p38 MAPK inhibition suppresses the TLR-hypersensitive phenotype in FANCC- and FANCA-deficient mononuclear phagocytes

Abstract

Fanconi anemia, complementation group C (FANCC)-deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNF␣, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNF␣ in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist-stimulated FANCCand Fanconi anemia, complementation IntroductionBM failure is a nearly universal complication of Fanconi anemia (FA), an inherited disease caused by biallelic inactivating mutations of any one of 15 genes. [1][2][3][4] FA gene products collectively facilitate responses to DNA damage, 1 and therefore it is often presumed (notwithstanding a lack of direct evidence supporting the idea) that hematopoietic defects simply reflect attrition of hematopoietic stem cells (HSCs) that have specifically suffered excessive DNA damage. An alternative explanation is that the FA proteins are multifunctional and play a direct role in stem cell maintenance, and therefore, DNA damage in FA HSCs is not necessarily required to suppress their function. [5][6][7][8][9] In normal cells, for example, Fanconi anemia, complementation group C (FANCC) modulates the hematopoietic inhibitory effects of TNF␣, IFN␥, and MIP-1␣, each of which normally function to suppress hematopoiesis. 6,10-12 FANCC influences TNF␣ responsiveness at least in part by modulating the activation state of the IFN-inducible double-stranded RNA activated protein kinase. 13 FANCC also suppresses the activation potential of certain TLR pathways in normal mononuclear phagocytes. 14 Therefore, in hematopoietic tissues, FANCC deficiency results in a TLR-dependent overproduction of TNF␣, one of the cytokines to which the stem-cell pool is uniquely intolerant. 10,[15][16][17][18][19] These abnormalities are important elements in the pathogenesis of BM failure. 6,20 There is also experimental evidence that this TNF␣-inhibitory loop is a selective pressure that enhances the ultimate emergence of TNF-resistant leukemic and preleukemic clones. [21][22][23] Therefore, interdiction of TNF␣-induced BM failure, particularly in ways that might have an additional favorable influence on IFN␥-and MIP-1␣-activated signaling pathways, may improve BM function and reduce the likelihood of clonal evolution by improving the fitness landscape and altering the coefficient of selection. 21 Seeking small molecules with these attributes, in the present study we exploited the TLR-hypersensitive phenotype as a screening tool to identify therapeutic agents that might suppress that pathway in FA cells. Using a TLR8-hypersensitive, FANCCdeficient mononuclear phagocyte cell line that we described previously, 14 we screened 75 small molecules, approximately 50 of which were kinase inhibitors. We identified 2 inhibitors, BIRB 796 and dasatinib, that functioned to suppress the TLR-dependent overproduction...

Elevated levels of STAT1 in Fanconi anemia group A lymphoblasts correlate with the cells' sensitivity to DNA interstrand crosslinking drugs

“…Indeed, a recent study was designed to find inhibitory molecules able to reduce the production of TNFα in FA patients and, therefore, retard the patients' progressive bone marrow failure. 8 In the present study, we found evidence of overexpression of STAT1 in FANCA-deficient cells and how this may explain the hypersensitivity of FA-A cells to both genotoxic agents and to inflammatory cytokines.…”

“…[4][5][6] Thus, two different mechanisms are nowadays considered to participate in the bone marrow failure of FA patients: on the one hand, FA cells accumulate DNA damage in each division, which results in exacerbated p53 activation and apoptosis of their hematopoietic progenitors 7 and, on the other hand, FA patients overproduce inhibitory cytokines, which eventually degrade their bone marrow. 8 Signal transducers and activators of transcription (STAT) are transcription factors activated by phosphorylation triggered by ligand-bound cell surface receptors. STAT factors mediate signaling initiated by many extracellular stimuli including cytokines and growth factors and induce responses such as cell proliferation, differentiation and apoptosis.…”

FANCA and FANCC modulate TLR and p38 MAPK–dependent expression of IL-1β in macrophages

“…The development and culture conditions of the derivative cell lines we established (T-shNT, T-shFA, and T-shFC) were described previously. 16,17 BM-derived macrophages (BMDM) from wild-type and Fancc 2/2 mice were prepared as described previously. 17 Murine macrophages were cultured at a concentration of 50 000 cells per mL for 24 hours in RPMI 1640 medium (Life Technologies, Carlsbad, CA) with 10% fetal bovine serum (FBS; Thermo Scientific Hyclone, Logan, UT) in the presence of the indicated doses of R848.…”

“…16,17 BM-derived macrophages (BMDM) from wild-type and Fancc 2/2 mice were prepared as described previously. 17 Murine macrophages were cultured at a concentration of 50 000 cells per mL for 24 hours in RPMI 1640 medium (Life Technologies, Carlsbad, CA) with 10% fetal bovine serum (FBS; Thermo Scientific Hyclone, Logan, UT) in the presence of the indicated doses of R848. All murine studies were approved by the Portland Veteran's Administration Institutional Animal Care and Use Committee.…”

“…8 The FA gene products participate in DNA damage response pathways and may protect hematopoietic cells from damage induced by the production of endogenous aldehydes. 9,10 There is also evidence that at least some of the FA proteins function in hematopoietic cells to modulate responses to suppressive cytokines in progenitor cells, [11][12][13][14][15] and Toll-like receptor (TLR) responses in macrophages, 16,17 even in the absence of DNA damage. [18][19][20] Thus, deficiency of either FANCA or FANCC in macrophages results in the overproduction of at least 1 growthsuppressive cytokine, tumor necrosis factor a (TNF-a), to which FA BM progenitors are inherently hypersensitive.…”