Lost from the KMT2A half: the WIN motif and the whole WRAD interface (aa 3762-3767, Arg3765)
also called WDR5 interaction motif · WIN motif · WRAD · MLL1 core complex assembly · R3765
KMT2A only works as an enzyme when four helper proteins clamp onto it, and they all attach through a single arginine near the far end of the protein. Every leukemic fusion is cut long before that point, so the fusion protein cannot assemble the enzyme at all.
The same thing, for a scientist
The MLL1 core complex requires WDR5, RBBP5, ASH2L and DPY-30 (WRAD), assembled onto MLL1 through the WDR5-interaction (WIN) motif at aa 3762-3767 with Arg3765 in the WDR5 arginine pocket [E1, E2, E4], and mutating Arg3765 abolishes WRAD co-immunoprecipitation from cells [E2]. Residue 3762 is ~2,400 residues C-terminal to the breakpoint cluster region, and consistently the founding biochemical survey found that oncogenic MLL fusions retain menin but not ASH2L, WDR5 or RBBP5 [E3]. The WRAD interface is therefore **wild-type-only**. **A live tension, stated cycle 31.** MM-401 blocks this interface and selectively kills MLL-rearranged cells [E5], while MLL1's SET activity - the output that interface assembles - is genetically dispensable for MLL-AF9 leukemogenesis [see `subtraction-kmt2a-set-domain`, PMID 24813891]. Both results are verified and in this graph, and the tension is resolved neither by this entry nor by the field. Later small-molecule WIN-site inhibitors sharpen it in an uncomfortable direction: they displace WDR5 from a small cohort of loci dominated by **ribosome protein genes**, and in MLL-rearranged cells the dominant response is ribosome attrition, nucleolar stress and p53/MDM4 activation rather than loss of an MLL1 programme [E6, E7]. So the entry's strategic point - a module absent from the fusion can still be a dependency of the cell - survives, but it now rests on genetics [E8] rather than on the pharmacology, because the pharmacology may not be reporting on MLL1 at all.
The subtraction, stated exactly
One arginine carries the whole assembly. Patel et al. solved WDR5 bound to the MLL1 Win peptide at 1.7 A and found Arg3765 of MLL1 bound in the same arginine-binding pocket on WDR5 previously proposed to bind histone H3, with the MLL1 Win peptide preferentially recognised over H3 1. Dharmarajan et al. then showed Arg3765 is required to co-immunoprecipitate WRAD from mammalian cells, and solved WDR5 bound to six naturally occurring Win motif sequences, finding that binding-energy differences come mostly from non-conserved residues C-terminal to the motif 2.
UniProt's independent annotation agrees: WIN motif 3762-3767, site 3765 'Important for WDR5-recognition and binding' 4. So this is a confirmed coordinate, by structure and by curation.
Because KMT2A breakpoints cluster around residues 1,100-1,400, no fusion protein contains residue 3765. Yokoyama et al.'s complex purification is the empirical confirmation: fusion MLL proteins coprecipitate menin and not ASH2L, WDR5 or RBBP5 3.
The twist: the interface the fusion lost is a validated drug target in the same disease1 deeper
This is the single most counter-intuitive thing in the KMT2A-side subtraction beat, and it cuts against a naive reading of 'lost means irrelevant'.
MM-401 is a peptidomimetic that blocks the MLL1-WDR5 interaction and thereby MLL1's H3K4 methyltransferase activity. It specifically blocks proliferation of MLL-rearranged cells, inducing cell-cycle arrest, apoptosis and myeloid differentiation, without general toxicity to normal bone marrow or non-MLL cells; its transcriptome changes resemble MLL1 deletion 5.
The fusion has no WIN motif. So MM-401 cannot be acting on the fusion. It must be acting on wild-type MLL1 - the other allele - and that is enough to kill KMT2A-rearranged cells selectively.
The implication for this beat is sharp: a module being absent from the fusion does not make it therapeutically irrelevant, because the leukemic cell still owns a copy. Subtraction is about the fusion protein; dependency is about the cell. The graph should keep those separate.
And the tension this creates with the SET-domain story
MM-401 works by disabling MLL1's catalysis (via assembly), and it kills MLL-rearranged cells 5. Mishra et al. deleted the SET domain and found MLL1 catalysis dispensable for MLL-AF9 leukemogenesis [see subtraction-kmt2a-set-domain]. These two results are in genuine tension, and the graph should not quietly pick one.
Possible reconciliations, none established: MM-401 blocks assembly and therefore removes WRAD-dependent scaffolding as well as catalysis; the genetic deletion allows compensation over developmental time that an acute pharmacological block does not; the models differ (human MLL cell lines vs mouse MLL-AF9). Each is testable and none has been tested head to head.
The live tension this entry has to state (cycle 31)
Three results, all verified, all in this graph, that do not sit together:
- MM-401 blocks the MLL1-WDR5 interaction and selectively kills MLL-rearranged cells, with cell-cycle arrest, apoptosis and myeloid differentiation, no general toxicity to normal bone marrow or non-MLL cells, an enantiomer control, and transcriptome changes resembling MLL1 deletion 5.
- MLL1's SET-domain methyltransferase activity is dispensable for MLL-AF9 leukemogenesis and for haematopoiesis (PMID 24813891, recorded in
subtraction-kmt2a-set-domain). The WIN motif's entire documented job is to assemble the complex that licenses that activity. - WIN-site inhibitors do something else. Potent small molecules from an independent group show the WIN site tethers WDR5 to a small cohort of loci dominated by ribosome protein genes; blocking it displaces WDR5, collapses ribosome protein gene transcription, and in MLL-rearranged cells produces ribosome subunit attrition, a broad translational choke, MDM4-inactivating splicing changes and p53 activation 67. Those authors state directly that the HSC-expansion mechanism originally proposed for peptidomimetic WINi is not what their data support.
How they might be reconciled, and why the entry does not pick one.
- If (3) is the whole story, MM-401's selectivity for MLL-rearranged cells is a p53/ribosome-stress sensitivity of those cells, not a statement about MLL1 at all - and this entry's inference that MM-401 must be acting on the wild-type allele is simply the wrong question. The interface is still wild-type-only; it just is not being blocked as an MLL1 interface.
- If (1) is right and (2) is right, then the WIN motif matters for something other than SET activity - complex integrity, residence time, or the location of WDR5 - and the SET domain being dispensable does not discharge the interface.
- The two agents are not the same chemotype. MM-401 is a peptidomimetic; 67 use small molecules. Nobody has run the ribosome-protein-gene and p53 readouts on MM-401, which is the obvious missing experiment and would cost very little.
What survives regardless. The strategic claim this entry exists for - a module being absent from the fusion does not make it therapeutically irrelevant, because the leukemic cell still owns a copy - is unaffected, but its best support is no longer MM-401. It is the genetic result that the wild-type Mll allele is required for MLL-AF9 leukemogenesis and maintenance 8. That is a cleaner argument, and it is the one the entry should lead with.
| module | WIN motif / WRAD assembly interface |
|---|---|
| uniprot | Q03164 |
| start | 3762 |
| end | 3767 |
| key residue | 3765 |
| fate in fusion | lost |
| demonstrated functional consequence of loss to the fusion | none - the fusion never had it; no separation-of-function experiment restores it |
| demonstrated relevance of the module to the disease | high, but through the WILD-TYPE allele, and best supported genetically (wild-type Mll required for MLL-AF9 leukemogenesis, PMID 20159607) rather than pharmacologically, since the WIN-site pharmacology may not report on MLL1 at all |
| loss class | wild-type-only module; absent from the fusion yet druggable in the same cell |
| mechanistic status | contested. MM-401's selective killing of MLL-rearranged cells (PMID 24389101) is interpreted in this entry as action on wild-type MLL1, but independent small-molecule WIN-site inhibitors act in MLL-rearranged cells through ribosome protein gene suppression, nucleolar stress and p53/MDM4 (PMIDs 30865883, 38682900), and MLL1 SET activity is genetically dispensable for MLL-AF9 leukemogenesis (PMID 24813891). |
| combination note | WIN-site inhibitors are reported synergistic with venetoclax and with BET-bromodomain inhibitors (PMID 38682900) - both already in this graph's therapeutic space. |
partners lost
[ "WDR5 (gene-wdr5)", "RBBP5 (gene-rbbp5)", "ASH2L (gene-ash2l)", "DPY30 (gene-dpy30)" ]
position confirmed by
[ "crystal structure PMID 18829459", "cellular co-IP PMID 22665483", "UniProt Q03164 motif + site features" ]
- Any WIN-motif or WDR5-directed agent acts on wild-type KMT2A, which is under extreme human loss-of-function constraint (LOEUF 0.07) and whose heterozygous loss causes Wiedemann-Steiner syndrome; the therapeutic window is a question about normal haematopoiesis and development, not about the fusion.
- WDR5 is a subunit of many complexes beyond MLL1, so a WDR5-pocket ligand is not an MLL1-selective agent even if a WIN-peptide mimetic is.
- If MM-401-type activity depends on the retained wild-type allele, cases that have lost it would be intrinsically resistant from the outset - a resistance mechanism that exists before treatment and is invisible unless allele status is measured.
- If the WIN-site phenotype in MLL-rearranged cells is ribosome stress and p53 activation, then the whole 'target the wild-type allele' strategy loses its best example, and the graph should not present MM-401 as a proof of concept for allele-directed therapy. The concept survives on genetics; the drug does not carry it.
- A p53-dependent mechanism is a poor fit for relapsed disease, where TP53 lesions are enriched - and it would be defeated by exactly the population a KMT2A-rearranged programme most needs to treat.
- Does MM-401 produce the ribosome-protein-gene, nucleolar-stress and p53 signature that small-molecule WIN-site inhibitors produce? Running those readouts on MM-401 would tell the field in one experiment whether two chemotypes hitting one pocket share a mechanism, and nobody has done it.
- If WIN-site inhibitors act through p53, are they inactive in TP53-mutant KMT2A-rearranged disease? That is a directly testable stratifier with an obvious clinical shape, and it is a very different biomarker from the wild-type-allele-status one this entry previously proposed.
- How can MM-401 kill MLL-rearranged cells by blocking assembly of a complex whose catalytic output is genetically dispensable in the same disease? Either the WIN motif does something besides license SET activity, or one of the two results is not about what it appears to be about.
- Could the fusion be engineered to carry a WIN motif, and would WRAD assembly onto a fusion be oncogenic, neutral or suppressive? Still unattempted, and still the reciprocal of the PHD3 add-back.
- E1established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper
A 1.7 A crystal structure of WDR5 bound to a peptide from the MLL1 WDR5-interaction (Win) motif shows Arg-3765 of MLL1 bound in the same arginine-binding pocket on WDR5 previously suggested to bind histone H3, and thermodynamic binding experiments show the MLL1 Win peptide is preferentially recognised by WDR5.
Structure of WDR5 bound to mixed lineage leukemia protein-1 peptide · Patel A, Dharmarajan V, Cosgrove MS · Journal of Biological Chemistry 283(47):32158-32161 2008What this evidence cannot show
ABSTRACT ONLY. A peptide-bound crystal structure and isothermal titration data with isolated components; it establishes the residue and the pocket, not the behaviour of the full-length 3,969-residue protein on chromatin. **The universal is false and the qualification matters.** The SET domain and its WRAD contacts are lost from the **direct 5' product** only. Two classes keep them: the **reciprocal der(4) product AFF1::KMT2A**, which carries KMT2A exons 12-37 including the WIN motif and SET domain and is expressed in a large minority of t(4;11) infants; and **NUP98::KMT2A** (inv(11)(p15q23)), which deletes the exon-1 menin motif while keeping WIN and SET, and is menin-inhibitor resistant. Both counterexamples were already in this graph when the universal was written — a linking failure, not a knowledge gap. Corrected 2026-09-20 after the Q23 adjudication; verified by the coordinator against both abstracts.
Evidence that argues against this (2)
- A 1.7 A peptide-bound structure establishes how the Win motif engages WDR5's arginine pocket; it does not establish that this interface is the rate-limiting one in a leukaemic cell. The same arginine pocket is used by the SET1-family paralogues, so the structure describes a shared mode rather than an MLL1-specific target. Journal of Biological Chemistry 2012
- The reciprocal AFF1::KMT2A product is the exact mirror of the one this entry describes: **WRAD-competent and menin-blind**. In the subset of t(4;11) infants expressing it, the cell contains a second KMT2A-derived oncoprotein with an intact methyltransferase module that a menin inhibitor cannot engage by construction. Any claim about what 'the fusion' retains has to say which one. Oncogene 2021
- E2established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper
Arginine 3765 of the MLL1 Win motif is required to co-immunoprecipitate WRAD (WDR5, RbBP5, Ash2L, DPY-30) from mammalian cells, indicating that the WDR5-Win motif interaction is important for assembly of the MLL1 core complex in vivo; peptides mimicking SET1-family Win motifs inhibit H3K4 dimethylation by the MLL1 core complex.
Structural basis for WDR5 interaction (Win) motif recognition in human SET1 family histone methyltransferases · Dharmarajan V, Lee JH, Patel A, Skalnik DG, Cosgrove MS · Journal of Biological Chemistry 287(33):27275-27289 2012What this evidence cannot show
ABSTRACT ONLY. Co-immunoprecipitation from HEK293 cells with overexpressed constructs plus in-vitro methylation assays and six crystal structures; 'in vivo' here means in cultured cells. No haematopoietic or leukemia model.
Evidence that argues against this (1)
- Co-immunoprecipitation of WRAD after an R3765 mutation shows the Win motif is required to ASSEMBLE the complex in cells, which is a necessary step and not evidence that assembly is what matters for the leukaemia. The fusion lacks this region entirely, so the experiment speaks to wild-type MLL1 biology. Journal of Biological Chemistry 2012
- E3established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper
In the purified 1-MDa wild-type MLL complex, ASH2L, WDR5, RBBP5, HCF-1 and HCF-2 are present alongside menin, but menin is the only identified component that associates with oncogenic mutant (fusion) MLL proteins.
Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression · Yokoyama A, Wang Z, Wysocka J, Sanyal M, Aufiero DJ, Kitabayashi I, Herr W, Cleary ML · Molecular and Cellular Biology 24(13):5639-5649 2004What this evidence cannot show
FULL TEXT read via PMC480881. 'Only identified component' means only among the components this 2004 purification identified; later-discovered partners of the retained N-terminus (LEDGF/PSIP1, the PAF complex) were not in scope. Fusion work used MLL-p300 in transfected 293 cells and endogenous MLL-ENL in HB cells.
Evidence that argues against this (1)
- Menin being the only identified component contacting the retained N-terminus is a statement about a 2004 purification and its detection limits. Later work added HCF-1 and LEDGF to the retained-side interaction map, so 'the only identified component' is a snapshot of what that preparation resolved rather than a structural fact about the fusion. Molecular and Cellular Biology 2004
- E4established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.database
UniProt Q03164 annotates a WDR5-interaction motif (WIN) at residues 3762-3767 and a site at residue 3765 described as important for WDR5 recognition and binding, with the SET domain at 3829-3945.
UniProtKB entry Q03164 (KMT2A_HUMAN) - motif and site features, retrieved via the UniProt REST API · UniProt Consortium · UniProtKB 2026What this evidence cannot show
Accession verified by reading back the primary gene name (KMT2A) and length (3,969 aa) from the fetched record rather than trusting a gene-symbol search. Curatorial annotation derived from the cited structural literature, so E4 is not fully independent of E1/E2; it is used here as a coordinate cross-check, not as a second experiment.
Evidence that argues against this (1)
- A UniProt feature table is a curated model rather than a measurement. Profile-based domain calls for KMT2A differ between UniProt, Pfam, SMART, InterPro and CATH, and the sibling atlas beat was corrected last cycle for quoting one source's range as though it were observed. Quoting coordinates without the spread states a choice as a fact. Journal of Biological Chemistry 2008
- E5strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
MM-401 specifically inhibits MLL1 activity by blocking the MLL1-WDR5 interaction and thus core-complex assembly, without affecting other MLL-family methyltransferases, and blocks proliferation of MLL-rearranged cells by inducing cell-cycle arrest, apoptosis and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells; its transcriptome changes resemble those of MLL1 deletion.
Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia · Cao F, Townsend EC, Karatas H, et al. (Wang S, Dou Y, senior authors) · Molecular Cell 53(2):247-261 2014What this evidence cannot show
ABSTRACT ONLY. A tool-compound study from the laboratory that made the compound; cell lines and mouse cells, with an enantiomer control (MM-NC-401) which is a good control. MM-401 is a peptidomimetic with poor drug-like properties and was never developed clinically. The inference that its target must be the wild-type allele (because the fusion lacks a WIN motif) is this Queen's inference from the combination of E1/E3/E5, not a statement made in the paper.
Evidence that argues against this (1)
- The mechanistic reading of MM-401 in this entry - that blocking the MLL1-WDR5 interaction kills MLL-rearranged cells by inhibiting MLL1 - is contradicted by later small-molecule work from an independent group. WIN-site inhibitors displace WDR5 from a small cohort of loci dominated by ribosome protein genes, and in MLL-rearranged cells the dominant response is ribosome subunit attrition, nucleolar stress and p53/MDM4 activation, not loss of an MLL1-dependent transcription programme. Those authors say explicitly that the HSC-expansion mechanism originally proposed for peptidomimetic WINi is not what their data show. Howard GC et al. 2024
- E6strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
Potent small-molecule WIN-site inhibitors show that the WIN site tethers WDR5 to chromatin at only a small cohort of loci, including a specific subset of ribosome protein genes; the inhibitors rapidly displace WDR5 from chromatin and decrease expression of those associated genes, causing translational inhibition, nucleolar stress and p53 induction.
Displacement of WDR5 from Chromatin by a WIN Site Inhibitor with Picomolar Affinity · Aho ER, Wang J, Gogliotti RD, Howard GC, Phan J, Acharya P, Macdonald JD, Cheng K, Lorey SL, Lu B, Wenzel S, Foshage AM, Alvarado J, Wang F, Shaw JG, Zhao B, Weissmiller AM, Thomas LR, Vakoc CR, Hall MD, Hiebert SW, Liu Q, Stauffer SR, Fesik SW, Tansey WP · Cell Reports 26(11):2916-2928.e13 2019What this evidence cannot show
ABSTRACT ONLY. Chemical biology in cancer cell lines from the group that made the compounds, with a defined structural series. It does not disprove that the WIN site matters in MLL-rearranged cells; it reassigns the primary consequence of blocking it, and the reassignment is not to MLL1 at all.
Evidence that argues against this (1)
- This result bounds the therapeutic reading of the WIN site rather than supporting it: a picomolar WIN-site inhibitor displaces WDR5 from only a small cohort of loci, chiefly ribosome protein genes. If the WIN site tethers WDR5 at a narrow and largely ribosomal set, the interface is not a general controller of the MLL1 programme. Cell Reports 2019
- E7strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
In MLL-rearranged cancer cells specifically, small-molecule WIN-site inhibitors act by suppressing ribosome protein gene transcription, inducing nucleolar stress and activating p53; they deplete the ribosome stock with a broad but modest translational choke and alternative-splicing changes that inactivate the p53 antagonist MDM4; and the authors state directly that peptidomimetic WIN-site inhibitors were ORIGINALLY proposed to act in MLL-rearranged cells through dysregulation of genes connected to haematopoietic stem cell expansion - the mechanism their own data do not support. WIN-site inhibitors are synergistic with venetoclax and with BET-bromodomain inhibitors.
Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition · Howard GC, Wang J, Rose KL, Jones C, Patel P, Tsui T, Florian AC, Vlach L, Lorey SL, Grieb BC, Smith BN, Slota MJ, Reynolds EM, Goswami S, Savona MR, Mason FM, Lee T, Fesik SW, Liu Q, Tansey WP · eLife 12:e90683 2024What this evidence cannot show
ABSTRACT ONLY (peer-reviewed eLife version; an earlier bioRxiv version, PMID 37546802, carries the same title and is not cited separately). Human MLL-rearranged cell lines, no in-vivo arm in the abstract, and the compounds are the authors' own small-molecule series rather than MM-401 itself - so this is strictly an argument about what WIN-site blockade does, not a re-run of the MM-401 experiment. It nonetheless directly contradicts the mechanistic interpretation this entry built on MM-401.
Evidence that argues against this (1)
- And the mechanism of WIN-site inhibitors in MLL-rearranged cells turns out to be ribosome protein gene suppression with nucleolar stress and a p53-MDM4 response - not disruption of the fusion's programme. That is an on-target effect at the wrong node for this entry's argument, and it predicts the efficacy would be p53-dependent. eLife 2024
- E8strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
The wild-type Mll allele is required for MLL-AF9-induced leukemogenesis and for maintenance of MLL-AF9-transformed cells, with menin recruiting both wild-type MLL and the fusion to HOX loci.
MLL-AF9-induced leukemogenesis requires coexpression of the wild-type Mll allele · Thiel AT, Blessington P, Zou T, Feather D, Wu X, Yan J, Zhang H, Liu Z, Ernst P, Koretzky GA, Hua X · Cancer Cell 17(2):148-159 2010What this evidence cannot show
ABSTRACT ONLY; mouse genetics in one fusion. Cited here because it is the independent support for this entry's central inference - that a wild-type-only module can be a real dependency - and it is much stronger support than the MM-401 pharmacology, which turns out to be mechanistically ambiguous.
Evidence that argues against this (1)
- The wild-type requirement this item states is contradicted by a later study in the same journal reporting endogenous MLL1 dispensable and MLL2 as the sustaining paralogue. A Win-WRAD entry that leans on the wild-type allele mattering needs to say which of the two it follows. Cancer Cell 2017
If WDR5 is partitioned, a WIN-site inhibitor acts on every WDR5-binding motif at once, including KANSL1's and SETD1B's - a different pharmacology from a KMT2A-only model.
residue-level geometry and measured affinity for the interface that entry records as lost
- ← refutesWDR5 — the partner that is not a partner: it binds the half of KMT2A the fusion throws away
[contested_by] WIN-site inhibitors act in MLL-rearranged cells through ribosome protein gene suppression and p53, not an MLL1 programme
[in_tension_with] MM-401 kills MLL-r cells by blocking assembly of a complex whose catalytic output is genetically dispensable in the same disease
- part of →KMT2A / MLL1 — the gene that breaks
WIN motif aa 3762-3767, Arg3765; ~2,400 residues past the breakpoint cluster
Arg3765 occupies the WDR5 arginine pocket; absent from every fusion
- related to →ASH2L — the WRAD subunit that has DPY30's essentiality profile and none of its co-dependency signal
WRAD subunit that cannot assemble on the fusion
WRAD subunit that cannot assemble on the fusion
- related to →DPY30 — the smallest COMPASS subunit, and the only one that behaves like a selective dependency
WRAD subunit that cannot assemble on the fusion
supplies the assembly requirement (R3765) that subunit-level discrimination has to work around
module absent from the fusion, yet druggable on the wild-type allele in the same cell
SET catalysis requires WRAD assembly through R3765; both are lost together
the clearest instance: a wild-type-only interface that is nonetheless a validated dependency in KMT2A-r cells
row: WIN 3762-3767
The WIN/WRAD interface is indeed absent from the direct fusion; this entry supplies the correct evidence class for that claim, which is structural rather than a measured fusion co-IP negative.
Constrains what the WIN-site dossier can claim: WIN-site engagement is shared with KMT2B, SETD1A and SETD1B and its published cellular signature is ribosomal and p53-dependent.