DNA STRAND BREAKS AND THE GENETIC BASIS OF LYMPHOMAS

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Mammalian cells respond to DNA double-strand breaks (DSBs) by activating pathways for cell cycle arrest and DNA repair. The signal for cell cycle arrest requires the ATM and p53 genes, which are mutated in at ataxia telangiectasia and Li-Fraumeni syndrome. DSB repair requires four genes: XRCC4, XRCC5 (Ku86), XRCC6 (Ku70), and XRCC7 (DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase), the latter of which is mutated in the scid mouse. Ataxia telangiectasia and Li-Fraumeni patients and the scid mouse are highly susceptible to lymphoma. Thus, a small fraction of lymphomas must arise from germ line mutations in one of the DSB response genes. This proposal will test the hypothesis that a significant fraction arises from somatic mutations in these genes. The specific aims are to: 1.1. Test lymphoma tumors for biochemical abnormalities in pathways responding to DSBs. Lymphomas will be screened for biochemical abnormalities in the known DSB response genes. The assays are rapid and sensitive to mutations in these and other genes in the DSB response pathway. The assays will test DNA end-binding activity for Ku, assembly of DNA-PK on DNA ends and its enzymatic activity. Immunoblots will evaluate the Ku, DNA-PKcs, p53, and ATM proteins, which are often altered in stability of size by mutations. 1.2. Test lymphoma tumors for genetic abnormalities in pathways responding to DSBs. Lymphoma tumors will be tested for mutations in the DSB response genes. The hunt for mutations will be facilitated by new technology, which consists of denaturing high performance liquid chromatography and is capable of detecting mutations with greater than 98 percent sensitivity much more rapidly than conventional methods. 1.3. Correlate abnormalities in pathways responding to DSBs with clinical and other lab findings. Surprisingly, most diffuse lymphomas utilize the VH4.21 immunoglobulin gene. Since ATM mutations lead to aberrant V(D)J recombination, this proposal will test if they also lead to biased VH4.21 usage. Since many lymphomas do not have p53 mutations, this proposal will test whether the remaining lymphomas have mutations in ATM, which acts in the same signaling pathway. Since DSB response genes confer resistance to key anticancer agents, this proposal will test whether mutations in these genes affect clinical outcome. The long term goal is to define the genetic changes that mediate malignant progression of lymphomas. Hopefully, molecular analysis of individual lymphomas will some day lead to the cure of more patients.