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644 Amplification of 5FU Resistance by DNA Mismatch Repair Deficiency and Subsequent MBD4 Frameshift Mutation
AGA Abstracts
643
reduction of tumor growth in mAb-treated mice (Shapira et al, Gastro 2011). Aim: To engineer anti-CD24 chimeric and humanized full-length IgGs, smaller derivatives (scFv and Fab) and toxin-immunconjugates. Methods: Edman-degradation, cDNA synthesis, sequence and computational analysis (Ig-blast) were performed to reveal the entire DNA sequence of the murine Ab. Replacement of the Fc with the human IgG1 resulted in a mouse-human chimera. Human donor (Ig-blast) was chosen as scaffold human Ab for grafting critical sequences of the murine antibody into it. The immunotoxins were constructed first by noncovalent linking via the ZZ protein (derived from Staphylococcus aureus). Then the bacterial system allowed a covalent linkage between the toxic molecule and the Ab. Sophisticated and high-efficiency engineering-technologies were established to construct, express and purify 15 novel anti-CD24 derivatives (Fig. 1) in tissue or bacterial cultures. Their bindingaffinity, selective-targeting and cytotoxic activities were confirmed in several CD24-expressing GI tumors cells. Results: The chimeric and humanized forms have higher affinity than the murine Abs. Anti-CD24 antibodies work additively with standard chemotherapies in inhibiting cancer cell growth. The immunotoxins were superior to the unarmed Ab with lower IC50 values by several orders of magnitude. Antibody targeting and accumulation within the tumor and its excess clearance was clearly demonstrated using direct imaging (SWA11ZZ-mCherry-HIS fluobody derivative). The scFv-toxin killed CD24-expressing cells with an IC50 of 1μg/ml, while the values for the IgG-ZZ-toxin were much lower (0.02-0.05μg/ml) (Fig. 2). The values of the humanized anti-CD24(di/tetra)-PE38 were similar to those of the scFv (Fig. 3), suggesting that the absence of post-translational modifications, in the E.coliproducing IgG, affected reactivity. MTD studies in dozens of mice confirmed no toxicity at high dose of 1mg/mouse. Conclusion: Targeting CD24 may be a promising treatment for GI malignancies in combination with chemotherapy. Humanized mAb (unarmed and toxinconjugated) to CD24 are more effective than the murine parental Ab. Following toxicology studies in big animals we aim for a Phase 1 study in 2012.
In Esophageal Epithelium, the Transcription Factor FOXA2 is a Hedgehog Target Gene That May Mediate Columnar Metaplasia, as Occurs in Barrett's Esophagus David H. Wang, Monica Kim, Anjana Tiwari, Xi Zhang, Nicholas J. Clemons, David M. Berman, Stuart J. Spechler, Rhonda F. Souza Introduction: Cells change their phenotype during the process of metaplasia, often by reverting to a phenotype that was present in the organ during embryonic development. The embryonic esophagus is initially lined by columnar cells that are later replaced by squamous cells, and Barrett's metaplasia involves the change of esophageal squamous cells back into columnar cells in the setting of GERD. We have reported that mouse embryonic esophageal columnar epithelium is characterized by active Hedgehog signaling, and that forced expression of Sonic hedgehog (Shh) in the adult mouse esophagus leads to a columnar metaplasia. Our microarray analyses of mouse embryonic columnar esophageal epithelium have identified robust expression of Foxa2, an endodermal transcription factor involved in the development of intestinal goblet cells. We also have demonstrated that Hedgehog is activated in Barrett's metaplasia, which expresses functional FOXA2, and that FOXA2 is expressed in telomeraseimmortalized esophageal squamous cell lines (NES-B3T, NES-B10T) from patients with Barrett's esophagus. Now, using those cell lines and genetically-engineered mouse models, we have explored whether FOXA2 is a direct Hedgehog pathway target gene. Methods: We generated Shh knockout and Shh over-expressing transgenic mice. Shh knockout embryos were harvested from timed pregnant females, and adult mouse esophageal epithelium was cultured in a novel In Vivo transplant culture system previously described by our group. NES-B3T and NES-10T cells were treated either with the recombinant ligand SHH or induced to express GLI1, a downstream mediator of the Hedgehog pathway. FOXA2 protein expression was determined by immunohistochemistry, and FOXA2 mRNA levels were assessed by quantitative real-time PCR. Results: In wild type mice with intact Hedgehog signaling, Foxa2 protein is expressed in the columnar cells of the esophageal epithelium from embryonic day 11.5 through postnatal day 2. In contrast, Shh knockout mice have undetectable levels of Foxa2 protein at embryonic days 14.5 and 17.5. In In Vivo transplant cultures of adult mouse esophageal squamous epithelium, Foxa2 protein was not detected in cells of wild type mice, but was detected in cells of Shh over-expressing transgenic mice. In NES-B3T and NES-B10T human esophageal squamous cell lines, both treatment with recombinant SHH and overexpression of GLI1 substantially increased FOXA2 mRNA expression levels. Conclusions: Foxa2 is a target of Hedgehog signaling in the embryonic mouse columnar esophageal epithelium. In human esophageal squamous epithelial cells from patients with Barrett's esophagus, activation of Hedgehog signaling increases FOXA2 mRNA levels. These data suggest that FOXA2 is a downstream target of Hedgehog signaling that might contribute to the development of Barrett's metaplasia. 644 Amplification of 5FU Resistance by DNA Mismatch Repair Deficiency and Subsequent MBD4 Frameshift Mutation Moriya Iwaizumi, Stephanie Tseng-Rogenski, John M. Carethers Background and Aims. We and others have demonstrated recognition of 5-fluorouracil (5FU) in DNA by the DNA mismatch repair (MMR) system, which triggers a DNA MMRdependent cell death. Loss of DNA MMR contributes to 5FU resistance for colorectal cancer patients. MBD4 is a DNA glycosylase that can remove 5FU from DNA, but is a target gene for frameshift mutation with DNA MMR deficiency (A10→A9 in exon 3) forming a truncated MBD4 (MBD4tru). Here we explored if MBD4tru as well as wild type MBD4 (MBD4WT) and MMR proteins recognize 5FU within DNA. Methods. Utilizing HT29 (MMR-proficient, MBD4A10/A10), HCT116 (hMLH1-/-, MBD4A10/A9), and HCA7 (hMLH1-/-, MBD4A9/A9) cells, nuclear localization and expression of both MBD4WT (expressed in HT29 and HCT116, not in HCA7) and MBD4tru (expressed in HCT116 and HCA7, not in HT29) were confirmed by indirect immunofluorescence analysis and Western blotting. To determine relative repair protein binding to 5FU incorporated into DNA, biotin-labeled 5FdU:G heteroduplex dsDNA (5FU dsDNA) with or without unlabeled 5FU dsDNA (competitor 5FU) or C:G homoduplex dsDNA (control competitor) were incubated with nuclear lysate. Probe-bound proteins were collected with Streptavidin magnetic beads and eluted. Eluted proteins were utilized for Western blotting to analyze 5FU binding affinity. Results. MBD4tru bound to 5FdU:G, and was competed off by competitor 5FU but not by control competitor. Within MBD4A10/A9 cells, the 5FU binding affinity of MBD4tru was higher than that of MBD4WT (competed off rate by 5FU competitor; 79.3% for MBD4tru, 45.9% for MBD4WT). Moreover, between MBD4A9/A9 (only MBD4tru expression) and MBD4A10/A10 (only MBD4WT expression) cells, the 5FU binding affinity of MBD4tru was higher than that of MBD4WT cells (competed off rate by 5FU competitor; 90.2% for MBD4tru of MBD4A9/A9 cells, 42.71% for MBD4WT of MBD4A10/A10 cells), whereas that of MMR proteins were lower in MBD4A9/A9 cells (competed off rate by 5FU competitor; 40.2% for MSH2, 1.5% for MSH3, 0.3% for MSH6) than that of MBD4A10/A10 cells (competed off rate by 5FU competitor; 44.8% for MSH2, 23.4% for MSH3, 10.3% for MSH6). Conclusion. 5FU incorporated in DNA is recognized by and has higher affinity for MBD4tru than MBD4WT, and MBD4tru reduces the 5FU affinity of the DNA MMR proteins. Frameshift mutation of MBD4 forming MBD4tru amplifies the 5FU resistance of DNA MMR deficiency, possibly be preventing access to 5FU within DNA by repair proteins.
Figure 1. Schematic representation of the novel anti-CD24 derivatives that were developed in our lab.
Figure 2. (A) scFv(SWA11)-Toxin was tested for its cell killing activity by the MTT assay in CD24-expressing (HT29) and not-expressing cells (HCT116). (B) The cytotoxic activities of the PE38-based immunotoxins were evaluated by inhibition of protein synthesis as measured by the [3H]-Leucine incorporation. (C) Human anti-CD24-di(H)-PE38, Human anti-CD24-di(H)-PE38-di(L)-PE38 and scFv(SWA11)-PE38 derivatived were evaluated for specific targeting of CD24-expressing tumor cells in female athymic nude mice bearing xenografts of CD24-expressing human CRC cells
645 From Murine to Humanized, Big and Small, Unarmed and Conjugated: Novel and Promising Biological Treatment Tools for CD24-Expressing Malignancies Shiran Shapira, Sarah Kraus, Diana Kazanov, Itai Benhar, Nadir Arber Background: CD24 is a cell-surface heavily glycosylated GPI-anchored mucin-like protein. We had shown that CD24 is a valid target in GI malignancies (Gastro 2006, Clin Can Res 2007, Can Res 2008). Anti-CD24 mAb treatment induces a significant growth inhibition of colorectal (CR) and pancreatic (PC) cells, in a time- and dose-dependent manner as well as
AGA Abstracts
S-128
643
reduction of tumor growth in mAb-treated mice (Shapira et al, Gastro 2011). Aim: To engineer anti-CD24 chimeric and humanized full-length IgGs, smaller derivatives (scFv and Fab) and toxin-immunconjugates. Methods: Edman-degradation, cDNA synthesis, sequence and computational analysis (Ig-blast) were performed to reveal the entire DNA sequence of the murine Ab. Replacement of the Fc with the human IgG1 resulted in a mouse-human chimera. Human donor (Ig-blast) was chosen as scaffold human Ab for grafting critical sequences of the murine antibody into it. The immunotoxins were constructed first by noncovalent linking via the ZZ protein (derived from Staphylococcus aureus). Then the bacterial system allowed a covalent linkage between the toxic molecule and the Ab. Sophisticated and high-efficiency engineering-technologies were established to construct, express and purify 15 novel anti-CD24 derivatives (Fig. 1) in tissue or bacterial cultures. Their bindingaffinity, selective-targeting and cytotoxic activities were confirmed in several CD24-expressing GI tumors cells. Results: The chimeric and humanized forms have higher affinity than the murine Abs. Anti-CD24 antibodies work additively with standard chemotherapies in inhibiting cancer cell growth. The immunotoxins were superior to the unarmed Ab with lower IC50 values by several orders of magnitude. Antibody targeting and accumulation within the tumor and its excess clearance was clearly demonstrated using direct imaging (SWA11ZZ-mCherry-HIS fluobody derivative). The scFv-toxin killed CD24-expressing cells with an IC50 of 1μg/ml, while the values for the IgG-ZZ-toxin were much lower (0.02-0.05μg/ml) (Fig. 2). The values of the humanized anti-CD24(di/tetra)-PE38 were similar to those of the scFv (Fig. 3), suggesting that the absence of post-translational modifications, in the E.coliproducing IgG, affected reactivity. MTD studies in dozens of mice confirmed no toxicity at high dose of 1mg/mouse. Conclusion: Targeting CD24 may be a promising treatment for GI malignancies in combination with chemotherapy. Humanized mAb (unarmed and toxinconjugated) to CD24 are more effective than the murine parental Ab. Following toxicology studies in big animals we aim for a Phase 1 study in 2012.
In Esophageal Epithelium, the Transcription Factor FOXA2 is a Hedgehog Target Gene That May Mediate Columnar Metaplasia, as Occurs in Barrett's Esophagus David H. Wang, Monica Kim, Anjana Tiwari, Xi Zhang, Nicholas J. Clemons, David M. Berman, Stuart J. Spechler, Rhonda F. Souza Introduction: Cells change their phenotype during the process of metaplasia, often by reverting to a phenotype that was present in the organ during embryonic development. The embryonic esophagus is initially lined by columnar cells that are later replaced by squamous cells, and Barrett's metaplasia involves the change of esophageal squamous cells back into columnar cells in the setting of GERD. We have reported that mouse embryonic esophageal columnar epithelium is characterized by active Hedgehog signaling, and that forced expression of Sonic hedgehog (Shh) in the adult mouse esophagus leads to a columnar metaplasia. Our microarray analyses of mouse embryonic columnar esophageal epithelium have identified robust expression of Foxa2, an endodermal transcription factor involved in the development of intestinal goblet cells. We also have demonstrated that Hedgehog is activated in Barrett's metaplasia, which expresses functional FOXA2, and that FOXA2 is expressed in telomeraseimmortalized esophageal squamous cell lines (NES-B3T, NES-B10T) from patients with Barrett's esophagus. Now, using those cell lines and genetically-engineered mouse models, we have explored whether FOXA2 is a direct Hedgehog pathway target gene. Methods: We generated Shh knockout and Shh over-expressing transgenic mice. Shh knockout embryos were harvested from timed pregnant females, and adult mouse esophageal epithelium was cultured in a novel In Vivo transplant culture system previously described by our group. NES-B3T and NES-10T cells were treated either with the recombinant ligand SHH or induced to express GLI1, a downstream mediator of the Hedgehog pathway. FOXA2 protein expression was determined by immunohistochemistry, and FOXA2 mRNA levels were assessed by quantitative real-time PCR. Results: In wild type mice with intact Hedgehog signaling, Foxa2 protein is expressed in the columnar cells of the esophageal epithelium from embryonic day 11.5 through postnatal day 2. In contrast, Shh knockout mice have undetectable levels of Foxa2 protein at embryonic days 14.5 and 17.5. In In Vivo transplant cultures of adult mouse esophageal squamous epithelium, Foxa2 protein was not detected in cells of wild type mice, but was detected in cells of Shh over-expressing transgenic mice. In NES-B3T and NES-B10T human esophageal squamous cell lines, both treatment with recombinant SHH and overexpression of GLI1 substantially increased FOXA2 mRNA expression levels. Conclusions: Foxa2 is a target of Hedgehog signaling in the embryonic mouse columnar esophageal epithelium. In human esophageal squamous epithelial cells from patients with Barrett's esophagus, activation of Hedgehog signaling increases FOXA2 mRNA levels. These data suggest that FOXA2 is a downstream target of Hedgehog signaling that might contribute to the development of Barrett's metaplasia. 644 Amplification of 5FU Resistance by DNA Mismatch Repair Deficiency and Subsequent MBD4 Frameshift Mutation Moriya Iwaizumi, Stephanie Tseng-Rogenski, John M. Carethers Background and Aims. We and others have demonstrated recognition of 5-fluorouracil (5FU) in DNA by the DNA mismatch repair (MMR) system, which triggers a DNA MMRdependent cell death. Loss of DNA MMR contributes to 5FU resistance for colorectal cancer patients. MBD4 is a DNA glycosylase that can remove 5FU from DNA, but is a target gene for frameshift mutation with DNA MMR deficiency (A10→A9 in exon 3) forming a truncated MBD4 (MBD4tru). Here we explored if MBD4tru as well as wild type MBD4 (MBD4WT) and MMR proteins recognize 5FU within DNA. Methods. Utilizing HT29 (MMR-proficient, MBD4A10/A10), HCT116 (hMLH1-/-, MBD4A10/A9), and HCA7 (hMLH1-/-, MBD4A9/A9) cells, nuclear localization and expression of both MBD4WT (expressed in HT29 and HCT116, not in HCA7) and MBD4tru (expressed in HCT116 and HCA7, not in HT29) were confirmed by indirect immunofluorescence analysis and Western blotting. To determine relative repair protein binding to 5FU incorporated into DNA, biotin-labeled 5FdU:G heteroduplex dsDNA (5FU dsDNA) with or without unlabeled 5FU dsDNA (competitor 5FU) or C:G homoduplex dsDNA (control competitor) were incubated with nuclear lysate. Probe-bound proteins were collected with Streptavidin magnetic beads and eluted. Eluted proteins were utilized for Western blotting to analyze 5FU binding affinity. Results. MBD4tru bound to 5FdU:G, and was competed off by competitor 5FU but not by control competitor. Within MBD4A10/A9 cells, the 5FU binding affinity of MBD4tru was higher than that of MBD4WT (competed off rate by 5FU competitor; 79.3% for MBD4tru, 45.9% for MBD4WT). Moreover, between MBD4A9/A9 (only MBD4tru expression) and MBD4A10/A10 (only MBD4WT expression) cells, the 5FU binding affinity of MBD4tru was higher than that of MBD4WT cells (competed off rate by 5FU competitor; 90.2% for MBD4tru of MBD4A9/A9 cells, 42.71% for MBD4WT of MBD4A10/A10 cells), whereas that of MMR proteins were lower in MBD4A9/A9 cells (competed off rate by 5FU competitor; 40.2% for MSH2, 1.5% for MSH3, 0.3% for MSH6) than that of MBD4A10/A10 cells (competed off rate by 5FU competitor; 44.8% for MSH2, 23.4% for MSH3, 10.3% for MSH6). Conclusion. 5FU incorporated in DNA is recognized by and has higher affinity for MBD4tru than MBD4WT, and MBD4tru reduces the 5FU affinity of the DNA MMR proteins. Frameshift mutation of MBD4 forming MBD4tru amplifies the 5FU resistance of DNA MMR deficiency, possibly be preventing access to 5FU within DNA by repair proteins.
Figure 1. Schematic representation of the novel anti-CD24 derivatives that were developed in our lab.
Figure 2. (A) scFv(SWA11)-Toxin was tested for its cell killing activity by the MTT assay in CD24-expressing (HT29) and not-expressing cells (HCT116). (B) The cytotoxic activities of the PE38-based immunotoxins were evaluated by inhibition of protein synthesis as measured by the [3H]-Leucine incorporation. (C) Human anti-CD24-di(H)-PE38, Human anti-CD24-di(H)-PE38-di(L)-PE38 and scFv(SWA11)-PE38 derivatived were evaluated for specific targeting of CD24-expressing tumor cells in female athymic nude mice bearing xenografts of CD24-expressing human CRC cells
645 From Murine to Humanized, Big and Small, Unarmed and Conjugated: Novel and Promising Biological Treatment Tools for CD24-Expressing Malignancies Shiran Shapira, Sarah Kraus, Diana Kazanov, Itai Benhar, Nadir Arber Background: CD24 is a cell-surface heavily glycosylated GPI-anchored mucin-like protein. We had shown that CD24 is a valid target in GI malignancies (Gastro 2006, Clin Can Res 2007, Can Res 2008). Anti-CD24 mAb treatment induces a significant growth inhibition of colorectal (CR) and pancreatic (PC) cells, in a time- and dose-dependent manner as well as
AGA Abstracts
S-128