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Supplementary Figure Legends

Supplementary Figure 7. INPP4B loss does not produce any morphological or cytoskeletal differences.

PDF - 14407K, Supporting histology for Olaparib treatment in vivo.

Supplementary Figure 3. INPP4B's promoter is not directly methylated.

Supplementary Figure S3. ERG binding to CHK1 promoter.

Supplementary Figure S5. ERG overexpression induces p53 activation through excessive DNA damage.

Supplementary Figure S2. ETV1 represses CHK1 expression.

PDF - 632K, Preclinical assessment of Olaparib and BKM120 treatment in vivo.

Supplementary Figure Legends

PDF - 546K, Olaparib induces a differing response in vitro depending on the genetic background.

PDF - 2951K, Olaparib induces a differing response in vivo depending on the genetic background.

Supplementary Figure 2. Validation of INPP4B antibody.

Supplementary Figure 1. Generation of Pten+/-Inpp4b+/- and Pten+/-Inpp4b-/- mice.

PDF - 114k, Supporting western blot analysis and drug treatment in prostate cancer cell lines.

Supplementary Figure S1. ERG downregulation of CHK1 is AR independent.

PDF - 116K, Supplementary Materials and Methods and Supplementary Figure Legends.

Supplementary Figure 4. Loss of Inpp4b in MEFs lead to increased Akt activation.

Supplementary Figure 5. Loss of INPP4B results in increased AKT2 activation.

PDF - 138K, Survival curves of the in vivo treatment of Olaparib and BKM120.

Depletion of FZR1 leads to BRAF stabilization and subsequent ERK activation. Supplementary Figure S2. Depletion of FZR1 triggers the onset of premature senescence in primary melanocytes. Supplementary Figure S3. APCFZR1 promotes BRAF ubiquitination and subsequent degradation in a D-box dependent manner. Supplementary Figure S4. FZR1 disrupts the BRAF dimerization process. Supplementary Figure S5. Phosphorylation of FZR1 N-terminus by ERK and CYCLIN D1/CDK4 inhibits the APCFZR1 E3 ubiquitin ligase activity. Supplementary Figure S6. Pharmacologically inhibiting BRAF/ERK and CDK4 restores the APCFZR1 E3 ligase activity. Supplementary Figure S7. Depletion of FZR1 co-operates with PTEN deficiency to promote co-activation of BRAF/ERK and AKT oncogenic signaling both in vitro and in vivo. Supplementary Table S1. Mutation and deletion of FZR1 and 14 APC core complex subunits identified in melanoma patients from the TCGA melanoma dataset (cbioportal.org).

Aims: We sought to determine whether KLF5 was involved, at either phenotypic or functional levels, in the genesis and progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC), and whether this involvement was related to sonic hedgehog (SHH) signaling. In human adults, KLF5 is expressed in the less differentiated cells of the intestinal crypts, where its activity promotes cell proliferation, contributes to intestinal epithelial homeostasis, and is necessary for villus formation. KLF5 has been found to be highly active in a number of other cancer types, including gastric cancer; moreover, the known role of KLF5 in intestinal epithelial differentiation may contribute to metaplasia and dysplasia in BE and EAC development. We also sought to ascertain whether KLF5 activity was connected to the sonic hedgehog (SHH) pathway, since this pathway is already known to be highly active in BE and EAC. Methods and results: Quantitative reverse transcriptase PCR (qRT-PCR) performed on RNA obtained from BE cell lines (GhTRT and QhTRT) and EAC cell lines (SKGT4 and OE33) revealed that KLF5 is underexpressed in BE cell lines and overexpressed in EAC cell lines relative to normal primary squamous esophageal epithelial cells (HEEpic). Moreover, qRT-PCR performed on clinical BE and EAC tissue specimens showed the same pattern, with downregulation in BE tissues and upregulation in EAC tissues (relative to matched normal esophageal control tissues). Next, we determined KLF5 functional effects in EAC in vitro. The EAC cell line SKGT4 was transfected with KLF5 siRNA, and WST-1 cell proliferation assays revealed significantly lower cell proliferation at 72 hours (p Conclusion: These tissue phenotypic and in vitro functional data identify KLF5 as a potential oncogene in esophageal adenocarcinogenesis. KLF5 is a potential therapeutic and/or chemopreventive target that can be evaluated in future studies of EAC or BE, such as with metformin, which is known to inhibit KLF5 activity. We also identify a potential new connection between KLF5 and SHH pathway activity in the development of EAC. This potential connection deserves further exploration in other malignancies, as well as in physiologic functions such as human development or adult stem cell maintenance. Citation Format: Christopher K. Ng, Yulan Cheng, John M. Abraham, Stephen J. Meltzer. Krüppel-like factor 5 and its connection to Sonic Hedgehog signaling in Barrett's esophagus and esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3077.

The discovery that bifunctional alkylating agents have antitumor activity led to the development of cancer chemotherapy over 50 years ago. While these compounds form a variety of cellular lesions, DNA interstrand cross-links are believed to be the most lethal, impeding both replication and expression of the genetic material. We are characterizing the mechanisms by which diepoxybutane (DEB) and the structurally related compound epichlorohydrin (ECH) exert their cytotoxic effects in cultured cells. Our first goal is to determine the relationship between interstrand cross-linking and cytotoxicity. We are assaying cross-linking ability in cultured cells using Hoechst 33258 to determine the amount of duplex DNA following alkaline denaturation. Only cross-linked DNA reanneals rapidly and interacts with the dye, which is highly fluorescent when bound to duplex DNA. Preliminary results in chicken 6C2 and human HL60 cells suggest a correlation between cross-linking and LD50 values for these compounds. Our second goal is to characterize the pathways by which these compounds induce apoptosis. Reverse-transcriptase real-time PCR analysis of HL-60 cells treated with DEB and ECH under conditions determined to induce apoptosis suggests up-regulation of several key genes involved in the mitochondrial apoptotic pathway, including BAK1, BAX, DIABLO, PUMA and APAF1. Finally, we are identifying the covalent structure of the ECH-cross-linked lesion via electrospray LC/MS. Characterization of the DNA modifications induced by these agents is an important step in understanding how these compounds exert their cytotoxic effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4904. doi:10.1158/1538-7445.AM2011-4904

The bifunctional alkylating agents are an important class of drugs for the treatment of cancer, psoriasis, and some anemias. These compounds are widely believed to exert their cytotoxic effects through reactions with biological nucleophiles, with DNA interstrand cross-links considered to be the most lethal lesions. Indeed, for many families of bifunctional alkylators, DNA interstrand cross-linking correlates with cytotoxicity. Moreover, apoptotic potential may play a role in antitumor chemotherapeutic utility. We have established previously that diepoxybutane (DEB) and the structurally related compounds epichlorohydrin (ECH) and 1-(chloroethenyl)oxirane (CEO) form DNA interstrand cross-links in vitro. With the goal of elucidating the molecular and cellular mechanisms by which this family of epoxides exerts its cytotoxic effects, we are determining the relationships between interstrand cross-linking, LD50 values, and apoptotic potentials for DEB, ECH, and CEO in chicken 6C2 and human HL-60 cells. Preliminary results in 6C2 cells suggest that cytotoxicity follows the order DEB > CEO > ECH. Furthermore, flow cytometry with Annexin V-FITC/PI dual staining has revealed that the apoptotic potentials follow the order DEB >> ECH > CEO. Reverse-transcriptase real-time PCR analysis of HL-60 cells treated with DEB under conditions known to induce apoptosis suggests up-regulation of several key genes involved in the mitochondrial apoptotic pathway, including BAK1, BAX, DIABLO, and APAF1. Further work is underway to characterize the pathways by which ECH and CEO exert their apoptotic effects. 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 1695.