Compared to other temporary aquatic ecosystems, we know relatively little about how inundation frequency and duration (i.e. hydrologic regime) influences the structure of aquatic communities in northern peatlands. In this study, we examined patterns in nutrient availability and aquatic community structure during a natural flooding event in an Alaskan fen where water‐table position had been manipulated in three large‐scale experimental plots during previous years to simulate both drought (lowered water‐table treatment) and flooding (raised water‐table treatment) conditions relative to a control without manipulation. Although the natural flood disrupted the long‐term experimental manipulation, it provided an opportunity to evaluate how variation in past hydrologic regime influences nutrient dynamics and aquatic food web structure during periods of inundation in a northern boreal peatland. Despite similar water depth among experimental plots during the time of sampling (i.e. water was above the peat surface in all plots), water‐column nutrient concentrations were significantly greater in the drought treatment (where water table had been lowered during the previous growing season) compared to the raised water‐table treatment and the control. Algal production increased with enhanced nutrient availability across all water‐table treatments and was most elevated following the rewetting of dry sediments in the drought treatment. Consumer biomass (heterotrophic bacteria and macroinvertebrates) increased with algal production and was significantly greater in the drought treatment compared to the raised water‐table treatment and the control. Consumer biomass decreased into the second year of constant inundation as algal production was constrained by reduced nutrient availability. Stable isotope analysis (13C and 15N) showed that elevated levels of periphyton (i.e. the intact biofilm) associated with enhanced nutrient availability promoted energy transfer to higher trophic levels (grazers and predators) rather than living or dead mosses or vascular plant material. Consumption of algal material by grazers altered the size and composition of the algal community. The algal community shifted from coccoid (edible) to filamentous (inedible) growth forms with increased grazer abundance in the drought treatment, possibly owing to selective grazing. Conversely, there was a similar proportion of edible and inedible taxa in the control and raised treatments where grazers were lower in abundance. Our results show that the legacy effects of drought can regulate aquatic community structure in northern peatlands. Within a predictive context, our findings suggest that conditions of more variable hydrology expected with climate change (i.e. increased frequency of drought) occurring across northern latitudes will promote energy flow to higher trophic levels by releasing nutrient constraints on microalgae during periods of inundation.
Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.
Dysregulation of innate immune signaling is a hallmark of hematologic malignancies. Recent therapeutic efforts to subvert aberrant innate immune signaling in MDS and AML have focused on the kinase IRAK4. IRAK4 inhibitors have achieved promising, though moderate, responses in pre-clinical studies and in clinical trials for MDS and AML. The reasons underlying the limited responses to IRAK4 inhibitors remain unknown. Here, we reveal that inhibiting IRAK4 in leukemic cells elicits functional complementation and compensation by its paralog, IRAK1. Using genetic approaches, we demonstrate that co-targeting IRAK1 and IRAK4 is required to suppress leukemic stem/progenitor cell (LSPC) function and induce differentiation in cell lines and patient-derived cells. While IRAK1 and IRAK4 are presumed to function primarily downstream of the proximal adapter MyD88, we found that complimentary and compensatory IRAK1 and IRAK4 dependencies in MDS/AML occur via non-canonical MyD88-independent pathways. Genomic and proteomic analyses revealed that IRAK1 and IRAK4 preserve the undifferentiated state of MDS/AML LSPCs by coordinating a network of pathways, including ones that converge on the PRC2 complex and JAK-STAT signaling. To translate these findings, we implemented a structure-based design of a potent and selective dual IRAK1 and IRAK4 inhibitor KME-2780. MDS/AML cell lines and patient-derived samples showed significant suppression of LSPCs in vitro and in xenograft studies when treated with KME-2780 as compared to selective IRAK4 inhibitors. Our results provide a mechanistic basis and rationale for co-targeting IRAK1 and IRAK4 for the treatment of cancers, including MDS/AML.
Hematopoietic stem and progenitor cells (HSPC) from MDS and AML patients exhibit overexpression of TRAF6 and related innate immune pathway genes, suggesting a dependency of leukemic HSPC on activated innate immune signaling. Unfortunately, inhibiting TRAF6 directly has proven difficult, as few binding pockets on TRAF6 exist for small molecule targeting. UBE2N/Ubc13, a cofactor of TRAF6 and key enzyme in innate immune signaling, is an ubiquitin-conjugating E2 enzyme that catalyzes lysine 63 (K63)-linked ubiquitin chains on TRAF6 and its substrates. Importantly, a commercially available compound and our own chemical series of UBE2N inhibitors are available. In this study we evaluated the cellular and molecular effects of pharmacologic and genetic inhibition of UBE2N in MDS and AML cells. Pharmacologic inhibition of UBE2N with NSC697923 or genetic inhibition with shRNAs reduced the clonogenic capacity of MDSL/AML cell lines and primary cells while not significantly affecting normal HSPC. Treatment of MDS/AML cells with NSC697923 reduced the cellular metabolic activity, induced a G2/M cell cycle arrest, and increased cell death. Moreover, xenotransplantation of an MDS-derived patient cell line (MDSL) into immunodeficient mice (NSG-SGM3) showed a 50-70% reduced graft upon UBE2N knockdown relative to a non-silencing control. The cellular effects of UBE2N inhibition correspond with suppression of TRAF6-induced NF-kB activation of target genes. In addition, we found that NSC697923 treatment results in a dramatic loss of TRAF6 protein expression, which is rescued by inhibition of the proteasome. Intriguingly, our molecular analysis revealed that UBE2N inhibition shifts the stoichiometry of TRAF6 ubiquitin chains from K63-linked to K48-linked ubiquitin, resulting in proteasome-mediated degradation. To identify the molecular basis of UBE2N inhibition, we performed a global ubiquitin screen for changes in ubiquitinated substrates and gene expression profiling by RNA sequencing. For the ubiquitin screen, K63 ubiquitinated proteins were immunoprecipitated from MDSL cells upon pharmacologic inhibition of UBE2N, followed by mass spectrometry analysis. UBE2N inhibition significantly altered the ubiquitination of ~140 proteins involved in innate immune signaling, glycolysis, cell survival, RNA splicing, and DNA damage response. In parallel, RNA sequencing of MDSL cells treated with NSC697923 revealed expression changes in genes involved in mRNA processing, cell cycle and glycolysis. Several components of the E3 ligase anaphase-promoting complex APC/CDC20 were downregulated after UBE2N inhibition. As expected, increased expression of APC/CDC20 substrates (i.e., cyclin B1) were observed following treatment with NSC697923, suggesting that UBE2N inhibition in MDS/AML blocks degradation of APC/CDC20 targets and leads to mitotic alterations and apoptosis. One substrate identified in NSC697923-treated MDSL cells by the ubiquitin screen is DDB1, a component of the CUL4-CRBN E3 ligase complex targeted by Lenalidomide (LEN). LEN has shown encouraging results in del(5q) MDS patients; however, its effects are limited in other cytogenetic subtypes of MDS or AML. Therefore, the identification of molecular targets that can enhance or extend the use of LEN in a broader spectrum of patients is desired. As such, we explored the possibility of a cooperative effect of LEN and NSC697923 on MDS/AML cells. As compared to individual treatments, the combination of LEN and NSC697923 or UBE2N shRNAs significantly suppressed the function and viability of MDS/AML cell lines and patient samples in vitro. More striking, treatment of LEN and NSC697923 impaired MDS/AML cells that are refractory to treatment of LEN or NSC697923 alone. These findings suggest that UBE2N is a promising target to extend the use of LEN to other subtypes of MDS/AML. In summary, our data reveal a novel therapeutic target, an E2 ubiquitin conjugating enzyme (UBE2N), in MDS/AML. UBE2N inhibition suppresses the function and viability of MDS/AML cell lines and patient samples, due in part to degradation of TRAF6, suppressing innate immune signaling, and inducing mitotic alterations. Lastly, we show that inhibition of UBE2N alters ubiquitination of DDB1, a component of the CRBN complex, and cooperates with LEN to target MDS/AML cells. Disclosures No relevant conflicts of interest to declare.
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