTP levels. Taken together, these data show that LP1 is a potent KRAS activity inhibitor and a potential candidate for clinical use against KRAS-driven cancers. Citation Format: Arsheed Ahmad Ganaie, Hifzur R. Siddique, Ishfaq Sheikh, Lei Wang, Aijaz Parray, Jayanth panyam, Peter Villalta, Joshua Liao, Yibin deng, Mohammad saleem. Development of a novel KRAS-targeting agent: systematic validation using in silico, in solution, cell models, PDX and transgenic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1246. doi:10.1158/1538-7445.AM2017-1246

Predicting tumor-immune response to checkpoint inhibitors using a novel patient-derived live tumor explant model.

e20035 Background: Immunotherapy has emerged as a powerful treatment paradigm wherein therapies primarily target immune components. For example, blockade of PD-1 and PD-L1 offers effective treatment options for patients with aggressive tumors such as head and neck squamous cell carcinoma (HNSCC) and in non-small cell lung carcinoma (NSCLC). However, clinical responses to immotherapy vary widely among patients. There is an unmet need to understand these disparities at the individual patient level. Rationally combining checkpoint inhibitors may address many of these underlying challenges. Methods: Here, we describe a patient-derived ex-vivo platform technology CANscript™, which captures the 3D profiles of native tumor microenvironment by incorporating tumor tissue, autologous immune cells, and immune-targeted agents. Utilizing late stage HNSCC and NSCLC patient tumors we interrogated the phenotypic changes in the tumor-immune contexture in response to standard-of-care agents, PD-1 and PD-L1 inhibitors. Flow cytometry and immunohistochemistry profiling of CD8, CD45, FOXP3, CXCR4, CD68, PDL1, PD1), cytokine profile (IL6, IL8, IFN-g, IL12 and others), and tumor proliferation/apoptosis were measured. Results: The data suggest that PD-1 and PD-L1 blockade induced patient-specific response, which was characterized by differential distribution and infiltration of CD8+ and CD4+ lymphocytes, distinct patterning of cytokines linked to functional dysregulation, and changes in tumor proliferation and apoptosis. Interestingly, the data demonstrated unique immune signatures associated with single agent vs. combination therapy that imply functionally distinct mechanisms of orchestration of response. Conclusions: Our data highlights the translational underpinnings of of CANScript™ as an ex vivo platform for predicting patient driven therapeutic response of immune checkpoint inhibitors where distinct tumor-immune networks influence clinical response to therapy. Information obtained from this study can re-shape our understanding of patient selection and rational combinations for novel immune checkpoint inhibitors.

Abstract 1019: Investigation of cabozantinib, a MET and VEGFR2 inhibitor, on tumor metabolism and efficacy in a colorectal cancer patient-derived tumor explant model

Abstract Background: Anti-angiogenic therapy is commonly used for the treatment of metastatic colorectal cancer (mCRC). Although patients derive some clinical benefit, treatment resistance inevitably occurs. Upregulation of MET in response to anti-VEGF therapy may play an essential role in treatment resistance. Based on this premise, we investigated cabozantinib, an inhibitor of kinases including MET and VEGFR2, in mCRC patient-derived tumor explant (PDTX) models. Based on initial observations of anti-tumor activity, we hypothesized that cabozantinib may alter tumor metabolism concurrent with its antitumor effects, and compared its efficacy with regorafenib in our models. Material and Methods: Ten CRC PDTX models were treated with cabozantinib (30 mg/kg daily) or regorafenib (10 mg/kg daily) and treatment responses were determined after 28 days. The tumor growth inhibition index (TGII) was calculated to compare treatment effects on tumor growth between cabozantinib and regorafenib. RNA Seq was used to assess gene expression and pathways modulated by cabozantinib treatment. Proteins involved in metabolism and autophagy were evaluated by immunoblotting at day 3, 7 and 28. The effects of cabozantinib on tumor glucose metabolism were investigated by 18FDG-PET at baseline, 7 and 28 days following treatment. Lastly, combination effects of cabozantinib and an ULK1 inhibitor, an autophagy inhibitor, were evaluated on the HCT116 cell line by a clonogenic assay. Results: Cabozantinib (average TGII: 3.202) demonstrated antitumor effects among all 10 CRC explants that compared favorably to regorafenib (average TGII: 48.48). In addition, cabozantinib significantly reduced glucose uptake measured by 18FDG-PET at days 7 and 28. A comprehensive pathway analysis using RNA Seq post-cabozantinib treatment revealed a significant reduction in pyruvate metabolism and the TCA cycle in the most sensitive tumors. Protein analyses showed downregulation of hexokinase 1 and pyruvate dehydrogenase and a marked increase in many components of oxidative phosphorylation and autophagy (LC3 and Beclin) at day 7 and 28 following cabozantinib treatment. The combination of an ULK1 inhibitor and cabozantinib enhanced the activity of cabozantinib in the HCT116 CRC cell line. Conclusions: Cabozantinib showed significant antitumor activity compared to regorafenib in our CRC PDTX models. Alterations in glucose metabolism and autophagy were identified in cabozantinib sensitive tumors, suggesting that a shift in cellular metabolism facilitates the survival of tumor cells following treatment. The combination effect of cabozantinib and an ULK1 inhibitor supports the hypothesis that induction of autophagy may be a mechanism of cabozantinib resistance. These findings support further evaluation of cabozantinib in patients with mCRC. Acknowledgements: Exelixis for providing cabozantinib. Citation Format: Aaron J. Scott, Rachel Yahn, Stacey Bagby, Kendra Huber, Natalie Serkova, Wells Messersmith, John Arcaroli. Investigation of cabozantinib, a MET and VEGFR2 inhibitor, on tumor metabolism and efficacy in a colorectal cancer patient-derived tumor explant model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1019.

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

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. 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. 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. 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.

Neuregulin 1 expression is a predictive biomarker for response to AV-203, an ERBB3 inhibitory antibody, in human tumor models.

ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/κ ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic. We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models. A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models. NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203.

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, BRAF(V600E) 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.

The c-Met Inhibitor MSC2156119J Effectively Inhibits Tumor Growth in Liver Cancer Models.

The mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase with hepatocyte growth factor (HGF) as its only high-affinity ligand. Aberrant activation of c-Met is associated with many human malignancies, including hepatocellular carcinoma (HCC). We investigated the in vivo antitumor and antimetastatic efficacy of the c-Met inhibitor MSC2156119J (EMD 1214063) in patient-derived tumor explants. BALB/c nude mice were inoculated with MHCC97H cells or with tumor fragments of 10 patient-derived primary liver cancer explants selected according to c-Met/HGF expression levels. MSC2156119J (10, 30, and 100 mg/kg) and sorafenib (50 mg/kg) were administered orally as single-agent treatment or in combination, with vehicle as control. Tumor response, metastases formation, and alpha fetoprotein (AFP) levels were measured. MSC2156119J inhibited tumor growth and induced complete regression in mice bearing subcutaneous and orthotopic MHCC97H tumors. AFP levels were undetectable after 5 weeks of MSC2156119J treatment, and the number of metastatic lung foci was reduced. Primary liver explant models with strong c-Met/HGF activation showed increased responsiveness to MSC2156119J, with MSC2156119J showing similar or superior activity to sorafenib. Tumors characterized by low c-Met expression were less sensitive to MSC2156119J. MSC2156119J was better tolerated than sorafenib, and combination therapy did not improve efficacy. These findings indicate that selective c-Met/HGF inhibition with MSC2156119J is associated with marked regression of c-Met high-expressing tumors, supporting its clinical development as an antitumor treatment for HCC patients with active c-Met signaling.

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

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. 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. 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. 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.

Abstract LB-302: Potent antitumor activity of XL184 (cabozantinib), a c-MET and VEGFR2 inhibitor, in colorectal cancer patient-derived tumor explant models.

Abstract Background: The c-MET signaling pathway plays an essential role in tumorigenesis and is recognized as an important mediator of angiogenesis by inducing the expression of VEGF. In colorectal cancer (CRC), c-MET overexpression is a marker of local migration and invasion and may have prognostic significance. In addition, anti-VEGF therapy has been shown to promote tumor cell metastasis by a c-MET dependent mechanism. The objective of this study was to evaluate the activity of XL184 (cabozantinib), a potent inhibitor of both c-MET and VEGFR2, in CRC patient-derived tumor explant (PDTX) models. Experimental Design: We used CRC PDTX models to investigate the efficacy of XL184 in spheroid cultures and in vivo in athymic nude mice. Spheroids with retained cell-cell contact were formed by culturing incompletely digested PDTXs in suspension. Spheroids were incubated for 3 days with 1-10 μM XL184. Spheroid area was measured by microscopy. In the mouse models, a total of 10 CRC PDTXs (3 KRAS wild-type and 7 KRAS mutant) were treated with XL184 (30 mg/kg daily, oral gavage) for 28 days. Mice were monitored daily for signs of toxicity and tumor size was evaluated twice per week by caliper measurements. Results: Spheroids from PDTXs were entirely composed of tumor cells but, unlike cell lines, spheroids recruited stroma when implanted in mice. Although 10 μM XL184 did not inhibit spheroid formation, spheroid area significantly decreased following incubation for 3 days with 10 μM or 5 μM XL184 (p= 0.0012 and p= 0.0056, respectively). In our CRC PDTX in vivo models, XL184 was well tolerated and resulted in a significant decrease in tumor growth in CRC explants from all 10 patients, independent of KRAS status. Strikingly, two of the CRC explants exhibited tumor regression after 28 days of treatment with XL184. Conclusion: XL184 showed potent antitumor activity in PDTX spheroid and mouse models. PDTX models may be particularly valuable for preclinical testing of inhibitors of stroma-modulated targets such as XL184, where activity is not reflected in cell lines. These promising data have contributed to the rationale and design of a clinical trial of XL184 in patients with advanced CRC. Our upcoming studies will focus on identifying predictive biomarkers of sensitivity or resistance to XL184. Citation Format: Chloe E. Atreya, Eun-Kee Song, Wells Messersmith, Alicia Purkey, Stacey Bagby, Kevin Quackenbush, Robin K. Kelley, Eunice Kwak, David Ryan, Alan Venook, John J. Arcaroli. Potent antitumor activity of XL184 (cabozantinib), a c-MET and VEGFR2 inhibitor, in colorectal cancer patient-derived tumor explant models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-302. doi:10.1158/1538-7445.AM2013-LB-302

414 The Anti-tumor Activity of the ERBB3 Inhibitory Antibody AV-203 in Patient Derived Tumor Explant Models

overexpression or shRNA. Further biochemical analysis demonstrated that unlike doxorubicin, which causes DNA damage mediated phosphorylation and stabilization of p53 and phosphorylation of H2AX, CLTX1 is a potent HdmX-specific inhibitor capable of rapidly stabilizing p53 in a DNA damage independent manner. An in vitro ELISA using recombinant p53, HdmX or Hdm2 demonstrated that CLTX1 disrupted HdmX-p53, but not Hdm2– p53 interactions. Moreover, while CLTX1 exhibits some anti-cancer activity alone when used to treat wild-type p53 expressing cell lines, particularly those that do not overexpress Hdm2, we have observed that the combined inhibition of HdmX (with CLTX1) and Hdm2 (with Nutlin 3) synergize to induce apoptosis. Overall, CLTX1 is a promising new anti-cancer agent that is capable of targeting cancer cells through the induction of p53 in a non-genotoxic manner.

Activity of MK1775, a selective Wee1 inhibitor, alone or in combination with gemcitabine, in sarcomas.

10084 Background: Sarcomas consist of more than 50 subtypes of mesenchymal tumors. Doxorubicin alone or in combination has been the primary therapy for treatment of sarcomas; however, the response rates are suboptimal in many of the more common adult subtypes of soft tissue sarcoma. Accordingly, new agents are needed for the treatment of this heterogeneous group of diseases. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Methods: MK1775 treatment of multiple sarcoma preclinical models at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death. In our current study we have investigated the therapeutic efficacy of MK1775 in sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo in a xenograft model of osteosarcoma both alone and in combination with gemcitabine. Results: In patient-derived bone and soft tissue sarcoma samples ex vivo treatments show MK1775 in combination with gemcitabine causes significant apoptotic cell death suggesting that this treatment may represent a novel approach in the treatment of sarcomas. The cytotoxic effect of Wee1 inhibition on sarcoma cells appears to be independent of p53 mutational status. Furthermore, in a patient-derived osteosarcoma xenograft mouse model we show the therapeutic efficacy of MK1775 in vivo by utilizing magnetic resonance imaging (MRI) and diffusion MRI methods. Our data shows MK1775 in combination with gemcitabine dramatically slows tumor growth, increases apoptotic cell death and increases CDC2 activity. Cell viability, a clinically established prognostic indicator of survival, was lowest with the combination and very low in animals treated with MK1775 alone. This was mainly due to increased mineralization of the tumors. Caspase-3 was increased in MK1775 treated animals by immunohistochemistry as well. Conclusions: These results together with the promising safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent alone or in combination with gemcitabine in the treatment of both adult as well as pediatric sarcoma patients.

Abstract 3047: MK1775, a selective Wee1 inhibitor shows antitumor activity against sarcoma cells

Abstract Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10-20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible. Accordingly, new agents are needed for the treatment of this heterogeneous group of diseases. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by a selective small molecule inhibitor MK1775 has been reported to to enhance the cytotoxic effect of DNA damaging agents. However, its role in the treatment of mesenchymal tumors has not been explored. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines and patient-derived tumor explants ex vivo both alone and in combination with other chemotherapeutic drugs frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of the DNA damaging agents, gemcitabine and doxorubicin. In MK1775 treated cells CDC2 activity was enhanced and G2/M ceheckpoint was impaired as assessed by increased expression of phosphorylated histone H3, a marker of mitotic entry. The cytotoxic effect of Wee1 inhibition on sarcoma cells appears to be independent of p53 status as all sarcoma cell lines with different p53 mutation were highly sensitive to treatment. Additionally, in patient-derived bone and soft tissue sarcoma samples we showed that MK1775 in combination with gemcitabine causes significant apoptotic cell death suggesting that this treatment may represent a novel approach in the treatment of sarcomas. In patient-derived xenograft mouse models of sarcoma we are currently analyzing the therapeutic efficacy of MK1775 in vivo by utilizing magnetic resonance imaging (MRI) and diffusion MRI methods. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3047. doi:1538-7445.AM2012-3047

MK1775, a Selective Wee1 Inhibitor, Shows Single-Agent Antitumor Activity against Sarcoma Cells

Wee1 is a critical component of the G(2)-M cell-cycle checkpoint control and mediates cell-cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by a selective small molecule inhibitor MK1775 can abrogate G(2)-M checkpoint, resulting in premature mitotic entry and cell death. MK1775 has recently been tested in preclinical and clinical studies of human carcinoma to enhance the cytotoxic effect of DNA-damaging agents. However, its role in mesenchymal tumors, especially as a single agent, has not been explored. Here, we studied the cytotoxic effect of MK1775 in various sarcoma cell lines and patient-derived tumor explants ex vivo. Our data show that MK1775 treatment at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. In MK1775-treated cells, CDC2 activity was enhanced, as determined by decreased inhibitory phosphorylation of tyrosine-15 residue and increased expression of phosphorylated histone H3, a marker of mitotic entry. The cytotoxic effect of Wee1 inhibition on sarcoma cells seems to be independent of p53 status as all sarcoma cell lines with different p53 mutation were highly sensitive to MK1775 treatment. Finally, in patient-derived sarcoma samples, we showed that MK1775 as a single agent causes significant apoptotic cell death, suggesting that Wee1 inhibition may represent a novel approach in the treatment of sarcomas.

Abstract B178: Molecular determinants of response to the investigational small molecule inhibitor of Nedd8-activating enzyme (NAE) MLN4924 in melanoma cell lines and patient-derived tumor explant models.

Abstract Background: The ubiquitin proteasome system (UPS) regulates the ubiquitination, and thus degradation and turnover, of many proteins vital to proper functioning of the cell. Recent studies have shown that the UPS is more complex than previously thought, and its role in cell cycle regulation, signal transduction, DNA replication, and thus the evolution of the malignant phenotype of many cancers, is now well appreciated. The proteasome inhibitor bortezomib has provided validation of interfering with the UPS for clinical benefit in cancer patients and has provided the impetus for the development of new drugs that target the UPS more specifically. MLN4924 (Millennium Pharmaceuticals, Inc.) is an investigational first-in-class small molecule inhibitor of the key enzyme in the Nedd8 conjugation pathway, NAE. Inhibition of NAE results in in S-phase arrest, DNA re-replication and apoptosis. In this study, we used both in vitro and in vivo models of melanoma to assess the preclinical efficacy of MLN4924 in conjunction with gene array and gene set enrichment analysis (GSEA) to identify differentially expressed genes and pathways as potential molecular determinants of response to this novel compound. Methods &amp; Results: A panel of thirty-six melanoma cell lines was exposed to MLN4924 and IC50s were determined using the CellTiter Glo method. The cell lines were then classified as sensitive (S) or resistant (R) based on an IC50&amp;lt;0.03μM or &amp;gt;2.59μM, respectively. The most extreme S and R lines were also grown as subcutaneous flank xenograft tumors in nude mice to confirm in vitro responses to MLN4924. Additionally, twelve patient-derived melanoma tumors were implanted in mice and both the cell line and explant xenograft mice were treated s.c. with MLN4924 at 90mg/kg. The compound was well-tolerated and no significant weight loss or lethality was observed. Tumors were measured three times weekly by caliper and tumor growth inhibition (TGI) was analyzed. Of the explant models, five responded favorably to MLN4924, with TGI&amp;gt;50%, with one model exhibiting tumor regression. Pharmacodynamic studies were also performed on melanoma explant tumors from mice exposed to a single dose of MLN4924 which were then harvested over a seven point time course ranging from 30 min to 24 hr post-drug. Immunoblotting of these tumors revealed rapid increases in the levels of caspase 3 as well as phospho-Chk-1 at 1 hr and decreases in neddylated cullin levels beginning at 8 hr, consistent with the known mode of action of MLN4924. Affymetrix 1.0 ST gene array analysis was performed on 6 S and 6 R cell lines in order to identify differentially expressed genes that could be used as predictive biomarkers. GSEA and KEGG pathway analysis of this data identified several DNA repair pathways as being enriched in the S lines while ABC drug transporter pathways were enriched in the R lines. Conclusion: MLN4924 demonstrated strong antitumor activity in a subset of melanoma cell lines and patient-derived explants. Based on these preclinical results, MLN4924 demonstrates promising data supportive of further patient-selective clinical development in melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B178.

Comparison of 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) and 17-allylamino-17-demethoxygeldanamycin (17AAG) in vitro: effects on Hsp90 and client proteins in melanoma models

The heat shock protein Hsp90 is a potential target for drug discovery of novel anticancer agents. By affecting this protein, several cell signaling pathways may be simultaneously modulated. The geldanamycin analog 17AAG has been shown to inhibit Hsp90 and associated proteins. Its clinical use, however, is hampered by poor solubility and thus, difficulties in formulation. Therefore, a water-soluble derivative was desirable and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) is such a derivative. Studies were carried out in order to evaluate the activity and molecular mechanism(s) of 17DMAG in comparison with those of 17-allylamino-demethoxygeldanamycin (17AAG). 17DMAG was found to be more potent than 17AAG in a panel of 64 different patient-derived tumor explants studied in vitro in the clonogenic assay. The tumor types that responded best included mammary cancers (six of eight), head and neck cancers (two of two), sarcomas (four of four), pancreas carcinoma (two of three), colon tumors (four of eight for 17AAG and six of eight for 17DMAG), and melanoma (two of seven). Bioinformatic comparisons suggested that, while 17AAG and 17DMAG are likely to share the same mode(s) of action, there was very little similarity with standard anticancer agents. Using three permanent human melanoma cell lines with differing sensitivities to 17AAG and 17DMAG (MEXF 276L, MEXF 462NL and MEXF 514L), we found that Hsp90 protein was reduced following treatment at a concentration associated with total growth inhibition. The latter occurred in MEXF 276L cells only, which are most sensitive to both compounds. The depletion of Hsp90 was more pronounced in cells exposed to 17DMAG than in those treated with 17AAG. The reduction in Hsp90 was associated with the expression of erbB2 and erbB3 in MEXF 276L, while erbB2 and erbB3 were absent in the more resistant MEXF 462NL and MEXF 514L cells. Levels of known Hsp90 client proteins such as phosphorylated AKT followed by AKT, cyclin D1 preceding cdk4, and craf-1 declined as a result of drug treatment in all three melanoma cell lines. However, the duration of drug exposure needed to achieve these effects was variable. All cell lines showed increased expression of Hsp70 and activated cleavage of PARP. No change in PI3K expression was observed and all melanoma cells were found to harbor the activating V599E BRAF kinase mutation. The results of our in vitro studies are consistent with both 17AAG and 17DMAG acting via the same molecular mechanism, i.e. by modulating Hsp90 function. Since 17DMAG can be formulated in physiological aqueous solutions, the data reported here strongly support the development of 17DMAG as a more pharmaceutically practicable congener of 17AAG.