The DOT1L-AF10 octamer - the second route to H3K79 methylation, required in MLL-AF10 and absent from KMT2A::AFF1
also called DOT1L-AF10 coiled coil · AF10 OM-LZ · MLLT10-DOT1L recruitment · DotCom AF10 arm
DOT1L reaches the genome by two different handshakes. One is with AF9 or ENL; the other is with AF10, and that second one only works when four copies of the pair assemble into a larger unit. The commonest infant leukaemia fusion contains neither AF9 nor AF10, so claims about how DOT1L gets recruited do not transfer between fusion partners.
The same thing, for a scientist
AF10/MLLT10 recruits DOT1L through its **octapeptide motif and leucine zipper (OM-LZ)**, which binds the DOT1L coiled-coil domains. The structural work shows the interaction is not simply a heterodimer: the C-terminal end of the LZ and the OM mediate **tetramerisation of the binary complex into a DOT1L-AF10 octamer**, and that oligomerisation is **essential** for MLL1-AF10's leukaemogenic function [E1]. Crystal structures of the coiled-coil regions have since supported a structure-optimised inhibitory peptide that lowers *HOXA* expression in human cells [E2]. Functionally AF10 is the cofactor that drives the **me1-to-me2 conversion**: H3K79me2 is converted to me1 at *HOX* loci as haematopoietic cells mature, AF10 inactivation reverses leukaemia-associated epigenetic profiles and impairs transformation by MLL-AF9, MLL-AF6 **and NUP98-NSD1** [E3]. CRISPR-ChIP places MLLT10 as the director of the **endogenous** DOT1L complex, distinct from the menin-dependent fusion-directed one [E4], and AF10 fusions themselves are recruited to chromatin by a MOZ/ENL complex whose KAT activity maintains their retention [E5]. **The policing point for this graph: AF10 is not in KMT2A::AFF1.** Any DOT1L-recruitment claim must name the partner it was established in.
Two interfaces, three fusions, and the transfer error to avoid
| route | interface | fusions that carry it |
|---|---|---|
| AHD | AF9/ENL ANC1 homology domain binds DOT1L | KMT2A::MLLT3, KMT2A::MLLT1 |
| OM-LZ octamer | AF10 leucine zipper binds the DOT1L coiled coils, tetramerising | KMT2A::MLLT10, PICALM::MLLT10 |
| neither | - | KMT2A::AFF1 |
KMT2A::AFF1 is the commonest fusion in infant ALL and contains no AHD and no AF10. Its route to DOT1L runs through complexes assembled around AFF1 rather than through either characterised direct interface, and this graph should not import an AF9- or AF10-derived recruitment claim into an AFF1 entry without saying so. The same applies in reverse: mechanism-dot1l-ppi-inhibition-modality describes AHD-pocket compounds that cannot touch the AF10 route.
AF10's own domain logic adds a second policing point. In both KMT2A::MLLT10 and PICALM::MLLT10 the OM-LZ is retained and the N-terminal PZP module is lost, so a fusion keeps the DOT1L-binding half and discards the histone-reading half - which is recorded in gene-mllt10 and protein-mllt10-domains and is contested there.
Why oligomerisation matters more than affinity
The octamer result reframes what a DOT1L-recruitment inhibitor has to achieve 1. A binary interface can be competed by a molecule with sufficient affinity. A tetramerised complex has avidity: four copies of a two-protein interface, so the effective off-rate of the assembly is far slower than that of any single contact, and a competitor must disrupt several contacts at once.
That is the structural reason micromolar peptide-based inhibitors of this interface 2 are further from a drug than their IC50 alone suggests, and it is a general warning for this project's interface-targeting programme: oligomerisation state, not affinity, is the variable that decides whether an interface is competable. The menin-KMT2A interface is a binary contact with a defined pocket, which is exactly why it yielded to chemistry where this one has not.
It is also a falsifiable structural claim with a therapeutic reading: if oligomerisation is essential, then an agent that merely caps the octamer - a monovalent LZ mimetic that blocks tetramerisation without displacing the dimer - would be sufficient, and would be a far easier molecule than a full interface disruptor.
| mechanism class | recruitment / complex assembly |
|---|---|
| enzyme | gene-dot1l |
| cofactor | gene-mllt10 |
| interface | AF10 octapeptide motif + leucine zipper (OM-LZ) binding the DOT1L coiled-coil domains |
| stoichiometry | octamer - tetramerisation of the DOT1L-AF10 binary complex, mediated by the LZ C-terminus and the OM |
| oligomerisation required for leukaemogenesis | true |
| upstream recruiter of af10 fusions | MOZ/ENL complex, via the ENL YEATS domain and MOZ/MORF KAT activity (PMID 37031220) |
| chemical matter | structure-optimised inhibitory peptide against the AF10 LZ (PMID 33993518); no drug-like small molecule reported in this beat's reading |
| functional role | controls the H3K79me1-to-me2 conversion at HOX loci |
fusions using this route
[ "fusion-kmt2a-mllt10", "PICALM::MLLT10 (CALM-AF10)" ]
fusions using the AHD route instead
[ "fusion-kmt2a-mllt3", "fusion-kmt2a-mllt1" ]
fusions using neither
[ "fusion-kmt2a-aff1" ]
- An interface held together by avidity across four copies is much harder to compete than its per-contact affinity suggests, so an inhibitor programme priced on a measured Kd will be systematically over-optimistic here.
- In KMT2A::MLLT10 and PICALM::MLLT10 the endogenous and oncogenic DOT1L routes converge on the same protein, so any strategy that gains selectivity by sparing the MLLT10-directed unit has no selectivity in exactly the fusions where AF10 is the partner.
- Blocking AF10-DOT1L also removes the cofactor that normal maturing progenitors use to set their HOX-locus methylation state, so this interface has less inherent tumour-versus-host margin than the AF9 interface, which is dispensable in normal haematopoiesis.
- What is the stoichiometry of the DOT1L-AF10 complex on chromatin in a leukaemic cell, as opposed to on purified coiled-coil fragments? The therapeutic argument for capping rather than disrupting the interface depends on the answer.
- Would a monovalent LZ mimetic that blocks tetramerisation without displacing the heterodimer be sufficient to abolish leukaemogenesis? That is a far easier molecule than a full interface disruptor and the octamer result predicts it would work.
- By what interface does KMT2A::AFF1 reach DOT1L, given that it has neither an AHD nor AF10? This graph asserts DOT1L dependence for KMT2A-rearranged leukaemia generally without a mapped route for its commonest infant fusion.
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
The MLL1-AF10 fusion causes leukaemia by recruiting DOT1L via AF10's octapeptide and leucine-zipper (OM-LZ) motifs; the AF10 LZ domain binds the coiled-coil domains of DOT1L through a conserved binding mode, and the C-terminal end of the LZ together with the OM domain mediate formation of a DOT1L-AF10 octamer by tetramerisation of the binary complex. The oligomerisation ability of the DOT1L-AF10 complex is essential for MLL1-AF10's leukaemogenic function.
A higher-order configuration of the heterodimeric DOT1L-AF10 coiled-coil domains potentiates their leukemogenenic activity. · Song X, Yang L, Wang M, et al. · Proc Natl Acad Sci U S A 2019What this evidence cannot show
Abstract read from the eutils record; full text not read. Structural and functional analysis of isolated coiled-coil domains; an octamer formed by purified fragments need not be the stoichiometry of the full-length complex on chromatin, and the oligomerisation state in cells is not established by the abstract. Functional essentiality is shown for MLL1-AF10 specifically. Note the title contains a typographical error in the original ('leukemogenenic'), reproduced here as PubMed stores it.
Evidence that argues against this (1)
- An oligomerisation requirement demonstrated with mutations that abolish tetramerisation cannot distinguish a structural requirement for avidity from a requirement for the surface those mutations also destroy. Independent crystal structures of the same coiled-coil regions support a different recognition pattern for an inhibitory peptide, which shows the interface tolerates more than one binding mode. Zhou Z et al. 2021
- E2moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper
Crystal structures of the coiled-coil regions of DOT1L-AF10 and of an AF10-inhibitory peptide complex show the inhibitory peptide forms a compact complex with AF10 via a different recognition pattern; a structure-based optimised inhibitory peptide decreases HOXA gene expression in a human cell line.
Structural characteristics of coiled-coil regions in AF10-DOT1L and AF10-inhibitory peptide complex. · Zhou Z, Kang S, Huang Z, et al. · J Leukoc Biol 2021What this evidence cannot show
Abstract read from the eutils record; full text not read. A peptide, not a small molecule, with no affinity, no cellular potency and no selectivity data in the abstract, and a single human cell line readout. The abstract contains an apparent error - 'catalysing H3K79 monomethylation to demethylation' - which should read as the conversion from mono- to di-methylation; this entry does not rely on that sentence. Structures of isolated coiled-coil regions do not report on the octamer.
Evidence that argues against this (1)
- If the functional unit is a tetramerised octamer, a peptide that occupies one AF10 LZ contact faces the avidity of three others and is unlikely to disassemble the complex at achievable concentrations - which is the structural reason to doubt that this chemotype reaches a drug. Song X et al. 2019
- E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
H3K79me2 is converted to H3K79me1 at HOX loci as haematopoietic cells mature, coinciding with decreased HOX expression; H3K79 methyltransferase activity and the me1-to-me2 conversion are regulated by the DOT1L cofactor AF10; AF10 inactivation reverses leukaemia-associated epigenetic profiles, precludes abnormal HOXA expression and impairs the transforming ability of MLL-AF9, MLL-AF6 and NUP98-NSD1, and NUP98-NSD1-transformed cells are sensitive to small-molecule DOT1L inhibition.
AF10 regulates progressive H3K79 methylation and HOX gene expression in diverse AML subtypes. · Deshpande AJ, Deshpande A, Sinha AU, et al. · Cancer Cell 2014What this evidence cannot show
Abstract read from the eutils record; full text not read. Mouse models throughout. The three fusions tested are myeloid (MLL-AF9, MLL-AF6, NUP98-NSD1); no lymphoid fusion and no KMT2A::AFF1. Endogenous AF10 is the subject, which is a different question from AF10 as a fusion partner. The 'conversion' language is inferred from distributions across maturation rather than from pulse-chase.
Evidence that argues against this (1)
- Under distributive kinetics, states cannot be generated independently, so what looks like a regulated me1-to-me2 conversion is what a change in enzyme flux looks like. AF10 may set how much methylation happens rather than which state is produced, and the two models are not distinguished by the distributions reported. Frederiks F et al. 2008
- E4strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
CRISPR-ChIP partitions H3K79 methylation into an oncogenic DOT1L complex directed by the MLL fusion protein in a menin-dependent manner, and a separate endogenous DOT1L complex whose catalytic activity is directed by MLLT10.
CRISPR-ChIP reveals selective regulation of H3K79me2 by Menin in MLL leukemia. · Gilan O, Talarmain L, Bell CC, et al. · Nat Struct Mol Biol 2023What this evidence cannot show
Abstract read from the eutils record; full text not read. The pooled screen is reporter-based, but the partition is not reporter-only: GSE192555 holds genome-wide H3K79me2 ChIP-seq for six arms plus FLAG-MLLAF9 occupancy, so the partition is supported at endogenous loci as well. **The fusion is now resolved and it is not the index fusion: murine MLL-AF9.** It is not named in the abstract, and the full text is unreachable, but the study's GEO depositions name it per sample. In GSE192555 all eight H3K79me2 ChIP-seq samples are Mus musculus with `cell type: MLLAF9`, including both DOT1L-KO guides, both MLLT10-KO guides and **the menin-inhibitor arm** (GSM5751590). GSE192561 is 24/24 murine. Across all 65 samples in the three series, **none mentions AFF1, AF4 or any AFF-family gene**. The human arm is K562 — a CML line with no KMT2A rearrangement — carrying H3K4me3, Pol II, and five H3K79me2 samples used as a mouse/human library-mix species-spike normalisation under a DOT1L inhibitor, not as a fusion system. So this result reaches KMT2A::AFF1 only by import across fusion, species and lineage at once. Verified by the coordinator directly against the GEO SOFT records on 2026-09-19. A functional-genomic requirement is not a biochemical mechanism.
Evidence that argues against this (2)
- Assigning MLLT10 to the endogenous unit and the fusion to the oncogenic one cannot hold in KMT2A::MLLT10 or PICALM::MLLT10, where AF10 IS the fusion partner. In those diseases the two units are not separable by targeting MLLT10, which bounds the generality of the partition. Deshpande AJ et al. 2014
- The fusion behind this result is murine MLL-AF9, not KMT2A::AFF1 — established from the study's own GEO depositions, since the full text is unreachable. Every H3K79me2 sample in GSE192555 is Mus musculus, cell type MLLAF9, and no sample in any of the three series mentions an AFF-family gene. The menin-dependence of the oncogenic unit, which is the part this graph leans on hardest, was measured in mouse myeloid MLL-AF9 cells. KMT2A::AFF1 engages DOT1L through a different architecture — AFF1 carries neither the AHD that MLLT3 and MLLT1 use nor the AF10 OM-LZ — so the partition may not transfer, and `hyp-index-fusion-h3k79-is-entirely-endogenous` is the sharper alternative. NCBI Gene Expression Omnibus 2022
- E5strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
AF10 fusion proteins such as CALM-AF10 cause aberrant self-renewal via ENL, which binds MOZ/MORF lysine acetyltransferases; the ENL-MOZ interaction through the ENL YEATS domain is critical for CALM-AF10-mediated transformation, the MOZ/ENL complex recruits DOT1L/AF10 fusion complexes and maintains their chromatin retention via KAT activity, MOZ/MORF KAT inhibitors suppress AF10 fusion function with strong antitumour effects, and combined MOZ/MORF and DOT1L inhibition cooperatively induces differentiation of CALM-AF10 leukaemia cells.
MOZ/ENL complex is a recruiting factor of leukemic AF10 fusion proteins. · Komata Y, Kanai A, Maeda T, et al. · Nat Commun 2023What this evidence cannot show
Abstract read from the eutils record; full text not read. The fusion studied is CALM-AF10 (PICALM::MLLT10), not KMT2A::MLLT10, and the abstract generalises to 'AF10 fusion proteins'. Mouse transformation assays and cell lines. KAT inhibitors have broad substrate scope, so 'directly suppress the functions of AF10 fusion proteins' is an inference from phenotype plus chromatin retention rather than from a selective perturbation.
Evidence that argues against this (1)
- This places ENL upstream of AF10 fusions, which means an ENL-directed agent would act on both DOT1L-recruitment routes at once - and therefore that the two routes this entry separates are not independent in AF10-fusion disease. The clean two-interface picture is a simplification. Gilan O et al. 2023
the same axis, written from the non-KMT2A side
AF10 OM-LZ is the interface this axis runs on
AF10 LZ binds the DOT1L coiled coils; the complex tetramerises into an octamer
oligomerisation of DOT1L-AF10 is essential for MLL-AF10 leukaemogenesis
AF10 controls the me1-to-me2 conversion at HOX loci
OM-LZ retained and PZP lost in both MLLT10 fusions
- related to →NUP98::NSD1, t(5;11)(q35;p15.5)
AF10 inactivation impairs NUP98-NSD1 transformation and those cells are DOT1L-inhibitor sensitive
ENL YEATS-MOZ complex recruits AF10 fusion/DOT1L complexes to chromatin
the AF10 axis is one of the two interfaces, and the index fusion has neither
the existing entry for this axis
caveat can now name the system
The DOT1L-AF10 axis is the highest-stoichiometry, core-flagged contact in this atlas pulldown, independent of the leukaemia literature
the normal-tissue arm of the same DOT1L-AF10 module, in intestinal crypts