No abstract

SUMMARYBasal nuclear factor κB (NF-κB) activation is required for hematopoietic stem cell (HSC) homeostasis in the absence of inflammation; however, the upstream mediators of basal NF-κB signaling are less well understood. Here, we describe TRAF6 as an essential regulator of HSC homeostasis through basal activation of NF-κB. Hematopoietic-specific deletion of Traf6 resulted in impaired HSC self-renewal and fitness. Gene expression, RNA splicing, and molecular analyses of Traf6-deficient hematopoietic stem/progenitor cells (HSPCs) revealed changes in adaptive immune signaling, innate immune signaling, and NF-κB signaling, indicating that signaling via TRAF6 in the absence of cytokine stimulation and/or infection is required for HSC function. In addition, we established that loss of IκB kinase beta (IKKβ)- mediated NF-κB activation is responsible for the major hematopoietic defects observed in Traf6-deficient HSPC as deletion of IKKβ similarly resulted in impaired HSC self-renewal and fitness. Taken together, TRAF6 is required for HSC homeostasis by maintaining a minimal threshold level of IKKβ/NF-κB signaling.

No abstract

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.

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.