TLR8-dependent TNF-α overexpression in Fanconi anemia group C cells

Abstract

Tumor necrosis factor alpha (TNF-␣) production is abnormally high in IntroductionThe Fanconi anemia (FA) proteins play an important role in regulating genome stability, 1 but there is little evidence that the loss of the genoprotection per se in FA cells accounts for the molecular pathogenesis of the bone-marrow failure characteristic of this disease. In fact there is evidence that at least some of these proteins are multifunctional 2 and participate in canonical signaling pathways in hematopoietic cells. [2][3][4][5][6][7][8] Fanconi anemia, complementation group C (FANCC)-deficient cells, for example, are hypersensitive to the apoptotic effects of tumor necrosis factor-␣ (TNF-␣). [4][5][6][7][8][9] In addition, FA cells overproduce TNF-␣ for reasons that have not yet been fully explained. [10][11][12] Most importantly, there is clear evidence that overproduction of and hypersensitivity to TNF-␣ in hematopoietic cells of Fancc Ϫ/Ϫ mice results in bone marrow hypoplasia 13,14 and that long-term ex vivo exposure of murine Fancc Ϫ/Ϫ hematopoietic cells to both growth factors and TNF-␣ results in the evolution of cytogenetically marked preleukemic clones. 9 Therefore, the hematopoietic phenotype of FA may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive. We designed gene expression microarray experiments by using marrow cells from both patients with FA and normal volunteers in part to seek potential clues to the mechanisms by which FA cells overproduce TNF-␣.Recognizing that transcriptomal analysis would not reveal aspects of the FA phenotype that were controlled translationally or posttranslationally, we also conducted a proteomics analysis. We based our experimental design on an accepted function of the FA "nuclear core complex," that is, its capacity to facilitate monoubiquitinylation of both Fanconi anemia, complementation group I and Fanconi anemia, complementation group D2 (FANCD2). 15,16 Although it is clear that monoubiquitinylation, at least of FANCD2, is required for the avoidance of genotoxicity, 17 it seemed to us unlikely that 8 individual FA genes encoding the "core complex proteins" should have evolved to control the monoubiquitinylation of merely 1 or 2 nuclear proteins. Therefore, reasoning that ubiquitinylation of a variety of other proteins might also be influenced by the core FA proteins, we designed a proteomics survey of ubiquitinylated proteins in FA-C cells and isogenic controls. We reasoned that this approach might lead to the identification of other proteins underubiquitinylated in mutant cells. As reported herein, the gene expression microarray analysis revealed a significant overrepresentation of overexpressed ubiquitin pathway genes in the mutant cells. We therefore took into account the alternative possibility that some ubiquitinylated proteins might be found uniquely in the mutant cells.Indeed, one such protein, Toll-like receptor 8 (TLR8), did appear in the ubiquitin-positive fractions only in FANCC-mutant cells. Given that TLR8 activ...

TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms

“…FA patients are hypersensitive to and have elevated levels of TNF␣, which promotes clonal evolution and leukemogenesis. [22][23][24][25] The combination of genetic instability and cytokine hypersensitivity creates an environment that supports the selection of leukemic over nonleukemic stem cells. 26,27 Malignant clones that arise are not only TNF␣-resistant, but give rise to progeny that over-produce TNF␣, further augmenting the selective pressure for expansion of the malignant clone over the hypersensitive parental stem cell pool.…”

Fancd2 −/− mice have hematopoietic defects that can be partially corrected by resveratrol

“…34 This system was suitable for smallmolecule screening and permitted us to test the capacity of resveratrol to influence that phenotype in FANCC-deficient cells. In the absence of resveratrol, imidazoquinoline resiquimod R848 induced the overexpression of TNF-␣ in FANCC-deficient THP1 cells, as quantified by TNF-␣ ELISA.…”

“…on April 3, 2019. by guest www.bloodjournal.org From ELISA TNF-␣ levels were measured with enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) as previously described. 34 Fancc shRNAtreated or nontargeted control shRNA-treated THP1 cells were stably integrated with the shRNA-expressing cassette. R848 (imidazoquinoline resiquimod; AXXORA) was used to induce TNF-␣ production.…”

FANCL ubiquitinates β-catenin and enhances its nuclear function

“…1 Although FANC proteins are known to execute a normal DNA damage response to crosslinking agents, emerging evidence points to alternative functions for these proteins in hematopoietic stem cells (HSCs), and the loss of these alternative functions may represent a driving force behind the common FA complications of myelodysplasia and acute myeloid leukemia. [11][12][13][14][15][16] Functional defects in FA HSCs exist, including decreased repopulating ability, reduced numbers of HSCs, defective homing capacity, tumor necrosis factor (TNF)-␣ hypersensitivity, and limited replicative and survival potential compared with normal HSCs. [17][18][19][20][21][22][23][24][25][26] Collectively these studies support the argument that the FA pathway has a role in maintaining the HSC pool and regulating stem cell fitness.…”