Kevin Quackenbush

Common PIK3CA Mutants and a Novel 3′ UTR Mutation Are Associated with Increased Sensitivity to Saracatinib

Purpose: Dysregulation of the phosphoinositide 3-kinase (PI3K) and Src signaling pathways commonly occur in colorectal cancer. Mutations in the PIK3CA gene are associated with an increase in severity of disease and worse clinical outcomes. Elevated levels of Src have been identified in premalignant lesions and are suggested to play a central role in tumor progression. Because these pathways appear to enhance tumor growth and metastasis, molecularly targeted agents for both pathways are currently being evaluated in early-phase clinical trials. Experimental Design: We used colorectal cancer cell lines and a patient-derived explant model to investigate the efficacy of saracatinib. Mutations in the PIK3CA were evaluated to examine the association between mutations in the PIK3CA gene and sensitivity to saracatinib. Results: We have identified a subset of patients with a PIK3CA (exon 9 and 20) mutation with increased sensitivity to saracatinib. A novel 3′ untranslated region (UTR) mutation was also shown to be associated with increased sensitivity to saracatinib and have a reduced affinity for miR-520a and miR-525a. Importantly, we show that Src inhibition reduces the interaction between Src and p85, subsequently decreasing Akt-dependent signaling. Conclusion: These results indicate that a personalized approach in targeting Src in PIK3CA-mutant patients with colorectal cancers may prove effective in a subset of patients with this genetic alteration. Clin Cancer Res; 18(9); 2704–14. ©2012 AACR.

ALDH+ tumor-initiating cells exhibiting gain in NOTCH1 gene copy number have enhanced regrowth sensitivity to a γ-secretase inhibitor and irinotecan in colorectal cancer

The Notch signaling pathway has been shown to be upregulated in colorectal cancer (CRC) and important for the self‐renewal of cancer stem cells. In this study, we evaluated the efficacy of PF‐03084014, a γ‐secretase inhibitor, in combination with irinotecan to identify the effects of treatment on tumor recurrence and the tumor‐initiating population in our CRC preclinical explant model. The combination of PF‐03084014 and irinotecan had the greatest effect at reducing tumor growth on four CRC tumors when compared with treatment with PF‐03084014 or irinotecan alone. The combination significantly reduced tumor recurrence in two CRC explants (CRC001 and CRC036) after treatment was discontinued. Both of these tumors exhibited elevated baseline levels of Notch pathway activation as well as an increase in NOTCH1 gene copy number when compared with the two CRC explants (CRC026 and CRC027) where tumors reappeared quickly after termination of treatment. Isolation and injection of aldehyde dehydrogenase (ALDH+ and ALDH−) cells in an in vivo explant model demonstrated that the ALDH+ cell population were tumorigenic. Evaluation of the ALDH+ cells after 28 days of treatment showed that the combination reduced the ALDH+ population in the tumors that did not regrow. Furthermore, ALDH+ cells from CRC001 and CRC027 were injected in vivo and treated immediately for 28 days. Two months after treatment, tumors were evident in the combination treatment group for CRC027 but not for CRC036. These results indicate the combination of PF‐03084014 and irinotecan may be effective in reducing tumor recurrence in CRC patients whose tumors exhibit elevated levels of the Notch pathway.

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.

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.

Biomarker‐driven trial in metastatic pancreas cancer: feasibility in a multicenter study of saracatinib, an oral Src inhibitor, in previously treated pancreatic cancer

Src tyrosine kinases are overexpressed in pancreatic cancers, and the oral Src inhibitor saracatinib has shown antitumor activity in preclinical models of pancreas cancer. We performed a CTEP‐sponsored Phase II clinical trial of saracatinib in previously treated pancreas cancer patients, with a primary endpoint of 6‐month survival. A Simon MinMax two‐stage phase II design was used. Saracatinib (175 mg/day) was administered orally continuously in 28‐day cycles. In the unselected portion of the study, 18 patients were evaluable. Only two (11%) patients survived for at least 6 months, and three 6‐month survivors were required to move to second stage of study as originally designed. The study was amended as a biomarker‐driven trial (leucine rich repeat containing protein 19 [LRRC19] > insulin‐like growth factor‐binding protein 2 [IGFBP2] “top scoring pairs” polymerase chain reaction [PCR] assay, and PIK3CA mutant) based on preclinical data in a human pancreas tumor explant model. In the biomarker study, archival tumor tissue or fresh tumor biopsies were tested. Biomarker‐positive patients were eligible for the study. Only one patient was PIK3CA mutant in a 3′ untranslated region (UTR) portion of the gene. This patient was enrolled in the study and failed to meet the 6‐month survival endpoint. As the frequency of biomarker‐positive patients was very low (<3%), the study was closed. Although we were unable to conclude whether enriching for a subset of second/third line pancreatic cancer patients treated with a Src inhibitor based on a biomarker would improve 6‐month survival, we demonstrate that testing pancreatic tumor samples for a biomarker‐driven, multicenter study in metastatic pancreas cancer is feasible.

Aldehyde Dehydrogenase 1B1 as a Modulator of Pancreatic Adenocarcinoma.

Objectives The aim of the current study was to examine expression and the role, if any, of aldehyde dehydrogenase (ALDH)1B1 in pancreatic adenocarcinoma. Methods A tissue microarray of 61 pancreatic cancer patients were evaluated for protein expression of ALDH1B1 by immunohistochemistry. The ALDH1B1 small interfering (RNA) was used to assess the contribution of ALDH1B1 on proliferation of pancreatic cancer cells. Results In normal human pancreas, ALDH1B1 is abundantly expressed in glandular cells, but sparsely in the ducts (ALDH1B1 immunopositivity = 16.7 ± 1.7). In pancreatic ductal carcinoma, we found high ALDH1B1 expression in ductal cancerous tissues (ALDH1B1 immunopositivity = 197.2 ± 29.4). Analysis of ALDH1B1 expression in a human pancreatic adenocarcinoma tissue microarray showed the greatest expression in tumors that were more invasive. A variation in ALDH1B1 expression was also observed in 16 human pancreatic cancer cell lines. Knockdown of ALDH1B1 caused a 35% reduction in cell growth in the high ALDH1B1-expressing cell lines. Conclusions Our data show for the first time that ALDH1B1 is expressed at very high levels in human pancreatic cancer, and it contributes to proliferation in these tumor cells. These data suggest a potential modulatory role for ALDH1B1 in pancreatic cancer.

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

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

Evaluation of the efficacy of dasatinib, a Src/Abl inhibitor, in colorectal cancer cell lines and explant mouse model

Background Dysregulation of the Src pathway has been shown to be important at various stages of cancer. Dasatinib is a potent Src/Abl inhibitor and has demonstrated to have anti-proliferative and anti-invasive activity in many preclinical models. The objective of this study was to determine the anti-tumor activity of dasatinib using in vitro and in vivo preclinical colorectal (CRC) models. Methods CRC cell lines and patient-derived tumor explant (PDX) models were used to investigate the efficacy of dasatinib. We treated 50 CRC cell lines with dasatinib for 72 hours and proliferation was assayed by a sulforhodamine B (SRB) assay; an IC50 ≤ 0.08 μmol/L was considered sensitive. We treated 17 patient-derived CRC explants with dasatinib (50 mg/kg/day, administered once-daily) for 28 days to determine in vivo efficacy. Tumor growth inhibition (TGI) ≥ 50% was considered sensitive. Results We found that 8 out of 50 CRC cell lines reached an IC50 ≤ 0.08 μmol/L with dasatinib treatment. In addition, of 17 CRC explants grown in the xenograft mouse model, 2 showed sensitivity to dasatinib. The anti-tumor effects observed in this study were a result of G1 cell cycle arrest as the dasatinib sensitive CRC cell lines exhibited G1 inhibition. Moreover, those CRC cell lines that were responsive (0.08 μmol/L) to treatment demonstrated a significant baseline increase in Src and FAK gene expression. Conclusion Dasatinib demonstrated significant anti-proliferative activity in a subset of CRC cell lines in vitro, especially in those with increased Src expression at baseline, but only showed modest efficacy in CRC explants. Dasatinib is currently being studied in combination with chemotherapy in patients with advanced CRC, as its use as a single agent appears limited.