Experiment: spike-in-normalised H3K4me3, menin and IgG CUT&Tag in SEM under VTP-50469 and EPZ-5676 - the one missing experiment that turns every ratio in this beat into an absolute number
Repeat the key experiment in infant-leukaemia cells with a measuring stick added: a fixed amount of DNA from another species mixed into every sample. That converts 'the mark moved around' into 'the mark went up or down by this much', which is the question nobody can currently answer.
The same thing, for a scientist
**Design.** SEM cells, 30 uM VTP-50469, 30 uM EPZ-5676 and DMSO, 72 h, n=3 biological replicates per arm. CUT&Tag for **H3K4me3, menin and IgG in every arm** (9 libraries per replicate), with a defined **exogenous spike-in** - either the E. coli carry-over from pA-Tn5 quantified explicitly, or heterologous chromatin added at a fixed cell ratio. Sequence to matched depth; report fraction-of-reads-in-peaks per library **before** any normalisation. **Readouts.** (1) Absolute H3K4me3 change at menin-bound vs H3K4me3-matched menin-free sites. (2) Whether menin actually leaves chromatin in SEM under this dose - currently unmeasured in any infant-derived line in this deposit. (3) Whether the peak-share collapse seen in the deposited VTP libraries reproduces, which decides whether it was drug or library.
Why this specific experiment and not a bigger one
Three separate uncertainties in this beat collapse into one experiment, and each is currently unresolvable:
| uncertainty | what the spike-in arm settles |
|---|---|
| compositional normalisation | whether H3K4me3 fell globally or redistributed |
| the 2.5-fold peak-share drop in the VTP libraries | drug effect or library quality, by reproducing it or not |
| menin eviction assumed, never measured in SEM | whether the drug did what it is supposed to do in this line |
The IgG-under-drug arm is the cheapest part and the most often omitted: without it, background cannot be separated from signal in exactly the arm where background is suspected to have risen.
Controls that must be in the design, not added later
- Housekeeping promoters reported alongside targets, pre-specified. The pre-registered control in this beat killed the headline result and it cost three lines to write.
- The site classes defined on enrichment over IgG, not raw occupancy, with the IgG value inside each class printed beside every result. Raw-occupancy classes failed here at 8-36x.
- EPZ-5676 with an H3K79me2 readout, so the comparator's target engagement is demonstrated rather than assumed. Its absence is the weakest point of the current negative control.
| design | SEM; DMSO / 30 uM VTP-50469 / 30 uM EPZ-5676, 72 h, n=3 biological replicates; CUT&Tag for H3K4me3, menin and IgG in every arm with exogenous spike-in; H3K79me2 in the EPZ arm for target engagement |
|---|---|
| readout | spike-in-normalised absolute signal; within-library menin-bound vs H3K4me3-matched menin-free class ratio; fraction of reads in peaks reported pre-normalisation |
| controls | IgG in every arm; pre-specified housekeeping promoters (GAPDH, ACTB, RPL13A, TUBB, PGK1); site classes defined on enrichment over IgG with the IgG value in each class reported |
| cost tier | med |
| timeline | 6-10 weeks including sequencing |
| replaces | a 5 kb reanalysis of 2 libraries per arm |
- A spike-in tells you the total changed; it does not tell you the change is on the causal path to cell death. Pairing this with a transcriptional and a viability readout in the same cells is what stops it becoming another correlate.
- If menin turns out not to leave chromatin in SEM at 72 h, the entire interpretation of the deposited experiment - and of this beat - changes, and that is the most likely single result to overturn everything written here.
- Is 30 uM the right dose? It is the deposit's dose, but VTP-50469's cellular IC50 in MLL-r lines is far below that, and a supra-pharmacological dose may produce chromatin effects that a clinical exposure would not.
- Would a degron-tagged MEN1 be a cleaner perturbation than the inhibitor, removing the off-target question entirely at the cost of no longer modelling the drug?
- E1established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.dataset
GSE159608 contains no spike-in normalisation in any of its 419 samples and no menin, IgG or second-mark library under VTP-50469 in SEM, so none of these three readouts currently exists.
GSE159608: Automated CUT&Tag profiling of chromatin heterogeneity in mixed-lineage leukemia (419 samples, hg19 fragment BEDs) · Janssens DH, Meers MP, Wu SJ, Babaeva E, Meshinchi S, Sarthy JF, Ahmad K, Henikoff S · NCBI Gene Expression Omnibus 2021What this evidence cannot show
Enumerated from the deposit's sample records and data-processing fields on 2026-09-23. Scoped to this series.
Evidence that argues against this (2)
- This absence is bounded to ONE deposit. It says these readouts do not exist in GSE159608, not that they do not exist; this project found in the same cycle that its paper index had missed 73.7% of the corpus's citations and that a deposit index had to be built separately, so any 'none of these three readouts currently exists' should be read as 'none was found in the place searched'.
- Spike-in absence is not fatal in the way this framing implies. This corpus already holds entries establishing that within-sample shares preserve the SHAPE of a distribution even when the level is unanchored, so several of the questions listed are answerable without a spike-in, and demanding one may defer analysis that could be done now.
- E2moderate confidence 3 of 5: One strong study, or several smaller consistent ones.dataset
The measurements this experiment would replace - a within-library site-class ratio of 0.766 under VTP-50469 and 1.010 under EPZ-5676 - rest on two libraries per arm whose peak share differs 2.5-fold from controls.
sem-arm-identity-from-signal: run2.py and run3.py (this repository) · Q140-sem-menin-arm · Oncofusion knowledge graph 2026What this evidence cannot show
Our own reanalysis; the confound it identifies is the experiment's primary justification.
Evidence that argues against this (1)
- If a spike-in re-run reproduces the peak-share collapse, the deposited result was biology after all and this beat's caution was excessive. That outcome is as informative as the alternative and the experiment is worth running either way. Janssens DH et al. 2021
- E3established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper
VTP-50469 displaces menin and reduces MLL occupancy at select genes in MLL-rearranged models, which is the effect this experiment would measure directly in an infant-derived KMT2A::AFF1 line for the first time in this deposit.
A Menin-MLL Inhibitor Induces Specific Chromatin Changes and Eradicates Disease in Models of MLL-Rearranged Leukemia · Krivtsov AV, Evans K, Gadrey JY, Eschle BK, Hatton C, Uckelmann HJ, et al., Armstrong SA · Cancer Cell 36:660-673.e11 2019What this evidence cannot show
The original demonstration is in other models and by ChIP; the gap being filled is the cell type, not the mechanism.
Evidence that argues against this (2)
- The effect this experiment would measure has now been measured in a different fusion by the same cycle's q141 beat, which found menin retention of 0.503 at fusion-occupied promoters in KMT2A::MLLT3. The proposed experiment is therefore a transfer test rather than a first measurement, which is a weaker justification than 'for the first time' implies.
- 'Displaces menin and reduces MLL occupancy at select genes' is the vendor-adjacent summary of the compound. The clinical record is more equivocal: menin inhibitors produce responses in a minority of patients and resistance arises through MEN1 mutations that prevent eviction, so eviction is necessary but demonstrably not sufficient.
The spike-in experiment measures whether the residual exists in absolute terms
The experiment exists because the deposit has no spike-in and no menin or IgG arm under drug
Reproducing or failing to reproduce the peak-share collapse decides drug effect versus library quality
Measures menin eviction and H3K4me3 in the same cells under VTP-50469
SEM is the system, chosen because it is the infant-derived index-fusion line