Oncofusion

start / graph / exp-q128-is-menin-an-scf-fbxw7-substrate-one-week-with-existing-reagents

Experimentspeculative confidence 1 of 5: Our own inference or analogy. Not shown to be true.Q128-the-menin-interactome-that-is-not-chromatin

Is menin an SCF-FBXW7 substrate? A one-week immunoblot and a free desk scan that settle Arm A

also called menin FBXW7 degradation test · menin CPD scan

A simple test of whether menin is destroyed by a specific cellular disposal system: remove the disposal component, and see if menin protein goes up. If it does not, one whole idea about menin can be closed cheaply.

The same thing, for a scientist

Designed to separate the two arms of `hyp-q128-three-of-menins-nine-declined-partners-are-e3-ligase-substrate-receptors`. Three steps in ascending cost. **Step 0 (free, do first):** scan UniProt O00255 against the CDC4 phosphodegron consensus, restricted to regions with AlphaFold pLDDT below 50, since degrons are disordered. No match in a disordered region and Arm A is dead on the desk [E1]. **Step 1 (one week):** quantitative immunoblot for menin in an FBXW7-null versus FBXW7-wild-type isogenic human leukaemia pair, n>=3, loading-normalised, with MEN1 transcript by qPCR as the specificity control - a protein-level rise with no transcript change is the signature of stabilisation [E2]. **Step 2 (same week):** 1 uM pevonedistat for 6 h in the parental line; a cullin-RING substrate stabilises, a non-substrate does not [E2]. **Step 3, only if 1 or 2 fire:** menin immunoprecipitation followed by ubiquitin immunoblot under MG132. Readout thresholds are pre-registered in the falsifiers of the hypothesis entry and are restated here so the experiment cannot be scored after the fact [E3].

Go deeper
Why this design and not a co-IP

The obvious experiment - co-immunoprecipitate menin and FBXW7 - is the wrong first experiment, for two reasons.

It answers a less useful question. A ligase-substrate contact is transient by design and a negative co-IP is nearly uninformative; a positive one still does not show that menin abundance depends on it. The abundance question is the one with a clinical consequence, because menin inhibitors are occupancy drugs and occupancy scales with target level.

It is more expensive and less decisive. Step 0 costs nothing and can kill Arm A before any reagent is ordered. Step 1 uses a single antibody and an isogenic pair. Only if those fire is a contact experiment worth doing, and by then the question is sharpened.

Order matters and is part of the design: 0, then 1 and 2 in parallel, then 3. A project that runs step 3 first will spend a month and learn less.

Pre-registered thresholds, restated so they cannot move
stepfires ifdoes not fire if
0 CPD scan>=1 consensus match in a region with AlphaFold pLDDT < 50no match in any disordered region
1 FBXW7-null blotmenin >= 1.5x parent, loading-normalised, n >= 3, with MEN1 mRNA within 1.2xmenin within 1.2x parent
2 pevonedistatmenin >= 1.5x vehicle at 6 h, 1 uMmenin within 1.2x vehicle

The 1.2x-to-1.5x band is deliberately left as neither-fires-nor-dies. An equivocal result should be recorded as equivocal, not rounded into whichever arm the reader prefers. This project has recorded a falsifier that gave two correct opposite answers depending on an interval nobody had named; naming the dead band here is the remedy.

What would make this experiment uninformative: if the chosen isogenic pair differs in more than FBXW7 - a common problem with commercially derived knockouts - then a menin change is not attributable, and the experiment measures the clone rather than the gene. Mitigation: two independent knockout clones, or an acute degron rather than a stable knockout.

Facts and figures
designStep 0: CPD consensus scan of O00255 restricted to AlphaFold pLDDT<50 regions. Step 1: quantitative anti-menin immunoblot, FBXW7-null versus isogenic wild-type human leukaemia line, n>=3, loading-normalised, with MEN1 qPCR as specificity control. Step 2: 1 uM pevonedistat 6 h versus vehicle in the parental line. Step 3 (conditional): menin IP, ubiquitin immunoblot, +/- MG132.
readoutmenin protein fold-change versus control, with MEN1 transcript fold-change as the discriminator between stabilisation and transcriptional change
controlsisogenic parent; two independent knockout clones or an acute degron to avoid clone-specific effects; vehicle arm for pevonedistat; MEN1 qPCR; a known FBXW7 substrate (e.g. a cyclin E or MYC blot) as a positive control that the knockout is functional
cost tierlow
timelinestep 0 same day; steps 1 and 2 one week in parallel; step 3 a further two weeks only if 1 or 2 fire
equivocal band1.2x to 1.5x, recorded as equivocal and not rounded
uninformative ifthe isogenic pair differs in more than FBXW7, in which case the experiment measures the clone and not the gene; mitigated by two clones or an acute degron
absence classnever-measured
absence class noteNo published experiment reports menin protein level as a function of FBXW7 status, in any cell type. A GEO and PRIDE check for an FBXW7-null proteomics dataset with menin quantified was NOT run by this beat, so the honest statement is that this beat did not look in the archives; a future agent should before commissioning the bench work.
fires if
{
  "step0": ">=1 CPD consensus match in a pLDDT<50 region",
  "step1": "menin >=1.5x parent with MEN1 mRNA within 1.2x",
  "step2": "menin >=1.5x vehicle"
}
dies if
{
  "step1_and_step2": "menin within 1.2x in both"
}
How this could bite us back
  • A positive result would mean menin inhibitor dosing should account for target abundance, which no trial currently measures and which would complicate every reported occupancy figure.
  • A positive result would also mean that proteasome and neddylation inhibitors, both used in haematological malignancy, raise the target of a menin inhibitor - a drug-drug interaction in the antagonistic direction that nobody is looking for.
  • A negative result closes a line cheaply, which is the main value here. The risk is that a bulk-blot negative gets quoted as 'menin turnover is not ubiquitin-regulated' when it only excludes a bulk, steady-state, FBXW7-dependent effect.
Still open
  1. Does a deposited FBXW7-knockout proteomics dataset already quantify menin? This beat did not query PRIDE or ProteomeXchange, and if such a dataset exists step 1 is already done and needs only a lookup.
  2. What is menin's protein half-life? Without it, the size of effect to expect from any degradation experiment is unknown.
  3. Should the same three-step design be run for SPOP and ZC3HC1? It is the same reagents and the same week, and doing all three at once is barely more expensive than doing one.
Every source behind this entry (3)
  1. E1established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.database

    FBXW7 (UniProt Q969H0, 707 aa) is the substrate-recognition WD40 subunit of an SCF E3 ubiquitin ligase and is annotated to nucleoplasm, nucleolus, chromosome and cytoplasm with experimental evidence. Menin (O00255, 610 aa) is annotated to Nucleus with experimental evidence. A substrate relationship is therefore compartmentally possible and is what a genuine FBXW7-menin association would most naturally mean.

    What this evidence cannot show

    This establishes plausibility, not the relationship. UniProt does not list menin among FBXW7's substrates and this entry does not claim it does. Rule 91 applies: the ECO codes behind these location lines point at papers that were not opened here.

    Evidence that argues against this (1)
    • Compartmental plausibility is nearly free - a large fraction of the nuclear proteome satisfies it - so it carries almost no evidential weight and is included only to show the design is not compartmentally incoherent. UniProt Consortium 2026
  2. E2established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.database

    The IntAct evidence that motivates this experiment is four MEN1-FBXW7 BRET records from PMID 35512704 (EBI-63685884, EBI-63690742, EBI-63695122, EBI-63699348), all in HEK293T, all annotated to supplementary Table S2(B), all intact-miscore 0.27, carrying FBXW7 p.Arg465Cys, p.Arg465His, p.Arg479Gln and p.Arg505Cys and all typed 'mutation with no effect'. These are the WD40 phosphodegron-contacting arginines.

    What this evidence cannot show

    Four records are four alleles of one BRET assay, not four experiments. The motivating evidence is therefore a single low-scoring measurement, which is why this experiment's step 0 is a free desk check rather than a bench step.

    Evidence that argues against this (1)
    • That all four substrate-cleft mutants left the signal unchanged argues that whatever BRET is detecting is not degron recognition, which is an argument against running even step 1. The counter-counter is that step 0 is free and step 1 is a week, and a clean negative would let the graph close the FBXW7 line permanently rather than leaving it open. IntAct (EMBL-EBI) 2026
  3. E3moderate confidence 3 of 5: One strong study, or several smaller consistent ones.database

    The readout thresholds for this experiment are pre-registered in the falsifiers field of hyp-q128-three-of-menins-nine-declined-partners-are-e3-ligase-substrate-receptors: Arm A dies if menin is within 1.2-fold of parent in an FBXW7-null line AND within 1.2-fold after 6 h of 1 uM pevonedistat; it fires at 1.5-fold or more; 1.2 to 1.5-fold is recorded as equivocal.

    What this evidence cannot show

    The 1.5-fold threshold is a convention, not a power calculation. No pilot variance for menin immunoblot in this system was available to set it from, so it is chosen to be detectable by quantitative immunoblot at n=3 and should be revised if pilot data say otherwise. Declaring it in advance is the point; its exact value is negotiable and its post-hoc movement is not.

    Evidence that argues against this (1)
    • A fold-change threshold on a blot is a weak instrument: menin abundance may be regulated in a subpopulation or a cell-cycle phase that a bulk blot averages away. A negative bulk result would not exclude a cell-cycle-restricted degradation, which is exactly the regime SCF ligases usually work in. UniProt Consortium 2026
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