Targeting Recruitment of Disruptor of Telomeric Silencing 1-like (DOT1L): CHARACTERIZING THE INTERACTIONS BETWEEN DOT1L AND MIXED LINEAGE LEUKEMIA (MLL) FUSION PROTEINS*

The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L_865–874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia.     

Computational modeling reveals key molecular properties and dynamic behavior of disruptor of telomeric silencing 1-like (DOT1L) and partnering complexes involved in leukemogenesis

Disruptor of telomeric silencing 1-like (DOT1L) is the only non-SET domain histone lysine methyltransferase (KMT) and writer of H3K79 methylation on nucleosomes marked by H2B ubiquitination. DOT1L has elicited significant attention because of its interaction or fusion with members of the AF protein family in blood cell biology and leukemogenic transformation. Here, our goal was to extend previous structural information by performing a robust molecular dynamic study of DOT1L and its leukemogenic partners combined with mutational analysis. We show that statically and dynamically, D161, G163, E186, and F223 make frequent time-dependent interactions with SAM, while additional residues T139, K187, and N241 interact with SAM only under dynamics. Dynamics models reveal DOT1L, SAM, and H4 moving as one and show that more than twice the number of DOT1L residues interacts with these partners, relative to the static structure. Mutational analyses indicate that six of these residues are intolerant to substitution. We describe the dynamic behavior of DOT1L interacting with AF10 and AF9. Studies on the dynamics of a heterotrimeric complex of DOT1L1-AF10 illuminated describe coordinated motions that impact the relative position of the DOT1L HMT domain to the nucleosome. The molecular motions of the DOT1L–AF9 complex are less extensive and highly dynamic, resembling a swivel-like mechanics. Through molecular dynamics and mutational analysis, we extend the knowledge previous provided by static measurements. These results are important to consider when describing the biochemical properties of DOT1L, under normal and in disease conditions, as well as for the development of novel therapeutic agents.     

Design,synthesis and anti leukemia cells proliferation activities of pyrimidylaminoquinoline derivatives as DOT1L inhibitors

A series of novel pyrimidylaminoquinoline derivatives 8(a-i) and 9(a-i) containing amino side chain, and the bisaminoquinoline analogs 3(b-e) have been designed and synthesized by structural modifications on a lead DOT1L inhibitor, 3a. All the compounds have been evaluated for their DOT1L inhibitory activities. The results showed that most of the compounds have strong anti DOT1L activities. Compounds 3e, 8h and 9e are the most potential ones from each category with the IC₅₀ values of 1.06 ± 0.35 μM, 5.72 ± 1.56 μM and 3.55 ± 1.28 μM, respectively. Such inhibitors expressed significant binding interactions with DOT1L by surface plasmon resonance (SPR)-based binding assay. The results of molecular docking experiments suggested that they could occupy the SAM binding pocket of DOT1L. Compounds 8h and 9e exhibited better inhibitory activities but poor selectivities against the both MLL-rearranged MV4-11 cells and the non MLL-rearranged Kasumi-1 cells than those of 3a and 3e, which suggested that the introduction of the amino side chain would be beneficial for their anti leukemia cells proliferation activities, possibly due to the improvement of the fat solubility. Additionally, the direct cellular inhibition activities were found that compound 9e could effectively down-regulate both the level of H3k79 methylation and MLL-rearranged leukemia gene expression of Hoxa9 and Meis1 in MV4-11 in the qRT-PCR and western blot studies. These observations suggested DOT1L was one of the potential targets but perhaps not the most pivotal one for these compounds, which made their poor selectivities against leukemia cells proliferation.     

CBX8 interacts with chromatin PTEN and is involved in regulating mitotic progression

ObjectivesBesides its role in regulating phosphatidylinositol‐3 kinase (PI3K) signalling in the cytosol, PTEN also has a nuclear function. In this study, we attempted to understand the mechanism of chromatin PTEN in suppressing chromosomal instability during cell division.Materials and methodsImmunocoprecipitation, ectopic expression, and deletional analyses were used to identify the physical interaction between Chromobox Homolog protein 8 (CBX8) and PTEN, as well as the functional domain(s) of PTEN mediating the interaction. Cell synchronization followed by immunoblotting was employed to study cell cycle regulation of CBX8 and the functional interaction between chromatin PTEN and CBX8. Small interfering RNAs (siRNAs) were used to study the role of PTEN and CBX8 in modulating histone epigenetic markers during the cell cycle.ResultsPolycomb group (PcG) proteins including CBXs function to repress gene expression in a wide range of organisms including mammals. We recently showed that PTEN interacted with CBX8, a component of Polycomb Repressing Complex 1 (PRC1), and that CBX8 co‐localized with PTEN in the nucleus. CBX8 levels were high, coinciding with its phosphorylation in mitosis. Phosphorylation of CBX8 was associated with monoubiquitinated PTEN and phosphorylated‐BubR1 on chromatin. Moreover, CBX8 played an important role in cell proliferation and mitotic progression. Significantly, downregulation of either PTEN or CBX8 induced H3K27Me3 epigenetic marker in mitotic cells.ConclusionCBX8 is a new component that physically interacts with chromatin PTEN, playing an important role in regulating mitotic progression.     

Regulation of epithelial sodium channel activity by SARS-CoV-1 and SARS-CoV-2 proteins

Severe acute respiratory syndrome (SARS) coronavirus (CoV) 2 (SARS-CoV-2), which causes the coronavirus disease 2019, encodes several proteins whose roles are poorly understood. We tested their ability either to directly form plasma membrane ion channels or to change functions of two mammalian plasma membrane ion channels, the epithelial sodium channel (ENaC) and the α3β4 nicotinic acetylcholine receptor. In mRNA-injected Xenopus oocytes, none of nine SARS-CoV-2 proteins or two SARS-CoV-1 proteins produced conductances, nor did co-injection of several combinations. Immunoblots for ORF8, spike (S), and envelope (E) proteins revealed that the proteins are expressed at appropriate molecular weights. In experiments on coexpression with ENaC, three tested SARS proteins (SARS-CoV-1 E, SARS-CoV-2 E, and SARS-CoV-2 S) markedly decrease ENaC currents. SARS-CoV-1 S protein decreases ENaC currents modestly. Coexpressing the E proteins but not the S proteins with α3β4 nicotinic acetylcholine receptors significantly reduces acetylcholine-induced currents. ENaC inhibition does not occur if the SARS-CoV protein mRNAs are injected 24 h after the ENaC mRNAs, suggesting that SARS-CoV proteins affect early step(s) in functional expression of channel proteins. Consistent with the hypothesis that the SARS-CoV-2 S protein-induced ENaC inhibition involves competition for available protease, mutating the furin cleavage site in SARS-CoV-2 S protein partially relieves inhibition of ENaC currents. Extending previous suggestions that SARS proteins affect ENaC currents via protein kinase C (PKC) activation, PKC activation via phorbol 12-myristate 13-acetate decreases ENaC and α3β4 activity. Phorbol 12-myristate 13-acetate application reduced membrane capacitance ～5%, presumably via increased endocytosis, but this decrease is much smaller than the SARS proteins’ effects on conductances. Also, incubating oocytes in Gö-6976, a PKCα and PKCβ inhibitor, did not alter E or S protein-induced channel inhibition. We conclude that SARS-CoV-1 and SARS-CoV-2 proteins alter the function of human plasma membrane channels, via incompletely understood mechanisms. These interactions may play a role in the coronavirus 2019 pathophysiology.     

Structure and dynamics of human Nedd4-1 WW3 in complex with the αENaC PY motif

Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) is an E3 ubiquitin ligase that interacts with and negatively regulates the epithelial Na⁺ channel (ENaC). The WW domains of Nedd4-1 bind to the ENaC subunits via recognition of PY motifs. Human Nedd4-1 (hNedd4-1) contains four WW domains with the third domain (WW3*) showing the strongest affinity to the PY motif. To understand the mechanism underlying this binding affinity, we have carried out NMR structural and dynamics analyses of the hNedd4-1 WW3* domain in complex with a peptide comprising the C-terminal tail of the human ENaC α-subunit. The structure reveals that the peptide interacts in a similar manner to other WW domain–ENaC peptide structures. Crucial interactions that likely provide binding affinity are the broad XP groove facilitating additional contacts between the WW3* domain and the peptide, compared to similar complexes, and the large surface area buried (83 Å²) between R430 (WW3*) and L647′ (αENaC). This corroborates the model-free analysis of the ¹⁵N backbone relaxation data, which showed that R430 is the most rigid residue in the domain (S² = 0.90 ± 0.01). Carr–Purcell–Meiboom–Gill relaxation dispersion analysis identified two different conformational exchange processes on the μs–ms time-scale. One of these processes involves residues located at the peptide binding interface, suggesting conformational exchange may play a role in peptide recognition. Thus, both structural and dynamic features of the complex appear to define the high binding affinity. The results should aid interpretation of biochemical data and modeling interfaces between Nedd4-1 and other interacting proteins.     

AF17 facilitates Dot1a nuclear export and upregulates ENaC-mediated Na+ transport in renal collecting duct cells

Our previous work in 293T cells and AF17(-/-) mice suggests that AF17 upregulates expression and activity of the epithelial Na(+) channel (ENaC), possibly by relieving Dot1a-AF9-mediated repression. However, whether and how AF17 directly regulates Dot1a cellular distribution and ENaC function in renal collecting duct cells remain unaddressed. Here, we report our findings in mouse cortical collecting duct M-1 cells that overexpression of AF17 led to preferential distribution of Dot1a in the cytoplasm. This effect could be blocked by nuclear export inhibitor leptomycin B. siRNA-mediated depletion of AF17 caused nuclear accumulation of Dot1a. AF17 overexpression elicited multiple effects that are reminiscent of aldosterone action. These effects include 1) increased mRNA and protein expression of the three ENaC subunits (α, β and γ) and serum- and glucocorticoid inducible kinase 1, as revealed by real-time RT-qPCR and immunoblotting analyses; 2) impaired Dot1a-AF9 interaction and H3 K79 methylation at the αENaC promoter without affecting AF9 binding to the promoter, as evidenced by chromatin immunoprecipitation; and 3) elevated ENaC-mediated Na(+) transport, as analyzed by measurement of benzamil-sensitive intracellular [Na(+)] and equivalent short circuit current using single-cell fluorescence imaging and an epithelial Volt-ohmmeter, respectively. Knockdown of AF17 elicited opposite effects. However, combination of AF17 overexpression or depletion with aldosterone treatment did not cause an additive effect on mRNA expression of the ENaC subunits. Taken together, we conclude that AF17 promotes Dot1a nuclear export and upregulates basal, but not aldosterone-stimulated ENaC expression, leading to an increase in ENaC-mediated Na(+) transport in renal collecting duct cells.     

DOT1L Inhibition Sensitizes MLL-Rearranged AML to Chemotherapy

DOT1L, the only known histone H3-lysine 79 (H3K79) methyltransferase, has been shown to be essential for the survival and proliferation of mixed-linkage leukemia (MLL) gene rearranged leukemia cells, which are often resistant to conventional chemotherapeutic agents. To study the functions of DOT1L in MLL-rearranged leukemia, SYC-522, a potent inhibitor of DOT1L developed in our laboratory, was used to treat MLL-rearranged leukemia cell lines and patient samples. SYC-522 significantly inhibited methylation at H3K79, but not H3K4 or H3K27, and decreased the expression of two important leukemia-relevant genes, HOXA9 and MEIS1, by more than 50%. It also significantly reduced the expression of CCND1 and BCL2L1, which are important regulators of cell cycle and anti-apoptotic signaling pathways. Exposure of MLL-rearranged leukemia cells to this compound caused cell cycle arrest and promoted differentiation of those cells, both morphologically and by increased CD14 expression. SYC-522 did not induce apoptosis, even at 10 µM for as long as 6 days. However, treatment with this DOT1L inhibitor decreased the colony formation ability of primary MLL-rearranged AML cells by up to 50%, and promoted monocytic differentiation. Notably, SYC-522 treatment significantly increased the sensitivity of MLL-rearranged leukemia cells to chemotherapeutics, such as mitoxantrone, etoposide and cytarabine. A similar sensitization was seen with primary MLL-rearranged AML cells. SYC-522 did not affect chemotherapy-induced apoptosis in leukemia cells without MLL-rearrangement. Suppression of DOT1L activity inhibited the mitoxantrone-induced increase in the DNA damage response marker, γH2AX, and increased the level of cPARP, an intracellular marker of apoptosis. These results demonstrated that SYC-522 selectively inhibited DOT1L, and thereby altered gene expression, promoted differentiation, and increased chemosensitivity by preventing DNA damage response. Therefore, inhibition of DOT1L, in combination with DNA damaging chemotherapy, represents a promising approach to improving outcomes for MLL-rearranged leukemia.     

DOT1—— 一类新的组蛋白赖氨酸甲基转移酶

组蛋白甲基化是一种重要的组蛋白共价修饰, 在染色质结构和基因表达的调控过程中起着重要的、多样化的作用。DOT1催化核心球体部位的组蛋白H3第79位赖氨酸(H3K79)使其发生甲基化, 是首个被发现的无SET结构域的组蛋白赖氨酸甲基转移酶, 代表了一类新的组蛋白赖氨酸甲基转移酶。DOT1及H3K79甲基化的特点决定了其可能具有重要的、特殊的生物学功能。文章重点综述了DOT1蛋白的结构及特点, DOT1及H3K79甲基化的生物学功能以及组蛋白泛素化修饰对H3K79甲基化的反式调控。