Does KMT2A::AFF1 deliver DOT1L to the AHD or displace it? A competition experiment in t(4;11) cells
also called AF4_int competition test · inverted AHD prediction test
The infant fusion sticks to the same patch of AF9 that the H3K79-writing enzyme uses. This experiment asks whether the fusion brings the enzyme along or pushes it off, which are opposite predictions nobody has tested.
The same thing, for a scientist
Reconstitution says DOT1L and AF4 do not co-occupy AF9 [E1], and the AHD binds AF4, AFF4 or DOT1L as alternatives [E2]. If that holds in cells, KMT2A::AFF1 - which reaches AF9/ENL through the AF4_int motif [E3] - should **reduce** DOT1L occupancy on AF9/ENL rather than increase it at its targets. The prediction has the opposite sign to the one this graph has been carrying by import from MLL-AF9, and it is testable. **Design.** In a human KMT2A::AFF1 line with a degron-tagged fusion: (i) endogenous AF9 (MLLT3) and ENL (MLLT1) immunoprecipitation with quantitative mass spectrometry, before and after fusion degradation, measuring the DOT1L : fusion : AFF4 stoichiometry on the AHD-bearing proteins; (ii) DOT1L ChIP/CUT&RUN genome-wide before and after degradation; (iii) a structure-guided AF4_int point mutant of the fusion (guided by the 2LM0 interface) expressed in a fusion-degron background, to separate AHD binding from every other fusion function. **Discriminating outcome.** Fusion degradation *increasing* DOT1L on AF9/ENL supports competition; *decreasing* it supports delivery; no change supports the transcription-coupled model in which the AHD is irrelevant to H3K79 at these loci.
Why a degron and not a knockdown
The competition, if it exists, is an occupancy equilibrium, and occupancy equilibria re-establish within minutes. Knockdown takes days, during which the transcriptional programme collapses, cells differentiate or die, and DOT1L occupancy changes for reasons unrelated to the AHD. A degron with a 30-minute to 2-hour window separates the direct occupancy question from the downstream programme - the same argument this graph already makes in exp-kmt2a-aff1-degron-occupancy-titration.
The AF4_int point mutant is the cleaner experiment of the two, because it changes one interface and nothing else, but it requires that the interface be mutatable without abolishing P-TEFb recruitment - which may not be possible, since AEP assembly runs through the same contact 4. If it is not, the degron series is the only route.
What a negative result would establish
If fusion degradation does not change DOT1L occupancy on AF9/ENL at all, then the AHD is not where the index fusion's H3K79 story happens, and both the imported model (fusion delivers DOT1L) and the inverted model (fusion displaces DOT1L) are wrong. That would leave the transcription-coupled and endogenous-AF10 models standing, and would make hyp-index-fusion-h3k79-is-entirely-endogenous the leading account.
A negative here is therefore informative, which is not true of most occupancy experiments.
| design | human KMT2A::AFF1 line with degron-tagged fusion; endogenous MLLT3 and MLLT1 IP-quantitative MS before and after degradation; DOT1L CUT&RUN genome-wide; plus a structure-guided AF4_int point mutant expressed in a fusion-degron background |
|---|---|
| readout | DOT1L : fusion : AFF4 stoichiometry on AHD-bearing proteins, and genome-wide DOT1L occupancy change |
| controls | short degradation windows (30 min - 2 h) to separate occupancy from programme collapse; AFF4 as the competing endogenous ligand; a KMT2A::MLLT3 line where the fusion supplies the AHD, as the opposite-sign control |
| cost tier | high |
| timeline | 6-9 months including degron line construction |
| decision rule | DOT1L on AF9/ENL rises after fusion degradation -> competition; falls -> delivery; unchanged -> the AHD is not where the index fusion's H3K79 story happens |
- If the fusion displaces DOT1L from AF9/ENL, then removing the fusion therapeutically raises DOT1L availability, and a menin-inhibitor plus DOT1L-inhibitor combination would be antagonistic in exactly the setting the two-unit model predicts synergy.
- A degron system reports on a tagged, over- or under-expressed fusion, and occupancy equilibria are concentration-dependent, so the answer could be an artefact of tag expression level rather than of biology.
- Can an AF4_int point mutation be found that abolishes AHD binding while preserving P-TEFb recruitment? If AEP assembly runs through the same contact, the answer may be no.
- What is the absolute stoichiometry of fusion, endogenous AFF1, AFF4 and DOT1L on AF9/ENL in a t(4;11) cell? Nobody appears to have measured it, and the competition argument is a stoichiometry argument.
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
In reconstitution from defined components, DOT1 forms a stable complex with AF9 and does not interact with AF9-AF4-P-TEFb complexes; AF4 and AFF4 independently mediate association of P-TEFb with AF9 through direct contacts with AF9 and cyclin T1.
Function of leukemogenic mixed lineage leukemia 1 (MLL) fusion proteins through distinct partner protein complexes. · Biswas D, Milne TA, Basrur V, Kim J, Elenitoba-Johnson KS, Allis CD, Roeder RG · Proc Natl Acad Sci U S A 2011What this evidence cannot show
Abstract fetched from PubMed and read on 2026-09-19; full text not read. Minimal purified complexes in the absence of other factors; a sufficiency test that can miss cellular stabilisation by chromatin or bridging proteins. This is the single result the experiment exists to test in cells.
Evidence that argues against this (1)
- The same paper reports these assemblies are dynamic and interconverting, so an in-cell experiment may find partial co-occupancy that the tube experiment could not, without either result being wrong. Leach BI et al. 2013
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
The AHD of AF9/ENL binds AF4, AFF4 or DOT1L, and a small-molecule inhibitor of these interactions reduces SEC assembly and DOT1L-mediated H3K79 methylation together.
Small-molecule inhibitor of AF9/ENL-DOT1L/AF4/AFF4 interactions suppresses malignant gene expression and tumor growth. · Wu F, Nie S, Yao Y, Huo T, Li X, Wu X, Zhao J, Lin YL, Zhang Y, Mo Q, Song Y · Theranostics 2021What this evidence cannot show
Abstract fetched from PubMed and read on 2026-09-19; full text not read. A micromolar probe with a broad transcriptional footprint; the 'or' is the authors' framing of a shared site rather than a structural demonstration of exclusivity. If this compound were used in the experiment it could not distinguish the two models, which is why the design uses a degron and a point mutant instead.
Evidence that argues against this (1)
- A compound that reduces both outputs is equally consistent with two independent sites being blocked by one promiscuous molecule, so this evidence constrains the design more than it constrains the biology. Kuntimaddi A et al. 2015
- E3established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.database
AFF1 retains the AF4 interaction motif (Pfam PF18875 / InterPro IPR043639), the element that binds the AF9 and ENL ANC1 homology domains, and the AF9 AHD-AF4 complex is structurally defined.
UniProtKB entries P51825 (AFF1_HUMAN), P42568 (AF9_HUMAN) and Q03111 (ENL_HUMAN): Pfam/InterPro cross-references and feature tables · UniProt Consortium · UniProtKB 2026What this evidence cannot show
Accessions fetched directly on 2026-09-19 with primary gene names and lengths read back. The residue assignment of the conserved core is inherited from this graph's protein-aff1-domains entry and was not re-derived in this cycle, so a point mutant should be designed from the PDB entity annotation rather than from that range.
Evidence that argues against this (1)
- Whether the fusion, rather than endogenous full-length AFF1 or AFF4, is the dominant AHD ligand in a t(4;11) cell is unmeasured; if AFF4 dominates, the competition exists but the fusion is not the competitor and the experiment's premise shifts. Yokoyama A et al. 2010
- E4established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper
AF4 and ENL family proteins form a higher-order complex with P-TEFb (AEP), and MLL oncoproteins fused with AEP components constitutively form MLL/AEP hybrid complexes to sustain target expression.
A higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription. · Yokoyama A, Lin M, Naresh A, Kitabayashi I, Cleary ML · Cancer Cell 2010What this evidence cannot show
Abstract fetched from PubMed and read on 2026-09-19; full text not read. Biochemical purification plus murine myeloid transformation. Cited here as a design constraint: AEP assembly runs through the same AF4-ENL-family contact, so an AF4_int mutant may not be separable from loss of P-TEFb recruitment.
Evidence that argues against this (1)
- If AEP assembly and DOT1L exclusion are inseparable, then the index fusion's elongation function and its putative DOT1L displacement are one event, and no point mutant can dissect them - which would make the degron series the only informative design. Biswas D et al. 2011
tests the interface's sign in cells
a negative result here promotes the endogenous model
measures DOT1L occupancy on AHD-bearing proteins
AF9 is the immunoprecipitated receptor
- tests →Import class 2: AHD-dependent mechanism, which cannot transfer because KMT2A::AFF1 has no AHD
tests the inverted prediction this import class generates