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PDF file - 68K, S5. Methylation of Sox9-promoter, Sox9 and VEGF expression in patient samples.

PDF file - 163K

PDF file - 70K, S2. A) Differential methylation of the Sox9-promoter in NMET and HMET cells. B) Methylation and hypoxia. NMET and HMET cells were cultivated under hypoxia or normoxia, respectively. (C) Effect of methylation inhibition on VEGF expression. Relative transcription of VEGF in LMET and HMET cells is demonstrated. D) Effect of methylation inhibition on Sox9 expression.

PDF file - 130K, Supplemental Table 1. Differential expression of the genes associated to cancer relevant pathways.

PDF file - 50K, S4. Sox9 does not influence stability of HIF1alpha.

Detailed patient characteristics

Mutation profile of the cell lines MM-1 and MM-6

Summary patient characteristics

Supplementary Tables 1-7 Supplementary Materials and Methods Suppl. Table 1. WNT target gene sets used for GSEA Suppl. Table 2. Gene Expression (real-time RT-PCR) primers used in this study. Primers were selected from the Universal Probe Library (Roche). Suppl. Table 3. Antibodies used for immunoblotting and immunohistochemistry. Suppl. Table 4. Fold change (F.C.) of consistently deregulated genes in three gene expression data sets of colon cancer cells with high vs. low WNT activity, and F.C. in differential expression of these genes in TCGA data in colon cancer vs. normal mucosa. P values are t test results. Suppl. Table 5. Proteome analysis results upon ADNP depletion. Proteins with significant up- or downregulation are listed (P < 0.05; abs. fold change >1.5) Suppl. Table 6. Clinical/pathological data and ADNP expression in colorectal cancer. Suppl. Table 7. Multivariate analysis of cancer specific survival.

Purpose: Constitutively active WNT signaling is a hallmark of colorectal cancers and a driver of malignant tumor progression. Therapeutic targeting of WNT signaling is difficult due to high pathway complexity and its role in tissue homeostasis. Here, we identify the transcription factor ADNP as a pharmacologically inducible repressor of WNT signaling in colon cancer.Experimental Design: We used transcriptomic, proteomic, and in situ analyses to identify ADNP expression in colorectal cancer and cell biology approaches to determine its function. We induced ADNP expression in colon cancer xenografts by low-dose ketamine in vivo. Clinical associations were determined in a cohort of 221 human colorectal cancer cases.Results: ADNP was overexpressed in colon cancer cells with high WNT activity, where it acted as a WNT repressor. Silencing ADNP expression increased migration, invasion, and proliferation of colon cancer cells and accelerated tumor growth in xenografts in vivo. Treatment with subnarcotic doses of ketamine induced ADNP expression, significantly inhibited tumor growth, and prolonged survival of tumor-bearing animals. In human patients with colon cancer, high ADNP expression was linked to good prognosis.Conclusions: Our findings indicate that ADNP is a tumor suppressor and promising prognostic marker, and that ketamine treatment with ADNP induction is a potential therapeutic approach that may add benefit to current treatment protocols for patients with colorectal cancer. Clin Cancer Res; 23(11); 2769–80. ©2016 AACR.

Suppl. Figure 1. (A) Immunostaining for ADNP and β-Catenin on serial sections of two colon cancers. (B) Staining intensities of ADNP in colon cancer cells (n = 500, 5 different tumors) with high or low nuclear β- Catenin expression. P value is t test result. (C) Immunostaining for ADNP in normal mucosa. Right panels aremagnifications of areas boxed in left panel.Scale bars, 100 μm. Suppl. Figure 2. (A-B) Immunoblotting of indicated proteins on whole cell lysates in ADNP wild-type (WT) or knockout (KO) cell lines. Numbers below immunoblots indicate fold change by densitometry. (C) Representative micrographs (left panels) and quantification (right panels) of migrated or invaded tumor cells in transwell assays for indicated ADNP WT and KO cell lines, treated with indicated siRNAs or control siRNA (siCtrl). (D) Representative proliferation kinetics based on cell quantification by impedance measurements. Data are mean, n {greater than or equal to} 3. β-Cat = β-Catenin. Suppl. Figure 3. Representative micrographs (left panels) and quantification (right panels) of migrated or invaded HCT116 or SW1222 tumor cells with transient ADNP overexpression by transfection with p4.2-hADNP compared to empty p4.2 vector for 24 h in transwell assays. Suppl. Figure 4. (A) Immunoblotting of indicated proteins on whole cell lysates of SW1222, HCT116 and primary (P-Tu) colon cancer cells. Numbers below immunoblots indicate fold change by densitometry. (B) Immunostaining of SW1222, HCT116 and primary colon cancer (P-Tu) xenografts for β-Catenin. Scale bars, 50 μm. Suppl. Figure 5. (A-B) Dual-luciferase assays for indicatedADNPwild-type (WT) or knockout (KO) colon cancer cells transfected with TOPflash reporter constructs. Treatments with PBS, ketamine or the WNT inhibitor XAV939, as indicated. (C) Representativemicrographs (left panels) and quantification (right panels) ofmigrated or invaded HCT116 or SW1222 tumor cells in transwell assays, treated with PBS or 100 μM ketamine for 24 h, as indicated.Data aremean {plus minus}SD, Pvalues are t test results, n {greater than or equal to} 3. Suppl. Figure 6. Assessment of nuclear β-catenin staining in primary human colorectal cancers. Tumors were assigned scores from 0 (no nuclear β-catenin) to 3 (most tumor cells with strong nuclear β-catenin) and accordingly categorized as β-catenin low (score 0-1) and β-catenin high (score 2-3). Scale bars, 100 μm.

Figure S1 Recurrently mutated genes and comparison to TCGA cohort. Figure S2 Stabililty of recurrently mutated genes over disease course. Figure S3 Variant allele frequency plots from diagnosis to relapse for each individual patient. Figure S4 Mutation patterns of individual genes. Lines represent mutations in individual patients. Figure S5 Detection of the pre-existence of gained variants at initial diagnosis. Figure S6 Mutations in AML-associated functional pathways. Figure S7 Deletion of exons 3-10 of the KDM6A gene in the MM-6 cell line. The sister cell line MM-1 is not affected. Deletions were detected by quantitative MLPA analysis and the peak ratio for each KDM6A exon specific probe is shown. Graph represents the results from two independent experiments. Figure S8 H3K27 methylation in the AML cell lines MM-1 and MM-6. The global mono-, di- and tri-methylation of H3K27 in MM-1 and MM-6 was analyzed by Western blot and normalized to H3. The median of three independent experiments is shown. P-Values were calculated using a two-tailed, unpaired Student's t-test. Figure S9 The MM-6 cell line is resistant to cytarabine (Ara-C) but not to Daunorubicin or AC220. Error bars indicate mean {plus minus} s.d. of at least three independent experiments. **P75th percentile) with clinical outcome according to gender in the AMLCG-99 trial (NCT00266136). Figure S11 Proportion of transversions from diagnosis to relapse. Transversion frequencies are shown for lost (blue), stable (orange) and gained (red) mutations. Dots represent individual patients. Figure S12 Median coverage in targeted sequencing of persistent and non-persistent DNMT3A variant positions at complete remission (CR). Figure S13 Clinical outcome according to mutation persistence at remission.

Somatic variants

Purpose: To study mechanisms of therapy resistance and disease progression, we analyzed the evolution of cytogenetically normal acute myeloid leukemia (CN-AML) based on somatic alterations.Experimental Design: We performed exome sequencing of matched diagnosis, remission, and relapse samples from 50 CN-AML patients treated with intensive chemotherapy. Mutation patterns were correlated with clinical parameters.Results: Evolutionary patterns correlated with clinical outcome. Gain of mutations was associated with late relapse. Alterations of epigenetic regulators were frequently gained at relapse with recurring alterations of KDM6A constituting a mechanism of cytarabine resistance. Low KDM6A expression correlated with adverse clinical outcome, particularly in male patients. At complete remission, persistent mutations representing preleukemic lesions were observed in 48% of patients. The persistence of DNMT3A mutations correlated with shorter time to relapse.Conclusions: Chemotherapy resistance might be acquired through gain of mutations. Insights into the evolution during therapy and disease progression lay the foundation for tailored approaches to treat or prevent relapse of CN-AML. Clin Cancer Res; 24(7); 1716–26. ©2018 AACR.

Purpose: To study mechanisms of therapy resistance and disease progression, we analyzed the evolution of cytogenetically normal acute myeloid leukemia (CN-AML) based on somatic alterations. Experimental Design: We performed exome sequencing of matched diagnosis, remission, and relapse samples from 50 CN-AML patients treated with intensive chemotherapy. Mutation patterns were correlated with clinical parameters. Results: Evolutionary patterns correlated with clinical outcome. Gain of mutations was associated with late relapse. Alterations of epigenetic regulators were frequently gained at relapse with recurring alterations of KDM6A constituting a mechanism of cytarabine resistance. Low KDM6A expression correlated with adverse clinical outcome, particularly in male patients. At complete remission, persistent mutations representing preleukemic lesions were observed in 48% of patients. The persistence of DNMT3A mutations correlated with shorter time to relapse. Conclusions: Chemotherapy resistance might be acquired through gain of mutations. Insights into the evolution during therapy and disease progression lay the foundation for tailored approaches to treat or prevent relapse of CN-AML. Clin Cancer Res; 24(7); 1716–26. ©2018 AACR.

Background: The understanding of invasion, angiogenesis and metastasis is essential for the development of new targeted molecular therapy against pancreatic cancer. The aim of this study is to analyse the regulation of differentially expressed genes in highly proangiogenic and prometastatic compared to low metastatic and low angiogenic pancreatic cancer cells Materials and methods: The transcriptome of the low metastatic pancreatic cancer cell line and its highly angiogenic and metastatic derivate L3.6pl have been compared under the normoxic and hypoxic conditions using Affymetrix HGU133plus 2.0 arrays. The hierarchic cluster analysis has been performed using SOTA algorithm. Promoters of the differentially expressed genes have been analysed using oPOSSUM 2 program. Affymetrix data have been validated via qRT-PCR and via ELISA. Chromosomal DNA containing promoters of the relevant genes has been amplified via PCR and sequenced. Results: The Affymetrix analysis revealed clusters of differentially regulated genes with regard to the cell line and the oxygen level. While VEGF expression was upregulated in FG cells under hypoxic condition, it was constitutively upregulated already under normoxic conditions in L3.6pl and no further stimulation was observed under hypoxic conditions. This regulation pattern was similar in a cluster of genes that share a promoter sequence coding for a binding site for the transcription factor Sox9. Sox9 itself shows a similar regulation, leading to the assumption that expression changes of Sox9 are involved in the different hypoxic gene expressions. Promoter analysis of Sox9 in FG and L3.6pl cells showed mutations in the promoter region in L3.6pl, while the VEGF promoter was unchanged. Further analysis has shown that this mutation is not relevant for biological function. Refined analysis revealed differential Sox9 promoter methylation as reason for observed phenomenon. Conclusions: Proangiogenic genes such as VEGF are upregulated in the more aggressive cell line L3.6pl, compared to FG. We identified a gene cluster that is constitutively upregulated in L3.6pl, independent of hypoxia. These genes are controlled by the transcription factor Sox9. A Sox9 promoter differential methylation may be the reason for the escape from hypoxic regulation in L3.6pl cells and thus may contribute to the higher angiogenic and metastatic potential. Taken together, Sox9 may be an interesting new diagnostic and therapeutic target in pancreatic cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3911.

The understanding of invasion, angiogenesis and metastasis is essential for the development of new molecular therapy against cancer. Inflammation plays important role in tumour initiation and progression. Here we report the role of the transcription factor Sox9 for regulation of IFIT3 an inflammation-related and tumour-promoting protein in pancreatic cancer. For in vivo and in vitro experiments we utilized the following human pancreatic cancer cell lines: low metastatic FG, high metastatic L3.6pl, and the stable transfected cell line FG-IFIT3. To demonstrate effects on primary tumor growth and metastases in vivo we orthotopically injected the different cell lines in the pancreas of nude mice. To evaluate the VEGF depending angiogenic capacity of the different cell lines ELISA technology was used. By One STrEP technology we were able to identify IFIT3-binding partners. Chromosomal immunoprecipitation (ChIP) using anti-Sox9 antibody, followed by PCR amplifying the IFIT3-promoter was used to identify the interaction of the IFIT3 promoter with the transcription factor Sox9. To investigate Sox9-depending expression of IFIT3 (protein and RNA) we used stable transfected L3.6pl-Sox9-shRNA cells under control of the Tet-CMV promoter in presence or absence of tetracycline, respectively. Analysis of differential gene expression by gene array technology demonstrated that the IFIT3 gene is up-regulated in L3.6pl cells as compared to FG cells. Results of animal experiment and in vitro experiments clearly demonstrated tumor-promoting, pro-metastatic and pro-angiogenic features of IFIT3. RT-PCR has revealed that both treatment with IFna as well as NFkB led to up-regulation of IFIT3-RNA expression. One STrEP experiments identified JNK and STAT1 as binding partners of IFIT3. ChIP has demonstrated binding of the transcription factor Sox9 to the IFIT3 promoter. RT-PCR and immunoblot data demonstrated constitutive up-regulation of Sox9 expression in L3.6pl cells. By Western blotting and RT-PCR we could show that diminishing of Sox9 expression in stable transfected L3.6pl Sox9-shRNA cells leads to a significant down-regulation of IFIT3-epression on the RNA and protein level. The inflammation associated protein IFIT3 is up-regulated in metastatic L3.6pl human pancreatic cancer cells and is in part responsible for the aggressive primary pancreatic tumor growth in vivo. This gene is up-regulated by IFna and NFkB. Interestingly Sox9 binds to the IFIT3P and activates its expression. Since in L3.6pl cells Sox9 is constitutively over-expressed, IFIT3 is up-regulated independent on the presence of the cytokine IFna. Therefore, the pro-inflammatory IFna-signaling pathway is activated even without actual inflammation in absence pro-inflammatory cytokine. The activation of such a “pseudo-inflammatory pathway” seems to be in part responsible for pancreatic cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3901.