Spike-in-normalised menin CUT&RUN with paired N- and C-terminal KMT2A antibodies in an infant KMT2A::AFF1 sample, on and off drug, in the same disease state
The experiment that would settle this: take one baby's KMT2A::AFF1 leukaemia cells, split them, treat half with a menin inhibitor, and measure menin and both ends of the KMT2A protein with a counting standard added to every tube - so the numbers are amounts, not proportions.
The same thing, for a scientist
Every limitation in this beat has one of four causes and this design removes all four. **(1) No spike-in** - so 'undetectable', 0.871 and 0.037 are all true of the same libraries; fix with a defined exogenous chromatin spike in every reaction. **(2) No IgG** - so occupancy is confounded with accessibility; fix with an IgG arm per condition, in the same protocol. **(3) Drug exposure confounded with clonal replacement** - fix by splitting one aliquot rather than comparing diagnosis to relapse. **(4) Wrong fusion** - fix by using KMT2A::AFF1, the index fusion, from an infant. Read out menin, KMT2A-N (two reagents), KMT2A-C (two reagents), ENL, DOT1L and H3K4me3, all in **one protocol on one day**, at n >= 3 biological replicates per arm.
Design
Material. A primary infant KMT2A::AFF1 B-ALL sample, or an infant-derived PDX, with enough cells for 3 replicates x 2 arms x 8 channels. A PDX is the realistic route and it costs the primary-sample claim, which should be stated.
Arms. DMSO and revumenib at a stated concentration and exposure, chosen to bracket a clinically achievable plasma level - and recorded in the deposition, which the existing dataset does not do.
Channels. menin (Bethyl A300-115A, matching the existing deposit), KMT2A-N1 (Millipore 05-764), KMT2A-N2 (CST D2M7U), KMT2A-C1 (Santa Cruz H10), KMT2A-C2 (Millipore 05-765), ENL (CST D9M4B), DOT1L (CST D4O2T), H3K4me3, and IgG in every arm. Same protocol for every channel, so no comparison crosses CUT&RUN and CUT&Tag.
Normalisation. A defined exogenous spike-in in every reaction, reported per sample, so that a global change in menin on chromatin is measurable rather than definitionally zero.
Primary readout. Spike-in-normalised menin occupancy at promoters stratified by the N-minus-C difference, DMSO versus drug, with the IgG arm as the accessibility floor.
What each arm decides
| question | what answers it |
|---|---|
| does menin leave in absolute terms, or only redistribute? | the spike-in, and nothing else |
| is menin's loss fusion-site-specific or global? | the N-minus-C stratification against the IgG floor |
| does the fusion itself move under the drug? | the N channel, with the C channel as the wild-type internal control |
| does ENL persist where menin goes? | the ENL channel in the same tubes |
| does the KMT2A::MLLT1 result transfer to the index fusion? | the fusion choice |
Cost, and the cheaper thing to do first
This is a medium-to-high cost experiment requiring primary infant material and about 48 libraries. Do the no-wet-work analysis first: the same N-versus-C comparison on the untreated KMT2A::AFF1 samples already public in GSE252378 (p279, p454, SEM) costs compute only and scores falsifier F1 of the companion hypothesis. If F1 fires, this experiment should not be run in this form.
| design | one primary infant KMT2A::AFF1 sample (or infant PDX) split into DMSO and revumenib arms; menin, KMT2A-N x2, KMT2A-C x2, ENL, DOT1L, H3K4me3 and IgG in a single protocol; n>=3 per arm |
|---|---|
| readout | spike-in-normalised menin occupancy at promoters stratified by the N-minus-C difference, against an in-arm IgG floor |
| controls | IgG in every arm; two reagents per KMT2A terminus; H3K4me3 as a chromatin-quality control; defined exogenous spike-in in every reaction |
| cost tier | high |
| timeline | 3-4 months given material access |
| do first | the no-wet-work N-versus-C analysis on the untreated KMT2A::AFF1 samples already in GSE252378, which scores falsifier F1 for free |
| record in the deposition | compound, concentration, exposure duration, spike-in organism and amount - none of which the existing dataset records |
fixes
[ "no spike-in", "no IgG", "drug confounded with clonal replacement", "wrong fusion", "cross-protocol comparison" ]
does not fix
[ "unrecorded blast fraction - needs flow-sorted blasts", "in-vitro exposure is not in-vivo dosing" ]
- This experiment is proposed partly to measure how completely a menin inhibitor clears menin in the index fusion. If the answer is 'not very', that is an argument for combination therapy in infants who currently have few options - and it should not be allowed to become an argument against menin inhibition, which eliminated the B-ALL clone in the patient this beat analyses.
- Infant primary material is scarce and consent-constrained. An experiment that consumes it should be pre-registered and should publish its negative result, which the field does not reliably do.
- Which infant KMT2A::AFF1 PDX models are available with enough cells for 48 libraries, and does any group hold a primary sample large enough to avoid a PDX?
- What revumenib concentration brackets a clinically achievable exposure in an infant? The existing literature's 1 uM figure is a cell-line convention, not a derived value.
- Is there a spike-in that behaves comparably in CUT&RUN and CUT&Tag, in case both protocols are needed?
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.dataset
Every quantitative limitation in the existing infant pair traces to the absence of a spike-in, the absence of an IgG arm, the diagnosis-versus-relapse design, or the fusion background.
analyses/menin-under-revumenib-in-an-infant-patient-pair - pre-registered re-analysis of GSE252378 samples 148752 and 152985 · Q143-menin-under-drug-in-infants (Oncofusion Knowledge Graph) · this repository 2026What this evidence cannot show
This is an assessment of one deposit, not a general claim about the field. A fifth limitation - unrecorded blast fraction - is not fixed by this design either and would need flow-sorted blasts.
Evidence that argues against this (1)
- A split-aliquot in-vitro drug exposure is not the same perturbation as weeks of oral dosing in a child, so the proposed experiment trades the clonal confound for an exposure-realism problem, and its concentration choice becomes the load-bearing assumption. Issa GC et al. 2023
- E2established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.dataset
The existing deposit uses two protocols on different days for the channels that must be compared, which this design removes by running one protocol.
GSE252378_family.soft.gz - the per-sample SOFT records for GSE252378, read raw with line numbers · Janssens DH et al. · NCBI GEO FTP 2024What this evidence cannot show
Read from the extract-protocol and title-batch fields of the sample records.
Evidence that argues against this (1)
- Running every channel in one protocol removes the cross-assay confound and introduces a different one: CUT&RUN and CUT&Tag do not perform equally on every epitope, so forcing all eight channels into one protocol may cost signal on the channels the other protocol suits better. Kaya-Okur HS et al. 2019
- E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
Revumenib is clinically active and dosed intermittently with CYP3A4-dependent adjustment, so a single in-vitro concentration is an approximation of the in-vivo exposure.
The menin inhibitor revumenib in KMT2A-rearranged or NPM1-mutant leukaemia · Issa GC, Aldoss I, DiPersio J, Cuglievan B, Stone R, Arellano M, Thirman MJ, Patel MR, Dickens DS, Shenoy S, Shukla N, Kantarjian H, Armstrong SA, Perner F, Perry JA, Rosen G, Bagley RG, Meyers ML, Ordentlich P, Gu Y, Kumar V, Smith S, McGeehan GM, Stein EM · Nature 615:920-924 2023What this evidence cannot show
Phase 1 trial; the pharmacokinetic detail is summarised rather than quoted here, and the correct bracketing concentration for this design was not derived.
Evidence that argues against this (1)
- Bracketing a clinically achievable plasma concentration assumes free drug in marrow tracks plasma, which is unestablished for revumenib in infants; an in-vitro concentration chosen on that basis could be well above or below the exposure the leukaemic blast actually sees. Perner F et al. 2023
the experiment that scores falsifier F3
the design that would make the falsifier's quantities measurable
the control gap this design fixes
the confound this design removes by splitting one aliquot
- related to →KMT2A::AFF1 (MLL-AF4), t(4;11)(q21;q23)
the index fusion this experiment would use
the drug arm
the primary readout