The Wnt–β-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 Wnt–β-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of β-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 β-catenin content. Nuclear β-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 Wnt–β-catenin signaling. In the presence of high nuclear β-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 β-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.

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/b-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/b-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 b-catenin content. High FOXO3A activity conferred sensitivity to NVP-TNKS656 treatment. Thirteen of 40 patients presented high nuclear b-catenin content and progressed earlier upon PI3K/AKT/ mTOR inhibition. Nuclear b-catenin levels predicted drug response, whereas clinicopathologic traits, gene expression profiles, or frequent mutations (KRAS, TP53, or PIK3CA) did not.Conclusions: High nuclear b-catenin content independently predicts resistance to PI3K and AKT inhibitors. Combined treatment with a Wnt/tankyrase inhibitor reduces nuclear b-catenin, reverts such resistance, and represses tumor growth. FOXO3A content and activity predicts response to Wnt/b-catenin inhibition and together with b-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/b-catenin inhibitors.

Purpose: Within the aim of advancing precision oncology, we have generated a collection of patientderived 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.

Activation of the Wnt pathway promotes the progressive phosphorylation of coreceptor LRP5/6 (low-density lipoprotein receptor-related proteins 5 and 6), creating a phosphorylated motif that inhibits glycogen synthase kinase 3␤ (GSK-3␤), which in turn stabilizes ␤-catenin, increasing the transcription of ␤-catenin target genes. Casein kinase 1 (CK1) kinase family members play a complex role in this pathway, either as inhibitors or as activators. In this report, we have dissected the roles of CK1 isoforms in the early steps of Wnt signaling. CK1 is constitutively bound to LRP5/6 through its interaction with p120-catenin and E-cadherin or N-cadherin and is activated upon Wnt3a stimulation. CK1␣ also associates with the LRP5/6/p120-catenin complex but, differently from CK1, only after Wnt3a addition. Binding of CK1␣ is dependent on CK1 and occurs in a complex with axin. The two protein kinases function sequentially: whereas CK1 is required for early responses to Wnt3a stimulation, such as recruitment of Dishevelled 2 (Dvl-2), CK1␣ participates in the release of p120-catenin from the complex, which activates p120-catenin for further actions on this pathway. Another CK1, CK1␥, acts at an intermediate level, since it is not necessary for Dvl-2 recruitment but for LRP5/6 phosphorylation at Thr1479 and axin binding. Therefore, our results indicate that CK1 isoforms work coordinately to promote the full response to Wnt stimulus.The Wnt pathway plays diverse roles in embryonic development and has been implicated in human diseases, including cancer (9). A key element in this pathway is the Ecadherin-associated protein ␤-catenin. When released from the junctional complex, ␤-catenin translocates to the nucleus, where it interacts with the Tcf family of transcriptional factors and regulates the expression of a variety of genes. The translocation of ␤-catenin to the nucleus is tightly controlled by the activity of a complex involved in ␤-catenin degradation. This complex includes the product of the tumor suppressor adenomatous polyposis coli (APC) gene, axin, and the associated Thr/Ser protein kinases, CK1␣ and glycogen synthase kinase 3␤ (GSK-3␤) (12). As a result of the activity of this complex, ␤-catenin is phosphorylated and degraded by the proteasome. The activity of the degradation complex is blocked by canonical Wnt factors, which activate a signaling pathway leading to the stabilization of cytosolic ␤-catenin (12, 13).Wnt ligands form a complex with low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and Frizzled (Fz) receptors (7). Upon Wnt ligand binding, the LRP5/6 cytosolic domain gets phosphorylated in different residues by the action of several protein kinases (15). For, instance Thr1479 is phosphorylated by casein kinase 1␥ (CK1␥), a modification that is also dependent on the Fz-associated protein Dishevelled (Dvl) (1,4,28). Other members of this family, such as CK1ε and CK1␣, also contribute to the phosphorylation of LRP5/6 and Dvl (17,21,27). Phosphorylation of LRP5/6 promotes the recruitment of ...

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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.

BackgroundThird-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as osimertinib are the last line of targeted treatment of metastatic non-small-cell lung cancer (NSCLC) EGFR-mutant harboring T790M. Different mechanisms of acquired resistance to third-generation EGFR-TKIs have been proposed. It is therefore crucial to identify new and effective strategies to overcome successive acquired mechanisms of resistance.MethodsFor Amplicon-seq analysis, samples from the index patient (primary and metastasis lesions at different timepoints) as well as the patient-derived orthotopic xenograft tumors corresponding to the different treatment arms were used. All samples were formalin-fixed paraffin-embedded, selected and evaluated by a pathologist. For droplet digital PCR, 20 patients diagnosed with NSCLC at baseline or progression to different lines of TKI therapies were selected. Formalin-fixed paraffin-embedded blocks corresponding to either primary tumor or metastasis specimens were used for analysis. For single-cell analysis, orthotopically grown metastases were dissected from the brain of an athymic nu/nu mouse and cryopreserved at −80°C.ResultsIn a brain metastasis lesion from a NSCLC patient presenting an EGFR T790M mutation, we detected MET gene amplification after prolonged treatment with osimertinib. Importantly, the combination of capmatinib (c-MET inhibitor) and afatinib (ErbB-1/2/4 inhibitor) completely suppressed tumor growth in mice orthotopically injected with cells derived from this brain metastasis. In those mice treated with capmatinib or afatinib as monotherapy, we observed the emergence of KRAS G12C clones. Single-cell gene expression analyses also revealed intratumor heterogeneity, indicating the presence of a KRAS-driven subclone. We also detected low-frequent KRAS G12C alleles in patients treated with various EGFR-TKIs.ConclusionAcquired resistance to subsequent EGFR-TKI treatment lines in EGFR-mutant lung cancer patients may induce genetic plasticity. We assess the biological insights of tumor heterogeneity in an osimertinib-resistant tumor with acquired MET-amplification and propose new treatment strategies in this situation.

Purpose: The limited knowledge of the molecular alterations that characterize poorly differentiated neuroendocrine carcinomas has limited the clinical development of targeted agents directed to driver mutations. Here we aim to identify new molecular targets in colon neuroendocrine carcinomas (co-NEC) and proof the efficacy of matching drugs. Experimental Design: We performed a multi-omic analysis of co-NEC to identify genetic or epigenetic alterations that could be exploited as effective drug targets. We compared co-NEC samples with colorectal carcinomas (CRC) to identify neuroendocrinespecific traits. Patients with co-NEC and patient-derived xenografts were treated with a BRAFV600E-blocking drug to demonstrate sensitivity. Results: co-NEC and CRC are similar in their mutational repertoire, although co-NECs are particularly enriched in BRAFV600E mutations. We report for the first time that V600EBRAF-mutant co-NECs may benefit from BRAF inhibition in monotherapy and how EGFR status is essential to predict innate sensitivity and acquired resistance by a differential methylation of its gene regulatory regions. Conclusions: The identification of V600E BRAF mutations in high-grade co-NECs has allowed the description of radiological responses to combination therapy of BRAF and MEK inhibitors in basket clinical trials. However, the molecular rationale for this treatment combination was based on the presence of the BRAF mutation and the efficacy observed in other cancer types such as melanoma. Future drug development in this setting should test BRAF inhibitors upfront and the addition of anti-EGFR antibodies instead of MEK inhibitors for an efficient blockade of acquired resistance.