Pathogenic Mechanisms of Cancer Causing MLH1 Mutations Functional Relationship between DNA Mismatch Repair and Cancer-Risk Eddie O’Donnell Laboratory of Dr. Andrew B. Buermeyer Department of Environmental and Molecular Toxicology Image: Ribbon diagram of E. Coli MutL Protein (PDB)

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DNA Mismatch Repair Deficiencies in Colorectal Cancer Causes of Cancer Mutations within cells cause uncontrolled cell growth Risk of cancer development can be inherited Most cancers are sporadic (no family history) Colorectal Cancer Approximate percentages of occurrence 15 % - Mismatch Repair (MMR) deficiency observed 90 % of sporadic cases linked to MMR deficiency are MLH1 deficient (loss of expression) 2-5 % - Lynch Syndrome (HNPCC) Discoveries involving Lynch Syndrome 1993 – MSH2 mutations linked to HNPCC 1994 – MLH1 mutations linked to HNPCC *Account for majority of HNPCC occurrences

DNA mismatches arise from errors during DNA Replication MMR corrects replication errors MMR Stimulates apoptosis in response to DNA damage Basic Mechanism: Mismatch recognition MutS family MSH2/MSH6 MSH2/MSH3 G T * MLH1/MLH3 Strand choice MutL family MLH1/PMS2 MLH1/PMS1 Excision Exonucleases PCNA RPA T Resynthesis Replicative DNA polymerase A T Mechanism & Functions of DNA MMR G T ATP Dependant

DNA Synthesis Error Mutation Mutations Prevented by MMR Successful Repair Dinucleotide Loop Insertion via slip-mispairing AC TG No Repair, Additional Replication Insertion Mutation Insertion / Deletion Loops  Microsatellite Instability (MSI) GTGT No Repair, Additional Replication ATAT ATAT GCGC Incorrect insertion of base Successful Repair Base Mismatches  Base Substitution Mutations

Implications of MMR Deficiency for Cancer Screening & Treatment Chemotherapy Microsatellite Instability - An Effective Screening Tool Clinical Relevance of MLH1: HNPCC cases without MSI? 36, (2004) D132H MLH1 amino acid site 132 changed from D (Aspartic Acid) to H (Histidine) Loss of repeats Normal Tumor

Hypothesis: Initial Data Data from recent publications D132H apparently associated with 5-fold increased cancer risk Modest decrease in ATPase function in D132H Increased mutation rate not dramatic enough for MSI detection Base substitutions more affected than microsatellites Apoptosis signaling function more affected than error correction Attenuated MLH1 function of D132H increases cancer risk

Is there an observable phenotype associated with MLH1-D132H? Central Question Research Goals 2. Determine in vitro repair capabilities for MLH1 mutant D132H using biochemical assays 1. Use Cellular assays to evaluate the effect of the MLH1 mutation D132H in vivo

Project Outline Mutant MLH1 & Repair proteins І ІІ ІІІ Cellular Assays In Vitro repair Research will involve in vitro MMR reactions to model presumed replication errors and score repair efficiency of MMR proteins hMLH1- expressing cells Mlh1 -/- MEFs Cellular Assays Measure Repair Efficiency GTGT + Mismatch Substrates In Vitro Repair 1. Forward Mutation Rate 2. Cytotoxic Response Indirect Measurement of MLH1 activity Direct Measurement of MLH1 activity Cell-free extracts

Identification of Cell Lines Expressing MLH1 Mutants Western Blot Analysis of Extract Preparation Cell-free extracts Transfection hMLH1 Neo-R MLH1 Drug Resistance Mlh1 -/- MEFs Drug Selection D132H-8 D132H-9 MLH1 MLH1-13 (+) MLH1-2 (+) MC2A (-) PMS2 Screen for MLH1 Expression with Western Blotting Isolate and Expand Expressing Cell Lines for extract 2 D132H Lines identified. Expression is less than MLH1 wildtype lines.

Fluctuation Analysis: Forward Mutation to Ouabain R Cell Line Events/Cell/Generation (Rate) MLH1 (-/-)60 x WT hMLH1* ~ 1 x Hmlh1- D132H 0.7 ± 0.2 x ** * - ** - 12 Cultures (1000 Ouabain S cells) Expansion, Accumulation of Mutants Exposure to Ouabain Count number of Ouabain Resistant Clones, Calculate Rate of mutation * Rates in MEF cell line determined by Dr. Andrew Buermeyer, ** Assay Repeated Twice ~5 x 10 6 cells, includes some Ouabain R cells Conclusion: Expression of D132H decreased rate of base substitution

Response to Cytotoxic Agents : 6-Thioguanine Response Cells 24 Hours 6-Thioguanine 0-6 uM Doses 24 Hours Remove 6-Thioguanine Count Surviving Colonies 6-10 Days Conclusion: Expression of D132H increased cytotoxic response to 6-Thioguanine

In Vitro Mismatch Repair Assay CT T GAG GA G CTC Mismatch substrate incubated with repair factors from extracts - Mismatch Blocks activity of Restriction Endonuclease - 3’ Nick initiates repair, facilitates Strand choice nick Mismatch dependant nick directed excision Xho1 CT C GAG GA G CTC Pvu1 Resynthesis leads to restoration of Xho1 site Pvu1 Site used to facilitate analysis

Substrate preparation protocol developed in the Hay’s Laboratory, OSU Gels 1% TAE 8 cm, 170V, 30’ w/Stain & w/Destain (10’,30’ ) Preparation of Mismatch Substrates Xho1 CT T GAG GA G CTC nick A – Closed Circular Substrate B – Double Digest Conclusions  Substrate Preparation yields >95% Mismatch Substrate  Successful Preparation for G/T and CT Loop mismatches Starting Plasmid A B A B A B G/T Mismatch -CT- Loop Linear (Pvu1 Cut) Xho1 & Pvu1 Cut Pvu1 A

Results & Discussion I.Expression of D132H in MLH1 deficient cells: 1) Reduced mutation rate similar to wildtype expressing cells, suggesting good repair activity in vivo 2) Increased cytotoxic response to 6-Thioguanine with a modest decrease in response relative to wildtype expressing cells -Protein Expression? II. In Vitro Repair 1) Substrates Prepared, Assays in Progress Future Work  Repair Assays  Additional D132H expressing lines for cellular assays

Acknowledgments Dr. Andrew Buermeyer Buermeyer Lab Group Xin Huo Hays Lab Group Pete Hoffman Huixian Wang Howard Hughes Medical Institute Dr. Kevin Ahern