Deficiency of the c-JUN Inhibitor DACH1 in FA Macrophages Accounts for the Cytokine Overproduction Phenotype

The inflammatory cytokines TNFα and IL-1β contribute to the bone marrow failure phenotype in Fanconi anemia (FA) as well as to clonal evolution to MDS and AML. Mononuclear phagocytes deficient in FANCA or FANCC overproduce these cytokines in response to toll-like receptor agonists (TLRa), but the precise molecular mechanisms by which FA proteins suppress cytokine production remains enigmatic. We show here that pretreatment of control macrophages with interferon (IFN) α enhances (by 3-fold) TLRa-induced production of both TNFα and IL-1β. FANCC-deficient cells produce more of these proteins (by 3-fold) after exposure to TLRa alone and IFNα does not enhance production of these cytokines. We therefore hypothesized that TLR stimulation of FANCC-deficient cells activates an IFNα-like pathway, one constrained by FANCC in normal cells. To test this notion, we performed gene expression microarray analysis (Affymetrix HTA 2.0) using RNA from FANCC-deficient (T-shFC) and control (T-shNT) THP-1 human mononuclear phagocytes treated with IFNα, the TLR7/8 agonist R848, or a combination of IFNα plus R848. We found that treatment of T-shFC cells with R848 alone was sufficient to enhance expression of 49 genes that were activated by IFNα in control cells. Thirteen of these genes were not induced by R848 in control cells but were induced by R848 in T-shFC cells (one of which, IFNgamma-inducible protein 30 (IFI30), is known to be overexpressed in Fancd2 -/- progenitor cells). These results support our hypothesis that FANCC functions normally to constrain IFNα pathway activation in TLRa-activated macrophages. Of greater functional importance vis-à-vis cytokine production, DACH1 mRNA was suppressed by 11-fold and DACH1 protein was barely detectable in FANCC-deficient cells under all conditions when compared to control cells. DACH1 is known to bind to and suppress the activities of a variety of transcription factors, notably c-JUN in fibroblasts. We tested the hypothesis that DACH1 deficiency is sufficient to account for the TLRa hypersensitivity of FA macrophages using gain- and loss-of-function studies. Knockdown of DACH1 in control (T-shNT) macrophages resulted in a 7-fold enhancement of R848-induced TNFα production, increased c-JUN protein (1.6-fold) and c-JUN phosphorylation (2.6-fold). Reporter gene expression (secreted embryonic alkaline phosphatase [SEAP]) from constructs containing both AP-1 and NF-κB sites was activated by DACH1 knockdown but deletion of the AP-1 site completely abrogated activation. Results of gain-of-function experiments with wild-type and mutant DACH1 cDNA sequences now taking place will be reported. Taken together, these results suggest that FANCC directly or indirectly enhances ground-state expression of DACH1 and thereby suppresses the TLR pathway in normal cells specifically by inhibiting R848-dependent activation of c-JUN. Conversely, loss of DACH1 expression in FANCC-deficient cells leads to unconstrained c-JUN activity, resulting in overproduction inflammatory cytokines. In light of the contribution of such cytokines to both bone marrow failure and clonal evolution, activation of DACH1 gene expression is a rational therapeutic objective in the management of patients with Fanconi anemia and is worthy of investigating in preclinical (animal) models.