Oncofusion

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Proteinstrong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.Q5c-interactome2

RBBP5 — a WRAD subunit that binds the one part of KMT2A every fusion throws away

also called RBBP5 · RbBP5 · retinoblastoma-binding protein 5 · RBQ3

RBBP5 is part of the four-protein core that switches on KMT2A's enzyme activity. That enzyme sits in the SET domain, and the SET domain is lost in every KMT2A fusion — so RBBP5 is not a partner of the fusion, it is a partner of the normal protein the fusion left behind.

The same thing, for a scientist

RBBP5 (Q15291, 538 aa) is a WD40-repeat WRAD subunit shared by all six human SET1/MLL complexes. Its contact with MLL1 is made on the SET domain, which is absent from every KMT2A fusion protein, so RBBP5 cannot be a direct interactor of KMT2A::AFF1. Its relevance to this disease is indirect, through the retained wild-type KMT2A allele that must pre-bind HOXA9 for the fusion to be stably recruited. RBBP5 is DepMap common-essential (-1.10, 97.6% of lines depleted) and among the most constrained genes in this beat (LOEUF 0.146).

What it looks like
loading AF-Q15291-F1…
AlphaFold AF-Q15291-F1 ↗
colourshape

This is a prediction, not a measurement. No one has seen this protein in this shape. The colours say how much the software trusts itself, and that is worth reading before anything else — the orange stretches are places the model is effectively saying this part has no fixed shape.

very high (90+)confident (70–90)low (50–70)very low (<50)
I have never read one of these pictures before

A protein is one long chain of amino acids that folds up. The ribbon traces that chain from one end to the other; it is not a wire, it is a simplification of where the backbone runs. Drag to rotate, scroll to zoom.

Domains are the stretches of chain that do a particular job, and they are what the coloured blocks above the picture mark out. Click one and the view flies to it. In this project the important question about any domain is usually whether the fusion still has it — the labels say lost where it does not.

Anything in yellow sticks is a bound small molecule, which is usually the drug.

Go deeper
Scope correction: WRAD is wild-type KMT2A, not the fusion

Scope correction (coordinator, 2026-09-18). This entry is filed in the KMT2A::AFF1 interactome. The primary complex-purification paper says the opposite for WRAD: "Oncogenic mutant forms of MLL retain an ability to interact with menin but not other identified complex components" — the identified components being ASH2L, WDR5, RBBP5, HCF-1 and HCF-2 [E_yokoyama2004].

The reason is positional. WRAD engages KMT2A near the SET domain, at the extreme C-terminus, which lies far beyond the breakpoint cluster region (exons 8-13, roughly residues 1100-1400) and is therefore absent from every fusion protein. The same applies to HCF-1/HCF-2 (aa 1799-1802), CyP33 at PHD3, and CBP at the TAD (2829-2883).

So WRAD membership is a property of wild-type KMT2A, not of the fusion. That does not make this entry worthless — wild-type KMT2A biology matters here, the remaining wild-type allele is retained in patients, and WRAD is where the H3K4 methyltransferase question lives. But any claim that this protein is part of the fusion's interactome needs a primary source showing WRAD on a fusion, and none was found.

This also reframes what the field's most-cited menin paper actually shows. It is read as evidence for menin. It is equally a statement about everything else, and that asymmetry is the strongest available argument that menin is the only handle on the retained fragment rather than the best of several.

Narrowing that correction: discarded from the fusion, required in trans

The scope correction above was right but over-applied, and is narrowed here (coordinator, 2026-09-18).

What Yokoyama 2004 licenses is precisely this: WRAD does not stably co-purify with fusion MLL under the detergent conditions of a 2004 affinity purification. It does not license the stronger reading this coordinator propagated — that the wild-type KMT2A literature describes a protein the leukemia does not make, and can therefore be set aside. Three results say otherwise, and all three were verified directly:

1. The wild-type allele is required. Menin recruits both wild-type MLL and the MLL-AF9 fusion to HOX loci, and "the wild-type Mll allele is required for MLL-AF9-induced leukemogenesis and maintenance of MLL-AF9-transformed cells" [E_thiel]. Every patient keeps an intact allele.

2. The fusion depends on the wild-type complex in trans. The essential MLL1 recruitment domain spans CXXC and the PHD fingers; "MLL1 fusion proteins lack the PHD fingers and require prebinding of a wild-type MLL1 complex ... for stable HoxA9 association" [E_milne]. So a module deleted from the fusion can still be load-bearing for the fusion — supplied in trans by the other allele.

3. This interface already has a selective compound. MM-401 blocks the MLL1-WDR5 interaction and "blocks proliferation of MLL cells ... without general toxicity to normal bone marrow cells or non-MLL cells" [E_cao]. An interface absent from the fusion nonetheless yields MLL-selective pharmacology.

The enumeration's two-category scheme is therefore incomplete. Retained and discarded are not exhaustive; there is a third category — discarded from the fusion, required in trans through the intact allele. This entry belongs in it. The positional claim stands; the strategic inference drawn from it does not.

Unresolved and recorded as such: MM-401 works by blocking MLL1 methyltransferase activity, while MLL1 SET activity is reported dispensable for MLL-AF9 leukemogenesis (PMID 24813891). Both are in this graph and they do not sit comfortably together.

Correction: MM-401 is not proof that an absent interface is druggable

The section above used MM-401 to argue that an interface absent from the fusion nonetheless yields MLL-selective pharmacology, and treated that as support for targeting wild-type-only modules. That argument does not survive the WIN-site literature, and the coordinator verified both papers directly.

The group that developed small-molecule WIN-site inhibitors states the position plainly: "Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene transcription, induce nucleolar stress, and activate p53" [E_howard]. The earlier work had already shown WIN-site inhibitors displace WDR5 from a small cohort of loci including a specific subset of ribosome protein genes, causing translational inhibition, nucleolar stress and p53 induction [E_aho].

So the selectivity for MLL-rearranged cells may not run through MLL1 biology at all. If it runs through ribosome depletion and p53, then:

  • MM-401 is not evidence that disrupting a wild-type-only MLL1 interface is a viable strategy against the fusion, and this graph should stop presenting it that way;
  • the mechanism predicts failure in TP53-mutant disease, which is exactly the relapsed setting where a new agent would be used;
  • and it is a genuinely different and possibly better idea — a ribosome-directed therapy — that happens to have been found through a chromatin protein.

What survives from the original point is narrower and still worth keeping: the wild-type allele is genetically required for MLL-AF9 leukemogenesis and maintenance, and the fusion requires a prebound wild-type complex at its target loci. That case now rests on genetics alone, not on a compound.

The correction: RBBP5 is not a fusion interactor1 deeper

Interactome lists routinely place the WRAD module in the KMT2A::AFF1 interactome. For RBBP5 this is a category error, and the structural reason is simple.

RBBP5 and ASH2L form a heterodimer that binds a non-active-site surface of the MLL1 SET domain, and that binding is what licenses H3K4 dimethylation 12. In the nucleosome-bound cryo-EM structures, RBBP5 additionally makes the direct contact with H2B-conjugated ubiquitin that orients the whole complex on chromatin 3.

Every KMT2A fusion breaks in the breakpoint cluster region and retains the N-terminus — AT-hooks, CXXC, menin-binding motifs — while losing the PHD fingers, the bromodomain, FYRN/FYRC, the transactivation domain and the SET domain. The surface RBBP5 binds is therefore not present in the fusion protein.

The correct annotation is: RBBP5 is an interactor of wild-type KMT2A/MLL1 COMPASS, not of KMT2A::AFF1. Any edge in this graph asserting a direct RBBP5–fusion contact should be retyped.

Why RBBP5 is still relevant to this disease

The retained wild-type KMT2A allele matters. Milne and colleagues showed that wild-type MLL1 must pre-bind the HOXA9 locus for the fusion protein to be stably recruited 4. If that is right, then everything that makes wild-type MLL1 competent at HOXA9 — including its WRAD core and therefore RBBP5 — is upstream of the fusion's own occupancy.

That is a real dependency relationship. It is just a different one from complex membership, and conflating the two would mislead any structural or pharmacological effort aimed at the fusion itself.

Dependency, constraint, and the null correlation

RBBP5 is DepMap common-essential: mean Chronos -1.105, leukemia -1.118, depleted in 97.6% of 1,258 lines 5. Leukemia-restricted correlation with MEN1 is -0.081, crossing zero 6 — and this project's own WRAD discrimination run was written partly to close the gap that RBBP5 had never been tested 6.

gnomAD gives pLI ~1.0 and LOEUF 0.146, with 5 observed loss-of-function variants against 72.2 expected — the tightest constraint in this beat and a well-powered estimate. Expression is flat and low (bone marrow 8.9, thymus 12.3 nTPM), offering no selectivity 5.

RBBP5 is therefore RED on every axis this project uses. It is not a target, and the reason is structural rather than chemical.

Facts and figures
uniprotQ15291
gene symbolRBBP5
ensemblENSG00000117222
length AA538
alphafold IDAF-Q15291-F1
PDB count27
domain noteNo UniProt-annotated domain feature; RBBP5 is a WD40-repeat beta-propeller resolved in MLL1 core-complex structures.
complexWRAD (WDR5, RBBP5, ASH2L, DPY30), shared by all six human SET1/MLL complexes
fusion relationshipNOT a direct interactor of KMT2A::AFF1. RBBP5 binds the MLL1 SET domain, which is lost at every KMT2A breakpoint. Relevance is via the retained wild-type KMT2A allele.
druggabilityNone. Pan-essential, maximally constrained, and binding a domain the fusion does not have.
subnuclear localizationunpublished - no subnuclear or nuclear-body observation of RBBP5 exists in any source read for this beat
shown for fusionNO - RBBP5 has never been localised in a KMT2A-rearranged cell
PDB verified
[
  "6KIU",
  "7UD5"
]
domains
[]
essentiality
{
  "depmap_common_essential": true,
  "chronos_mean": -1.1048,
  "chronos_mean_leukemia_lines": -1.118,
  "n_lines": 1258,
  "fraction_lines_depleted": 0.976,
  "gnomad_pLI": 1,
  "gnomad_loeuf": 0.1456,
  "gnomad_obs_lof": 5,
  "gnomad_exp_lof": 72.2,
  "mouse_ko": "Not established; no Rbbp5 knockout citation verified and none asserted",
  "hpa_ntpm": {
    "thymus": 12.3,
    "lymph_node": 10.6,
    "liver": 10.2,
    "bone_marrow": 8.9,
    "spleen": 8.7,
    "naive_B_cell": 6.1,
    "heart_muscle": 5.9,
    "testis": 5.7,
    "classical_monocyte": 4.9
  },
  "hpa_specificity": "Low tissue specificity"
}
codependency men1
{
  "r_leukemia_lines": -0.0809,
  "ci_leukemia": [
    -0.332,
    0.1642
  ],
  "crosses_zero": true,
  "p_permutation": 0.56249
}
localization
{
  "subcellular": "nucleus (chromatin, as a COMPASS/WRAD subunit). The only compartment experiment located for this beat is a negative: the authors of PMID 19651892 could not detect Golgi localisation of RBBP5 with multiple antibodies or GFP fusions, although RBBP5 knockdown reproduces the CIMPR endosome-to-TGN redistribution seen on DPY30 depletion.",
  "subnuclear": "unpublished - no subnuclear or nuclear-body observation of RBBP5 exists in any source read for this beat",
  "how_shown": "stated (unpublished-data) negative for Golgi localisation by multiple antibodies and GFP fusions; siRNA knockdown with a CIMPR trafficking readout (PMID 19651892)",
  "systems": "HeLa and CV-1; no haematopoietic or leukaemic system",
  "node": "localization-dpy30-trans-golgi-network-pool"
}
cell type
{
  "evidence_class_key": "concept-expressed-required-maintains-three-evidence-classes",
  "expressed_in": {
    "class": 1,
    "summary": "ubiquitous"
  },
  "required_in": {
    "class": 2,
    "floor_compressed": true,
    "pct_all_lines_depleted": 97.7,
    "all_1258_lines_mean_gene_effect": -1.107,
    "kmt2a_rearranged_lines_mean_gene_effect": -1.16,
    "myeloid_kmt2ar_mean": -1.151,
    "blineage_kmt2ar_mean": -1.175,
    "verdict": "PAN-ESSENTIAL: depleted below -0.5 in 97.7% of 1,258 screened lines, KMT2A-rearranged arm -0.053 relative to all lines. DepMap carries no cell-type information for RBBP5, and its apparent KMT2A-r selectivity is the floor.",
    "source": "DepMap via Open Targets fetched 2026-09-20; ana-q60-cell-type-versus-fusion"
  },
  "context": "wild-type protein housekeeping",
  "graph_links": []
}
cell state
{
  "maintains": {
    "class": 3,
    "states": [],
    "claim": "not assessed in this beat"
  },
  "note": "RBBP5 was carried as a reference-axis gene in the Q60 DepMap panel, not as a worklist gene, so only the class-2 measurement was made. No bounded cell-state query was run for RBBP5 and no absence is claimed."
}
depmap kmt2a selectivity
{
  "chronos_mean_kmt2a_rearranged": -1.1602,
  "chronos_mean_other_leukaemia": -1.2089,
  "difference": 0.0487,
  "pan_essentiality_all_lines": -1.1048,
  "selectivity_rank": 14622,
  "rank_denominator": 17728,
  "provenance": "Joined 2026-09-21 from analyses/depmap-kmt2a-selective-scan-full (generated 2026-09-19T10:12:06Z), the project's own genome-wide scan over 11 KMT2A-rearranged and 43 other leukaemia DepMap lines. NOT re-fetched and NOT independent of that scan: every caveat it carries applies here, including that the KMT2A-rearranged arm is a small number of lines drawn from nine individuals, that the rank denominator is 17,728 genes, and that a difference is not a dependency. Added because only 26 of 308 gene entries carried this number, which made any KMT2A-selectivity question answerable for 8% of the corpus.",
  "how_to_read": "A negative difference means MORE required in KMT2A-rearranged lines than in other leukaemia lines. A rank near 17,728 is ANTI-selective. Neither is evidence of a fusion-driven dependency on its own: check pan_essentiality_all_lines before calling anything selective, and note that a paralogue-redundant gene can score near zero for reasons that have nothing to do with this disease - see ana-the-paralogue-redundancy-excuse-is-not-tested-and-my-proxy-decided-the-answer."
}
How this could bite us back
  • Essential-function toxicity: required by 97.6% of cell lines and the most LoF-constrained gene in this beat. There is no version of RBBP5 inhibition that spares normal cells.
  • Conceptual risk: carrying RBBP5 as a fusion interactor would send a structural campaign after a contact that does not exist in the fusion protein. This is the kind of error that costs a project a year.
Still open
  1. Does RBBP5 contact KMT2A::AFF1 through any surface other than the lost SET domain? A proteomic pull-down from a KMT2A::AFF1 line, reported at subunit resolution, would confirm or kill the correction made in this entry.
  2. How much of the fusion's dependency on the wild-type KMT2A allele runs through WRAD? If WRAD-dependent wild-type MLL1 activity is what licenses fusion recruitment, then a WRAD-directed agent would act on the fusion indirectly, which nobody has framed this way.
  3. How many other entries in this graph's interactome are complex-level inferences that do not survive the question 'is that domain still in the fusion?'
Every source behind this entry (12)
  1. E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    WRAD (WDR5, RbBP5, Ash2L, DPY-30) forms a non-SET-domain multi-subunit methyltransferase required for sequential nucleosomal H3 methylation by the MLL1 core complex, with the minimal active module being WDR5 plus RbBP5 plus Ash2L.

    PMID 21106533DOI
    What this evidence cannot show

    Reconstituted biochemistry. The claim that WRAD has intrinsic methyltransferase activity independent of the SET domain has been contested in the field and should not be treated as settled.

    Evidence that argues against this (1)
    • This is the decisive objection to the whole WRAD thread and it should be read before any of it. The SET domain of MLL1 -- the catalytic activity that WRAD exists to stimulate -- is dispensable for maintaining haematopoietic stem cells AND for supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein in mouse. On Mll1 deletion, what is lost at MLL1 target genes is H4K16 acetylation, and inhibiting SIRT1 is sufficient to restore both the acetylation and target-gene expression. The conclusion the authors draw is that recruited MOF activity, not MLL1's intrinsic methyltransferase activity, is what maintains these genes. If H3K4 methylation by MLL1 is not required for MLL-fusion leukemia, then the subunit whose job is to enable that methylation is not a target in this disease, whatever its biochemistry. Mishra BP et al. 2014
  2. E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    A non-active-site surface of the MLL1 SET domain is crucial for the interaction of MLL1 with the RbBP5/Ash2L heterodimer within MLL family core complexes.

    A non-active-site SET domain surface crucial for the interaction of MLL1 and the RbBP5/Ash2L heterodimer within MLL family core complexes. · Shinsky SA, Hu M, Vought VE, Ng SB, Bamshad MJ, Shendure J, Cosgrove MS · Journal of Molecular Biology 2014
    PMID 24680668DOI
    What this evidence cannot show

    In vitro mapping on MLL1 constructs. The inference that this excludes RBBP5 from the KMT2A fusion complex is ours, and rests on the well-established fact that the SET domain is lost at every KMT2A breakpoint. It remains formally possible that RBBP5 contacts the fusion through a second, unmapped surface.

    Evidence that argues against this (1)
    • The RbBP5/ASH2L contact is on the MLL1 SET domain, which KMT2A fusions do not retain. RBBP5 is wild-type KMT2A biology in this context. Cell Reports 2014
  3. E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    RBBP5 directly binds the H2B-conjugated ubiquitin on the nucleosome and orients the association between MLL1 or MLL3 and the nucleosome.

    Structural basis of nucleosome recognition and modification by MLL methyltransferases. · Xue H, Yao T, Cao M, Zhu G, Li Y, Yuan G, Chen Y, Lei M, Huang J · Nature 2019
    PMID 31485071DOI
    What this evidence cannot show

    Cryo-EM of reconstituted complexes on designed nucleosomes, with catalytic modules rather than full-length proteins.

    Evidence that argues against this (1)
    • Orienting the complex on the nucleosome is only worth doing if the catalysis it orients is required, and in MLL-fusion leukemia it is not: the MLL1 SET domain is dispensable for MLL-AF9 leukemogenesis in mouse. The H2Bub-RBBP5 contact remains real biochemistry, but as a therapeutic rationale for KMT2A::AFF1 it is load-bearing on an assumption that has been tested and failed. Note also that the RBBP5 contact is with a nucleosomal mark and the MLL1 SET domain, neither of which the fusion retains, so the entry's own framing already concedes the fusion is not the thing being oriented. Mishra BP et al. 2014
  4. E4strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    Multiple interactions recruit MLL1 and MLL1 fusion proteins to the HOXA9 locus, with wild-type MLL1 pre-binding required for stable recruitment of the fusion.

    Multiple interactions recruit MLL1 and MLL1 fusion proteins to the HOXA9 locus in leukemogenesis. · Milne TA, Kim J, Wang GG, Stadler SC, Basrur V, Whitcomb SJ, Wang Z, Ruthenburg AJ, Elenitoba-Johnson KSJ, Roeder RG, Allis CD · Molecular Cell 2010
    PMID 20541448DOI
    What this evidence cannot show

    Single-locus mechanics in cell models; the wild-type-allele requirement has not been tested pharmacologically and the extension to RBBP5 specifically is our inference.

    Evidence that argues against this (1)
    • Wild-type MLL1 pre-binding as a requirement for fusion recruitment is contested by the later finding that endogenous MLL1 is dispensable with MLL2 sustaining the disease. Cell Reports 2014
  5. E5strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.database

    RBBP5 is DepMap common-essential with mean Chronos -1.105 across 1,258 lines and depletion in 97.6% of lines; gnomAD reports pLI ~1.0 and LOEUF 0.146 with 5 observed against 72.2 expected loss-of-function variants; HPA reports low tissue specificity with bone marrow 8.9 nTPM.

    What this evidence cannot show

    DepMap release version not stated by the API.

    Evidence that argues against this (1)
    • Pan-essentiality is the finding here and it argues against the target rather than for it. A gene depleted in more than 90% of all cell lines is required by the patient's normal marrow as much as by the leukaemia, so its appearance in a KMT2A complex is a statement about the complex and not a therapeutic opening. RBBP5 at 97.6% of lines depleted and LOEUF 0.146 is among the most essential genes in the genome. gnomAD, Broad Institute 2026
  6. E6moderate confidence 3 of 5: One strong study, or several smaller consistent ones.dataset

    Across 54 DepMap leukemia lines RBBP5 correlates with MEN1 at r = -0.081, confidence interval -0.332 to 0.164, crossing zero; RBBP5 had never been tested in this project's earlier co-dependency panels.

    Leukemia-restricted co-dependency with MEN1, bootstrap and permutation testing; analyses/wrad-discrimination/ in this project · Broad Institute DepMap; Open Targets Platform; analysis run by this project · Database and derived analysis 2026
    What this evidence cannot show

    Our own computation in 54 lines. RBBP5 is pan-essential, so its flat profile cannot correlate with anything and this null carries no biological information.

    Evidence that argues against this (1)
    • Searched and nothing contradicting found. A targeted search for a primary study reporting RBBP5 as a selective or fusion-specific dependency in KMT2A-rearranged leukemia, or for a published RBBP5 co-dependency with MEN1, returned no result. The null here is therefore a genuine null rather than an untested field: no published work asserts what this entry's DepMap analysis fails to find. That is consistent with RBBP5's profile as a pan-essential scaffolding subunit (97.6% of lines depleted, LOEUF 0.146) whose flat dependency profile across lineages leaves no variance for a co-dependency to be detected in. Broad Institute DepMap; Open Targets Platform; search performed by this project 2026
  7. E_yokoyama2004strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    Biochemical purification of MLL identified a SET1-like complex containing ASH2L, WDR5, RBBP5, HCF-1, HCF-2 and menin. Oncogenic mutant (fusion) forms of MLL retain the ability to interact with menin but not the other identified complex components.

    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 24:5639-49 2004
    PMID 15199122DOI
    What this evidence cannot show

    Read as abstract only by the coordinator; the interactome beat reports reading the full text via PMC480881. 2004 affinity-purification mass spectrometry, at depths well below current practice, so a negative here is a failure to detect rather than a demonstration of absence. No proximity-labelling study of any KMT2A fusion has ever been published, so this negative has never been re-tested with a method capable of catching transient or substoichiometric contacts. **The founding negative is an inference from a deletion mutant, not a measured fusion co-immunoprecipitation.** Read in full 2026-09-20: WRAD subunits were mapped using a C-terminal deletion construct of otherwise wild-type MLL in 293 cells. The paper's only fusion experiments blot for HIS, MLL-C, HCF-1, menin, MLL-N and ENL — **no WRAD subunit is blotted on any fusion anywhere in it**. It is a measured negative for HCF-1 only.

    Evidence that argues against this (4)
    • A 2004 co-purification negative is weak evidence of non-interaction. Modern proximity labelling routinely recovers contacts that affinity purification misses, particularly transient ones, and the entire composition literature for this fusion rests on stable-complex purifications from 2004-2012. The claim here should be read as 'not detected as a stable complex member', not 'does not interact'. Yokoyama A et al. 2004
    • CORRECTED 2026-09-21 (cycle 44), and the previous version of this note was too strong. It said "zero published experiments test WRAD-versus-KMT2A-fusion association in either direction" and cited PMID 15199122 for that - a 2004 paper, which cannot establish an absence as of today, in a note whose own next sentence warned against citing that paper for more than it shows. Re-bounded by query today: the subunit-by-fusion co-occurrence search is NOT empty (WDR5 26 records, ASH2L 6, DPY30 3, RBBP5 2), and reading all 26 WDR5 titles returns two that genuinely bear on the question. PMID 27462455 shows MLL1 and MLL-AF9 occupy DISTINCT chromatin regions and attributes MLL1's enhancer recruitment to a C-terminal WDR5 interaction - hedged as "probably" by its own authors - which is an experiment discriminating wild-type from fusion with WDR5 as the proposed discriminator. PMID 36402263 shows genetic Wdr5 deletion attenuates MLL-AF9 leukemogenesis, acting through ribosomal genes rather than through fusion binding. THE SURVIVING ABSENCE IS NARROWER AND STILL WORTH STATING: no direct binding assay - co-IP, structure or proximity labelling - has tested whether WRAD associates with a KMT2A FUSION protein. The two experiments above are genomic occupancy and genetic requirement, neither of which is a binding measurement. The structural argument for exclusion is unchanged, because the WRAD contacts sit thousands of residues past the breakpoint. What this graph must not do is cite a 2004 deletion-mutant result as if it were a fusion co-IP negative. PubMed (bounded query, counts and date recorded) 2026
    • The nearest thing to a published test, and it cuts against the absence being total: MLL1 and MLL-AF9 occupy distinct chromatin regions, and the authors attribute MLL1's enhancer recruitment to a C-terminal WDR5 interaction that the fusion lacks. It is an occupancy experiment, not a binding assay, and the WDR5 attribution is hedged in the source. Xu J et al. 2016
    • And a functional requirement in the fusion context: genetic Wdr5 deletion attenuates MLL-AF9 leukemogenesis in vivo. That WDR5 is REQUIRED where the fusion drives disease does not show WRAD binds the fusion - the reported mechanism is ribosomal gene regulation - but it makes 'WRAD is irrelevant to the fusion' untenable. Liu L et al. 2023
  8. E_thielstrong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    Menin recruits both wild-type MLL and the oncogenic MLL-AF9 fusion protein to HOX gene loci to activate transcription, and the wild-type Mll allele is required for MLL-AF9-induced leukemogenesis and for maintenance of MLL-AF9-transformed cells.

    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 17:148-59 2010
    PMID 20159607DOI
    What this evidence cannot show

    Read as abstract only. Mouse genetics with a retroviral MLL-AF9 model; the requirement is shown for MLL-AF9, not for KMT2A::AFF1, and this graph has repeatedly found partner-specific differences. 'Required' here is allele-level and does not identify which wild-type MLL module carries the requirement.

    Evidence that argues against this (1)
    • A wild-type-allele requirement shown for MLL-AF9 in mouse need not hold for KMT2A::AFF1 in infant B-ALL, and this project has already been burned generalising across partners. The claim should be carried as strong for MLL-AF9 and untested for the fusion this project is about. Thiel AT et al. 2010
  9. E_milnestrong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    The essential MLL1 recruitment domain includes both the CXXC domain and the PHD fingers and is controlled by interactions with the PAF elongation complex and H3K4me2/3. MLL1 fusion proteins lack the PHD fingers and require prebinding of a wild-type MLL1 complex, together with CXXC recognition of DNA, for stable HoxA9 association.

    Multiple interactions recruit MLL1 and MLL1 fusion proteins to the HOXA9 locus in leukemogenesis · Milne TA, Kim J, Wang GG, Stadler SC, Basrur V, Whitcomb SJ, Wang Z, Ruthenburg AJ, Elenitoba-Johnson KSJ, Roeder RG, Allis CD · Molecular Cell 38:853-63 2010
    PMID 20541448DOI
    What this evidence cannot show

    Read as abstract only. Establishes a trans requirement at HoxA9 specifically; whether it generalises to the whole fusion target set is not addressed in the abstract. The authors themselves frame the distinct recruitment mechanisms as a therapeutic opportunity rather than an obstacle.

    Evidence that argues against this (1)
    • If the fusion needs the wild-type complex prebound, then wild-type MLL1 recruitment is a potential target — but it is also a normal function in every cell that expresses MLL1, so the therapeutic window argument that applies to menin does not automatically transfer here. Milne TA et al. 2010
  10. E_caostrong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    MM-401 inhibits MLL1 activity by blocking the MLL1-WDR5 interaction and preventing complex assembly, without affecting other MLL-family methyltransferases, and specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells.

    Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia · Cao F, Townsend EC, Karatas H, Xu J, Li L, Lee S, Liu L, Chen Y, et al. (Dou Y, senior author) · Molecular Cell 53:247-61 2014
    PMID 24389101DOI
    What this evidence cannot show

    Read as abstract only. A peptidomimetic tool compound characterised in cells; no clinical or in vivo efficacy is claimed in the abstract, and 'MLL cells' spans MLL-rearranged lines generally rather than KMT2A::AFF1 specifically. SUPERSEDED IN PART (2026-09-19): MM-401's stated mechanism is inhibition of MLL1 methyltransferase activity, but WIN-site inhibition in MLL-rearranged cells is reported to act through ribosome protein gene suppression and p53 activation (PMIDs 30865883, 38682900). This citation should no longer be used as evidence that a wild-type-only MLL1 interface is druggable through MLL1 biology.

    Evidence that argues against this (1)
    • MM-401's stated mechanism is inhibition of MLL1 H3K4 methyltransferase activity, yet the MLL1 SET domain is reported dispensable for maintaining HSCs and for MLL-AF9-driven leukemogenesis (Mishra 2014, PMID 24813891), which this graph also carries. Either the selectivity of MM-401 runs through something other than catalysis, or the two results are in tension. The graph should hold both rather than choose. Mishra BP et al. 2014
  11. E_ahostrong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    Potent WIN-site inhibitors show that the WIN site links WDR5 to chromatin at 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 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, et al. (Tansey WP, senior author) · Cell Reports 26:2916-2928.e13 2019
    PMID 30865883DOI
    What this evidence cannot show

    Read as abstract only. Establishes the mechanism of small-molecule WIN-site inhibitors across cancer contexts; it is not specific to KMT2A::AFF1 and does not directly test the peptidomimetic MM-401.

    Evidence that argues against this (1)
    • MM-401 and the small-molecule WIN-site inhibitors are different chemotypes, and this paper does not test MM-401. It remains formally possible that the peptidomimetic acts differently. But the burden has shifted: the mechanism cannot now be assumed to be MLL1-programme disruption without direct evidence, and this graph previously assumed exactly that. Howard GC et al. 2024
  12. E_howardstrong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    In MLL-rearranged cancer cells, WDR5 WIN-site inhibition acts by suppressing ribosome protein gene transcription, inducing nucleolar stress and activating p53, with depletion of the ribosome stock, a broad modest translational choke, and alternative-splicing changes that inactivate the p53 antagonist MDM4. The authors state that peptidomimetic WIN-site inhibitors were originally proposed to act via genes connected to haematopoietic stem cell expansion, and that their data show otherwise. WIN-site inhibitors are synergistic with venetoclax and 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, et al. (Tansey WP, senior author) · eLife 12:e90683 2024
    PMID 38682900DOI
    What this evidence cannot show

    Read as abstract only. Cell-line work in MLL-rearranged lines; the p53 dependence is inferred from the mechanism rather than demonstrated across TP53-mutant and wild-type isogenic pairs in the abstract. Does not address KMT2A::AFF1 infant B-ALL specifically.

    Evidence that argues against this (1)
    • A p53/ribosome mechanism does not make WIN-site inhibition useless in MLL-rearranged leukaemia - the same paper reports synergy with venetoclax and BET inhibitors, and a ribosome-directed strategy is novel in its own right. What it removes is the specific inference this graph drew: that MM-401 demonstrates an absent-from-the-fusion interface can be drugged through MLL1 biology. Howard GC et al. 2024
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