Stacey Bagby

Potent antitumor activity of cabozantinib, a c-MET and VEGFR2 inhibitor, in a colorectal cancer patient-derived tumor explant model.

Antiangiogenic therapy is commonly used for the treatment of colorectal cancer (CRC). Although patients derive some clinical benefit, treatment resistance inevitably occurs. The MET signaling pathway has been proposed to be a major contributor of resistance to antiangiogenic therapy. MET is upregulated in response to vascular endothelial growth factor pathway inhibition and plays an essential role in tumorigenesis and progression of tumors. In this study, we set out to determine the efficacy of cabozantinib in a preclinical CRC patient‐derived tumor xenograft model. We demonstrate potent inhibitory effects on tumor growth in 80% of tumors treated. The greatest antitumor effects were observed in tumors that possess a mutation in the PIK3CA gene. The underlying antitumor mechanisms of cabozantinib consisted of inhibition of angiogenesis and Akt activation and significantly decreased expression of genes involved in the PI3K pathway. These findings support further evaluation of cabozantinib in patients with CRC. PIK3CA mutation as a predictive biomarker of sensitivity is intriguing and warrants further elucidation. A clinical trial of cabozantinib in refractory metastatic CRC is being activated.

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.

Tumours with elevated levels of the Notch and Wnt pathways exhibit efficacy to PF-03084014, a γ-secretase inhibitor, in a preclinical colorectal explant model.

Background:Dysregulation of the Notch pathway has been identified to play an important role in the development and progression of colorectal cancer (CRC). In this study, we used a patient-derived CRC explant model to investigate the efficacy of the clinical γ-secretase inhibitor (GSI) PF-03084014.Methods:A total of 16 CRC explants were treated with PF-03084014. Knockdown of RBPjκ gene was used to determine the specificity of PF-03084014. Evaluation of the Notch and Wnt pathways in CRC explant tumours was performed by gene array and immunoblotting.Results:We identified a subset of CRC tumours that exhibited elevations of the Notch and Wnt pathways sensitive to PF-03084014. Treatment with the GSI resulted in a significant reduction in cleaved Notch, Axin2 (Wnt-dependent gene) and active β-catenin. In addition, knockdown of the RBPjκ gene showed that PF-03084014 has specificity for the Notch pathway in an HCT116 cell line xenograft model. Finally, an increase in apoptosis was observed in CRC001- and CRC021-sensitive tumours.Conclusion:This study provides evidence that inhibition of γ-secretase may be beneficial in a subset of patients with elevated levels of the Wnt and Notch pathways.

A NOTCH1 gene copy number gain is a prognostic indicator of worse survival and a predictive biomarker to a Notch1 targeting antibody in colorectal cancer

Dysregulation of the Notch1 receptor has been shown to facilitate the development and progression of colorectal cancer (CRC) and has been identified as an independent predictor of disease progression and worse survival. Although mutations in the NOTCH1 receptor have not been described in CRC, we have previously discovered a NOTCH1 gene copy number gain in a portion of CRC tumor samples. Here, we demonstrated that a NOTCH1 gene copy number gain is significantly associated with worse survival and a high percentage of gene duplication in a cohort of patients with advanced CRC. In our CRC patient‐derived tumor xenograft (PDTX) model, tumors harboring a NOTCH1 gain exhibited significant elevation of the Notch1 receptor, JAG1 ligand and cleaved Notch1 activity. In addition, a significant association was identified between a gain in NOTCH1 gene copy number and sensitivity to a Notch1‐targeting antibody. These findings suggest that patients with metastatic CRC that harbor a gain in NOTCH1 gene copy number have worse survival and that targeting this patient population with a Notch1 antibody may yield improved outcomes.

The novel tankyrase inhibitor (AZ1366) enhances irinotecan activity in tumors that exhibit elevated tankyrase and irinotecan resistance

Background Dysregulation of the canonical Wnt signaling pathway has been implicated in colorectal cancer (CRC) development as well as incipient stages of malignant transformation. In this study, we investigated the antitumor effects of AZ1366 (a novel tankyrase inhibitor) as a single agent and in combination with irinotecan in our patient derived CRC explant xenograft models. Results Six out of 18 CRC explants displayed a significant growth reduction to AZ1366. There was one CRC explant (CRC040) that reached the threshold of sensitivity (TGII ≤ 20%) in this study. In addition, the combination of AZ1366 + irinotecan demonstrated efficacy in 4 out of 18 CRC explants. Treatment effects on the WNT pathway revealed that tankyrase inhibition was ineffective at reducing WNT dependent signaling. However, the anti-tumor effects observed in this study were likely a result of alternative tankyrase effects whereby tankyrase inhibition reduced NuMA levels. Materials and Methods Eighteen CRC explants were treated with AZ1366 single agent or in combination for 28 days and treatment responses were assessed. Pharmacokinetic (AZ1366 drug concentrations) and pharmacodynamic effects (Axin2 levels) were investigated over 48 hours. Immunohistochemistry of nuclear β-catenin levels as well as western blot was employed to examine the treatment effects on the WNT pathway as well as NuMA. Conclusions Combination AZ1366 and irinotecan achieved greater anti-tumor effects compared to monotherapy. Activity was limited to CRC explants that displayed irinotecan resistance and increased protein levels of tankyrase and NuMA.

Kinase Gene Fusions in Defined Subsets of Melanoma.

Genomic rearrangements resulting in activating kinase fusions have been increasingly described in a number of cancers including malignant melanoma, but their frequency in specific melanoma subtypes has not been reported. We used break‐apart fluorescence in situ hybridization (FISH) to identify genomic rearrangements in tissues from 59 patients with various types of malignant melanoma including acral lentiginous, mucosal, superficial spreading, and nodular. We identified four genomic rearrangements involving the genes BRAF, RET, and ROS1. Of these, three were confirmed by Immunohistochemistry (IHC) or sequencing and one was found to be an ARMC10‐BRAF fusion that has not been previously reported in melanoma. These fusions occurred in different subtypes of melanoma but all in tumors lacking known driver mutations. Our data suggest gene fusions are more common than previously thought and should be further explored particularly in melanomas lacking known driver mutations.

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.

ALK inhibitor response in melanomas expressing EML4-ALK fusions and alternate ALK isoforms

Oncogenic ALK fusions occur in several types of cancer and can be effectively treated with ALK inhibitors; however, ALK fusions and treatment response have not been characterized in malignant melanomas. Recently, a novel isoform of ALK (ALKATI) was reported in 11% of melanomas but the response of melanomas expressing ALKATI to ALK inhibition has not been well characterized. We analyzed 45 melanoma patient-derived xenograft models for ALK mRNA and protein expression. ALK expression was identified in 11 of 45 (24.4%) melanomas. Ten melanomas express wild-type (wt) ALK and/or ALKATI and one mucosal melanoma expresses multiple novel EML4-ALK fusion variants. Melanoma cells expressing different ALK variants were tested for response to ALK inhibitors. Whereas the melanoma expressing EML4-ALK were sensitive to ALK inhibitors in vitro and in vivo, the melanomas expressing wt ALK or ALKATI were not sensitive to ALK inhibitors. In addition, a patient with mucosal melanoma expressing ALKATI was treated with an ALK/ROS1/TRK inhibitor (entrectinib) on a phase I trial but did not respond. Our results demonstrate ALK fusions occur in malignant melanomas and respond to targeted therapy, whereas melanomas expressing ALKATI do not respond to ALK inhibitors. Targeting ALK fusions is an effective therapeutic option for a subset of melanoma patients, but additional clinical studies are needed to determine the efficacy of targeted therapies in melanomas expressing wt ALK or ALKATI. Mol Cancer Ther; 17(1); 222–31. ©2017 AACR.

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.