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: Rationale: Tumor Necrosis Factor-alpha (TNF) is elevated in myeloproliferative neoplasm (MPN) and plays a key role in expansion of the JAK2V617F neoplastic clone. A high TNF environment, as is the case in MPN patients, gives TNF resistant JAK2V617F mutant cells a selective advantage over their TNF sensitive non-mutant counterparts resulting in expansion of the neoplastic clone. To efficiently target TNF production therapeutically it is necessary to identify the mechanism driving this excessive TNF production. TNF is classically produced by monocytes after stimulation through Toll-like receptors (TLR), crucial pattern recognition receptors for microbial products. Because TLR signaling plays an integral role in inflammation and TNF production we hypothesized that exaggerated signaling of the TLR pathway is the mechanism by which TNF is overproduced in MPN. To test this hypothesis we quantified TLR responses in monocytes from MPN patients and normal controls. Results: We compared the response of peripheral blood monocytes from MPN versus normal controls to the TLR agonists R848 (TLR7/8), LPS (TLR4), or zymosan (TLR2). After stimulation with each of these TLR agonists for 24 hours, CD14+ monocytes from MPN patients (n=18) produced increased amounts of TNF (measured by ELISA) as compared to normal controls (n=10) at all concentrations tested (p

Description: Fanconi anemia, complementation group C (FANCC)–deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNFα, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNFα in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist–stimulated FANCC- and Fanconi anemia, complementation group A (FANCA)–deficient macrophages containing an NF-κB/AP-1–responsive reporter gene (SEAP). Of the 75 small molecules screened, the p38 MAPK inhibitor BIRB 796 and dasatinib potently suppressed TLR8-dependent expression of the reporter gene. Fanconi anemia (FA) macrophages were hypersensitive to the TLR7/8 activator R848, overproducing SEAP and TNFα in response to all doses of the agonist. Low doses (50nM) of both agents inhibited p38 MAPK–dependent activation of MAPKAPK2 (MK2) and suppressed MK2-dependent TNFα production without substantially influencing TNFα gene transcription. Overproduction of TNFα by primary FA cells was likewise suppressed by these agents and involved inhibition of MK2 activation. Because MK2 is also known to influence production and/or sensitivity to 2 other suppressive factors (MIP-1α and IFNγ) to which FA hematopoietic progenitor cells are uniquely vulnerable, targeting of p38 MAPK in FA hematopoietic cells is a rational objective for preclinical evaluation.

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: Background: Stem cell (HSC) hypersensitivity to inflammatory cytokines and exaggerated TLR-dependent production of such cytokines contribute to bone marrow failure and clonal selection in Fanconi anemia (FA). Clonal neoplasms in FA patients and FA-deficient mice exhibit either resistance or paradoxical proliferative responses to tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN). Because FA MDS/AML shares cytogenetic and clinical features in common with secondary MDS/AML developing after prior MDS or exposure to cytotoxic chemotherapy (sMDS/sAML), we tested the idea that an FA-like TLR/cytokine hypersensitive phenotype might underlie clonal selection in sMDS/sAML. A pilot study of 4 hematologically normal individuals with histories of prior cytotoxic chemotherapy (including alkylating agents) revealed 2 whose committed progenitor cells were hypersensitive to TNF and IFN. Three patients with sMDS/sAML exhibited resistance to IFN and paradoxical growth responses to TNF. To determine the true prevalence of these FA-like phenotypes, we conducted a larger study of patients with sMDS/sAML, quantifying a) progenitor cell growth in response to inflammatory cytokines and b) TLR7/8 or TLR4-dependent cytokine production in peripheral blood monocytes. Methods: Patients with newly diagnosed sMDS or sAML were eligible. All potential subjects with active infections were excluded. Research bone marrow aspirates and peripheral blood (PB) were obtained at the same time. TNF was quantified (ELISA) in the supernatants of PB CD14+ cells cultured in the presence of 1 ng/ml lipopolysaccharide (LPS, a TLR-4 agonist) or 3-5 μM R848 (TLR-7/8 agonist) for 24-hours. BFU-E and CFU-GM were quantified in methylcellulose cultures of low density bone marrow mononuclear cells (LDBMCs) (SCF, IL-3, EPO +/- TNF or IFN). Progenitor responses were classified as resistant/paradoxical if either CFU-GM or BFU-E colonies were 〉120% of control (normal volunteer bone marrow cells) at TNF 1 ng/ml, or 〉100% at IFN 0.1 ng/ml; all other responses were categorized as sensitive. TNF production was classified as exaggerated if levels exceeded normal mean values by more than two standard deviations. Results: A total of 22 patients with sMDS/sAML had bone marrow and PB analysis, including 13 patients with complex karyotypes and 9 patients with non-complex karyotypes. An additional 18 patients had PB samples drawn without providing bone marrow samples. Progenitor responses: In colony assays, 18/22 patients demonstrated resistant/paradoxical growth responses to TNF/IFN, a phenomenon seen in patients with both complex and non-complex karyotypes. Interestingly, TNF resistance was observed only in samples that were also IFN resistant. Among patients with a complex karyotype, only 3 demonstrated normal progenitor sensitivity. None of these 3 had received prior chemotherapy (2 with radiation only). Macrophage responses: CD14+ cells from 63% of patients with resistant/paradoxical colony assay responses overproduced TNF in response to either R848 or LPS. Mean TNF levels from CD14+ cells of these patients were higher compared to patients with sensitive colony responses [1756 pg/ml (R848) and 1699 pg/ml (LPS), versus 783 pg/ml (R848) and 959 pg/ml (LPS), for resistant/paradoxical versus sensitive patients respectively]. Among all patients with PB samples (including those with/without marrow results), CD14+ cells from 68% of these patients overproduced TNF. Mean TNF levels in response to R848 or LPS stimulation of patient cells were elevated compared to normal donor controls [1730 pg/ml (R848) and 1795 pg/ml (LPS), versus 719 pg/ml (R848) and 555 pg/ml (LPS), from patient and normal donor CD14+ cells, respectively]. Conclusions: In patients with sMDS/sAML there is a high prevalence of TNF resistance in committed progenitors and TLR-hyper-reactivity in CD14+ cells. In FA the MDS/AML clonal resistance phenotype is selected because it possesses a selective advantage over the unfit pool of non-clonal FA HSC. We suggest that prior chemotherapy can create a fixed phenotype of HSC unfitness (e.g. hypersensitivity to inflammatory cytokines) and that the desultory fitness landscape in that pool permits the selection of cytokine resistant clones. Mechanistic studies should provide opportunities to identify patients at risk of sMDS/sAML and to develop pharmacological approaches to leukemia prevention. Disclosures No relevant conflicts of interest to declare.

Description: Tumor Necrosis Factor-alpha (TNF) is elevated in myeloproliferative neoplasm (MPN) and plays a key role in expansion of the JAK2V617F neoplastic clone. Because JAK2V617F cells are TNF resistant, a high TNF environment, as is the case in MPN patients, gives JAK2V617F mutant cells a selective advantage over their TNF sensitive non-mutant counterparts, resulting in expansion of the neoplastic clone. Targeting excessive TNF production therapeutically in MPN would reduce the competitive advantage of the JAK2V617F neoplastic clone and lead to its contraction. To efficiently target TNF production therapeutically in MPN it is necessary to first identify the mechanism driving this excessive TNF production. TNF is classically produced by monocytes after stimulation through Toll-like receptors (TLR), crucial pattern recognition receptors for microbial products. Upon TLR stimulation a signaling transduction cascade ensues, culminating in the production of inflammatory cytokines including TNF. Because TLR signaling plays an integral role in inflammation and TNF production we hypothesized that exaggerated signaling of the TLR pathway is the mechanism by which TNF is overproduced in MPN. To test this hypothesis we quantified TLR responses in monocytes from MPN patients and normal controls. We compared the response to the TLR 7/8 agonist R848 in peripheral blood monocytes from MPN versus normal controls. After stimulation with R848 for 24 hours, CD14+ monocytes from MPN patients (n=18) produced increased amounts of TNF (measured by ELISA) as compared to normal controls (n=10) at all concentrations tested (0.5, 1, 3, 5µM, p