Identification of critical cellular pathways triggered by mitomycins interstrand crosslinks

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This proposal’s objective is to identify critical cellular pathways triggered by the presence of DNA interstrand crosslinks (ICLs) produced by mitomycins, and to identify how the structure of mitomycins ICLs determines the cellular signaling leading to cell death/cell cycle arrest in the absence of a functioning p53. This study includes mitomycin C (MC), an anti-cancer drug currently used in the clinics, decarbamoyl mitomycin C (DMC), a derivative of mitomycinc (MC) and a novel MC-derivative, MC-Lex, synthesized in our laboratory. These compounds form highly cytotoxic interstrand crosslinks (ICLs) that share common structural features. MC forms the α-ICL with DNA, DMC the β-ICL and MC-Lex the γ-ICL. Representative conformations of the three ICLs indicate that the level of DNA perturbation induced by each ICL increases in the order α<β<γ, with the α-ICL inducing minimal DNA perturbation and the γ-ICL shows significant DNA distortion and widening of the minor groove. Previous research has shown that the three drugs (MC, DMC, MC-Lex) also differ in their cytotoxicity and p53 signaling. Since ICLs are the lesions primarily responsible for the cytotoxicity of mitomycins, this indicates that the three ICLs could trigger very diverse biochemical cellular mechanisms, mediated by specific mutations present in cancer cells, a hallmark of modern cancer therapy. The proposed research will establish how the ICLs behave as biological signals to trigger different cell death/cell cycle arrest pathways. Knowledge of how differences in the modification of the local DNA structure by mitomycins correlates with their cytotoxicity is crucial for understanding the action of clinically used drugs. Therefore, the study of these three structurally different ICLs (α/β/γ ICLs) provides an ideal model for identifying structural features determining the cell-signaling outcome in the presence or the absence of a functioning p53 pathway. Since p53 tumor suppressor is mutated in more than 50% of human cancers, the need to identify cellular pathways that induce cell death or cell cycle arrest independently of p53 deserves substantial attention. This has the potential to identify treatments that could be tailored to cancer cells with mutations in the p53 gene for personalized treatment consideration based on the genetic makeup of a tumor. To correlate ICL structures with signaling outcome, we will be pursue the following aims: Specific aim #1: Synthesis of MC, DMC and MC-Lex ICLs (α,β,γ-ICLs) using biomimetic methods. Specific aim #2: Determination of p53-dependent and independent DNA adducts (ICLs) response mechanisms using quantitative label free proteomics and bioinformatics Specific aim #3: Mechanistic of CHK1 mediated cell cycle arrest triggered by MC/DMC and α/β-ICL Expected outcomes: This study will identify structural ICL features that determine the cell signaling outcome in the presence or the absence of a functioning p53 pathway, a feature of particular interest for the development of personalized anticancer therapy.