Experiment: five-fraction subcellular blot of a t(4;11) line, with a menin-inhibitor arm - converting thirty years of unattributable images into attributable numbers using only existing antibodies
also called fractionate and blot KMT2A::AFF1 · salt-extraction series for MLL-AF4 · attributable localisation experiment
Instead of photographing the leukemia fusion protein, break the cells into five layers - cytoplasm, soluble nuclear liquid, loosely bound chromatin, tightly bound chromatin and the insoluble scaffold - and run each layer on a gel. The fusion and the normal protein separate by size in every lane, so for the first time you can say how much of each is where, with antibodies that already exist.
The same thing, for a scientist
The fusion-attribution audit found localisation to be the one claim class with no endogenous survivor, because every published localisation readout is immunofluorescence and immunofluorescence has no size dimension. This experiment removes the readout rather than the reagent. Fractionate SEM, RS4;11 and a non-rearranged control line into five fractions - cytoplasm, soluble nucleoplasm, low-salt chromatin eluate, high-salt chromatin eluate and the nuclease-resistant pellet - and blot each with the anti-AF4 and anti-MLL-N reagents already used in this field. **Every lane then carries its own internal control**, because wild-type AFF1 (125/145 kDa) and the fusion (~240 kDa) resolve on the same gel from the same antibody, and wild-type KMT2A appears as its taspase1-processed N320 and C180 fragments. Four numbers come out that nobody has: the fusion's chromatin-bound fraction, the wild-type AFF1 chromatin-bound fraction, the ratio between them, and the salt concentration at which each releases. A menin-inhibitor arm then answers the parked-versus-degraded question that the imaging experiment this graph already proposes would answer more slowly and more expensively [E1][E2].
Design
Cells. Two t(4;11) lines with different breakpoints, plus a KMT2A-germline B-ALL line as the negative control and a t(11;19) line as a partner control. Note the PubMed search caution rather than the biology one: SEM is unusable as a literature search token because it matches Structural Equation Modeling and Scanning Electron Microscopy, and RS4;11 translates to rs4 11. That is a retrieval hazard, not an experimental one.
Fractions, in increasing stringency:
- cytoplasm (hypotonic lysis, NP-40)
- soluble nucleoplasm (150 mM NaCl)
- low-salt chromatin eluate (300 mM NaCl)
- high-salt chromatin eluate (500 mM, then 2 M NaCl)
- nuclease-resistant pellet (benzonase, then SDS)
Blots. Anti-AF4 C-terminal (sees wild-type AFF1 and the fusion, resolved by size), anti-MLL-N (sees wild-type MLL-N320, the fusion, and any truncated N-terminal product), anti-MLL-C (sees the taspase1-released MLL-C180 fragment and, critically, cannot see the fusion - but it also cannot separate wild-type MLL-C180 from the identical fragment released by cleavage of the reciprocal AFF1::KMT2A product, so it is fusion-excluding rather than wild-type-specific, and in a reciprocal-positive line it reports the sum of two alleles).
Fraction-purity controls. GAPDH or tubulin for cytoplasm, a soluble nuclear factor for fraction 2, histone H3 for fractions 3-5, lamin B1 for fraction 5. Without these the experiment is uninterpretable, and their absence is the commonest defect in published fractionations.
Arms. (a) untreated; (b) menin inhibitor, timecourse 2, 6, 24, 48 h; (c) proteasome inhibitor plus menin inhibitor, to separate redistribution from degradation; (d) cycloheximide chase in each fraction, for fraction-specific half-life.
What each result would mean
| observation | interpretation |
|---|---|
| fusion enriched in fractions 3-5 relative to wild-type AFF1 in the same lane | the fusion is more chromatin-retained than its parent - the first quantitative fusion-versus-parent localisation statement in this literature |
| fusion and wild-type AFF1 distribute identically | the partner contributes no residence change, consistent with compartment being a retained-KMT2A property |
| fusion requires 2 M NaCl or nuclease | matrix-class association, which would put a number on this graph's nuclear-matrix entry |
| on menin inhibition, fusion moves 3-5 into 2 | parked - the drug is a displacer, and intermittent dosing permits re-loading |
| on menin inhibition, total fusion falls across all fractions, rescued by proteasome inhibition | degraded - the drug is a partial degrader, supporting the graph's standing hypothesis |
| menin protein falls but fusion does not | the known menin-destabilising effect without a fusion effect, which would isolate the two |
The fifth row is the one this graph most needs, because the degron-versus-menin analysis it already holds is an inference from transcriptional time-course shape, and this is a direct measurement.
One extra output comes free. Blotting the anti-MLL-N and anti-MLL-C lanes in the same five fractions also asks whether MLL-N320 and MLL-C180 co-fractionate. The 2003 work says they heterodimerise and that the complex confers MLL's subnuclear destination, and that breaking the FYRN-FYRC interaction redistributes C180 to a diffuse nuclear pattern 45; a 2019 study reports subcellular localisation unchanged when cleavage is blocked. A fraction-resolved measurement would adjudicate a disagreement that imaging has not settled in twenty-three years.
Why it has not been done, and why that is not a reason
The three historical fractionations of KMT2A chimeras all date from 1996-97 and all stopped at nucleus-versus-cytoplasm, because the question then was whether a chimera with a cytoplasmic partner was nuclear at all. Once that was settled the technique was not carried forward, and the field moved to ChIP, which answers a genomic question rather than a compartment one and has its own attribution problem.
Nothing about the experiment is hard. It needs no new antibody, no knock-in, no tag, and no mass spectrometry. Its cost tier is low and its timeline is weeks. It is the cheapest unrun experiment in this beat and probably in this graph.
| design | five-fraction subcellular fractionation (cytoplasm / soluble nucleoplasm / 300 mM eluate / 500 mM-2 M eluate / nuclease-resistant pellet) of two t(4;11) lines, one t(11;19) line and one KMT2A-germline control, blotted with anti-AF4 C-terminal, anti-MLL-N and anti-MLL-C |
|---|---|
| readout | band intensity of fusion versus wild-type parent within each fraction, on the same blot; plus salt concentration of release |
| controls | GAPDH/tubulin (cytoplasm), histone H3 (chromatin), lamin B1 (pellet), KMT2A-germline line, MLL-C180 as a fusion-excluding band (not wild-type-only: the reciprocal product releases the same fragment) |
| cost tier | low |
| timeline | 6-10 weeks |
| new reagents required | none |
arms
[ "untreated", "menin inhibitor timecourse 2/6/24/48 h", "menin inhibitor + proteasome inhibitor", "cycloheximide chase per fraction" ]
- A 'parked' result would predict rapid rebound on drug withdrawal and would argue against intermittent dosing schedules currently attractive for tolerability.
- A 'degraded' result would make the drug a partial degrader and raise the possibility that resistance arises by stabilising the fusion rather than by blocking the pocket, a mechanism no current resistance assay would detect.
- If the fusion is mostly soluble at baseline, occupancy-based pharmacodynamic biomarkers are reading a minority pool and could show a large fractional change for a small absolute one.
- Does the fusion's fraction distribution differ between two t(4;11) lines with different breakpoints?
- Do MLL-N320 and MLL-C180 co-fractionate, as the FYRN-FYRC re-association model predicts?
- Is the reciprocal AFF1::KMT2A product, where present, in a different fraction from the direct product?
- In a reciprocal-positive t(4;11) line, can the MLL-C180 band be decomposed into wild-type and reciprocal-derived contributions, or does the reciprocal product make that band uninterpretable as a control?
- Do MLL-N320 and MLL-C180 co-fractionate, and does blocking taspase1 cleavage change the fraction distribution even though it reportedly leaves the microscope image unchanged?
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
The menin-MLL inhibitor VTP-50469 displaces menin from protein complexes and alters MLL chromatin occupancy at target genes, inducing differentiation and apoptosis and eradicating disease in patient-derived xenografts - but no measurement of fusion protein abundance, stability or localisation is reported.
A Menin-MLL Inhibitor Induces Specific Chromatin Changes and Eradicates Disease in Models of MLL-Rearranged Leukemia · Krivtsov AV, et al. · Cancer cell 2019What this evidence cannot show
The occupancy readout is ChIP, which has no size dimension and cannot separate fusion from wild-type KMT2A without subtraction or a tag. The absence of a protein-fate measurement is the gap this experiment fills, and is an absence of testing rather than a negative result.
Evidence that argues against this (1)
- Menin inhibitors reduce menin protein levels and shorten menin's half-life through CHIP-mediated ubiquitination, so a fall in fusion signal after treatment could be secondary to loss of its adaptor rather than a direct effect, and the proteasome arm is needed to tell them apart. Wu Y et al. 2019
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
In one KMT2A-rearranged sample that lineage-switched during revumenib treatment the oncoprotein and menin were reduced to undetectable levels while ENL persisted at oncoprotein target loci, and across 36 samples oncoprotein genomic enrichment was highly variable and dynamically regulated.
MLL oncoprotein levels influence leukemia lineage identities · Janssens DH, et al. · Nature communications 2024What this evidence cannot show
A single confounded human observation - the lineage switch independently lowers oncoprotein level - which is exactly why a controlled in-vitro fractionation is needed. The readout is genomic occupancy, not fraction-resolved protein.
Evidence that argues against this (1)
- Variable and dynamic oncoprotein enrichment across samples means a single cell line's fractionation profile may not represent patient disease, so the experiment should be repeated in at least one patient-derived sample. Krivtsov AV et al. 2019
- E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
The same anti-AF4 reagents resolve MLL-AF4 at about 240 kDa from wild-type AF4 at 125 and 145 kDa on a blot of t(4;11) cells, which is the property that makes every lane of this experiment self-controlled.
AF4 encodes a ubiquitous protein that in both native and MLL-AF4 fusion types localizes to subnuclear compartments · Li Q, et al. · Blood 1998What this evidence cannot show
Abstract-level plus this graph's audit of the same paper; no PMCID and no DOI. Band positions depend on the patient's breakpoint and on gel percentage, so each line's expected size must be predicted from its own fusion transcript.
Evidence that argues against this (1)
- The fusion is the less abundant species, so fractions containing little fusion may fall below detection and an apparent absence from a fraction could be a sensitivity limit rather than a real exclusion; a loading series is required. Butler LH et al. 1997
- E4established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper
MLL is cleaved at two conserved sites generating N320 and C180 fragments which heterodimerise to stabilise the complex and confer its subnuclear destination - so blotting both fragments is itself a localisation readout, and the fusion, which lacks C180, cannot form that heterodimer.
Taspase1: a threonine aspartase required for cleavage of MLL and proper HOX gene expression · Hsieh JJ, et al. · Cell 2003What this evidence cannot show
The subnuclear-destination clause is the paper's own summary statement; the fragment complex was characterised biochemically and by localisation of transfected constructs, not in a t(4;11) cell. An anti-MLL-C reagent is fusion-excluding but not wild-type-specific: the reciprocal AFF1::KMT2A product carries the KMT2A taspase1 sites and releases a sequence-identical MLL-C fragment, so in a reciprocal-positive line the MLL-C180 band is the sum of two alleles.
Evidence that argues against this (1)
- Taspase1-dependent cleavage was later shown to DESTABILISE MLL1 rather than mature it, with taspase1 loss leaving MLL1 uncleaved, more degradation-resistant and markedly increased in chromatin occupancy while subcellular localisation was UNCHANGED - which argues the cleavage's localisation role is weaker than the 2003 summary implies. Zhao Z et al. 2019
- E5strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
Cleavage generates N-terminal p320 and C-terminal p180 fragments that form a stable complex localising to a subnuclear compartment; disrupting the FYRN-FYRC interaction markedly decreases N320 levels and redistributes C180 to a diffuse nuclear pattern; and MLL fusion proteins of leukaemia, which lose the ability to complex with C180, have their stability conferred instead by the fusion partners.
Proteolytic cleavage of MLL generates a complex of N- and C-terminal fragments that confers protein stability and subnuclear localization · Hsieh JJ, et al. · Molecular and cellular biology 2003What this evidence cannot show
Biochemistry and transfected-construct localisation in non-leukaemic cells. The 'diffuse nuclear pattern' result is the single clearest published demonstration that a defined KMT2A module change produces a defined subnuclear change, and it concerns the C-terminal half the fusion does not have.
Evidence that argues against this (1)
- A 2019 study found subcellular localisation UNCHANGED when cleavage was blocked, so the localisation consequence of the N320-C180 complex is contested and the fractionation experiment should treat it as a hypothesis to test rather than a premise. Zhao Z et al. 2019
the experiment that would fill the fractionation gap this entry documents
the menin-inhibitor plus proteasome arm separates parked from degraded directly
a cheaper, attributable alternative to the imaging time course, answering the same question without a knock-in
reads out what the menin inhibitor does to the fusion protein itself, which no published study measures
replaces an inference from transcriptional time-course shape with a direct fraction-resolved measurement
measures the fusion's chromatin-bound fraction against wild-type AFF1 in the same lane
the cheapest falsifier: fusion and wild-type in different fractions of the same lysate
the MLL-C180 control band is fusion-excluding but not wild-type-specific in a reciprocal-positive line
the anti-MLL-N blot lane is interpreted under that entry's rules
a fraction-resolved test of whether the N320-C180 complex really confers a subnuclear destination, which two studies disagree about
the fractionation is the readout that could adjudicate the 2003-versus-2019 disagreement