TNFα Overproduction in FANCC-Deficient Cells Is TLR8 Dependent.
Authors: Vanderwerf Scott , Svahn Johanna , Anur Praveen , Pasquini Ricardo , Bagby Grover C. 
Published: October 2019

Abstract
Abstract 494
The Fanconi anemia (FA) proteins play a role in regulating genome stability but it is not clear that loss of genoprotection in FA hematopoietic cells accounts for the molecular pathogenesis of bone marrow failure so characteristic of this disease. Other factors are known to influence survival and replication of FA stem cells. For example, not only are FA progenitors and stem cells hypersensitive to the apoptotic effects of TNFα, FA cells over-produce TNFα. Most importantly over-production of and hypersensitivity to TNFα in hematopoietic cells of Fancc-/- mice results in bone marrow hypoplasia 1;2 and long-term ex-vivo exposure of murine Fancc -/- hematopoietic cells to both growth factors and TNFα results in the evolution of cytogenetically marked preleukemic clones.3 Therefore, the hematopoietic phenotype of FA is likely multifactorial and may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive.

Methods:
We sought to clarify the molecular basis of aberrant TNFα-production. We conducted gene expression microarray experiments using RNA samples from low density marrow cells obtained from 11 normal volunteers and 22 Fanconi anemia patients with uncomplicated marrow hypoplasia without clonal cytogenetic defects. Because the FA complex is known to enhance ubiquitinylation of FANCD2, we reasoned that the ubiquitinylation state of proteins involved in the TNF pathways might also be influenced by core FA proteins. Therefore, we conducted in vitro ubiquitinylation assays using hexahistidine-tagged ubiquitin and an ATP-recycling system added to lysates of FANCC-deficient lymphoblasts (HSC536) and control cells (isogenic cells complemented with WT FANCC cDNA). Following the ubiquitinylation reaction, ubiquitinylated proteins were affinity purified, digested and analyzed by 2D capillary LC-MS/MS. Mass spectra were obtained and peptide precursor-MS/MS spectrum pairs were analyzed using SEQUEST and support vector machine learning.4 Peptides identified only in one or the other cell line were considered.

Results:
Initially we anticipated focusing on the set of proteins uniquely ubiquitinated in normal cells. However, the transcriptomal results indicated that genes encoding proteins in the ubiquitin pathway were over-represented in the list of genes that were over-expressed in FA samples. Consequently, we examined both differential ubiquitination lists and found that a major regulator of TNF-gene expression, TLR8, appeared in the ubiquitinylated fraction only in mutant cells. In co-immunoprecipitation studies we confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells, and using RNAi determined that high level TNFα synthesis in mutant cells depended upon TLR8 and its downstream signaling intermediates IRAK-1 and IKK-alpha/beta. FANCC deficient THP1 blue cells were created using lentiviral shRNA targeting FANCC. These cells exhibited the MMC hypersensitive phenotype and over-expressed both TNFα and an NF-kappaB reporter gene (secreted embryonic alkaline phosphatase) in response to TLR8 agonists but not to other TLR agonists. Primary splenic macrophages from Fancc-/- mice were also hypersensitive to the TLR8 agonist R848. TNFα production in FA-C cells was suppressed by inhibitors of TLR8, p38 MAPK, IRAK, and IKK. Engineered point mutants of FANCC were capable of complementing the mitomycin C hypersensitivity phenotype of FANCC mutant cells but did not suppress TNFα overproduction in FANCC mutant cells. In conclusion, TNF over-expression in FANCC-deficient cells reflects the loss of FANCC function as a suppressor of TLR8 activation. In addition, FANCC suppresses TLR8 dependent production of TNFα in normal mononuclear phagocytes at least in part by suppressing either TLR8 ubiquitinylation or by inhibiting its association with an ubiquitinylated protein. Finally, this function of FANCC is independent of its function in protecting the genome from cross-linking agent-induced damage. In light of the role of TNFα in bone marrow failure and clonal evolution in this disease, control of TNF-production by targeting the TLR8 pathway might provide an opportunity to enhance hematopoietic activity and forestall clonal evolution in patients with this disorder.
1. Sejas DP, et al, J Immunol 2007;178:5277-5287. 2. Zhang × et al, J.Cell Sci. 2007;120:1572-1583.
3. Li J, et al, J.Clin.Invest. 2007;117:3283-3295, 4. Anderson DC, et al, J Proteome.Res 2003;2:137-146.

Disclosures:
No relevant conflicts of interest to declare.