Experiment: blot menin in a SET-NUP214 immunoprecipitate, then run a menin inhibitor and selinexor alone and combined in LOUCY and MEGAL
also called the missing menin lane · menin inhibitor in LOUCY
One extra antibody on an experiment that has already been done would show whether the menin protein travels with SET::NUP214. If it does, the next step is to give the cells a menin drug and see whether the HOXA genes go down.
The same thing, for a scientist
**Two tiers, deliberately ordered so the cheap one can stop the expensive one.** **Tier 1 (low cost, days).** Nuclear extract from **LOUCY** and **MEGAL**; immunoprecipitate with anti-NUP214 (the antibody the founding study used to recover the fusion) and reciprocally with anti-menin; blot both for **menin, wild-type MLL1 N-terminus, LEDGF/PSIP1, CRM1 and DOT1L**. Controls: **SKW3** as the fusion-negative T-ALL line used in the founding paper, and a KMT2A::AFF1 line as the positive control for the menin blot. CRM1 in the anti-NUP214 lane is the internal positive control that the IP worked - it is a published, reproducible band in this exact experiment. **Tier 2 (medium cost, weeks), run only if Tier 1 is positive or equivocal.** Dose-response of a clinical menin inhibitor (**revumenib** and **ziftomenib**, both, because this graph records that they are not interchangeable), **selinexor**, and the combination, in LOUCY and MEGAL, with a KMT2A::AFF1 line and a HOXA-low T-ALL line as flanking controls. Readouts: HOXA9/HOXA10/HOXB transcript, CD differentiation markers and TCR-gamma-delta surface expression (the founding paper's own differentiation readout), viability, and **CUT&RUN for menin, MLL1-N, CRM1 and NUP214 at the HOXA and HOXB clusters** at a single early timepoint. **Why the CUT&RUN matters more than the viability curve:** viability in a line whose CRM1 is common-essential will move under selinexor whatever the mechanism. Occupancy separates the two arms; killing does not.
What each outcome would mean
| Tier 1 result | reading |
|---|---|
| menin in the anti-NUP214 IP, CRM1 also present | the MLL1 route is real; proceed to Tier 2 |
| CRM1 present, menin absent | hyp-set-nup214-hoxa-runs-through-recruited-wild-type-mll1 is falsified at its first prediction; SET::NUP214 becomes the clean second instance alongside CALM-AF10 and the menin-interface hypothesis is sharpened |
| neither present | the IP failed; nothing is learned, and this is why CRM1 is in the panel |
| Tier 2 result | reading |
|---|---|
| menin inhibitor lowers HOXA partially, selinexor lowers it further, combination lowest | two parallel arms; both are targets and neither is sufficient |
| menin inhibitor does nothing at exposures that work in the KMT2A::AFF1 control | menin dependence excluded in these lines; report as a bounded negative with the exposure stated |
| menin inhibitor removes fusion occupancy as well as menin | the MLL1 route is upstream of fusion recruitment, which no current model predicts and which would need its own beat |
Known obstacles, because three of them are real
- Two cell lines is the entire model space. There is no SET::NUP214 mouse model in the literature this beat searched, no PDX named, and the only other route to primary material is the GRAALL or Italian biobanks. A negative in two lines is a weak negative and must be written as one.
- Menin antibodies for IP in a lymphoid line are not a solved reagent problem, and a failed menin blot is indistinguishable from an absent protein without a positive-control lysate - hence the KMT2A::AFF1 arm.
- The MLL identity problem from
mechanism-set-nup214-recruits-wild-type-mll1propagates here. The blot must use an antibody whose epitope is in the KMT2A N-terminus specifically, and the entry should record the clone, because brief rule 74 applies: for an antibody readout the clone is the identifier, not the label. - LOUCY is not in DepMap, so there is no orthogonal genetic dependency datum to triangulate against, and a CRISPR arm would have to be generated rather than looked up.
| design | Tier 1: reciprocal co-immunoprecipitation (anti-NUP214 and anti-menin) from LOUCY and MEGAL nuclear/chromatin extract, blotted for menin, KMT2A N-terminus, PSIP1/LEDGF, CRM1 and DOT1L, with SKW3 as fusion-negative control and a KMT2A::AFF1 line as menin-blot positive control. Tier 2 (conditional on Tier 1): revumenib and ziftomenib dose-response, selinexor, and the combination, in LOUCY and MEGAL with a KMT2A::AFF1 line and a HOXA-low T-ALL line as flanking controls. |
|---|---|
| readout | Tier 1: presence or absence of a menin band in the anti-NUP214 lane at a sensitivity that recovers CRM1 in the same lysate. Tier 2: HOXA9/HOXA10/HOXB transcript by RT-qPCR, TCR-gamma-delta and membrane CD3 by flow cytometry (the founding paper's own differentiation readout), viability, and CUT&RUN for menin, KMT2A-N, CRM1 and NUP214 at the HOXA and HOXB clusters. |
| controls | SKW3 (fusion-negative T-ALL, the founding paper's own comparator); a KMT2A::AFF1 line for menin-inhibitor exposure calibration; CRM1 in the anti-NUP214 IP as the internal proof the IP worked; selinexor as the positive-control perturbation with a published effect size in this exact line; antibody clone recorded for every readout per brief rule 74. |
| cost tier | low |
| timeline | Tier 1: 2-3 weeks with existing reagents. Tier 2: 8-12 weeks. |
| kill criterion | If Tier 1 recovers CRM1 and no menin, stop. Do not run Tier 2. |
| system | human T-ALL and AML cell lines - NOT infant B-lineage KMT2A::AFF1, and any transfer of the result crosses lineage and age |
tests hypotheses
[ "hyp-set-nup214-hoxa-runs-through-recruited-wild-type-mll1" ]
- A positive co-IP would be over-read as evidence for menin-inhibitor eligibility in SET::NUP214 patients; the entry states explicitly that Tier 1 alone licenses nothing clinical.
- A negative Tier 2 in two cell lines does not exclude menin dependence in patients, and should be written as a bounded negative with the exposure and the control line's response stated.
- Selinexor's activity in these lines is confounded by CRM1 being common-essential, so a combination benefit could be additive toxicity rather than mechanistic synergy unless the CUT&RUN separates the arms.
- Is there a SET::NUP214 PDX anywhere? This beat found none named in the literature it searched, and two cell lines is a thin basis for a negative.
- Should CALM-AF10 lines be run in the same plate? The marginal cost is small and it would give the coordinator the two-instance comparison the beat was commissioned to produce, with one set of controls.
- Would a menin degrader rather than an inhibitor be the better tool here, given that the hypothesis is about displacing a recruited scaffold rather than blocking a pocket?
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
The founding study already performed immunoprecipitation in LOUCY with rabbit anti-NUP214, rabbit anti-PP32, mouse anti-SET, rabbit anti-CRM1 and rabbit anti-hDOT1L, and recovered SET-NUP214 in the NUP214, CRM1 and DOT1L immunoprecipitates by anti-SET western blot - so the assay and the positive control band already exist and only the menin lane is missing.
The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia. · Van Vlierberghe P, et al. · Blood 2008What this evidence cannot show
2008 reagents, several of them gifts (anti-CRM1 from M. Yoshida, anti-hDOT1L from Y. Zhang, anti-PP32 from J. Brody) and not necessarily obtainable now. The lysis is a single-detergent NP-40 buffer, which may not extract a chromatin-bound menin complex efficiently - a nuclear/chromatin fractionation step may be needed that the original protocol does not include. Full text read via the PMC HTML route (PMC2343598).
Evidence that argues against this (1)
- The same paper is internally inconsistent about whether a DOT1L immunoprecipitation was feasible in this system: the Figure 6C legend and the Methods describe one, while the Results state that reciprocal DOT1L IP experiments could not be performed for want of a suitable antibody. Anyone planning to reuse this protocol should expect the antibody situation to be worse than the figure panels imply, and should budget for validating every IP antibody rather than assuming the published panel is reproducible. Van Vlierberghe P et al. 2008
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
LOUCY and MEGAL are the only two cell lines of 141 human leukaemia and lymphoma lines screened that express SET-NUP214, and both express the 140 kDa fusion protein; SKW3 is the fusion-negative T-ALL comparator used in the founding ChIP and expresses neither the fusion nor HOXA promoter H3K79 methylation.
SET-NUP214 fusion in acute myeloid leukemia- and T-cell acute lymphoblastic leukemia-derived cell lines. · Quentmeier H, et al. · J Hematol Oncol 2009What this evidence cannot show
A 2009 panel; a line acquired by a repository since would not appear. The SKW3 half of the claim comes from the founding paper, not from this one. SKW3 is a T-ALL line but is not otherwise matched to LOUCY for maturation stage, so it controls for the fusion and not for cell state.
Evidence that argues against this (1)
- PUBLISHED CONFLICTING RESULT about the model space, and it changes the experiment being proposed. The 141-line panel is a 2009 snapshot, and the design it licenses - LOUCY plus MEGAL, both SET::NUP214 - omits the only NUP214-fusion model with a published in vivo arm. FKH-1 carries DEK::NUP214, shows a strong dependency on XPO1 by CRISPR deletion, and a DEK::NUP214 patient-derived xenograft responds to the second-generation SINE eltanexor with molecular clearance in marrow. Adding a DEK::NUP214 arm turns the selinexor half of this experiment from a two-cell-line observation into a comparison against an in vivo reference, and it tests whether anything here is about SET rather than about NUP214. Charles Cano F et al. 2025
- E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
In LOUCY, KPT-330 (selinexor) at 1,000 nM for 24 h almost completely removes CRM1 and Nup214 ChIP peaks at HOX clusters and lowers HOX gene expression, giving an established positive-control arm and an exposure to anchor a dose-response against.
Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells. · Oka M, et al. · eLife 2019What this evidence cannot show
1,000 nM for 24 h is a high, non-clinical exposure and is used here only as a mechanistic anchor, not as a dose proposal. CRM1 inhibition is pleiotropic across every NES cargo, so a selinexor arm is not a clean chromatin perturbation. Full text read via Europe PMC (PMC6874418).
Evidence that argues against this (1)
- METHODOLOGICAL DEFEATER on the anchor exposure. KPT-330 at 1,000 nM for 24 h also DEGRADES SET-Nup214 in this system, so the loss of CRM1 and Nup214 ChIP peaks at HOX is not a clean displacement measurement - part of it is loss of the protein being immunoprecipitated, and any dose-response anchored to it inherits that ambiguity. A cleaner reference exists in the sibling fusion: eltanexor reduces XPO1 expression, disrupts XPO1/DEK-NUP214 co-localisation at chromatin and downregulates fusion target genes, carried through to a xenograft. If this experiment anchors to 1 uM KPT-330 it must run a fusion-protein blot at the same timepoint, or the chromatin arm cannot be interpreted. Charles Cano F et al. 2025
Tier 1 co-IP falsifies the hypothesis at its first prediction if menin is absent
Tier 2 dose-response in LOUCY and MEGAL, with a KMT2A::AFF1 line for exposure calibration
positive-control perturbation arm with a published effect size in LOUCY
the experiment is designed around the two lines that exist, and its negatives are bounded by them