Inhibition of UBE2N As a Therapeutic Approach in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Abstract 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.

Download Full-text

IMMU-34. ATRX MUTATIONS PREDICT RESPONSE TO INNATE BASED THERAPY IN GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noz175.526 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi126-vi126

Author(s):

Michelle Bowie ◽

Seethalakshmi Hariharan ◽

Janell Hostettler ◽

Kristen Roso ◽

Yiping He ◽

...

Keyword(s):

Cell Lines ◽

Glioma Cell ◽

Innate Immune ◽

Type I ◽

Loss Of Function ◽

Who Grade ◽

Immune Signaling ◽

Innate Immune Signaling ◽

Recurrent Mutations ◽

Glioma Cell Lines

Abstract BACKGROUND Innate based immunotherapies are becoming increasingly important for treating brain tumor patients. Gliomas carry recurrent mutations in regulatory genes that control innate immune signaling responses. About 71% of adult WHO grade II and III gliomas and 57% of secondary glioblastomas also carry a loss-of-function mutation in the ATRX gene. ATRX is a SWI-SNF chromatin remodeling protein that has major roles in processes such as cell cycle regulation and maintenance of genomic stability. Recent studies have implicated ATRX in dysfunctional innate immune signaling in cancer cells. However, the role of ATRX in mediating innate immune responses has not been investigated in gliomas. METHODS AND RESULTS Human and mouse glioma cell lines from a variety of genetic contexts have been examined including models which carry IDH/ATRX mutations, IDH 1p-/19q- and ATRX -/- status. Additionally, using Crispr-Cas9 technology and cloning cell lines with ATRX deletions, we have derived a series of immune competent and nude mice models. Treating these cell lines with double-stranded RNA based innate stimuli led to an enhanced early induction in phospho-interferon regulatory factor 3 (IRF3) and late induction in phospho-STAT1 in the ATRX knockout (KO) cell lines. A differential increase in interferon-stimulated gene 15 (ISG15) release was also noted in the ATRX KO cell lines, further suggesting that ATRX deletion may enable a potent activation of type I interferon production. A combination of patient-derived glioma cell lines in xenograft models and syngeneic murine glioma models derived from ATRX KO cell lines and controls confirm a survival advantage in both immuno-competent mice and xenografts. Our models are under evaluation with PVSRIPO and other innate based RNA therapies. CONCLUSION Our data suggests that ATRX mutations may confer sensitivity to RNA-based innate immune signaling agonists in gliomas. This potential vulnerability can be targeted in future therapies.

Download Full-text

Therapeutic Targeting of the Ubiquitin Conjugating Enzyme UBE2N in Myeloid Malignancies

Blood ◽

10.1182/blood-2018-99-115359 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4050-4050

Author(s):

Laura Barreyro ◽

Avery Sampson ◽

Kathleen Hueneman ◽

Lyndsey C Bolanos ◽

Mario Pujato ◽

...

Keyword(s):

Cell Lines ◽

Active Site ◽

Hematologic Malignancies ◽

Innate Immune ◽

Primary Cells ◽

Rna Seq ◽

Immune Signaling ◽

Innate Immune Signaling ◽

Conjugating Enzyme

Abstract Inflammatory and innate immune signaling pathways are activated in leukemic stem and progenitor cells and contribute to the pathogenesis of hematologic malignancies, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). UBE2N is a ubiquitin (Ub) conjugating enzyme that catalyzes lysine 63 (K63)-linked Ub chains on substrates that are critical for signal transduction of broad innate immune signaling pathways. Here we report that UBE2N is required for leukemic cell function by mediating oncogenic innate immune signaling, and identified a novel chemical class of small molecule inhibitors that target UBE2N enzymatic activity. Upon UBE2N downregulation with two lentivirally expressed shRNAs, MOLM-13 and THP-1 cells lose their clonogenic potential and undergo cell death. Unlike for AML cells, UBE2N was dispensable for colony formation and viability of healthy cord blood CD34+ cells. The active site of UBE2N contains a cysteine (Cys) at position 87, which is essential for binding and transfer of Ub to its substrates. We performed in silico structure- and in vitro cell-based screens to identify small molecules that dock to the active site of UBE2N and covalently bind to Cys-87, as an approach to inhibit Ub transfer to substrates. Two structurally-related candidates, UC-764864 and UC-764865, emerged as inhibitors of UBE2N, as they specifically blocked the E1-UBE2N thioester transfer in vitro. Treatment of MDS/AML cell lines and patient-derived primary cells with UC-764864 and UC-764865 suppressed innate immune signaling and induced cytotoxic effects in MDS/AML cell lines and primary cells while sparing healthy hematopoietic cells in vitro and in vivo. To identify the molecular basis of UBE2N inhibition, we performed a global Ub screen for changes in ubiquitinated substrates by mass spectrometry and evaluated changes in gene expression by RNA-seq in MOLM-13 cells treated with vehicle control or the newly derived UBE2N inhibitors. RNA-seq of MOLM-13 cells treated with UC-764864 revealed that inhibition of UBE2N in leukemic cells targets oncogenic innate immune pathways, including NF-kB and Type I interferon signaling networks. UC-764864 and UC-764865 reduced the ubiquitination status of UBE2N, and altered the ubiquitination of proteins involved in innate immune signaling and the DNA damage response by primarily reducing K63-linked Ub modifications. Two substrates identified by the Ub screen, DDB1 and UBE2M, are components of the CUL4-CRBN E3 ligase complex and a target of the anti-leukemic therapy, 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 UBE2N inhibitors on MDS/AML cells. As compared to individual treatments, the combination of LEN and UC-764864, UC-764865 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 UBE2N inhibitors impaired MDS/AML cells that are refractory to treatment of LEN or UBE2N inhibitors alone. These findings suggest that UBE2N is a promising target to extend the use of LEN to other subtypes of MDS or AML. In summary, we implicate the Ub conjugating enzyme UBE2N as a target in MDS/AML, and identified novel small molecule inhibitors that target UBE2N and modify the function of Ub E3 ligases that are important for UBE2N-associated diseases, including autoinflammatory and autoimmune disorders, and hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

Download Full-text