Experiment: where do KMT2A::AFF1 blasts go when a menin inhibitor releases the block? Single-cell lineage destination under drug
also called menin inhibitor lineage destination experiment · serial single-cell CITE-seq under revumenib
Treat infant leukaemia cells with a menin inhibitor and watch, one cell at a time, what they turn into. The question is whether they mature into healthy B cells or drift toward myeloid cells, because only one of those outcomes is a cure.
The same thing, for a scientist
A serial single-cell experiment in human KMT2A::AFF1 patient-derived xenografts, with a parallel retrospective arm in banked clinical marrows, designed to determine the lineage destination of blasts under menin inhibition. Readout is combined single-cell transcriptome plus surface proteome (CITE-seq) with a B-and-myeloid antibody panel, plus immunoglobulin locus status, at baseline and at multiple on-treatment timepoints. The experiment directly tests hyp-menin-release-lineage-destination and is designed so that either outcome is publishable and decision-relevant.
Design
Arm 1, patient-derived xenografts (primary). At least 4 independent infant KMT2A::AFF1 PDX lines, chosen to span the differentiation-stage clusters where possible (a HOXA-type AFF1 case and, if obtainable, an IRX-type undifferentiated case) 1. Treat with a menin inhibitor at a clinically relevant exposure; vehicle and a cytotoxic comparator as controls. Sample marrow, spleen and CNS at baseline, at early response, at maximal response and at progression.
Arm 2, human retrospective (cheap, do first). Banked diagnostic and on-treatment marrows from B-lineage KMT2A-rearranged patients who received a menin inhibitor. Even stored flow-cytometry files would allow a myeloid-marker re-analysis. If any trial collected serial immunophenotype with myeloid markers, this arm costs a data request rather than an experiment.
Readout.
- Single-cell RNA plus surface protein (CITE-seq) with CD19, CD10, CD34, CD22, NG2/CSPG4, CD33, CD13, CD117, CD15, CD14, CD11b, HLA-DR and IL7R/CD127 23.
- Immunoglobulin heavy-chain locus status per cell, to detect whether any cells cross the pre-BCR checkpoint.
- Surface IgM as the unambiguous marker of genuine B maturation.
- Fusion-transcript detection per cell, so that any myeloid population can be proved to be the same clone rather than normal myelopoiesis recovering.
Controls that decide the interpretation. Recovering normal myelopoiesis under any effective therapy will produce myeloid cells. The experiment is only informative because per-cell fusion detection distinguishes fusion-positive myeloid cells (lineage switch) from fusion-negative myeloid cells (normal recovery). Without that, the result is uninterpretable, and that is the commonest reason clinical flow data cannot answer this question already.
Decision rules, set in advance
| observation | conclusion | action |
|---|---|---|
| fusion-positive cells acquire surface IgM and a mature B program | standard differentiation model holds | proceed with monotherapy development |
| fusion-positive cells acquire CD33/CD13/CD11b with loss of CD19 | lineage switch under chromatin-directed therapy | combination or sequencing strategy is mandatory |
| fusion-positive cells persist with no change in lineage program | release is incomplete or menin-independent | look at the IRX-type/RAS group and at non-menin mechanisms |
| only fusion-negative myeloid cells appear | normal recovery | reassuring; report as such |
Committing to these in advance matters, because the intermediate result (a small fusion-positive myeloid subpopulation) is the one most easily explained away.
| design | Serial single-cell CITE-seq (transcriptome plus surface proteome) of human infant KMT2A::AFF1 patient-derived xenografts under menin inhibition versus vehicle and cytotoxic comparator, sampled at baseline, early response, maximal response and progression from marrow, spleen and CNS; plus a retrospective arm re-analysing banked serial marrows or stored flow files from B-lineage KMT2A-r patients treated with a menin inhibitor. |
|---|---|
| readout | Per-cell lineage program assignment (B versus myeloid), surface IgM as the marker of genuine B maturation, immunoglobulin heavy-chain locus status, and per-cell fusion-transcript detection to distinguish fusion-positive lineage switch from fusion-negative normal myeloid recovery. |
| controls | Vehicle and cytotoxic comparator arms; per-cell fusion detection as the decisive control against normal myelopoiesis; at least 4 independent PDX lines stratified by integrative cluster where obtainable. |
| cost tier | high |
| timeline | Retrospective arm 2-4 months if data access is granted; PDX arm 12-18 months including PDX expansion |
| feasibility notes | The retrospective arm may be answerable with existing unanalysed trial data and should be attempted before committing to the PDX programme. |
tests hypotheses
[ "hyp-menin-release-lineage-destination" ]
panel
[ "CD19", "CD10", "CD34", "CD22", "NG2/CSPG4", "CD33", "CD13", "CD117", "CD15", "CD14", "CD11b", "HLA-DR", "IL7R/CD127", "surface IgM" ]
- PDX establishment selects for the most proliferative subclones, so the compartment most likely to switch lineage may be the one least represented in the model.
- A xenograft mouse has no normal human myelopoiesis to confound the readout, which makes the PDX arm cleaner but also removes the competitive context in which switch may actually occur in patients.
- If the answer is that a small fusion-positive myeloid subpopulation appears, that intermediate result is easy to dismiss and hard to act on unless decision rules are fixed in advance.
- Do any menin inhibitor trials in B-lineage KMT2A-r disease already hold serial marrow immunophenotype with myeloid markers? If so the first arm is a data request, not an experiment.
- Can per-cell fusion-transcript detection be made sensitive enough in 3-prime single-cell chemistry, or does this require targeted amplification of the KMT2A::AFF1 junction?
- Are infant KMT2A::AFF1 PDX lines available that represent the IRX-type undifferentiated cluster, or does PDX establishment systematically select against it?
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
Infant KMT2A-rearranged leukaemias fall into five integrative clusters at distinct differentiation stages, with the IRX-type undifferentiated group being the most refractory and universally RAS-pathway-mutated, so any lineage-destination experiment must be stratified by cluster rather than pooled.
Multi-omics analysis defines highly refractory RAS burdened immature subgroup of infant acute lymphoblastic leukemia · Isobe T, Takagi M, Sato-Otsubo A, Nishimura A, Nagae G, Yamagishi C, Tamura M, Tanaka Y, Asada S, Takeda R, Tsuchiya A, Wang X, Yoshida K, Nannya Y, Ueno H, Akazawa R, Kato I, Mikami T, Watanabe K, Sekiguchi M, Seki M, Kimura S, Hiwatari M, Kato M, Fukuda S, Tatsuno K, Tsutsumi S, Kanai A, Inaba T, Shiozawa Y, Shiraishi Y, Chiba K, Tanaka H, Kotecha RS, Cruickshank MN, Ishikawa F, Morio T, Eguchi M, Deguchi T, Kiyokawa N, Arakawa Y, Koh K, Aoki Y, Ishihara T, Tomizawa D, Miyamura T, Ishii E, Mizutani S, Wilson NK, Gottgens B, Miyano S, Kitamura T, Goyama S, Yokoyama A, Aburatani H, Ogawa S, Takita J · Nature Communications 2022What this evidence cannot show
Cluster assignment is inferred from multi-omics projection; obtaining PDX lines that faithfully represent each cluster may not be possible, and PDX establishment itself selects for aggressive, proliferative subclones.
Evidence that argues against this (1)
- The experiment's core premise — that a menin inhibitor releases the block and the cells then differentiate somewhere — may not hold in ALL. Testing revumenib in vitro on primary KMT2A-rearranged ALL and AML, ALL samples underwent rapid, dose-dependent cell death while it was the AML samples that responded slowly and by myeloid differentiation. If KMT2A-r ALL blasts die rather than mature, a single-cell lineage-destination readout will have very few surviving cells to track, and the experiment needs a death-versus-differentiation arm before it can be interpreted. Read: abstract only. Adriaanse FRS et al. 2024
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
NG2/CSPG4 is a near-specific surface marker of KMT2A rearrangement that is minimally expressed in healthy haematopoietic cells but is state-dependent rather than hierarchy-defining, being upregulated on extramedullary and CNS-infiltrating blasts, so it belongs in the panel as a state readout rather than as a clone identifier.
NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL · Prieto C, Lopez-Millan B, Roca-Ho H, Stam RW, Romero-Moya D, Rodriguez-Baena FJ, Sanjuan-Pla A, Ayllon V, Ramirez M, Bardini M, De Lorenzo P, Valsecchi MG, Stanulla M, Iglesias M, Ballerini P, Carcaboso AM, Mora J, Locatelli F, Bertaina A, Padilla L, Rodriguez-Manzaneque JC, Bueno C, Menendez P · Leukemia 2018What this evidence cannot show
NG2 antibody clones and thresholds vary between laboratories, so cross-site comparison of NG2 intensity is unreliable without a shared standard.
Evidence that argues against this (1)
- Same source, same point: the lineage-destination readout presumes survival. In KMT2A-rearranged ALL, revumenib produced rapid dose-dependent induction of leukaemic cell death rather than differentiation, so a surface panel scored on drug will be dominated by selection of resistant cells. The study also found revumenib resistance arising in KMT2A-r ALL both through MEN1 mutation and independently of MEN1, so 'where do the cells go' and 'which cells survive' will be confounded. Read: abstract only. Adriaanse FRS et al. 2024
- E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
MLL-Af4-transformed cells can assume a myeloid state under environmental pressure while retaining lymphoid potential, and t(4;11) patients were observed evading CD19-directed therapy by myeloid lineage switch, establishing that the phenotype the experiment looks for is biologically available.
Instructive Role of MLL-Fusion Proteins Revealed by a Model of t(4;11) Pro-B Acute Lymphoblastic Leukemia · Lin S, Luo RT, Ptasinska A, Kerry J, Assi SA, Wunderlich M, Imamura T, Kaberlein JJ, Rayes A, Althoff MJ, Anastasi J, O'Brien MM, Meetei AR, Milne TA, Bonifer C, Mulloy JC, Thirman MJ · Cancer Cell 2016What this evidence cannot show
The model uses murine Af4 and retroviral delivery, so the ease of the myeloid transition in the model may exceed that in endogenous human disease.
Evidence that argues against this (2)
- Resistance to menin inhibition arises in patients by a route that has nothing to do with lineage. Somatic MEN1 mutations at the drug-menin interface were recovered from patients with acquired resistance and reproduced in xenografts and a base-editor screen; they block drug binding without disturbing the menin-MLL1 interaction, so menin and MLL1 stay on chromatin. Blasts that persist under drug in this experiment may be MEN1-mutant escapees rather than cells that chose a lineage. Read: abstract only. Perner F et al. 2023
- Any myeloid phenotype seen under drug should not be assumed permanent. A KMT2A-rearranged infant whose leukaemia switched to AML under blinatumomab spontaneously reverted to the lymphoid phenotype within weeks, so a myeloid readout at a single timepoint may be a transient adaptation rather than a lineage destination. Read: abstract only. Wölfl M et al. 2018
supplies the destination outcomes against which the methylome score is scored
- related to →Lineage switch as antigen escape from CD19-directed therapy in KMT2A-rearranged leukemia
the myeloid-drift readout this experiment looks for is the same phenotype as therapy-driven lineage switch; counter-evidence notes that KMT2A-r ALL blasts die rapidly under revumenib rather than differentiating, so few cells may survive to be tracked
serial single-cell CITE-seq with per-cell fusion detection under menin inhibition
asks whether the arrested pro-B blast resolves to a mature B cell or to a myeloid cell under drug, using per-cell fusion detection to exclude normal myeloid recovery
the specific agent whose lineage pharmacodynamics are unmeasured in B-lineage KMT2A-r disease
serial single-cell CITE-seq under menin inhibition would give the rate and the destination
serial single-cell phenotyping under menin inhibition, with matched drug sensitivity, separates the three possible readings