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XLSX file - 768K, Nanostring CRC codeset.

PDF file - 674K, Histological characterization of patients' carcinomas and the correspondent xenograft tumors.

Supplementary Figures S1-S11. Figure S1. Apoptosis of CRC patient-derived cells upon treatment with API-2 or NVP-BKM120 in combination with XAV939. Figure S2. Evaluation of apoptosis in primary sphere cell cultures by immunofluorescent quantification of cleaved-CASPASE3. Figure S3. Frequent mutations in CRC do not determine the response to API2, NVP-BKM120 or NVP-TNKS656. Figure S4. NVP-TNKS656 is not toxic and reduces nuclear β-catenin in the skin and small intestine in NOD-SCID mice. Figure S5. Analyses of pharmacodynamic markers at endpoint of in vivo experiments with API2 and NVP-TNKS656. Figure S6. Nuclear FOXO3A content is preserved in PDX and primary sphere cultures. Figure S7. Nuclear FOXO3A in paired primary tumors and liver metastases. Figure S8. Immunohistochemical staining of β-catenin in CRC tumors. Figure S9. β-catenin in single-agent treatments, relevant mutations, TNM stage or tumor site do not associate with a differential response to PI3K/AKT/mTOR inhibitors. Figure S10. Gene expression clustering and mutational status of baseline tumor samples from patients treated with PI3K/AKT/mTOR inhibitors. Figure S11. Molecular prescreening scheme to select CRC patients eligible for the treatment with PI3K/AKT and/or Wnt/β-catenin inhibitory drugs.

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.

PDF file - 115K, 18-FDG is the best radiotracer for tumor growth evaluation.

Supplementary Tables S1-S15. Table S1. Genes and positions analyzed by amplicon sequencing. Table S2. Genes sequenced by Haloplex Target Enrichment System. Table S3. Antibodies. Table S4. Treatment-induced apoptosis in 10 primary patient-derived sphere cell cultures. Table S5. Genotype of patient-derived colorectal cancer models. Table S6. Exome sequencing of colorectal cancer patient-derived models. Table S7. Genes regulated by NVP-TNKS656 in PDX-P2. Table S8. Genes regulated by NVP-TNKS656 in PDX-P30. Table S9. Genes regulated by NVP-TNKS656 in PDX-P5. Table S10. Intestinal β-catenin/TCF signature in human CRC. Table S11. Genes regulated by β-catenin/FOXO3A signaling in human CRC. Table S12. Molecular profiling of 130 CRC tumors. Table S13. Clinicopathological data of 40 CRC patients treated with PI3K/AKT/mTOR inhibitors and their corresponding baseline tumor samples analyzed for mutations and nuclear β-catenin content. Table S14. Nuclear β-catenin content but not relevant mutations or TNM stage determine response of CRC patients to PI3K/AKT/mTOR inhibitors. Table S15. Multivariate analysis of baseline tumors from patients treated with PI3K/AKT/mTOR inhibitors.

Table S1. MiSeq 57-gene panel list

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/β-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/β-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 β-catenin content. High FOXO3A activity conferred sensitivity to NVP-TNKS656 treatment. Thirteen of 40 patients presented high nuclear β-catenin content and progressed earlier upon PI3K/AKT/mTOR inhibition. Nuclear β-catenin levels predicted drug response, whereas clinicopathologic traits, gene expression profiles, or frequent mutations (KRAS, TP53, or PIK3CA) did not.Conclusions: High nuclear β-catenin content independently predicts resistance to PI3K and AKT inhibitors. Combined treatment with a Wnt/tankyrase inhibitor reduces nuclear β-catenin, reverts such resistance, and represses tumor growth. FOXO3A content and activity predicts response to Wnt/β-catenin inhibition and together with β-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/β-catenin inhibitors. Clin Cancer Res; 22(3); 644–56. ©2015 AACR.

Supplementary Figures 1 and 2 and legends for Table S1, S2 and S3

Supplementary Table 2

Supplementary Table 1

Supplementary Figure 2

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. Clin Cancer Res; 19(24); 6787–801. ©2013 AACR.

Supplementary Figure and Table Legends. The text describes the panels and tables included as Supplementary Figure and Table Legends

Supplementary Table 4

Supplementary Table 3

Supplementary Table 3

Figure S2. Immunohistochemistry of p53 in tumors from CRC patients treated with MEK- plus PI3K-inhibitors

Aberrant Wnt signaling drives a number of cancers through regulation of diverse downstream pathways. Wnt/β-catenin signaling achieves this in part by increasing the expression of proto-oncogenes such as MYC and cyclins. However, global assessment of the Wnt-regulated transcriptome in vivo in genetically distinct cancers demonstrates that Wnt signaling suppresses the expression of as many genes as it activates. In this study, we examined the set of genes that are upregulated upon inhibition of Wnt signaling in Wnt-addicted pancreatic and colorectal cancer models. Decreasing Wnt signaling led to a marked increase in gene expression by activating ERK and JNK; these changes in gene expression could be mitigated in part by concurrent inhibition of MEK. These findings demonstrate that increased Wnt signaling in cancer represses MAPK activity, preventing RAS-mediated senescence while allowing cancer cells to proliferate. These results shift the paradigm from Wnt/β-catenin primarily as an activator of transcription to a more nuanced view where Wnt/β-catenin signaling drives both widespread gene repression and activation.Significance:These findings show that Wnt/β-catenin signaling causes widespread gene repression via inhibition of MAPK signaling, thus fine tuning the RAS-MAPK pathway to optimize proliferation in cancer.

Supplementary Table 1

PDF file - 1113K, Patient-derived cells preserve pluripotency.

Supplementary Figure 1

Supplementary Figure Legends

Supporting Information. Supplementary methods to complete the information for gene expression, genotyping, immunohistochemistry evaluation criteria and protein extraction. The file also contains legends for supplementary figures S1-S11 and supplementary tables S1-S15.

PDF file - 248K, Distant metastasis generated by cells derived from patient 5.

Supplementary Figure 3

Supplementary Table 2

PDF file - 290K, Histopathological and genetic traits of CRC PDX.

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 neuroendocrine-specific 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.

Dormant or slow-cycling tumor cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. Slow-cycling cancer cells (SCCC) represent a cellular status rather than a cell population present in a minor proportion, even in growing tumors. We have adapted the pulse-chase expression of histone H2B fused to enhanced GFP (H2BeGFP) for labelling and isolating SCCC. SCCC show cancer-initiation potential and enhanced chemoresistance, and present a distinctive nongenetic and cell-autonomous gene expression profile shared across different tumor types. The use of our H2BeGFP pulse-chase method opens the possibility to study live SCCC in any growing tissue either cancerous or normal.

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.

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.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 neuroendocrine-specific traits. Patients with co-NEC and patient-derived xenografts were treated with a BRAFV600E-blocking drug to demonstrate sensitivity.co-NEC and CRC are similar in their mutational repertoire, although co-NECs are particularly enriched inThe identification of

Patient-derived xenografts (PDXs) have emerged as an important platform to elucidate new treatments and biomarkers in oncology. PDX models are used to address clinically relevant questions, including the contribution of tumour heterogeneity to therapeutic responsiveness, the patterns of cancer evolutionary dynamics during tumour progression and under drug pressure, and the mechanisms of resistance to treatment. The ability of PDX models to predict clinical outcomes is being improved through mouse humanization strategies and the implementation of co-clinical trials, within which patients and PDXs reciprocally inform therapeutic decisions. This Opinion article discusses aspects of PDX modelling that are relevant to these questions and highlights the merits of shared PDX resources to advance cancer medicine from the perspective of EurOPDX, an international initiative devoted to PDX-based research.