The Fanconi Anemia (FA) pathway consists of proteins involved in repairing DNA damage, including interstrand cross-links (ICLs). The pathway contains an upstream multiprotein core complex that mediates the monoubiquitylation of the FANCD2 and FANCI heterodimer, and a downstream pathway that converges with a larger network of proteins with roles in homologous recombination and other DNA repair pathways. Selective killing of cancer cells with an intact FA pathway but deficient in certain other DNA repair pathways is an emerging approach to tailored cancer therapy. Inhibiting the FA pathway becomes selectively lethal when certain repair genes are defective, such as the checkpoint kinase ATM. Inhibiting the FA pathway in ATM deficient cells can be achieved with small molecule inhibitors, suggesting that new cancer therapeutics could be developed by identifying FA pathway inhibitors to treat cancers that contain defects that are synthetic lethal with FA.

A defining characteristic of FA cells is hypersensitivity to cross-linking agents, such as the chemotherapeutic agent cisplatin [2, 5]. Cisplatin (and other platinum-based compounds) has been used as a chemotherapeutic drug for over 30 years (for review see [18]). The toxicity of platinum-based chemotherapy (nephrotoxicity, neurotoxicity, and ototoxicity) and development of cisplatin resistance are limitations of the therapy [18–20]. Once inside the cell, cisplatin enters the nucleus and forms covalent DNA interstrand cross-links via platinum-DNA adducts. These cross-links block ongoing DNA replication, and in the absence of repair, activate apoptotic pathways [18, 19]. A functional FA pathway is required for processing damage after exposure to cisplatin and other crosslinking agents, and is at least partially responsible for resistance to cisplatin. Cell-free and cell-based assays have identified inhibitors of the FA pathway, and some of these inhibitors can resensitize platinum-resistant tumors and cell lines [19, 21, 22]. Further efforts to identify small molecule compounds that specifically inhibit the FA pathway could lead to improved resensitization from treatment-induced resistance.

In addition to sensitization, inhibiting the FA pathway may be an effective strategy to exploit synthetic lethal interactions aimed at improving targeted killing of tumor cells. Current approaches in cancer treatment are generally not selective, affecting both cancer cells and normal cells. However, inactivation of DNA repair pathways, an event that occurs frequently during tumor development [23], can make cancer cells overdependent on a reduced set of DNA repair pathways for survival. There is new evidence that targeting the remaining functional pathways by using a synthetic lethal approach can be useful for single-agent and combination therapies in such tumors. Two genes have a synthetic lethal relationship if mutants for either gene are viable but simultaneous mutations are lethal [20]. A successful example of this approach is specific targeting of BRCA-deficient tumors with PARP (poly (ADP-ribose) polymerase) inhibitors [24].

Inhibition of the FA pathway could occur at any point in the multistep FA protein network, but a key predictive readout for FA function and resistance to ICLs is the monoubiquitylation of FANCD2 [11, 55]. Several inhibitors of FANCD2 monoubiquitylation have been identified including proteasome inhibitors bortezomib and MG132, curcumin, and the curcumin analogs EF24 and 4H-TTD [19, 22, 56, 57]. Curcumin, a natural product derived from turmeric, was identified as a weak inhibitor of FANCD2 monubiquitylation in a cell-based screen [19]. We developed a cell-free assay in Xenopus egg extracts to screen small molecules for stronger and more specific inhibitors of FANCD2 monubiquitylation. Unlike cell-based screening assays for small molecules capable of inhibiting the FA pathway, the cell-free method uncouples FANCD2 monoubiquitylation from DNA replication, thus focusing more specifically on the key biochemical steps in a soluble context enriched for nuclear proteins and capable of full genomic replication [22]. Screening in egg extracts identified 4H-TTD, a compound with structural similarity to curcumin as an inhibitor, and this inhibitory effect was verified in human cells [22, 57]. A series of curcumin analogs were also tested, including EF24, a potent monoketone analog of curcumin [58, 59]. The prediction that an FA inhibitor would selectively kill ATM-deficient cells was tested in cell-based assays for synthetic lethality in ATM-proficient and ATM-deficient cells. ATM-deficient cells treated with EF24 demonstrated an increased sensitivity compared to ATM wt cells (see Figure 3) [22, 57]. The increased lethality in ATM-deficient cells provides evidence for future synthetic lethal approaches with FA pathway inhibitors in the treatment of ATM-deficient tumors, and other tumors with deficiencies in genes that are synthetically lethal with FA (see Table 1) [6, 52].

Understanding how the Fanconi anemia pathway functions in concert with other DNA damage response networks is essential for understanding genomic stability and for exploiting synthetic lethality for new cancer treatments. New chemotherapeutic agents could be developed by identifying potent and specific inhibitors of the FA pathway, for example, by screening for compounds that inhibit key FA pathway steps (e.g., monoubiquitylation and deubiquitylation of FANCD2/FANCI). While a long-term defect in the function of the FA pathway would result in genomic instability, short-term inhibition could provide a treatment strategy for tumors with deficiencies in certain other DNA repair pathways. Stringent identification of additional genes with synthetic lethal relationships with the FA pathway, and identification of malignancies with deficiencies or mutations in genes that are synthetic lethal with FA will be required for these tailored therapeutic approaches.