Oncofusion

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Explainerstrong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.Q23-wrad-adjudication

ASH2L: the only WRAD subunit ever perturbed in a KMT2A::AFF1 cell, once, weakly

also called ASH2L versus the fusion · ASH2L1 · ASH2L evidence split

ASH2L sits on the enzyme end of KMT2A with RBBP5, an end the usual fusion deletes. It is the one WRAD protein anyone has actually knocked down in a cell carrying the infant leukemia fusion - a single 2020 paper, one target gene, no growth measurement.

The same thing, for a scientist

**(a) Wild-type.** Established. ASH2L engages the MLL1 SET domain as a heterodimer with RBBP5 [E1]; with WDR5 and RBBP5 it forms the minimal module that confers sequential nucleosomal H3K4 methylation [E2]; its SPRY/B30.2 domain docks the DPY30 SDI motif [E3]. Loss of ASH2L reduces chromatin accessibility at promoters [E8] and it is required for haematopoietic stem and progenitor proliferation and differentiation [E9]. **(b) Fusion.** One paper, weak. Wu et al. show that ASH2L siRNA in the t(4;11) KMT2A::AFF1 line RS4;11 and in THP-1 lowers HOXC8 transcript and protein and reduces RBBP5, WDR5, MLL and BPTF occupancy at the HOXC8 promoter [E4]. That is the only WRAD perturbation ever reported in a KMT2A::AFF1 cell. It does not test association with the fusion, uses a non-HOXA target gene, and has no growth, survival or HOXA9/MEIS1 readout. The alias-expanded fusion query returns 6 records, of which this is the only primary experiment in a KMT2A-rearranged cell [E5]. **(c) Domain.** The MLL1 SET domain surface - LOST from every direct 5'-KMT2A fusion, RETAINED in reciprocal der products and in NUP98::KMT2A.

Go deeper
The correction this entry carries forward

This project's target register previously recorded that nobody had tested a non-catalytic WRAD requirement in KMT2A::AFF1 'in either direction'. That statement was withdrawn on 2026-09-19 because Wu et al. had done it 4. This entry restates the corrected position precisely, because the correction is easy to over-read: ASH2L has been knocked down once in a KMT2A::AFF1 line, with a single target gene as the readout, and never with a leukaemia-maintenance readout. The register's strike is not overturned; the strike's premise is narrowed.

ASH2L is the subunit that breaks this graph's own explanation

This graph's standing explanation for why WRAD subunits never surface as MEN1 co-dependencies is that a pan-essential gene has a flat profile and a flat profile cannot correlate with anything. ASH2L defeats it. ASH2L is NOT pan-essential - mean Chronos -0.333 across 1,258 lines, depleted in 21.4%, a distribution close to DPY30's -0.390 and 31.5% - and it still lands at r = -0.153 against MEN1 with a confidence interval of -0.433 to 0.118 6. Two subunits of the same four-protein module, comparable dependency variance, opposite answers. Whatever separates them is not statistical power.

The honest reading, given that DPY30's own co-dependency has since failed four independent variance-matched and within-subtype designs in this project, is that neither subunit has a real MEN1 co-dependency and the contrast being explained does not exist. That is a less interesting conclusion than the one the graph has been carrying and it is better supported.

Aliases, checked in both directions

ASH2L publishes under ASH2L, ASH2L1, ASH2L2 and ASH2. The bare symbol ASH2L returns 216 PubMed records; ASH2L1 returns 2. The permissive query (ASH2L2 OR "ASH2 like" OR Ash2l) AND (MLL OR KMT2A) AND (leukemia OR leukaemia) returns 61 records - a tenfold inflation over the 6-record fusion-specific query - because Ash2l case-insensitively matches everything the current symbol matches and MLL matches every mixed-lineage-leukaemia paper regardless of fusion. That inflation is the precision half of this project's alias rule and it is recorded here so the 6 is not mistaken for a recall failure. ASH2 alone is a Drosophila/yeast gene name and returns records about a different organism.

Facts and figures
subunitASH2L
uniprotQ9UBL3
wild type contactMLL1 SET-domain surface via the RBBP5/ASH2L heterodimer; SPRY/B30.2 domain (aa 360-583) docks the DPY30 SDI motif
contact retained in direct fusionfalse
contact retained in reciprocal fusiontrue
contact retained in nup98 kmt2atrue
fusion association evidencenone; one weak siRNA perturbation in a KMT2A::AFF1 line (PMID 32021283)
pubmed records subunit x fusion6
pubmed query(ASH2L OR ASH2L1 OR ASH2) AND (MLL-AF4 OR MLL-AF9 OR MLL-ENL OR MLL-AF10 OR MLL-AF6 OR MLL-ELL OR "MLL fusion" OR "MLL fusion protein" OR "KMT2A fusion" OR "MLL-rearranged" OR "KMT2A-rearranged")
verdictwild-type KMT2A SET-domain interactor; no measured association with any KMT2A fusion protein; the only WRAD subunit ever perturbed in a KMT2A::AFF1 cell, once, at the level of a single target gene
alias record counts
ASH2L12.000ASH2L1: 2permissive variant with M…61.0permissive variant with MLL and leukemia: 61ASH2L216.0ASH2L: 216
aliases searched
[
  "ASH2L",
  "ASH2L1",
  "ASH2L2",
  "ASH2",
  "Ash2l"
]
alias record counts — the numbers behind the chart
{
  "ASH2L": 216,
  "ASH2L1": 2,
  "permissive variant with MLL and leukemia": 61
}
How this could bite us back
  • Ash2l is required for normal haematopoietic stem and progenitor proliferation and differentiation, so an ASH2L-directed agent carries a direct marrow liability in a disease whose patients are already marrow-compromised infants.
  • ASH2L is shared across all six SET1/MLL complexes; disrupting it is a global H3K4-methylation intervention and cannot be made KMT2A-selective at the target level.
  • The one published perturbation in the right cell type used siRNA and reported no growth effect, which is as consistent with 'no maintenance requirement' as with 'not measured' - and building an intervention on it would be building on an unmeasured endpoint.
Still open
  1. Does ASH2L knockdown in a KMT2A::AFF1 line affect growth, HOXA9/MEIS1 or leukaemia maintenance? The only published perturbation stopped at one target gene's transcription, and the obvious next readout has never been reported.
  2. Does the ASH2L-dependent chromatin occupancy in RS4;11 come from the wild-type KMT2A allele, from SETD1A/B, or from the reciprocal AF4-MLL fusion? The experiment as published cannot say.
  3. If neither ASH2L nor DPY30 has a real MEN1 co-dependency once variance-matched nulls are applied, what exactly was the DPY30-versus-ASH2L contrast that three entries in this graph set out to explain?
Every source behind this entry (9)
  1. E1strong 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 direct KMT2A fusions do not retain.

    PMID 24680668DOI
    What this evidence cannot show

    Reconstituted in vitro system with recombinant fragments; maps a requirement in that system rather than the full set of contacts in a cell.

    Evidence that argues against this (1)
    • Multistate cryo-EM of MLL1-WRAD on H2B-ubiquitinated nucleosomes reports that portions of the ASH2L structure and the position of DPY30 remain ambiguous and that MLL1-WRAD docking on nucleosomes is variable, so the ASH2L contact is less fixed than a single mapped surface implies. Rahman S et al. 2022
  2. E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    WDR5 plus RBBP5 plus ASH2L is the minimal non-SET-domain module required for sequential nucleosomal H3 methylation by the MLL1 core complex.

    PMID 21106533DOI
    What this evidence cannot show

    In vitro reconstitution; establishes chemistry, not cellular or disease relevance.

    Evidence that argues against this (1)
    • The activity is genetically dispensable for MLL-AF9 leukaemogenesis in mouse, so the minimal module for catalysis is not the minimal module for disease. Mishra BP et al. 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.

    PMID 30270175DOI
    What this evidence cannot show

    Structural and biochemical work outside any leukaemia model; it does not address MEN1 or KMT2A fusions.

    Evidence that argues against this (1)
    • Whatever the division of labour within WRAD, the SET-domain catalysis the module exists to position is dispensable for MLL-AF9 leukaemogenesis, so a placement-versus-catalysis distinction does not by itself produce a target in this disease. Mishra BP et al. 2014
  4. E4weak confidence 2 of 5: Preliminary, small, indirect, or not yet peer reviewed.paper

    ASH2L siRNA knockdown in the t(4;11) KMT2A::AFF1 line RS4;11 and in the MLL-rearranged line THP-1 significantly reduces HOXC8 gene and protein expression and reduces occupancy of RBBP5, WDR5, MLL and BPTF at regions of the HOXC8 promoter by ChIP.

    PMID 32021283DOI
    What this evidence cannot show

    The only WRAD perturbation ever run in a KMT2A::AFF1 line. siRNA rather than a genetic or degron system; one non-HOXA target gene; no growth, survival, differentiation or HOXA9/MEIS1 readout; no comparison against a KMT2A-wild-type line; a low-impact venue. The MLL antibody used detects the wild-type allele, so the occupancy loss is most simply read as loss of wild-type KMT2A complex.

    Evidence that argues against this (1)
    • The wild-type KMT2A allele is itself required for KMT2A-fusion leukaemia maintenance, which supplies a complete explanation for this result that does not involve the fusion protein at all. Thiel AT et al. 2010
  5. E5moderate confidence 3 of 5: One strong study, or several smaller consistent ones.dataset

    An alias-expanded PubMed query pairing ASH2L, ASH2L1 or ASH2 with the named common KMT2A fusions returns 6 records, of which exactly one is a primary experiment perturbing ASH2L in a KMT2A-rearranged cell.

    What this evidence cannot show

    Counted absence bounded by ASH2L/ASH2L1/ASH2. The permissive variant returns 61 records and is noise, so the two numbers must not be conflated. Complex-level framing and supplementary-only data remain invisible to both.

    Evidence that argues against this (1)
    • A study framed entirely at the level of 'the MLL1 complex' in CEBPA-mutated AML would not be retrieved by any ASH2L query, which is the documented failure mode of subunit-only enumeration. Schmidt L et al. 2019
  6. E6moderate 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 its leukaemia-restricted correlation with MEN1 is r = -0.153 with a confidence interval of -0.433 to 0.118.

    What this evidence cannot show

    Restated from this project's own earlier run, not recomputed. n = 54 leukaemia lines, wide intervals, no lineage adjustment within the leukaemia subset, no infant KMT2A-rearranged primary samples, DepMap release version unverified.

    Evidence that argues against this (1)
    • Cell-line panels evolve genetically and transcriptionally in culture, so cross-line dependency correlations can reflect culture-acquired state rather than tumour biology. Ben-David U et al. 2018
  7. E7established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper

    Endogenous ASH2L coprecipitates with transfected full-length MLL in 293 cells and is lost when the SET domain is deleted; ASH2L was never blotted on any fusion protein in the paper that established this.

    PMID 15199122DOI
    What this evidence cannot show

    Transient overexpression in an epithelial line; the fusion panels in the same paper blot menin, HCF-1, MLL-N, MLL-C and ENL only.

    Evidence that argues against this (1)
    • The same paper notes that a previously reported 'MLL super complex' contained WDR5 and RBBP5 but lacked ASH2L, so ASH2L's membership was purification-dependent from the start. Yokoyama A et al. 2004
  8. E8strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    Loss of the ASH2L subunit of H3K4 methyltransferase complexes reduces chromatin accessibility at promoters.

    PMID 36513698DOI
    What this evidence cannot show

    Non-haematopoietic experimental system; establishes a general chromatin consequence of ASH2L loss rather than anything specific to KMT2A-rearranged leukaemia.

    Evidence that argues against this (1)
    • In a KMT2A::AFF1 line, ASH2L knockdown produced a measurable effect on one target gene's promoter occupancy without any reported effect on cell growth, so a general chromatin-accessibility phenotype does not imply a leukaemia-maintenance phenotype. Wu YJ et al. 2020
  9. E9strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    Haematopoietic stem and progenitor cell proliferation and differentiation requires Ash2l.

    PMID 31164666DOI
    What this evidence cannot show

    Mouse conditional genetics in normal haematopoiesis, not leukaemia; it bounds tolerability rather than establishing efficacy.

    Evidence that argues against this (1)
    • A normal-haematopoiesis requirement is a toxicity argument, not an efficacy argument, and the same is true of DPY30, whose conditional deletion causes severe pancytopenia - so the whole module carries the same on-target marrow liability. Yang Z et al. 2016
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