ALBERT

Description: Recent work has clarified a role for aldehyde dehydrogenases in protecting Fanconi anemia (FA) hematopoietic stem cells (HSC) and indicates that the increase in endogenous aldehydes that attends the genetic loss of ALDH function is sufficient to induce bone marrow failure in this disease. Studies in many laboratories have also documented that: (1) exposure of FA HSC to inflammatory cytokines suppresses stem cell self-replication and, in vivo, exhausts the stem cell pool and (2) FA macrophages exposed to specific toll-like receptor (TLR) ligands overproduce the very inflammatory cytokines that suppress the HSC pool. Given that aldehydes form adducts with and, in some cases, activate signaling proteins involved in cytokine production, we tested the hypothesis that overproduction of inflammatory cytokines by FA macrophages results from a loss of FA protein-dependent ALDH function. We treated THP-1 human monocytic leukemia cells expressing shRNA targeting FANCC (T-shFC) or a non-targeted shRNA (T-shNT) with the aldehyde 4-hydroxynonenal (4-HNE) before exposing them to the TLR-7/8 agonist R848. 4-HNE alone did not induce TNF production in either cell line but did enhance TLR-induced TNF overproduction by T-shFC cells (but not T-shNT cells), suggesting that FANCC-deficient macrophages lack factors (e.g., ALDH) that neutralize a signal-enhancing effect of this aldehyde. To directly test the effects of ALDH loss, we pretreated cells with the general ALDH inhibitor diethylaminobenzaldehyde and found that this agent enhanced R848-induced normal production of TNF by approximately 1.6-fold in control cells but did not enhance TNF production in T-shFC cells that were already overproducing TNF. Having determined that ALDH1A1 (but not ALDH2) was highly inducible in both THP-1 cells (by R848) and Lin-Sca-1+Kit+ (by TNF) murine marrow cells, we used siRNA to suppress expression of ALDH2 or ALDH1A1. Knockdown of ALDH1A1 (but not ALDH2) enhanced R848-induced normal production of TNF by approximately 1.8-fold in T-shNT but not TNF overproduction in T-shFC (fig 1) or T-shFA (FANCA knockdown) cells. Our results are consistent with the notion that ALDH1A1 is non-functional in FANCC-deficient macrophages and we confirmed that suspicion in gain-of-function analyses. Specifically, treatment of T-shNT and T-shFC cells with Alda-1 (a small molecule ALDH agonist known to enhance the activity of both ALDH1A1 and ALDH2) suppressed TLR-induced TNF production (fig 2), even in the presence of 4-HNE, by both T-shNT and T-shFC cells. In summary, (1) increasing the aldehyde load in normal macrophages has little influences on the inflammatory response induced by TLR activation but in FANCC-deficient cells aldehydes exacerbate the inflammatory response, (2) suppression of ALDH function with DEAB and specific suppression of ALDH1A1, (but not ALDH2) induces an FA-like phenotype in control macrophages, and (3) pharmacological enhancement of ALDH activity suppresses induced cytokine overproduction by FANCC-deficient macrophages. We conclude that: (1) optimal function of ALDH1A1 is FANCC-dependent in normal macrophages, (2) that the TLR-dependent overproduction of inflammatory cytokines by FANCC-deficient macrophages may result either from an increase in the aldehyde load or the loss of a non-canonical signal-suppressive function of ALDH1A1, and (3) enhancement of ALDH activity using small molecule agonists such as Alda-1 may alleviate the FA macrophage phenotype and may thereby protect HSC from inflammation-induced exhaustion. Disclosures No relevant conflicts of interest to declare.

Description: Key PointsTLR-activated FANCA- and FANCC-deficient macrophages overproduce IL-1β. IL-1β suppresses in vitro expansion of Fancc-deficient multipotent hematopoietic progenitor cells.

Description: Abstract 1263 The molecular basis for how a Fanconi anemia (FA) genetic background contributes to hematopoietic stem cell defects and hypoplastic organ development remains poorly understood. Protein modification by ubiquitination is a mechanism that diversifies the function and regulation of proteins. In light of this, we focus on the dysfunction of FANCL, the E3 ubiquitin ligase of the FA pathway, as a key molecular defect in Fanconi anemia. Here we report our studies investigating mechanisms of post-translational regulation of FANCL. We view these mechanisms as potential targets to augment the function of the FA core complex and correct hematopoietic stem cell defects. We provide evidence that FANCL is exquisitely regulated by ubiquitin-proteosome degradation. Ligase-inactive mutants (FANCL-C307A and -W341G) are less sensitive to this regulation, suggesting a role for auto-ubiquitination in directing lysine-48 polyubiquitination. This constitutive negative regulation of FANCL is partially reversed with an ATP-competitive glycogen synthase kinase-3beta (GSK-3beta) inhibitor. GSK-3beta is a serine/threonine kinase that phosphorylates proteins and marks them for ubiquitin-mediated proteolysis. Mitogenic and survival pathways, including Ras/MAPK and PI3K/Akt, negatively regulate GSK-3beta by serine-9 phosphorylation. We show that the regulation of FANCL by GSK-3beta is likely direct because FANCL and GSK-3beta co-immunoprecipitate in cell lysates and as GST-fusion proteins. To define the biochemical mechanisms of FANCL regulation, we generated N-terminal deletion mutants of FANCL and we show that the regulation of FANCL is dictated by a region at the N-terminus (aa1-78). Mutational analysis of FANCL (lysine to arginine) in this N-terminus region does not affect the overall protein level or ubiquitination of FANCL, suggesting that FANCL may be targeted for degradation by phosphorylation and/or in a complex with other proteins. The potential biological relevance of our findings, that FANCL is regulated by GSK-3beta is revealed in studies overexpressing constitutively active, myristoylated-Akt. This experimental condition increases FANCL protein levels and suggests a role for FANCL as a downstream effector of PI3K/Akt signaling. In turn, FANCL likely regulates non-canonical targets that alter the transcriptome profile favoring self-renewal and survival of hematopoietic stem cells. We recently published our studies identifying beta-catenin as one such downstream target (Blood 2012 Jul 12;120:323). Suppression of FANCL expression severely disrupts Wnt/beta-catenin signaling and expression of downstream Wnt-responsive targets MYC and CCND1. We also identified that GSK3B gene expression is approximately 5-fold higher in Fancc-deficient hematopoietic stem cells exposed to TNF-alpha compared to untreated cells or to wildtype cells with or without TNF-alpha. Our current studies show that inhibition of GSK-3beta preserves the number of murine Fancc-deficient hematopoietic stem cells exposed to TNF-alpha compared with no GSK-3beta inhibition. Taken together, we have accumulated evidence suggesting that GSK-3beta is a promising molecular target to improve the self-renewal and survival of FA hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1335 Fanconi anemia (FA) is associated with a hereditary predisposition to bone marrow failure. The proteins encoded by the FANC genes are primarily involved in DNA repair responses through the formation of a large, multisubunit complex that has E3 ubiquitin ligase activity (Annual Review of Genetics 2009;43:223). FA hematopoietic stem cells display defective stem cell properties and limited replicative potential. However, the molecular basis for how a FA genetic background contributes to those defects remains poorly understood. Here we provide evidence that FANCL, which has E3 ubiquitin ligase activity, enhances beta-catenin activity (Figure A) and protein expression. Beta-catenin is a nuclear effector of canonical Wnt signaling. The Wnt/beta-catenin pathway is active in normal hematopoietic stem cells in the native bone marrow environment and disruption of this signaling pathway results in defective hematopoietic stem cells (Nature 2003;423:409). To test whether FANCL positively regulates beta-catenin through its ubiquitination activity, we performed cell-based ubiquitination assays. We show that FANCL functionally ubiquitinates beta-catenin (Figure B) and that ubiquitin chain extension can occur via non-lysine-48 ubiquitin linkages. Accumulating evidence reveal diverse, non-proteolytic biological roles for proteins modified by atypical ubiquitin chains (EMBO Reports 2008;9:536). Our data suggests that FANCL may enhance the protein function of beta-catenin via ubiquitination with atypical ubiquitin chains. Importantly, we demonstrate that suppression of FANCL expression in human CD34+ cord blood stem cells reduces beta-catenin expression (Figure C) and multilineage progenitor expansion. These results demonstrate a role for the FA pathway in regulating Wnt/beta-catenin signaling. Therefore, diminished Wnt/beta-catenin signaling may be an important underlying molecular defect in FA hematopoietic stem cells leading to their accelerated loss. A, LEF-TCF-luciferase reporter assay showing increasing beta-catenin activity in 293FT cells with increasing FANCL expression compared with vector-control (VC) (n=4). B, Immunoprecipitation of beta-catenin in cells transfected with vector-control or FANCL and probed for hemagglutinin (HA)-tagged ubiquitin shows increased ubiquitinated forms of beta-catenin with FANCL expression (n=4). C, shRNA suppression of FANCL expression in CD34+ cord blood stem cells results in decreased beta-catenin expression compared with a scramble control (Scr) by immunofluorescence analysis (three different shRNA constructs, n=3 for each construct). Disclosures: No relevant conflicts of interest to declare.

Description: Fancc suppresses cross-linker–induced genotoxicity, modulates growth-inhibitory cytokine responses, and modulates endotoxin responses. Although loss of the latter function is known to account for endotoxin-induced marrow failure in murine Fancc (mFancc)–deficient mice, some argue that cytokine and endotoxin hypersensitivities devolve simply from genomic instability. Seeking to resolve this question, we planned to ectopically express instructive human FANCC (hFANCC) mutants in murine Fancc-deficient hematopoietic stem cells. To first assure that hFANCC cDNA was competent in murine cells, we compared hFANCC and mFancc in complementation assays for cross-linking agent hypersensitivity and endotoxin hypersensitivity. We found that mFancc complemented murine Fancc-deficient cells in both assays, but that hFANCC fully suppressed only endotoxin hypersensitivity, not cross-linking agent hypersensitivity. These results support the notions that Fancc is multifunctional and that structural prerequisites for its genoprotective functions differ from those required to constrain endotoxin responses known to lead to marrow failure in Fancc-deficient mice.

Description: Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34+ stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.