Spike-in-normalised menin CUT&RUN in a KMT2A::AFF1 degron line, with replicates and a PSIP1 arm
The experiment that would settle this: measure menin on chromatin in the fusion this project actually cares about, with a built-in ruler so the amounts are real, repeated enough times to trust, and with LEDGF knocked down in one arm to see whether that is what holds the leftover menin.
The same thing, for a scientist
Every limit this beat hit is fixed by one experiment. **System:** a KMT2A::AFF1 (t(4;11)) B-ALL line - SEM or RS4;11 - carrying an FKBP12(F36V) degron knocked into the fusion allele, so the degron is on the index fusion rather than on MLL::AF9. **Readout:** menin CUT&RUN with a **spike-in** (yeast or Drosophila chromatin, or a defined exogenous nucleosome standard), so occupancy is on an absolute scale and a uniform global change is visible. **n >= 3 per arm.** **Arms:** DMSO; dTAG 3 h; dTAG 24 h; revumenib at a clinically relevant exposure, 3 h and 24 h; shPSIP1 or dTAG-PSIP1; shPSIP1 + fusion degradation. **Companion channels in the same cells:** HA/fusion, PSIP1, wild-type-MLL1 C-terminal, DOT1L. **Analysis, pre-specified:** stratify on fusion occupancy in the DMSO arm and report the median spike-in-normalised ratio per stratum with a per-replicate confidence interval, plus the treated/control peak-width ratio beside every peak-derived figure.
What each design choice buys
- Spike-in converts every number in this beat from a within-sample share into an amount, and is the only way to distinguish menin redistribution from renormalisation.
- n >= 3 supplies the error bar that does not exist anywhere in GSE173599; the only replicate-derived spread available there is 4.5 points, measured on a near-total collapse, which does not bound a 20% effect.
- KMT2A::AFF1 removes the wrong-fusion caveat that limits every magnitude claim in this beat.
- A PSIP1 arm tests the hypothesis directly rather than by elimination.
- CUT&RUN rather than ChIP improves signal-to-noise at low-occupancy sites, which is where the peak instrument failed here.
What would make it worth running even if the answer is boring
The stratified menin ratio at fusion-occupied promoters is a candidate pharmacodynamic endpoint for menin inhibitors. At present no one knows its dynamic range, its variance across replicates, or whether it differs between fusion partners. Those three numbers are worth having independently of the tether question.
| design | Degron knock-in on the KMT2A::AFF1 allele in SEM or RS4;11; spike-in-normalised menin CUT&RUN; n>=3 per arm; arms DMSO / dTAG 3 h / dTAG 24 h / revumenib 3 h / revumenib 24 h / PSIP1 depletion / PSIP1 depletion + dTAG; companion HA, PSIP1, MLL1-Cterm and DOT1L channels in the same cells |
|---|---|
| readout | median spike-in-normalised menin occupancy ratio at fusion-occupied vs non-occupied promoters, with a per-replicate interval; peak-width ratio reported beside every peak-derived figure |
| controls | spike-in chromatin; IgG; a fusion-occupancy positive control (HA) that must separate the strata before any menin number is interpreted; H3K4me3 and wild-type-MLL1 negative controls |
| cost tier | med |
| timeline | degron knock-in 3-4 months if a line does not already exist, then 6-8 weeks for the CUT&RUN panel |
| pre registration required | declare in advance the stratum definition, the cut ladder, the resolvability threshold, and the peak-width re-score trigger |
- If the experiment shows menin tracks the fusion closely in KMT2A::AFF1 but not in KMT2A::MLLT3, then menin-inhibitor efficacy should differ by fusion partner - and the approved label does not distinguish them.
- A pharmacodynamic endpoint with a measured dynamic range can be used to declare a drug inactive as well as active; establishing it is a commitment to whatever it reports.
- Does a KMT2A::AFF1 degron line already exist in a published deposit? If so this becomes a reanalysis rather than an experiment.
- Is spike-in CUT&RUN quantitative enough at these occupancy levels, or is a calibrated ChIP with an internal standard the better instrument?
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.dataset
The deposited experiment has n=1 per arm for every channel except HA, no spike-in, and uses KMT2A::MLLT3 rather than KMT2A::AFF1.
GSE173599 - Chromatin state changes induced by targeted degradation of MLL-AF9 are phenocopied by combined DOT1L and MENIN inhibition [ChIP-Seq] · Olsen SN, Hatton C (Armstrong lab, Dana-Farber) · NCBI GEO (SubSeries of GSE173600) 2022What this evidence cannot show
Established by reading the sample list and the MACS command lines; a replicate sequenced but not deposited would not be visible this way.
Evidence that argues against this (1)
- A single-replicate experiment is not automatically uninformative - the deposit's two independent HA 3 h batches agree to within 4.5 points - so the n=1 objection bounds precision rather than invalidating the published conclusions. Olsen SN et al. 2022
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.dataset
Menin retains 50.3% of its signal at fusion-occupied promoters 3 h after the fusion is degraded, which is the quantity this experiment is designed to measure properly.
GSE173599_TSS_gene_body_signal_Figure3.xlsx - per-gene housekeeping-normalised TSS and gene-body ChIP signal, DMSO vs dTAG-VHL 3 h and 24 h, 16 channels · Olsen SN et al. · NCBI GEO supplementary file 2022What this evidence cannot show
Housekeeping-normalised, n=1; the point of the proposed experiment is that this number currently has no error bar and no absolute scale.
Evidence that argues against this (1)
- If the 50.3% residue is partly antibody background rather than menin, a spike-in adds an absolute scale but does not fix specificity; the experiment therefore needs a MEN1-knockout or degron-menin specificity arm that this design does not yet include. Yokoyama A et al. 2008
- E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
LEDGF/p75 is essential for MLL-rearranged leukaemogenesis and dispensable for steady-state haematopoiesis, which makes the PSIP1 arm both mechanistically informative and therapeutically relevant.
LEDGF/p75 is dispensable for hematopoiesis but essential for MLL-rearranged leukemogenesis · El Ashkar S, Schwaller J, Pieters T, Goossens S, Demeulemeester J, Christ F, et al., De Rijck J · Blood 131(1):95-107 2018What this evidence cannot show
Mouse conditional knockout; a human acute-depletion arm is what this design adds.
Evidence that argues against this (1)
- LEDGF/p75 was shown essential for MLL-fusion leukaemia INITIATION; if it is dispensable for maintenance, a PSIP1 depletion arm in an established line may show no menin change for reasons unrelated to tethering. El Ashkar S et al. 2018
spike-in menin CUT&RUN in KMT2A::AFF1 with a PSIP1 arm and n>=3, which is what every limit of this beat requires
- related to →KMT2A::AFF1 (MLL-AF4), t(4;11)(q21;q23)
moves the measurement into the index fusion, where no equivalent degron dataset exists
a PSIP1 depletion arm tests the tether directly rather than by elimination
the spike-in requirement in this design is the corpus-wide blocker that entry identified