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Supplementary Figures 1-4 from Deficiencies in the Fanconi Anemia DNA Damage Response Pathway Increase Sensitivity to HPV-Associated Head and Neck Cancer

DNA repair defects are frequently encountered in human cancers. These defects are utilized by traditional therapeutics but also offer novel cancer treatment strategies based on synthetic lethality. To determine the consequences of combined Fanconi anemia (FA) and mismatch repair pathway inactivation, defects in Fancd2 and Mlh1 were combined in one mouse model. Fancd2/Mlh1 double-mutant embryos displayed growth retardation resulting in embryonic lethality and significant underrepresentation among progeny. Additional inactivation of Trp53 failed to improve the survival of Fancd2/Mlh1–deficient embryos. Mouse fibroblasts were obtained and challenged with cross-linking agents. Fancd2-deficient cells displayed the FA-characteristic growth inhibition after mitomycin C (MMC) exposure. In primary fibroblasts, the absence of Mlh1 did not greatly affect the MMC sensitivity of Fancd2-deficient and Fancd2-proficient cells. However, in Trp53 mutant immortalized fibroblasts, Mlh1 deficiency reduced the growth-inhibiting effect of MMC in Fancd2 mutant and complemented cells. Similar data were obtained using psoralen/UVA, signifying that MLH1 influences the cellular sensitivity to DNA interstrand cross-links. Next, the effect of MLH1 deficiency on the formation of chromosomal aberrations in response to cross-linking agents was determined. Surprisingly, Mlh1 mutant fibroblasts displayed a modest but noticeable decrease in induced chromosomal breakage and interchange frequencies, suggesting that MLH1 promotes interstrand cross-link repair catastrophe. In conclusion, the combined inactivation of Fancd2 and Mlh1 did not result in synthetic lethality at the cellular level. Although the absence of Fancd2 sensitized Mlh1/Trp53 mutant fibroblasts to MMC, the differential survival of primary and immortalized fibroblasts advocates against systemic inactivation of FANCD2 to enhance treatment of MLH1-deficient tumors. [Cancer Res 2009;69(24):9431–8]

Supplementary Figure Legends 1-4 from Embryonic Lethality after Combined Inactivation of Fancd2 and Mlh1 in Mice

Supplementary Figure 1 from Embryonic Lethality after Combined Inactivation of Fancd2 and Mlh1 in Mice

Supplementary Figure 4 from Embryonic Lethality after Combined Inactivation of Fancd2 and Mlh1 in Mice

Supplementary Figure 2 from Embryonic Lethality after Combined Inactivation of Fancd2 and Mlh1 in Mice

Supplementary Figure 3 from Embryonic Lethality after Combined Inactivation of Fancd2 and Mlh1 in Mice

DNA repair defects are frequently encountered in human cancers. These defects are utilized by traditional therapeutics but also offer novel cancer treatment strategies based on synthetic lethality. To determine the consequences of combined Fanconi anemia (FA) and mismatch repair pathway inactivation, defects in Fancd2 and Mlh1 were combined in one mouse model. Fancd2/Mlh1 double-mutant embryos displayed growth retardation resulting in embryonic lethality and significant underrepresentation among progeny. Additional inactivation of Trp53 failed to improve the survival of Fancd2/Mlh1–deficient embryos. Mouse fibroblasts were obtained and challenged with cross-linking agents. Fancd2-deficient cells displayed the FA-characteristic growth inhibition after mitomycin C (MMC) exposure. In primary fibroblasts, the absence of Mlh1 did not greatly affect the MMC sensitivity of Fancd2-deficient and Fancd2-proficient cells. However, in Trp53 mutant immortalized fibroblasts, Mlh1 deficiency reduced the growth-inhibiting effect of MMC in Fancd2 mutant and complemented cells. Similar data were obtained using psoralen/UVA, signifying that MLH1 influences the cellular sensitivity to DNA interstrand cross-links. Next, the effect of MLH1 deficiency on the formation of chromosomal aberrations in response to cross-linking agents was determined. Surprisingly, Mlh1 mutant fibroblasts displayed a modest but noticeable decrease in induced chromosomal breakage and interchange frequencies, suggesting that MLH1 promotes interstrand cross-link repair catastrophe. In conclusion, the combined inactivation of Fancd2 and Mlh1 did not result in synthetic lethality at the cellular level. Although the absence of Fancd2 sensitized Mlh1/Trp53 mutant fibroblasts to MMC, the differential survival of primary and immortalized fibroblasts advocates against systemic inactivation of FANCD2 to enhance treatment of MLH1-deficient tumors. [Cancer Res 2009;69(24):9431–8]

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure and an increased susceptibility to cancer. FA is genetically heterogeneous, consisting of at least 11 complementation groups, FA-A through L, including FA-D1 (BRCA2) and D2. We have previously reported an increased incidence of epithelial tumors in Fancd2 knockout mice. To further investigate the role of the FA pathway in tumor prevention, Fancd2 mutant mice were crossed to mice with a null mutation in the tumor suppressor gene, Trp53. The tumor spectrum in Fancd2−/−/Trp53+/− mice included sarcomas expected in Trp53 heterozygotes, as well as mammary and lung adenocarcinomas that occur rarely in Trp53 heterozygotes. These tumors occurred earlier than in Fancd2−/− control mice. Therefore, the Fancd2−/−/Trp53+/− mice represent an improved model for the study of adenocarcinoma in FA. In addition, it was found that Fancd2−/− mouse embryonic fibroblasts but not Fancd2−/−/Trp53−/− mouse embryonic fibroblasts arrest following DNA damage. Therefore, Trp53 is required for the S phase checkpoint activation observed in Fancd2 mutant cells. Fancd2−/−/Trp53−/− cells showed an increase in aneuploidy and had multiple gross chromosomal rearrangements.

Experimental results generated from human pancreatic ductal adenocarcinoma (PDAC) specimens have produced a broad knowledge of pancreatic tumors. However, successful personalized treatment of pancreatic disease will require an exceptionally deep knowledge of the molecular and cellular diversity of tumors, as well as their evolution through the course of disease progression, therapeutics, relapse, and terminal disease burden. To address this need, we have conducted diverse but complementary experimental analyses on adjacent regions of fresh tumor specimens collected from over 70 primary pancreas specimens obtained during Whipple and RAMPS procedures. Coordination between surgical and research teams has ensured that only a minimum time elapses between surgical resection and specimen processing, thus limiting potential degradation by pancreatic enzymes. Researchers with diverse cancer biology expertise obtain adjacent dissections from three regions (i.e., tumor, dysplastic, and normal) within each pancreas specimen. An annotated image of the whole pancreas specimen records the location of each dissected portion relative to one another. Fresh tissues are immediately used to 1) characterize the phenotypic diversity of tumor and non-tumor cells in the microenvironment, 2) isolate and culture cancer stem cells, 3) perform high resolution imaging including 3D-SEM, 4) “bioprint” and culture three-dimensional, multi-cell-type structures, and 5) propagate tumors in mouse avatars. Genomics, epigenomics, and transcriptomics analyses are also performed on laser-captured specimens. In addition to primary PDAC tumors, other fresh pancreatic cancer specimens are being added to the collection including core needle biopsies obtained from treatment-naïve patients, metastases removed during primary tumor resection or recurrence, and tumor specimens from a rapid autopsy program. Importantly, all specimens are fully clinically annotated as patients are followed through the course of their disease. By comprehensively characterizing individual pancreatic tumors from many patients we hope to produce a unique body of information – an “Atlas” of pancreatic disease – that will inform the research community of new molecular and cellular features that contribute to the progression and therapeutic resistance of this devastating disease. Research data and clinical information in the Atlas will be accessible through a user-friendly customizable database. We hope that this resource will support the development of molecularly targeted early detection, therapeutics, and prevention in order to improve patient care. Funding for this initiative comes from philanthropic support to the Brenden-Colson Center for Pancreatic Care at Oregon Health and Science University. Citation Format: Jason M. Link, Brittany Allen-Petersen, Andrew Gunderson, Danielle Jorgens, Craig Dorrell, Jody Hooper, Philip Streeter, Markus Grompe, Lisa Coussens, Joe Gray, Hope Hardaker, Charles D. Lopez, Rosalie C. Sears, Brett C. Sheppard. Developing a molecular and cellular atlas of pancreatic disease. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B118.

Fanconi's anemia (FA) is a genetically heterogeneous inherited disorder characterized by bone marrow failure, birth defects and cancer susceptibility. At the cellular level, FA features hypersensitivity to DNA cross-linking agents, cell cycle abnormalities and abnormal responses to cytokines. Therapy for the hematopoietic defects of FA has consisted of supportive care, stem cell transplantation and administration of androgens such as oxymetholone. Unfortunately, even successfully transplanted FA patients have a very high risk for subsequent solid tumors, especially squamous cell carcinomas of oropharyngeal and urogenital areas. The pharmacological treatment of FA has been unchanged for several decades and for this reason our group has embarked on systematic search for small molecule therapeutics that could benefit FA patients. This effort has been made possible by the cloning of 16 FA genes and biochemical insights into the function of FA proteins. Mouse models, zebra fish models and FA patient cell lines, including IPSC are available for screening and validation. The specific goals of small molecule interventions are to delay or prevent hematopoietic failure and also to delay or prevent carcinogenesis. To date we have shown that the superoxide dismutase mimetic drug Tempol can significantly delay tumor formation in FA mice. Similarly, resveratrol clearly improved hematopoiesis in the same model. Medium throughput screens have revealed numerous new molecules which ameliorate the FA phenotypes of cells, mice and zebrafish respectively. Using genetic approaches we also have identified the p53 pathway as a therapeutic target in FA. These therapeutic opportunities and progress in FA small molecule screening will be discussed. Citation Format: Markus Grompe, John Postlethwait, Alan D'Andrea, Susan Olson, Qingshuo Zhang, Grover Bagby. Fanconi's anemia: Emerging therapeutic opportunities. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA05. doi:10.1158/1538-7445.CANSUSC14-IA05