Pharmacological disruption of the Notch transcription factor complex.

Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule γ-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the downstream mediators of Notch signaling, the Notch transcription activation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lymphoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrangements in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that interferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch-driven cancers.

Precision Oncology and Systemic Targeted Therapy in Pseudomyxoma Peritonei.

PURPOSE: Pseudomyxoma peritonei (PMP) is a rare and poorly understood malignant condition characterized by the accumulation of intra-abdominal mucin produced from peritoneal metastases. Currently, cytoreductive surgery remains the mainstay of treatment but disease recurrence and death after relapse frequently occur in patients with PMP. New therapeutic strategies are therefore urgently needed for these patients. EXPERIMENTAL DESIGN: A total of 120 PMP samples from 50 patients were processed to generate a collection of 50 patient-derived organoid (PDO) and xenograft (PDX) models. Whole exome sequencing, immunohistochemistry analyses, and in vitro and in vivo drug efficacy studies were performed. RESULTS: In this study, we have generated a collection of PMP preclinical models and identified druggable targets, including BRAFV600E, KRASG12C, and KRASG12D, that could also be detected in intra-abdominal mucin biopsies of patients with PMP using droplet digital PCR. Preclinical models preserved the histopathological markers from the original patient sample. The BRAFV600E inhibitor encorafenib reduced cell viability of BRAFV600E PMP-PDO models. Proof-of-concept in vivo experiments showed that a systemic treatment with encorafenib significantly reduced tumor growth and prolonged survival in subcutaneous and orthotopic BRAFV600E-PMP-PDX mouse models. CONCLUSIONS: Our study demonstrates for the first time that systemic targeted therapies can effectively control PMP tumors. BRAF signaling pathway inhibition represents a new therapeutic opportunity for patients with BRAFV600E PMP who have a poor prognosis. Importantly, our present data and collection of preclinical models pave the way for evaluating the efficacy of other systemic targeted therapies toward extending the promise of precision oncology to patients with PMP.

Identifying Cell Differentiation in Colorectal Cancer.

The intestinal epithelium is a rapid self-renewing tissue. Stem cells at the bottom of the crypts first give rise to a proliferative progeny that finally differentiates to a variety of cell types. These terminally differentiated intestinal cells are mostly present in the villi of the intestinal wall and serve as functional units to sustain the main purpose of the organ: food absorption. But for a balance homeostasis, the intestine is composed not only by absorptive enterocytes but also by other cell types such as goblet cells that secrete mucus to lubricate the intestinal lumen, Paneth cells that secrete antimicrobial peptides to control microbiome, and others. Many relevant conditions affecting the intestine including chronic inflammation, Crohn's disease, or cancer can alter the composition of these different functional cell types. As a consequence, they can lose their specialized activity as functional units and further contribute to disease progression and malignancy. Measuring the amount of these different cell populations in the intestine is essential to understand the bases of these diseases and their specific contribution to their malignancy. Interestingly, patient-derived xenograft (PDX) models faithfully recapitulate patients' tumors including the proportion of the different cell lineages present in the original tumor. Here we expose some protocols for evaluating the differentiation of intestinal cells in colorectal tumors.

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (µCT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

DPPA3-HIF1α axis controls colorectal cancer chemoresistance by imposing a slow cell-cycle phenotype.

Tumor relapse is linked to rapid chemoresistance and represents a bottleneck for cancer therapy success. Engagement of a reduced proliferation state is a non-mutational mechanism exploited by cancer cells to bypass therapy-induced cell death. Through combining functional pulse-chase experiments in engineered cells and transcriptomic analyses, we identify DPPA3 as a master regulator of slow-cycling and chemoresistant phenotype in colorectal cancer (CRC). We find a vicious DPPA3-HIF1α feedback loop that downregulates FOXM1 expression via DNA methylation, thereby delaying cell-cycle progression. Moreover, downregulation of HIF1α partially restores a chemosensitive proliferative phenotype in DPPA3-overexpressing cancer cells. In cohorts of CRC patient samples, DPPA3 overexpression acts as a predictive biomarker of chemotherapeutic resistance that subsequently requires reduction in its expression to allow metastatic outgrowth. Our work demonstrates that slow-cycling cancer cells exploit a DPPA3/HIF1α axis to support tumor persistence under therapeutic stress and provides insights on the molecular regulation of disease progression.

The target antigen determines the mechanism of acquired resistance to T cell-based therapies.

Despite the revolution of immunotherapy in cancer treatment, patients eventually progress due to the emergence of resistance. In this scenario, the selection of the tumor antigen can be decisive in the success of the clinical response. T cell bispecific antibodies (TCBs) are engineered molecules that include binding sites to the T cell receptor and to a tumor antigen. Using gastric CEA+/HER2+ MKN45 cells and TCBs directed against CEA or HER2, we show that the mechanism of resistance to a TCB is dependent on the tumor antigen. Acquired resistant models to a high-affinity-CEA-targeted TCB exhibit a reduction of CEA levels due to transcriptional silencing, which is reversible upon 5-AZA treatment. In contrast, a HER2-TCB resistant model maintains HER2 levels and exhibit a disruption of the interferon-gamma signaling. These results will help in the design of combinatorial strategies to increase the efficacy of cancer immunotherapies and to anticipate and overcome resistances.

Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR × LGR5 bispecific antibody with efficacy in epithelial tumors.

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.

Advanced Colorectal Cancer Orthotopic Patient-Derived Xenograft Models for Cancer and Stem Cell Research.

In the recent years has being a great expansion of new preclinical models of colorectal cancer (CRC) based on patient-derived cells, from ex vivo 2D cell lines, toward 3D tumoroids or animal xenografts. These new technologies have been key to overcome historical limitations in CRC research such as precision medicine, pharmacogenomic screenings, or investigating mechanism of drug resistance. Here we describe a method to generate metastatic CRC in mice with patient-derived cells and the evaluation of drug response with computerized tomography. CRC at this advanced stage is the most frequent situation in patients enrolled in therapies with novel drugs that in some cases are designed to target metastatic cells. Therefore, these orthotopic models could be considered the best to recapitulate advance CRC and are therefore becoming instrumental to investigate the biology behind drug-response in metastatic disease.

TET2 controls chemoresistant slow-cycling cancer cell survival and tumor recurrence.

Dormant or slow-cycling tumor cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. We have adapted the pulse-chase expression of H2BeGFP for labeling and isolating slow-cycling cancer cells (SCCCs). SCCCs showed cancer initiation potential and enhanced chemoresistance. Cells at this slow-cycling status presented a distinctive nongenetic and cell-autonomous gene expression profile shared across different tumor types. We identified TET2 epigenetic enzyme as a key factor controlling SCCC numbers, survival, and tumor recurrence. 5-Hydroxymethylcytosine (5hmC), generated by TET2 enzymatic activity, labeled the SCCC genome in carcinomas and was a predictive biomarker of relapse and survival in cancer patients. We have shown the enhanced chemoresistance of SCCCs and revealed 5hmC as a biomarker for their clinical identification and TET2 as a potential drug target for SCCC elimination that could extend patients' survival.

Tankyrase Inhibition Blocks Wnt/ß-Catenin Pathway and Reverts Resistance to PI3K and AKT Inhibitors in the Treatment of Colorectal Cancer.

PURPOSE: Oncogenic mutations in the KRAS/PI3K/AKT pathway are one of the most frequent alterations in cancer. Although PI3K or AKT inhibitors show promising results in clinical trials, drug resistance frequently emerges. We previously revealed Wnt/ß-catenin signaling hyperactivation as responsible for such resistance in colorectal cancer. Here we investigate Wnt-mediated resistance in patients treated with PI3K or AKT inhibitors in clinical trials and evaluate the efficacy of a new Wnt/tankyrase inhibitor, NVP-TNKS656, to overcome such resistance. EXPERIMENTAL DESIGN: Colorectal cancer patient-derived sphere cultures and mouse tumor xenografts were treated with NVP-TNKS656, in combination with PI3K or AKT inhibitors.We analyzed progression-free survival of patients treated with different PI3K/AKT/mTOR inhibitors in correlation with Wnt/ß-catenin pathway activation, oncogenic mutations, clinicopathological traits, and gene expression patterns in 40 colorectal cancer baseline tumors. RESULTS: Combination with NVP-TNKS656 promoted apoptosis in PI3K or AKT inhibitor-resistant cells with high nuclear ß-catenin content. High FOXO3A activity conferred sensitivity to NVP-TNKS656 treatment. Thirteen of 40 patients presented high nuclear ß-catenin content and progressed earlier upon PI3K/AKT/mTOR inhibition. Nuclear ß-catenin levels predicted drug response, whereas clinicopathologic traits, gene expression profiles, or frequent mutations (KRAS, TP53, or PIK3CA) did not. CONCLUSIONS: High nuclear ß-catenin content independently predicts resistance to PI3K and AKT inhibitors. Combined treatment with a Wnt/tankyrase inhibitor reduces nuclear ß-catenin, reverts such resistance, and represses tumor growth. FOXO3A content and activity predicts response to Wnt/ß-catenin inhibition and together with ß-catenin may be predictive biomarkers of drug response providing a rationale to stratify colorectal cancer patients to be treated with PI3K/AKT/mTOR and Wnt/ß-catenin inhibitors.

MEK plus PI3K/mTORC1/2 Therapeutic Efficacy Is Impacted by TP53 Mutation in Preclinical Models of Colorectal Cancer.

PURPOSE: PI3K pathway activation occurs in concomitance with RAS/BRAF mutations in colorectal cancer, limiting the sensitivity to targeted therapies. Several clinical studies are being conducted to test the tolerability and clinical activity of dual MEK and PI3K pathway blockade in solid tumors. EXPERIMENTAL DESIGN: In the present study, we explored the efficacy of dual pathway blockade in colorectal cancer preclinical models harboring concomitant activation of the ERK and PI3K pathways. Moreover, we investigated if TP53 mutation affects the response to this therapy. RESULTS: Dual MEK and mTORC1/2 blockade resulted in synergistic antiproliferative effects in cell lines bearing alterations in KRAS/BRAF and PIK3CA/PTEN. Although the on-treatment cell-cycle effects were not affected by the TP53 status, a marked proapoptotic response to therapy was observed exclusively in wild-type TP53 colorectal cancer models. We further interrogated two independent panels of KRAS/BRAF- and PIK3CA/PTEN-altered cell line- and patient-derived tumor xenografts for the antitumor response toward this combination of agents. A combination response that resulted in substantial antitumor activity was exclusively observed among the wild-type TP53 models (two out of five, 40%), but there was no such response across the eight mutant TP53 models (0%). Interestingly, within a cohort of 14 patients with colorectal cancer treated with these agents for their metastatic disease, two patients with long-lasting responses (32 weeks) had TP53 wild-type tumors. CONCLUSIONS: Our data support that, in wild-type TP53 colorectal cancer cells with ERK and PI3K pathway alterations, MEK blockade results in potent p21 induction, preventing apoptosis to occur. In turn, mTORC1/2 inhibition blocks MEK inhibitor-mediated p21 induction, unleashing apoptosis. Clin Cancer Res; 21(24); 5499-510. ©2015 AACR.

A personalized preclinical model to evaluate the metastatic potential of patient-derived colon cancer initiating cells.

PURPOSE: Within the aim of advancing precision oncology, we have generated a collection of patient-derived xenografts (PDX) characterized at the molecular level, and a preclinical model of colon cancer metastasis to evaluate drug-response and tumor progression. EXPERIMENTAL DESIGN: We derived cells from 32 primary colorectal carcinomas and eight liver metastases and generated PDX annotated for their clinical data, gene expression, mutational, and histopathological traits. Six models were injected orthotopically into the cecum wall of NOD-SCID mice in order to evaluate metastasis. Three of them were treated with chemotherapy (oxaliplatin) and three with API2 to target AKT activity. Tumor growth and metastasis progression were analyzed by positron emission tomography (PET). RESULTS: Patient-derived cells generated tumor xenografts that recapitulated the same histopathological and genetic features as the original patients' carcinomas. We show an 87.5% tumor take rate that is one of the highest described for implanted cells derived from colorectal cancer patients. Cecal injection generated primary carcinomas and distant metastases. Oxaliplatin treatment prevented metastasis and API2 reduced tumor growth as evaluated by PET. CONCLUSIONS: Our improved protocol for cancer cell engraftment has allowed us to build a rapidly expanding collection of colorectal PDX, annotated for their clinical data, gene expression, mutational, and histopathological statuses. We have also established a mouse model for metastatic colon cancer with patient-derived cells in order to monitor tumor growth, metastasis evolution, and response to treatment by PET. Our PDX models could become the best preclinical approach through which to validate new biomarkers or investigate the metastatic potential and drug-response of individual patients.

ß-catenin confers resistance to PI3K and AKT inhibitors and subverts FOXO3a to promote metastasis in colon cancer.

The Wnt­ß-catenin and PI3K-AKT-FOXO3a pathways have a central role in cancer. AKT phosporylates FOXO3a, relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. Simultaneous hyperactivation of the Wnt­ß-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of ß-catenin and FOXO3a, respectively, promoting cell scattering and metastasis by regulating a defined set of target genes. Indeed, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis of cells with high nuclear ß-catenin content. Nuclear ß-catenin confers resistance to the FOXO3a-mediated apoptosis induced by PI3K and AKT inhibitors in patient-derived primary cultures and in corresponding xenograft tumors in mice. This resistance is reversed by XAV-939, an inhibitor of Wnt­ß-catenin signaling. In the presence of high nuclear ß-catenin content, activation of FOXO3a by PI3K or AKT inhibitors makes it behave as a metastasis inductor rather than a proapoptotic tumor suppressor. We show that it is possible to evaluate the ß-catenin status of patients' carcinomas and the response of patient-derived cells to target-directed drugs that accumulate FOXO3a in the nucleus before deciding on a course of treatment. We propose that this evaluation could be essential to the provision of a safer and more effective personalized treatment.

Vitamin D receptor deficiency enhances Wnt/ß-catenin signaling and tumor burden in colon cancer.

Aberrant activation of the Wnt/ß-catenin pathway is critical for the initiation and progression of most colon cancers. This activation provokes the accumulation of nuclear ß-catenin and the induction of its target genes. Apc(min/+) mice are the most commonly used model for colon cancer. They harbor a mutated Apc allele and develop intestinal adenomas and carcinomas during the first months of life. This phenotype is caused by the mutation of the second Apc allele and the consequent accumulation of nuclear ß-catenin in the affected cells. Here we describe that vitamin D receptor (VDR) is a crucial modulator of nuclear ß-catenin levels in colon cancer in vivo. By appropriate breeding of Apc(min/+) mice and Vdr(+/-) mice we have generated animals expressing a mutated Apc allele and two, one, or none Vdr wild type alleles. Lack of Vdr increased the number of colonic Aberrant Crypt Foci (ACF) but not that of adenomas or carcinomas in either small intestine or colon. Importantly, colon ACF and tumors of Apc(min/+)Vdr(-/-) mice had increased nuclear ß-catenin and the tumors reached a larger size than those of Apc(min/+)Vdr(+/+). Both ACF and carcinomas in Apc(min/+)Vdr(-/-) mice showed higher expression of ß-catenin/TCF target genes. In line with this, VDR knock-down in cultured human colon cancer cells enhanced ß-catenin nuclear content and target gene expression. Consistently, VDR depletion abrogated the capacity of 1,25(OH)(2)D(3) to promote the relocation of ß-catenin from the nucleus to the plasma membrane and to inhibit ß-catenin/TCF target genes. In conclusion, VDR controls the level of nuclear ß-catenin in colon cancer cells and can therefore attenuate the impact of oncogenic mutations that activate the Wnt/ß-catenin pathway.