ASH2L — the WRAD subunit that has DPY30's essentiality profile and none of its co-dependency signal

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.

• Not a fusion interactor: targeting ASH2L would act on wild-type COMPASS across all six SET1/MLL complexes, not on the fusion.
• Broad chromatin toxicity: WRAD is shared by every H3K4 methyltransferase complex in the cell, so any ASH2L-directed agent is a global H3K4 intervention.
• The anomaly cuts both ways: if ASH2L's null result is the correct one and DPY30's positive is the artefact, then the most promising entry in this beat evaporates.

1. Does an ASH2L mutant that retains RBBP5 scaffolding but loses the DPY30 interface phenocopy DPY30 loss in a KMT2A::AFF1 line? This is the decisive experiment for the whole DPY30 question, the structures needed to design it exist (4RIQ, 6E2H), and it has not been done.
2. Why does ASH2L show no co-dependency with MEN1 despite having the variance to do so? Either the DPY30 signal is an artefact or WRAD subunits have genuinely separable functions, and those two possibilities have completely different consequences for this graph.
3. Does ASH2L contact KMT2A::AFF1 through any surface other than the lost SET domain?

1. E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

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

   A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex. · Patel A, Vought VE, Dharmarajan V, Cosgrove MS · Journal of Biological Chemistry 2011
   PMID 21106533DOI
   ▸What this evidence cannot show

   Reconstituted biochemistry; the claim of intrinsic WRAD methyltransferase activity has been contested and should not be treated as settled.

   ▸Evidence that argues against this (2)

   • 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
   • The 'minimal active module' is not minimal in the way the phrase implies, and WDR5 is not obligatory. Testing the Win motif across all six human SET1-family enzymes showed that in the absence of WDR5, MLL3 still interacts with RAD (RbBP5, Ash2L, DPY-30) and shows ENHANCED activity, and that MLL2/MLL4 and SETD1B complexes assemble and retain enzymatic activity in vitro despite Win-motif disruption. Only MLL1 and SETD1A are severely disrupted, and the reason given is that they have the weakest Win-motif-WDR5 interaction. So WRAD composition is not a fixed four-subunit requirement, and an ASH2L-centred model that treats the module as a unit will mispredict which complexes survive a given perturbation. Alicea-Velazquez NL et al. 2016
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, placing the ASH2L contact on a domain that KMT2A fusions do not retain.

   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 exclusion of ASH2L from the fusion complex is our inference from the loss of the SET domain at every breakpoint; a second unmapped contact surface cannot be formally excluded.

   ▸Evidence that argues against this (1)

   • This is the argument that should govern the entry: the RbBP5/ASH2L contact sits on the SET domain, which KMT2A fusions do NOT retain. ASH2L is therefore wild-type KMT2A biology, and its place in a fusion interactome needs an explicit justification the entry does not give. Cell Reports 2014
3. E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

   DPY30 contributes only modestly to allosteric stimulation of KMT2 activity in vitro while the extensive DPY30-ASH2L interface is critical for correct genome-wide placement of H3K4me2 and H3K4me3.

   Structural Analysis of the Ash2L/Dpy-30 Complex Reveals a Heterogeneity in H3K4 Methylation. · Haddad JF, Yang Y, Takahashi YH, Joshi M, Chaudhary N, Woodfin AR, Benyoucef A, Yeung S, Brunzelle JS, Skiniotis G, Brand M, Shilatifard A, Couture JF · Structure 2018
   PMID 30270175DOI
   ▸What this evidence cannot show

   Not performed in leukemia models and does not address MEN1. The division-of-labour explanation built on it in this entry is our hypothesis.

   ▸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
4. E4moderate confidence 3 of 5: One strong study, or several smaller consistent ones.dataset

   ASH2L is not DepMap common-essential (mean Chronos -0.333 across 1,258 lines, 21.4% of lines depleted, a distribution comparable to DPY30's -0.390 and 31.5%) yet correlates with MEN1 at r = -0.153 across 54 leukemia lines with a confidence interval of -0.433 to 0.118, while DPY30 reaches +0.446 and survives Holm correction; gnomAD gives ASH2L pLI 0.954 and LOEUF 0.502 with 30 observed against 81.1 expected loss-of-function variants.

   DepMap CRISPR (Chronos) gene effect via the Open Targets Platform GraphQL API and leukemia-restricted co-dependency with bootstrap and permutation testing; analyses/wrad-discrimination/ in this project; gnomAD v4 constraint via the gnomAD GraphQL API · Broad Institute DepMap; gnomAD; Open Targets Platform; analysis run by this project · Database and derived analysis 2026
   ▸What this evidence cannot show

   Our own computation in 54 leukemia lines with wide intervals; the DPY30 and ASH2L confidence intervals overlap slightly at their extremes, so the contrast is clear in point estimate and Holm outcome but not absolute. Similar essentiality distributions do not guarantee equal statistical power to detect a correlation.

   ▸Evidence that argues against this (2)

   • Searched and nothing contradicting found. A targeted search for a primary report of ASH2L as a dependency in KMT2A-rearranged leukemia, or of an ASH2L-MEN1 functional relationship, returned no result. The negative co-dependency this entry reports is therefore not contradicted by anything in the literature, but it is also not corroborated by anything: nobody has tested ASH2L individually in this disease. The honest reading is that the DepMap null is the only evidence there is, and it is our own unreviewed computation on 54 lines with an uncontrolled lineage confounder. Do not present r = -0.153 as a finding that ASH2L is irrelevant. Broad Institute DepMap; Open Targets Platform; search performed by this project 2026
   • This project's own deeper re-analysis withdraws the DPY30 result that this evidence item rests on. Against variance-matched nulls drawn from 2,743 usable random protein-coding genes across the same 54 leukemia lines, the Holm-corrected survivors are KMT2A (r 0.783), PSIP1 (0.686), DOT1L (0.681), FLT3 (0.645), MEF2C (0.526) and KAT7 (0.517). DPY30 is explicitly among those that do NOT clear correction, alongside MEIS1 (Holm 0.067), MLLT1 and BCL2. The earlier 16-gene panel gave DPY30 Holm p = 0.015 using an 'exceeds the maximum of the null' criterion that the deep-pool run shows to be unstable by construction, because the null maximum rises mechanically as the pool grows. The DPY30-versus-other-WRAD-subunits contrast is therefore a contrast between one uncorrected near-signal and three nulls, not between a survivor and three failures. Broad Institute DepMap; Open Targets Platform; analysis run by this project 2026
5. E5established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper

   ASH2L, RBBP5, WDR5 and DPY-30 constitute the subcomplex shared across all human SET1-like complexes, whereas PTIP, PA1 and UTX specifically associate with the MLL3/MLL4-containing complex.

   PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex. · Cho YW, Hong T, Hong S, Guo H, Yu H, Kim D, Guszczynski T, Dressler GR, Copeland TD, Kalkum M, Ge K · Journal of Biological Chemistry 2007
   PMID 17500065DOI
   ▸What this evidence cannot show

   Complex purification from cell lines; subunit stoichiometries reported here are qualitative, and the paper notes UTX associates substoichiometrically.

   ▸Evidence that argues against this (1)

   • Being shared across all human SET1-like complexes is the problem rather than the credential: a subunit common to six complexes cannot be inhibited without affecting all six, and the specificity would have to come from somewhere else. Cell Reports 2014
6. E6established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.database

   UniProt Q9UBL3 annotates human ASH2L as 628 residues with an atypical PHD-type zinc finger at 108-160 and a B30.2/SPRY domain at 360-583.

   UniProtKB Q9UBL3 (ASH2L_HUMAN), retrieved via the UniProt REST API · UniProt Consortium · UniProtKB 2026
   ▸What this evidence cannot show

   Curated annotation; the SDI-binding surface that DPY30 docks onto is a sub-feature of the SPRY region and is not separately annotated.

   ▸Evidence that argues against this (1)

   • A UniProt domain annotation is a curated inference from sequence and homology, revised between releases, and an 'atypical' PHD-type zinc finger is precisely the case where the assignment is least secure. UniProt Consortium 2026
7. E7established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.database

   Verified structures include the isolated ASH2L SPRY domain at 2.07 A (3TOJ) and the DPY-30 dimerisation/docking domain bound to the Ash2L SDI motif at 2.23 A (4RIQ).

   RCSB PDB entries 3TOJ and 4RIQ, verified against the RCSB data API for title, experimental method and resolution · RCSB Protein Data Bank · RCSB PDB 2026
   ▸What this evidence cannot show

   We verified accession, title, method and resolution only.

   ▸Evidence that argues against this (1)

   • Counting PDB entries measures how tractable a protein was to CRYSTALLISE, which correlates with being small, ordered and well-behaved rather than with being druggable. What decides design value is whether the deposited density covers the site that matters, at a resolution that supports modelling, in the relevant state — and this graph has repeatedly found structures described in the direction that overstates what is resolved. An isolated SPRY domain and a DPY-30 dimerisation module are the two parts that fold independently; neither is the SET-domain contact the entry's own E2 identifies as the functional interface. RCSB PDB 2026
8. 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
9. 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
10. 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
11. 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
12. 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
13. 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

• ← refutesThe bite-back of striking a target: a wrong strike costs a cure and generates no data that it was wrong

  2026-09-19: 'nobody has tested RBBP5/ASH2L in KMT2A::AFF1' withdrawn - ASH2L siRNA in RS4;11 lowers HOXC8 (PMID 32021283)

• ← related toThe leukaemia term hid the answer for DPY30 — a rule-77 demonstration with clean rule-67 controls

  DPY30 acts as an ASH2L-specific stabiliser, so the phenotype is a complex-level phenotype read out on one subunit

• ← part ofASH2L is studied in leukaemia and has never been placed in a cell state there — and the KMT2A-rearranged arm needs it LESS than average

  the bounded cell-state absence for this gene

• ← related toRepairing 85 defective citation blocks, and the finding that the queue's own numbers were wrong in three directions at once

  worked example of copy-propagation: a 1,100-character counter-note is byte-identical across gene-ash2l, gene-dpy30, gene-rbbp5 and gene-wdr5, and the truncated title inside it travelled with the prose

• ← depends onThe other arm of the crosstalk: H2Bub licenses H3K4 methylation through ASH2L and relieves a built-in COMPASS autoinhibition — and MLL3 does not respond at all

  ASH2L is essential for H2Bub-dependent H3K4 methylation; K99 of the winged-helix motif carries the receiving surface

• related to →KMT2A / MLL1 — the gene that breaks

  Counter-evidence: same objection as RBBP5 — MLL1 methyltransferase activity is not required for leukemogenesis, so the ASH2L catalytic role is not a target rationale here

• refutes →KMT2A::AFF1 (MLL-AF4), t(4;11)(q21;q23)

  correction: ASH2L binds the SET domain, lost at every KMT2A breakpoint

• binds →DPY30 — the smallest COMPASS subunit, and the only one that behaves like a selective dependency

  SPRY/SDI surface docks the DPY30 homodimer at 2:1; crystal 4RIQ

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

  RBBP5/ASH2L heterodimer engages the MLL1 SET-domain surface

• refutes →Do the other WRAD subunits behave like DPY30? (run, not planned)

  ASH2L has DPY30-like variance and no MEN1 correlation, breaking the flat-profile explanation

• ← related toKDM6A / UTX — a commonly asserted KMT2A partner that belongs to a different complex and points the opposite way

  UTX is the subunit that distinguishes MLL3/MLL4 complexes from the shared WRAD core

• ← related toDPY30 aims the H3K4 machinery while ASH2L powers it, which is why only DPY30 tracks menin

  ASH2L is the control case the hypothesis must explain

• ← refutesFirst systematic enumeration of the KMT2A fusion interactome, and the diff against this graph

  same discrepancy

• ← bindsASH2L SPRY and RBBP5 347-356: a phospho-reader contact gated by RbBP5 Ser350, and the only WRAD interface with a measured fold-change

  SPRY domain reads phospho-S350 on RBBP5

• ← bindsDPY30 D/D domain and the ASH2L SDI helix: four hydrophobic pockets, one load-bearing leucine - and a 94-residue numbering offset that relocates the whole motif

  SDI helix, Q9UBL3 603-618, load-bearing L607

• ← recruitsCARM1 / PRMT4 (Q86X55) - the H3R17me2a writer, and the enzyme whose product is read by the KMT2A C-terminus that every fusion deletes

  same ASH2L:MLL1/2:WDR5:RBBP5 recruitment, via arginine-methylated Pax7 read by the MLL C-terminus

• ← related to18% of this graph's evidence is the same finding said again

  One of the four WRAD subunit entries carrying identical copies of complex-level evidence.

• ← related to49 of 91 proteins in this graph have never had a drug-like molecule bound to them

  Covered end-to-end by deposited constructs across 32 PDB entries and never liganded. Structure is not the bottleneck for this target; chemical matter is.

• ← related toMLL1 stays on condensed mitotic chromosomes and tethers menin there — a menin-dependent function absent from this graph until cycle 34

  TETHERED TO MITOTIC CHROMATIN alongside menin and RbBP5.

• ← related toDPY30 has a second job at the trans-Golgi network, and the authors who found it could not put ASH2L or RBBP5 there

  ASH2L's nuclear annotation is a curator inference (ECO:0000305), and its Golgi localisation could not be detected

• ← inhibitsH3R2me2a - the asymmetric arginine mark two residues from H3K4 that LOCKS the WRAD module off the histone tail, and the first arginine mark node in this graph

  binding of an ASH2/WDR5/MLL-family methyltransferase complex to the H3 tail is prevented

• ← related toRe-bound at full text: ASH2L still has no haematopoietic cell-state result - the loose intersection is 429 records and the tight one 17, and every title read is a review, a complex-membership mention or a congress abstract book

  no full-text record places this protein in a haematopoietic cell state; the recall is its complex's literature

• ← bindsKMT2A-AKAP8 (AKAP95): graded 'prey-only' by this project, and it is a direct positive modulator of MLL H3K4 methyltransferase activity from a dedicated MLL paper - ADMITTED

  ASH2L IP co-precipitates AKAP95, reciprocally

• ← related toTwo thirds of the curated interactome of this project's six core proteins is spoke-expanded - and the pairs this graph chose to write are significantly better than the ones it did not

  ASH2L-KMT2A is 10 of 10 spoke-expanded with no MI:0407 record, corroborated by co-membership of the MLL1 core-complex structures rather than by a binary observation

• ← bindsAR / androgen receptor - the best-supported partner in the absence set by independent-publication count, and the reason a menin inhibitor is in prostate cancer trials

  co-IP and reverse co-IP in VCaP; ASH2L siRNA inhibits AR-driven KLK3 >7.5-fold

• ← related toLost from the KMT2A half: the WIN motif and the whole WRAD interface (aa 3762-3767, Arg3765)

  WRAD subunit that cannot assemble on the fusion

• ← refutesBOD1L (BOD1L1) — the top SETD1A co-dependency, and the only candidate in this cycle tested by CRISPR tiling in a KMT2A::AFF1 line

  same contrast: the WRAD strike rests on catalysis being dispensable, and BOD1L's reported function is not catalytic support

• ← testsAuditing the seven strikes before applying them: nine of ten decisions stand, four for different reasons than were given, and one clinical argument fails

  strike confirmed as argued (PMID 24813891, 27563068)

• ← related toThe founding WRAD negative was never run on a fusion protein

  Same evidence-class correction to E_yokoyama2004.

• ← related toASH2L: the only WRAD subunit ever perturbed in a KMT2A::AFF1 cell, once, weakly

  Splits this subunit's evidence into what is established for wild-type KMT2A and what, if anything, is established for a fusion.

• ← supportsASH2L: the only WRAD subunit ever perturbed in a KMT2A::AFF1 cell, once, weakly

  ASH2L is the only WRAD subunit ever perturbed in a KMT2A::AFF1 cell: siRNA in RS4;11 lowers HOXC8 and WRAD occupancy at its promoter.