Abstract
Abstract 3191
Poster Board III-128
The Fanconi Anemia (FA) proteins play an important role in regulating genome stability, 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. Indeed, there is evidence that at least some of these proteins are multifunctional and participate in canonical signaling pathways in hematopoietic cells. FANCC deficient cells, for example, are hypersensitive to the apoptotic effects of TNFαa. In addition, FA-C cells over-produce TNFαa at least in part because FANCC ordinarily suppress the activation potential of toll-like receptor 8 (TLR8) (abstract submitted to this meeting). There is clear evidence that over-production of TNFαa and hypersensitivity to TNFαa in hematopoietic cells of Fancc-/- mice results in bone marrow hypoplasia (Sejas et al, 2007 and Zhang et al 2007) and that long-term ex-vivo exposure of murine Fancc -/- hematopoietic cells to both growth factors and TNFαa results in the evolution of cytogenetically marked preleukemic clones (Li et al 2007). Because the hematopoietic phenotype of FA may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive, we reasoned that suppression of TNFαa production by FA cells might enhance hematopoiesis. So we sought to develop a strategy to permit high throughput screening of small molecules designed to suppress TNFαa production specifically in FANCC deficient cells.
THP1 Blue cells (THP1B) have a stably integrated NF-kappaB reporter gene, secreted embryonic alkaline phosphatase (SEAP) and express SEAP and TNFαa in response to TLR ligands including the TLR8 ligand (R848). Each of five samples of THP1B cells were transduced with one of five lentiviral vectors expressing FANCC targeted shRNA. One of these vectors suppressed FANCC expression (by immunoblotting and RT-RT-PCR), suppressed FANCD2 levels in MMC exposed THP1B cells, induced chromosomal instability in the MMC assay and markedly enhanced R848-induced TNFαa production when compared to THP1B cells transduced with a non-targeted shRNA lentiviral vector. In multiwell plates, THP1B-shFANCC cells were exposed to multiple doses one of 81 small molecules including steroid hormones and inhibitors of tyrosine or serine threonine kinases. TNFαa (ELISA) and SEAP (QUANTI-blue colorimetry) were quantified in the supernatant media 24 hours after exposure to R848.
15 agents suppressed SEAP production without cytotoxicity and all of these suppressed TNFαa production as well. The same agents suppressed TNFαa production in two patient-derived FANCC-deficient cell lines (HSC536 and PD149) both of which over-express TNFαa in the ground state. Four p38 inhibitors (100nM-10μM) were analyzed and at 500 nM all suppressed SEAP and TNFαa by 90% or more. The Src family kinase inhibitor, Dasatinib (500nM) was also effective. Using Fancc-deficient mice exposed to TLR activating agents, in vivo preclinical studies designed to test the effectiveness of Dasatinib and one p38 inhibitor are underway, as are mechanistically focused multiplex assays in which known target molecules of these agents are suppressed using RNAi.
We have developed a reliable screening tool based upon the TNFαa-overproduction phenotype of FANCC deficient cells. Using it, we have identified inhibitors of p38 MAPK and Src family kinases that suppress TNFαa-overproduction in patient derived FANCC-deficient cells. The identification of these agents provides not only an opportunity to discover novel biochemical roles played by FANCC in innate immunity but also a strong rationale for evaluating such agents in preclinical models for marrow failure in FA.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
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