Oncofusion

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Mechanismweak confidence 2 of 5: Preliminary, small, indirect, or not yet peer reviewed.Q11b-subtraction

AFF1 as the subtraction control: the refutation is WITHDRAWN - the degron is lost, not retained

also called AF4 control partner · AFF1 addition mechanism · SIAH degron retained

For the fusion this project cares most about, the fusion partner brings something new rather than losing something old. We tested the best candidate for a hidden loss — a self-destruct tag that could have been cut off — and found the tag is still there.

The same thing, for a scientist

KMT2A::AFF1 is the designated control for the subtraction audit, and it survives the audit. The AFF1 junction at exon 4 removes only residues 1-53; every named ALF-family module lies downstream and is retained. The best available subtraction hypothesis — that the fusion escapes SIAH1/2-mediated proteasomal degradation by losing the ALF degron — fails on coordinates: the Siah-binding degron maps to Af4 residues 268-290 (consensus PxAxVxP; the robotic-mouse mutation is V280A), roughly 215 residues downstream of the junction. The verdict is addition, and the retained-half framing is correct here for the reason the project has always given.

Go deeper
What is actually discarded, and where the junction falls

In the canonical KMT2A::AFF1 architecture the fusion joins KMT2A exon 9 or 11 to AFF1 exon 4. Working from the Ensembl canonical transcript (ENST00000395146): AFF1 exon 1 is entirely 5' UTR, the CDS begins inside exon 2 contributing 38 coding nucleotides, and exon 3 contributes 121 — so 159 nucleotides, or 53 codons, precede exon 4. The fusion therefore discards approximately AFF1 residues 1-53.

UniProt P51825 annotates residues 1-45 as disordered with a basic-and-acidic compositional bias at 9-35, and assigns no domain, motif or functional region anywhere in 1-53. There is no known function to lose.

(Numbering note: the Ensembl canonical translation is 1,218 aa against UniProt's 1,210, so residue positions differ by up to 8 between the two references. The conclusion is insensitive to that offset.)

The subtraction hypothesis worth testing, and why it fails1 deeper

AFF1 is not obviously an addition-only partner, because ALF-family proteins carry a built-in destruction signal. Bursen et al. showed that the oncogenic mechanism of the reciprocal AF4·MLL product 'involves the two E3 ubiquitin ligases SIAH1 and SIAH2, the N-terminal portion of AF4, and the protection of the AF4·MLL fusion protein against proteosomal degradation' 1. If the degron sat in the discarded leader, then KMT2A::AFF1 would be a stabilised protein by virtue of a deletion — a textbook subtraction, hiding in the project's flagship fusion.

It does not. Oliver et al. mapped the Siah-binding site to Af4 residues 268-290, containing the consensus PxAxVxP motif; the robotic-mouse mutation V280A lies inside it and reduces Siah-1a affinity roughly 15-fold with near-complete abolition of Siah-1a-mediated degradation and a significant increase in Af4 transcriptional activity 2. Residues 268-290 are ~215 residues downstream of the exon-4 junction and are therefore retained in KMT2A::AFF1.

The hypothesis is refuted on arithmetic. 'N-terminal portion of AF4' in Bursen et al. means the N-terminal third of a 1,210-residue protein, not the discarded leader — an ambiguity that would have produced a false positive had it not been checked against coordinates.

A residual question the refutation does not close

Retaining a degron is not the same as being degraded through it. Bursen et al. report that the AF4·MLL reciprocal product is protected from proteasomal degradation despite containing the AF4 N-terminus 1, which means SIAH-mediated turnover can be defeated by something other than losing the degron — fusion-induced conformational masking, for instance, or altered complex membership.

So the correct statement is narrow: the degron is present in KMT2A::AFF1, so degron deletion is not a mechanism. Whether the degron is functional in the forward fusion is a separate, untested question, and if it is silenced the effect would be indistinguishable from subtraction at the level of protein levels.

The addition, restated with its primary evidence

What AFF1 brings is well established and is exactly what the retained-half framing predicts. AF4-family and ENL-family proteins form a higher-order complex with P-TEFb, designated AEP, which is normally recruited to MLL target chromatin to facilitate transcription; MLL oncoproteins fused to AEP components form constitutive MLL/AEP hybrid complexes that cause sustained target-gene expression and transform haematopoietic progenitors 3.

The retained AFF1 modules recorded in this graph — the ALF/N-terminal homology domain, the pSER transactivation region and the CHD that binds AF9/ENL — are the components of that hybrid complex. This is addition in its cleanest documented form: a retained partner module supplying an activity to KMT2A target loci that intact KMT2A never had there.

The control therefore does its job. It confirms that the project's framing is right about its own flagship fusion, which is what makes the failures elsewhere in this beat informative rather than merely destructive.

Facts and figures
partnergene-aff1
fusionfusion-kmt2a-aff1
role in this beatcontrol
discarded regionAFF1 residues 1-53 (exons 2-3 coding portion); no annotated domain, motif or functional region
intact function of discarded regionnone known; UniProt annotates 1-45 as disordered with a basic/acidic compositional bias
subtraction hypothesis testedThat KMT2A::AFF1 escapes SIAH1/SIAH2-mediated proteasomal degradation by discarding the ALF-family degron
genuinely discarded at observed breakpointstrue
discarded region functionally significantfalse
loss shown to contribute to transformationfalse
verdictaddition
verdict confidencestrong
experiment to settleLargely settled. The residual test is a degron-function assay rather than a deletion assay: compare the proteasomal turnover of KMT2A::AFF1 carrying a wild-type degron versus a V280A-equivalent degron-dead mutant. If turnover is identical, the retained degron is non-functional in the fusion and the stabilisation question reopens by a different route.
subtraction hypothesis outcome WITHDRAWNrefuted on coordinates — the Siah-binding degron maps to Af4 residues 268-290 (PxAxVxP; robotic V280A), ~215 residues downstream of the junction and therefore retained
subtraction hypothesis outcomeREINSTATED, not refuted. The Siah degron maps to human AFF1 282-304 and the junction is at residue 347, so the degron is discarded with the AFF1 leader. Junction fixed at AFF1 residue 347: QTYSNEVH occurs exactly once in UniProt P51825 (1,210 aa), and the corpus independently reconstructed the junction peptide VDFK|QTYSNEVHCVEEILK from the siMA6 siRNA, recording that both KMT2A::AFF1 chimeras resume at QTYSNEVHCVEEILK so the AFF1 acceptor is constant across breakpoints. Degron located in human numbering by homology: mouse Af4 268-290 (KSAAMQQKPTAYVRPMDGQDQAP) aligns 22/23 to human P51825 282-304 (KSVAMQQKPTAYVRPMDGQDQAP), offset +14; the PxAxVxP motif PTAYVRP is unique in human at 290-296 and the robotic-equivalent valine is V294. The element ends at 304, 43 residues N-terminal to the junction.
discarded residues
{
  "start": 1,
  "end": 53,
  "uniprot": "P51825",
  "numbering": "Ensembl ENST00000395146 CDS arithmetic, mapped onto P51825 with an offset of up to 8 residues"
}
retained oncogenic modules
[
  "ALF / N-terminal homology domain",
  "pSER transactivation region",
  "CHD (C-terminal homology domain, binds AF9/ENL)"
]
How this could bite us back
  • Confirming that the control behaves as expected is reassuring about the framing, not about the therapy: the menin interface remains a single point of failure whose resistance routes (MEN1 M327I and relatives) are already documented in this graph.
  • If the retained degron is functional, a stabilising mutation anywhere in AFF1 residues 268-290 would raise fusion protein levels and could act as a resistance mechanism against dose-limited menin inhibition. No sequencing study of relapsed KMT2A::AFF1 has looked at the AFF1 half of the fusion for such variants.
Still open
  1. Is the retained SIAH degron functional in KMT2A::AFF1? Bursen et al. found the reciprocal product escapes degradation while containing the degron, so retention does not settle turnover — and fusion protein half-life is directly relevant to degrader strategies.
  2. WITHDRAWN 2026-09-19. This question previously began "About half of KMT2A::AFF1 cases are spliced fusions with the genomic break upstream of AFF1 exon 1" and asked what that implies for the control. The premise is false: spliced fusions in KMT2A::AFF1 are **1 of 1,262 cases (0.08%)**. The ~50% figure belongs to MLLT1 (241 of 460) and was mis-generalised. There is essentially no spliced-AFF1 population to ask the question about, so the question is withdrawn rather than rephrased. The reciprocal-free population that DOES exist in t(4;11) is a different one: the reciprocal AF4-MLL transcript is expressed in only about half of patients carrying an intact der(4).
  3. If AFF1's contribution is purely additive, why does KMT2A::AFF1 have the distinctive infant, HOXA-low, B-lymphoid phenotype rather than simply a stronger version of the common program? An addition mechanism explains potency better than it explains specificity.
Every source behind this entry (4)
  1. E1moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

    AF4 wild-type and the AF4-MLL fusion protein have growth-transforming potential, and the underlying oncogenic mechanism involves the E3 ubiquitin ligases SIAH1 and SIAH2, the N-terminal portion of AF4, and protection of the AF4-MLL fusion protein against proteasomal degradation.

    Interaction of AF4 wild-type and AF4.MLL fusion protein with SIAH proteins: indication for t(4;11) pathobiology? · Bursen A, Moritz S, Gaussmann A, Moritz S, Dingermann T, Marschalek R · Oncogene 2004
    PMID 15221006DOI
    What this evidence cannot show

    Concerns the RECIPROCAL AF4-MLL product, not the forward MLL-AF4 fusion that this graph's fusion-kmt2a-aff1 entry describes. The phrase 'N-terminal portion of AF4' is not given residue boundaries in the abstract, which is precisely the ambiguity that E2 resolves. Verified against PubMed on 2026-09-18.

    Evidence that argues against this (1)
    • The SIAH result is treated here as a hypothesis about the forward fusion's stability, but the same laboratory later showed the reciprocal product is a driver in its own right, which moves the question. Bursen et al. transduced purified Lin-/Sca1+ progenitors and found that AF4.MLL alone produced pro-B ALL, B/T biphenotypic acute leukaemia or mixed-lineage leukaemia on transplant, while MLL.AF4 alone produced no disease across 13 months of observation. If the disease-causing activity in that system sat in AF4-MLL, then degron arithmetic performed on KMT2A::AFF1 answers a question about the other protein, and this entry's verdict of 'addition' for the forward fusion survives only because the subtraction hypothesis it refutes was aimed at the reciprocal all along. Note also the direct tension with this graph's index framing: a model in which t(4;11) rests on two oncoproteins is not a model in which the forward fusion is the whole target. Bursen A et al. 2010
  2. E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

    Af4 binds the E3 ubiquitin ligases Siah-1a and Siah-2; the Siah-binding degron maps to Af4 residues 268-290 and contains the consensus PxAxVxP motif; the robotic-mouse V280A mutation inside this site reduces Siah-1a affinity about 15-fold, almost completely abolishes Siah-1a-mediated degradation of mutant Af4, and significantly increases its transcriptional activity.

    Mediation of Af4 protein function in the cerebellum by Siah proteins · Oliver PL, Bitoun E, Clark J, Jones EL, Davies KE · Proceedings of the National Academy of Sciences USA 2004
    PMID 15459319DOI
    What this evidence cannot show

    Residue numbering is mouse Af4, and the work is in cerebellum and Purkinje cells, not haematopoiesis. Human AFF1 differs slightly in length, so 268-290 should be treated as approximate in human numbering — but the margin between it and the exon-4 junction at residue ~53 is roughly 215 residues, far larger than any plausible alignment error. PMID, DOI, title, authors and abstract verified against PubMed on 2026-09-18; degron coordinates and the V280A detail taken from the full text on PMC the same day.

    Evidence that argues against this (1)
    • Bursen et al. report that the AF4-MLL reciprocal product is protected from proteasomal degradation despite containing the AF4 N-terminus. Degron retention therefore does not guarantee degron function in a fusion context, so the refutation here is of degron deletion specifically, not of every stabilisation mechanism. Bursen A et al. 2004
  3. E3established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper

    AF4-family and ENL-family proteins comprise a higher-order complex with P-TEFb (AEP) that is normally recruited to MLL-target chromatin to facilitate transcription; MLL oncoproteins fused with AEP components constitutively form MLL/AEP hybrid complexes causing sustained target-gene expression and transformation of haematopoietic progenitors.

    PMID 20153263DOI
    What this evidence cannot show

    Biochemistry plus murine transformation assays. Verified against PubMed on 2026-09-18.

    Evidence that argues against this (1)
    • Constitutive hybrid-complex formation is not the only published account of how an MLL fusion reaches its targets, and the competing account makes the fusion depend on wild-type MLL1 rather than on its partner. Milne et al. identified an MLL1 recruitment domain including CXXC and the PHD fingers, controlled by direct interactions with the PAF complex and H3K4me2/3, and showed that fusion proteins lacking the PHD fingers require prebinding of a wild-type MLL1 complex, plus CXXC-DNA recognition, for stable HoxA9 association. The two models predict different dependencies - one predicts that removing wild-type MLL1 disables the fusion, the other does not - and this graph's KMT2A::AFF1 entries have never chosen between them. Milne TA et al. 2010
  4. E4established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.database

    In the Ensembl canonical AFF1 transcript ENST00000395146, exon 1 is entirely 5' untranslated, the coding sequence begins within exon 2 contributing 38 coding nucleotides, and exon 3 contributes 121 nucleotides, so 53 codons precede exon 4; UniProt P51825 annotates residues 1-45 as disordered and assigns no domain, motif or functional region within residues 1-53.

    What this evidence cannot show

    The Ensembl canonical translation is 1,218 aa and UniProt P51825 is 1,210 aa, so positions can differ by up to 8 residues between references. Exon 4 is also not the only reported AFF1 junction. A break upstream of AFF1 exon 1, with the mRNA produced by splicing, would discard even less — but that happens in **1 of 1,262 cases (0.08%)**, so it is not a caveat on this arithmetic. (This previously read 'roughly half', which is MLLT1's figure; the entry's open question was withdrawn on that basis and this sentence was left standing.) Both records fetched and checked on 2026-09-18.

    Evidence that argues against this (1)
    • The junction arithmetic is computed on one reference transcript against one breakpoint, and the breakpoint is not one thing. Meyer et al. determined genomic breakpoints in 2,345 patients, characterised 94 partner genes at the molecular level, catalogued 135 distinct rearrangements, and documented an age-dependent shift of the MLL-side breakpoint (intron 11 in ALL and in younger patients, intron 9 in AML and older patients). A degron-versus-junction margin quoted from the Ensembl canonical AFF1 transcript describes the commonest configuration; it should be recomputed over the AFF1-side breakpoint distribution actually observed in infant t(4;11) before it is treated as a refutation rather than as an argument about the modal case. Meyer C et al. 2018
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