Histone H2B ubiquitin ligase RNF20 is required for MLL-rearranged leukemia Eric Wanga,1, Shinpei Kawaokaa,1, Ming Yub, Junwei Shia,c, Ting Nid, Wenjing Yangd, Jun Zhud, Robert G. Roederb,2, and Christopher R. Vakoca,2 aCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; bLaboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10065; cMolecular and Cellular Biology Program, Stony Brook University, Stony Brook, NY 11794; and dGenetics and Development Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by Robert G. Roeder, January 18, 2013 (sent for review December 19, 2012) Mixed-lineage leukemia (MLL) fusions are potent oncogenes that (4–6). The translocation partners of MLL can be highly diverse; initiate aggressive forms of acute leukemia. As aberrant tran- however, fusions with AF9 are the most common in AML (1). scriptional regulators, MLL-fusion proteins alter gene expression The aberrant recruitment of AF9-associated proteins to MLL- in hematopoietic cells through interactions with the histone H3 occupied genes (e.g., HOXA9) leads to transcriptional deregu- lysine 79 (H3K79) methyltransferase DOT1L. Notably, interference lation and, consequently, leukemia initiation. Several binding with MLL-fusion cofactors like DOT1L is an emerging therapeutic partners of AF9 are involved in MLL-AF9–mediated leukemo- strategy in this disease. Here, we identify the histone H2B E3 ubiq- genesis, including the super elongation complex (SEC), the uitin ligase ring finger protein 20 (RNF20) as an additional chromatin histone H3K79 methyltransferase DOT1L, and the chromodo- regulatorthatisnecessaryforMLL-fusion–mediated leukemo- main-containing protein CBX8 (7–10). genesis. Suppressing the expression of Rnf20 in diverse models Targeting of MLL-fusion–associated factors has emerged as a of MLL-rearranged leukemia leads to inhibition of cell proliferation, promising therapeutic strategy in this subtype of leukemia. Ge- under tissue culture conditions as well as in vivo. Rnf20 knockdown netic studies have validated that conditional inactivation Dot1l leads to reduced expression of MLL-fusion target genes, effects re- specifically inhibits progression of MLL-rearranged leukemias in sembling Dot1l inhibition. Using ChIP-seq, we found that H2B vivo, in association with reduced expression of MLL-AF9 target ubiquitination is enriched in the body of MLL-fusion target genes, genes (11, 12). Furthermore, a small-molecule inhibitor of DOT1L CELL BIOLOGY correlating with sites of H3K79 methylation and transcription elon- demonstrates antileukemia activity in MLL-rearranged disease gation. Furthermore, Rnf20 is required to maintain local levels of models (13). Genetic or pharmacological disruption of the MLL: H3K79 methylation by Dot1l at Hoxa9 and Meis1.Thesefindings Menin interaction has also been validated as means of sup- support a model whereby cotranscriptional recruitment of Rnf20 at pressing proliferation of MLL-fusion leukemias (5, 14). Target- MLL-fusion target genes leads to amplification of Dot1l-mediated ing of DOT1L or Menin in cells that lack MLL rearrangements H3K79 methylation, thereby rendering leukemia cells dependent leads to remarkably little toxicity, suggesting a potential thera- on Rnf20 to maintain their oncogenic transcriptional program. peutic window for this general approach (13, 14). Ring finger protein 20 (Rnf20) (also called Bre1a) is the major epigenetics | cancer H2B-specific ubiquitin ligase in mammalian cells that targets lysine 120 for monoubiquitination [H2B ubiquitination (H2Bub)] he mixed-lineage leukemia (MLL) protooncogene (also called (15–18). Rnf20 can be recruited to chromatin via the PAF com- TMLL1)wasfirst cloned based on its involvement in chromo- plex, resulting in the accumulation of H2Bub at genes in a tran- somal translocations found in leukemia, occurring at a frequency scription-dependent manner (19–22). Although found broadly of 5–10% in acute myeloid leukemia (AML) and acute lympho- at active genes, H2Bub is not strictly required for transcription blastic leukemia (1). MLL translocations are especially common in elongation, but instead performs specialized roles in regulating infant acute leukemias and in secondary AMLs that arise follow- nucleosome dynamics (22), the DNA damage response (23, 24), ing cancer treatment with topoisomerase II inhibitors (1). In both and the activity of other histone-modifying enzymes (19, 21, of these clinical settings, MLL rearrangements are associated with 22). Regarding the latter, it is known that the presence of H2Bub a poor outcome (1). MLL-rearranged leukemias are notable for on nucleosomes can stimulate the activity of DOT1L in cata- (i) a paucity of additional gene mutations found in these cases lyzing H3K79 methylation in vitro and in vivo through apparent (although NRAS mutations are among the most common coop- allosteric regulation (19, 25). H2Bub also promotes H3K4 erating lesion) (2), (ii) a unique transcriptional signature char- methylation by the SET1 family of lysine methyltransferases (26). acterized by overexpression of homeobox A (HOXA) genes (1), The role of H2Bub in supporting histone methylation in mam- fi and (iii) an absence of available targeted therapies. Regarding malian cells appears to be dependent on the speci ccelltype fi the latter, the current clinical management of MLL-rearranged and/or on the speci c genomic region examined (17, 27, 28). leukemia with cytotoxic chemotherapy is similar to that of other Although substantial evidence indicates cross talk between genetic subtypes of leukemia that fall into the same risk category (1). Hence, a strong rationale exists to define unique therapeutic targets tailored to the molecular pathogenesis of this disease. Author contributions: E.W., S.K., R.G.R., and C.R.V. designed research; E.W., S.K., M.Y., MLL and J.S. performed research; E.W., S.K., M.Y., T.N., W.Y., J.Z., and C.R.V. analyzed data; encodes a chromatin regulator belonging to the SET1 and R.G.R. and C.R.V. wrote the paper. family of histone H3K4 methyltransferases, which form multi- The authors declare no conflict of interest. subunit protein complexes that maintain active transcription (1, Freely available online through the PNAS open access option. 3). MLL translocations found in leukemia generate fusions that Data deposition: The data reported in this paper have been deposited in the Gene Ex- encode an N-terminal fragment of MLL (which lacks the meth- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE43725). yltransferase domain) linked to a C-terminal fragment of various 1E.W. and S.K. contributed equally to this work. partner proteins (1). This N-terminal fragment of MLL physically 2 To whom correspondence may be addressed. E-mail: [email protected] or roeder@ interacts with Menin, CpG-rich DNA, and the polymerase rockefeller.edu. fi associated factor (PAF) complex, which collectively are suf - This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cient for chromatin occupancy at specific genes such as HOXA9 1073/pnas.1301045110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1301045110 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 s H2Bub and H3K79 methylation in various contexts, it has yet to 2Een A Ube2a be addressed whether mammalian Rnf20 supports the bi- Ube2b otsi Ube2e1 ological functions performed by Dot1l in vivo. h Dzip3 Here, we identify a role for Rnf20 in the pathogenesis of s 3Een Ring1 MLL-fusion leukemia. Suppression of Rnf20 leads to impaired Rnf2 o leukemia progression in vivo associated with reduced expres- tsih Rnf20 sion of MLL-AF9 target genes, a finding we link to a defect in Rnf40 maintenance of local H3K79 methylation. Hence, our findings sBUD Mysm1 Usp3 implicate Rnf20 as a key requirement for MLL-fusion leukemia e Usp16 n through regulatory cross talk with Dot1l. otsih Usp21 Usp22 Results slor Rpa3 t noc Ren luc Rnf20 Is Required for Proliferation of MLL-Fusion Leukemia Cells. 0102030 Based on the known role of H2Bub in stimulating H3K79 meth- Fold-decrease in GFP% ylation in various systems (19, 25, 29), we hypothesized that Rnf20 might support the leukemogenic function of Dot1l in MLL-rear- B MLL-AF9; NrasG12D AML d2 ranged disease. We approached this question in a systematic 1.0 d4 d6 fashion in which we suppressed expression of several histone d8 d10 monoubiquitination regulators and evaluated the impact on pro- d12 liferation of MLL-fusion+ leukemia cells. We constructed a set of 0.5 45 shRNA vectors targeting the known E2 conjugating enzymes, E3 ligases, and deubiquitinating enzymes with specificity for his- Relative GFP% tone H2A or H2B for monoubiquitination (13 genes in total) (30). 0.0 Each shRNA (linked to a GFP reporter) was retrovirally in- Rnf20 shRpa3 457 troduced into cells derived from a genetically engineered mouse β-actin model of MLL-AF9/NrasG12D AML, shown previously to reca- shRen 2853 2949 3718 1295 3277 1944 3595 713 pitulate an aggressive, chemotherapy-resistant disease subtype shRnf20 (31). The GFP positivity of partially infected cells was tracked C Immortalized fibroblast D shRNF20.2710 fl d2 d6 d10 d4 d12 d20 d28 by ow cytometry over 8 d to monitor the relative rate of accu- d4 d8 d12 d8 d16 d24 mulation of shRNA+/GFP+ cells relative to shRNA−/GFP− 1.0 1.0 cells. Four shRNAs targeting Rnf20 were found to inhibit leu- kemia proliferation/viability compared with a negative control 0.5 0.5 shRNA targeting Renilla luciferase and a positive control shRNA Relative GFP% A Relative GFP% targeting the replication protein A3 (Rpa3) (Fig. 1 ) (32). No- 0.0 0.0 tably, all of the other shRNAs targeting ubiquitination regulators shRen 3277 1944 3595 shRpa3 MOLM13 MV4-11 THP-1 CMK 713 shRnf20 457 human MLL-fusion+ AML led to negligible effects on leukemia proliferation (Fig. 1A). This includes shRNAs targeting Ube2a and Ube2b, which encode Fig. 1. Rnf20 is required for proliferation of MLL-AF9/NrasG12D leukemia Rad6a and Rad6b, respectively, the E2-conjugating enzymes that cells in vitro.