Therapeutic Targeting of the Ubiquitin Conjugating Enzyme UBE2N in Myeloid Malignancies

• Published in: Blood 2018-11, Vol.132 (Supplement 1), p.4050-4050
• Main Authors: Barreyro, Laura, Sampson, Avery, Hueneman, Kathleen, Bolanos, Lyndsey C, Pujato, Mario, Chutipongtanate, Somchai, Wyder, Michael, Haffey, Wendy, Choi, Kwangmin, Neelamraju, Yaseswini, Smith, Molly A, Niederkorn, Madeline, Ramesh, Vighnesh, Muto, Tomoya, O'Brien, Eric, Meydan, Cem, Christie, Susanne, Liu, Xiaona, Garret-Bakelman, Francine, Bartholdy, Boris, Will, Britta, Weirauch, Matthew T., Gul, Zartash, Medlin, Stephen, Perentesis, John Peter, Greis, Kenneth D, Seibel, William, Starczynowski, Daniel T.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2018-99-115359

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 ligas