The non-synchronisable state: five KMT2A-rearranged lines refuse mimosine, double thymidine and nocodazole, and the fusion protein does not fluctuate through the cycle
also called refractory to chemical cell-cycle synchronisation · checkpoint-defective state · constant fusion protein through the cell cycle · loss of biphasic MLL expression
Researchers normally line up cells at the same point in their division cycle using drugs, so that everyone in the dish is doing the same thing at the same time. Five of the standard KMT2A-rearranged leukaemia cell lines simply will not line up, and the fusion protein inside them stays at a constant level throughout division while the normal version rises and falls twice.
The same thing, for a scientist
This is a cell state that has been sitting unnamed in one figure of a 2007 paper for nearly twenty years, and it constrains the design of every kinetic experiment in this disease. Liu, Cheng and Hsieh established that wild-type MLL is degraded biphasically through the cell cycle — by SCF-Skp2 at S phase and APC-Cdc20 at late M — giving two protein peaks, at G1/S and G2/M [E1]. When they turned to leukaemia lines they reported, in one sentence of Results and one of Discussion, that they **"were unable to use mimosine, double thymidine, or nocodazole to synchronize available MLL leukemia cells, including MLL-AF4-bearing SEM and RS(4;11) and MLL-AF9-bearing THP-1, NOMO-1, and MOLM13 cell lines"**, and read this as **"inherent cell cycle defects"** and **"global defects in cell cycle checkpoints"** [E1]. They recovered phase-separated populations by **centrifugal elutriation**, a physical rather than chemical method, and found that **MLL-AF4 protein did not fluctuate through the cell cycle** in SEM while its level was not markedly increased [E1]. The same group later supplied a mechanism: MLL is an S-phase checkpoint effector phosphorylated at S516 by ATR, and **MLL fusions act as dominant negatives that abrogate ATR-mediated stabilisation of wild-type MLL**, producing radioresistant DNA synthesis [E2]. The state is therefore not an incidental culture quirk; it is a predicted consequence of the fusion. Two things follow. Methodologically, any experiment in this graph that assumes cells can be blocked and released — cell-cycle-resolved occupancy, synchronised degron washout, phase-stratified drug sensitivity — must use elutriation, counterflow or sorting, not a chemical block. Biologically, the constant fusion level across the cycle removes an entire regulatory layer that the wild-type protein has, and is a plainly fusion-attributable measurement made on an **endogenous** protein, which is rare in this literature.
What the paper actually says, in its own words
Two sentences carry the whole finding, and both are easy to miss because neither is in the abstract.
Results, Figure 6B: "Interestingly, unlike commonly utilized tissue culture cell lines such as HeLa, 293T, and U2OS cells, we were unable to use mimosine, double thymidine, or nocodazole to synchronize available MLL leukemia cells, including MLL-AF4-bearing SEM and RS(4;11) and MLL-AF9-bearing THP-1, NOMO-1, and MOLM13 cell lines. This surprising observation suggests inherent cell cycle defects of these MLL leukemia cells." 1
Discussion: "Most interestingly, we noticed that all of the five tested human MLL leukemia cell lines failed to be synchronized by drugs like mimosine, thymidine, and nocodazole, implicating global defects in cell cycle checkpoints." 1
The three agents block at three different places by three different chemistries — mimosine arrests in late G1, double thymidine at the G1/S boundary by nucleotide-pool imbalance, nocodazole in prometaphase by depolymerising microtubules. Failure of all three is not a single broken checkpoint; it is the observation the authors themselves call global.
What worked instead. Centrifugal elutriation separates cells by size and sedimentation velocity — physics, not signalling — and it did produce clean phase fractions confirmed by PI/FACS. So these cells do traverse the cycle and can be fractionated. What they will not do is stop where a drug tells them to and resume together.
The fusion does not oscillate, and the way that was measured is itself notable
In SEM, "we witnessed a loss of the unique biphasic expression of MLL-AF4... In contrast to the biphasic expression of wild-type MLL, MLL-AF4 protein did not fluctuate through the cell cycle while its level was not markedly increased" 1.
The assay deserves a sentence of its own, because this project's fusion-attribution audit found that almost nothing in this literature measures the fusion rather than wild-type MLL. Here, elutriated fractions were immunoprecipitated with an anti-AF4 antibody recognising the AF4 C terminus and the immunoprecipitate was blotted with an anti-MLL antibody recognising MLL amino acids 720–780 (Bethyl) 1. Both epitopes are retained in KMT2A::AFF1 — the breakpoint cluster region lies well C-terminal to residue 780, and AFF1's C terminus is the fusion's 3′ contribution. A molecule carrying both epitopes is the fusion.
The honest caveat is that IP-western detects complexes as well as molecules, and anti-AF4 will pull down AEP complexes that can contain wild-type MLL. What separates them here is the gel: the fusion runs at a distinct size. That is the same conclusion this project's attribution audit reached by a different route — the working attribution reagent in this literature is the size dimension, not the antibody.
Level versus activity. The measurement is of protein abundance. It says nothing about whether fusion chromatin occupancy, complex composition or activity vary through the cycle. NARROWED 2026-09-20: nobody has measured those FOR THE FUSION — but they have been measured for wild-type MLL1. PMID 20064463 reports that MLL occupies promoters on condensed mitotic chromosomes with a globally rearranged pattern, tethers menin, RbBP5 and ASH2L there, and accelerates transcriptional reactivation after mitotic exit while being dispensable for preserving H3K4 methylation. HeLa, wild-type protein, 2009. See [[mechanism-mll1-bookmarks-mitotic-chromatin-and-carries-menin-there]].
The mechanism the same group published three years later
In 2010 the Hsieh group placed MLL in the S-phase checkpoint network: ATR phosphorylates MLL at serine 516 under genotoxic stress in S phase, which disrupts its interaction with SCF-Skp2 and so blocks its degradation; stabilised MLL accumulates on chromatin, methylates H3K4 at late replication origins and inhibits CDC45 loading to delay replication 2. MLL-deficient cells show radioresistant DNA synthesis and chromatid-type abnormalities. Critically for this entry, the paper states that MLL fusions function as dominant-negative mutants that abrogate the ATR-mediated phosphorylation and stabilisation of wild-type MLL, and that Mll-CBP knock-in myeloid progenitors show a severe radioresistant DNA synthesis phenotype 2.
That is a mechanism for exactly the phenotype reported in 2007, published by the same laboratory, and the two are almost never cited together as the pair they are. It also predicts that the defect should be present in fusions other than MLL-AF4 and MLL-AF9 — the 2010 evidence is from MLL-CBP — which makes it a partner-independent property, unusual in this graph.
What this forbids, and what it makes possible
Forbidden, or at least unsound without adaptation:
- Any block-and-release protocol in SEM, RS4;11, THP-1, NOMO-1 or MOLM-13 — the five lines named. A published-looking synchronisation in one of these lines should be read with the efficiency figure in hand.
- Cell-cycle-resolved CUT&RUN or ChIP that uses a thymidine or nocodazole block to generate its phases.
- Degron washout or pulse-chase kinetics that assume a synchronised population.
Permitted: centrifugal elutriation (demonstrated in this very paper), counterflow centrifugation, FACS on Hoechst/Pyronin Y or on a FUCCI reporter, and single-cell methods that infer phase computationally rather than imposing it.
This matters directly to exp-cell-cycle-sorted-menin-sensitivity, which this graph proposes as a one-experiment test of whether menin-inhibitor sensitivity tracks cell-cycle state. That experiment is still doable — but it must be a sort, not a block, and the entry proposing it should say so.
Correction to the premise this entry was dispatched with
The dispatch brief stated that the paper says MLL-AF4 protein does not fluctuate through the cell cycle while wild-type MLL does, and that the five lines cannot be synchronised. Both are correct and are quoted verbatim above.
One qualification the brief did not carry: the wild-type biphasic pattern was established in HeLa, 293T and U2OS — none of them haematopoietic and none of them leukaemic. The comparison in Figure 6B is therefore between MLL-AF4 in SEM and wild-type MLL in an epithelial or osteosarcoma line, not between the fusion and the wild-type allele in the same cell. The 2007 paper does not show a side-by-side of fusion and wild-type MLL through the cycle within SEM, which would be the clean experiment and is still unpublished.
| state definition | A population-level state in which cells traverse the cell cycle but cannot be arrested at a defined phase by standard chemical synchronisation agents, and in which the fusion oncoprotein's abundance is constant across phases rather than oscillating as wild-type KMT2A does |
|---|---|
| fusion detection assay | anti-AF4 (C-terminal epitope) immunoprecipitation followed by anti-MLL (amino acids 720-780, Bethyl) immunoblot on elutriated fractions |
| mechanistic link | ATR phosphorylates MLL at S516 in S phase, blocking SCF-Skp2-mediated degradation; MLL fusions act as dominant negatives on this step (PMID 20818375) |
| monocytic confound | three of the five refractory lines (THP-1, NOMO-1, MOLM-13) are monocytic AML lines, which this project has separately flagged as a lineage confound in its DepMap work; the B-ALL arm is only SEM and RS4;11 |
lines reported refractory
[
{
"line": "SEM",
"fusion": "KMT2A::AFF1",
"lineage": "B-ALL"
},
{
"line": "RS4;11",
"fusion": "KMT2A::AFF1",
"lineage": "B-ALL"
},
{
"line": "THP-1",
"fusion": "KMT2A::MLLT3",
"lineage": "AML (monocytic)"
},
{
"line": "NOMO-1",
"fusion": "KMT2A::MLLT3",
"lineage": "AML (monocytic)"
},
{
"line": "MOLM-13",
"fusion": "KMT2A::MLLT3",
"lineage": "AML (monocytic)"
}
]agents that failed
[ "mimosine (late G1)", "double thymidine (G1/S)", "nocodazole (prometaphase)" ]
methods that worked
[ "centrifugal elutriation (size/sedimentation velocity)" ]
methods not tried in the paper
[ "FACS on Hoechst/Pyronin Y", "FUCCI reporter", "computational phase inference from single-cell RNA" ]
wild type reference lines
[ "HeLa", "HEK293T", "U2OS" ]
wild type mll degradation
{
"s_phase": "SCF-Skp2",
"late_m_phase": "APC-Cdc20",
"peaks": [
"G1/S transition",
"G2/M transition"
]
}atlas refs
[
{
"label": "Five MLL leukaemia lines refractory to mimosine, thymidine and nocodazole; MLL-AF4 constant through the cycle",
"pmid": "17908926",
"title": "Bimodal degradation of MLL by SCFSkp2 and APCCdc20 assures cell cycle execution: a critical regulatory circuit lost in leukemogenic MLL fusions.",
"authors": "Liu H, Cheng EH, Hsieh JJ",
"venue": "Genes Dev",
"year": 2007,
"title_provenance": "Title/authors/venue backfilled from PubMed esummary on 2026-09-20 because this citation stored a bare PMID with no title, which made it unscoreable by verify-citations.py. BACKFILLING IS NOT VERIFICATION: it confirms the PMID resolves to a real paper, not that this is the right paper for the claim. That still requires reading."
},
{
"label": "MLL as S-phase checkpoint effector; fusions dominant-negative on ATR-S516 stabilisation",
"pmid": "20818375",
"title": "Phosphorylation of MLL by ATR is required for execution of mammalian S-phase checkpoint.",
"authors": "Liu H, Takeda S, Kumar R, et al.",
"venue": "Nature",
"year": 2010,
"title_provenance": "Title/authors/venue backfilled from PubMed esummary on 2026-09-20 because this citation stored a bare PMID with no title, which made it unscoreable by verify-citations.py. BACKFILLING IS NOT VERIFICATION: it confirms the PMID resolves to a real paper, not that this is the right paper for the claim. That still requires reading."
}
]- Every phase-stratified experiment proposed in this graph, including exp-cell-cycle-sorted-menin-sensitivity, must be redesigned around sorting or elutriation rather than a chemical block, or it will report an artefact of incomplete synchronisation as a biological difference.
- If the S-phase checkpoint is genuinely compromised by the fusion, combining a menin inhibitor with a genotoxic agent could be worse than either alone: a cell that cannot pause to repair will either die or carry the damage forward, and the second outcome is how therapy-related second leukaemias arise in exactly this gene.
- A constant fusion level across the cycle removes the only natural window in which fusion abundance would be low. Any dosing strategy that hopes to catch the oncoprotein at a trough has no trough to catch.
- Three of the five lines named are monocytic, so a reader who takes 'KMT2A-rearranged cells cannot be synchronised' as a general fact may be importing a monocytic-lineage property into infant B-ALL, where the evidence is two lines and no quantification.
- Is the synchronisation failure reproducible, and with what efficiency? The claim has never been quantified. Running a thymidine block on SEM and MOLM-13 with a matched KMT2A-wild-type B-ALL and monocytic line, and reporting percent-in-phase rather than a narrative, would settle a twenty-year-old 'data not shown'.
- Is it the fusion or the line? Three of the five refractory lines are monocytic AML lines, and monocytic identity is a known confound in this project's own dependency work. An isogenic test — inducible KMT2A::AFF1 in a synchronisable B-progenitor line — would separate the lesion from the culture.
- Does fusion OCCUPANCY vary through the cycle even though fusion ABUNDANCE does not? Elutriated-fraction CUT&RUN with an MLL-N antibody, or better with an endogenous C-terminal tag, has never been done and would be the first cell-cycle-resolved chromatin measurement in this disease.
- Does wild-type KMT2A retain its biphasic oscillation in the same cells in which the fusion is flat? The 2007 comparison is across cell types; the within-cell comparison, which is the one that matters, is unpublished.
- If the S-phase checkpoint really is dominant-negatively suppressed, why is KMT2A-rearranged infant ALL among the most mutationally silent cancers measured? Either the defect is absent in patients or it is silent in consequence, and the two have different implications for combining a menin inhibitor with a genotoxic agent.
- E1moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper
Five human MLL leukaemia cell lines — MLL-AF4-bearing SEM and RS(4;11) and MLL-AF9-bearing THP-1, NOMO-1 and MOLM13 — could not be synchronised with mimosine, double thymidine or nocodazole, which the authors interpret as inherent cell cycle defects and global defects in cell cycle checkpoints; phases were instead separated by centrifugal elutriation, and in SEM the endogenous MLL-AF4 protein did not fluctuate through the cell cycle, in contrast to the biphasic expression of wild-type MLL that is degraded by SCF-Skp2 and APC-Cdc20.
Bimodal degradation of MLL by SCFSkp2 and APCCdc20 assures cell cycle execution: a critical regulatory circuit lost in leukemogenic MLL fusions. · Liu H, Cheng EH, Hsieh JJ · Genes Dev 2007What this evidence cannot show
Read in full via the PMC HTML route (PMC1993870, 91,692 characters of stripped text) after Europe PMC fullTextXML returned HTTP 500. The synchronisation failure is reported as a narrative observation with no figure, no efficiency number and no supplementary data — 'data not shown' in the strict sense — so it cannot be quantified from the paper. The five lines are established immortalised cultures, not primary blasts, and cell lines acquire cycle abnormalities for reasons unrelated to their founding lesion. The wild-type biphasic comparator is HeLa, 293T and U2OS, none haematopoietic, so the fusion-versus-wild-type contrast is across cell types rather than within one. The MLL-AF4 readout is anti-AF4 (C-terminal) IP followed by anti-MLL (aa 720-780) blot, which is fusion-selective by epitope pair but is an IP-western, so a co-precipitated species is discriminated by size rather than by the antibodies alone. n = 1 line for the fusion-level measurement.
Evidence that argues against this (2)
- The 'global checkpoint defect' reading is directly contradicted by a targeted test. CRISPR/Cas9 knock-in of MLL-AF4 or of the reciprocal AF4-MLL at the AAVS1 locus of HEK293 cells gave repair kinetics for etoposide- and irradiation-induced double-strand breaks identical to wild type by flow cytometry, gamma-H2AX immunoblot and comet assay; NHEJ, alt-NHEJ and homologous recombination protein levels and functional efficiencies were unchanged; and critically, 'treatment with etoposide consistently induced cell cycle arrest in S/G2/M independent of MA4/A4M expression, revealing a proper activation of the DNA damage checkpoints'. If the fusion were sufficient to break checkpoints globally, this isogenic system should have shown it. The obvious reconciliation is that HEK293 is not a haematopoietic cell and an AAVS1 transgene is not the endogenous allele in its own chromatin, but the result means the 2007 interpretation is an inference from a culture observation and has never been tested where it was made. Read: abstract only. Castano J et al. 2016
- NARROWS THIS ENTRY'S ABSENCE CLAIM. Chromatin occupancy and complex composition through the cell cycle HAVE been measured for wild-type MLL1: it occupies promoters on condensed mitotic chromosomes in a globally rearranged pattern and tethers menin, RbBP5 and ASH2L there, while being dispensable for preserving H3K4 methylation. That is HeLa and wild-type protein rather than a fusion or a leukaemia cell, so the fusion-specific absence survives — but the unqualified 'nobody has measured those' did not. Blobel GA et al. 2009
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
MLL is an effector of the mammalian S-phase checkpoint: ATR phosphorylates MLL at serine 516 in response to genotoxic stress in S phase, disrupting its interaction with and degradation by SCF-Skp2, and the stabilised protein accumulates on chromatin, methylates H3K4 at late replication origins and inhibits CDC45 loading to delay replication; MLL-deficient cells show radioresistant DNA synthesis and chromatid-type genomic abnormalities, and MLL fusions function as dominant-negative mutants that abrogate ATR-mediated phosphorylation and stabilisation of wild-type MLL, with Mll-CBP knock-in murine myeloid progenitors showing a severe radioresistant DNA synthesis phenotype.
Phosphorylation of MLL by ATR is required for execution of mammalian S-phase checkpoint. · Liu H, Takeda S, Kumar R, Westergard TD, Brown EJ, Pandita TK, et al., Hsieh JJ · Nature 2010What this evidence cannot show
Same laboratory as E1, so the mechanism and the phenotype are not independent observations. The fusion evidence is an Mll-CBP knock-in, modelling t(11;16), not KMT2A::AFF1 or KMT2A::MLLT3; the dominant-negative claim for MLL fusions generally is an extrapolation from that allele plus biochemistry. Radioresistant DNA synthesis is a specific S-phase checkpoint assay and does not itself establish that G1 or mitotic checkpoints are defective, which is what would be needed to explain failure of mimosine and nocodazole blocks. Read: abstract only.
Evidence that argues against this (1)
- A dominant-negative S-phase checkpoint defect predicts genomic instability, and KMT2A-rearranged infant ALL is the least mutated leukaemia measured: the same group that tested repair kinetics notes that 'the genome-wide mutational landscape in MLL-rearranged infant B-ALL has been reported silent'. A cell whose S-phase checkpoint is broken from the founding event should accumulate damage; these cells characteristically do not. That does not refute the biochemistry, but it does mean the checkpoint defect, if real in patients, has no detectable mutational consequence, which weakens any argument that it contributes causally to the disease rather than being a property of the cultured lines. Read: abstract only. Castano J et al. 2016
- E3moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper
Conditional graded expression of MLL-AF4 in the U937 myelomonocytic line produced a dosage-dependent decrease in growth rate and in the fraction of S-phase cells with accumulation in G1 and prolonged doubling time, paralleled by increased CD11b expression; t(4;11) leukaemic cells have prolonged doubling time and a lower percentage of cells in cycle than non-t(4;11) B-lineage lines, and the authors propose that this cell-cycle control underlies resistance to etoposide-mediated cytotoxicity and to death-promoting stimuli.
Modulation of cell cycle by graded expression of MLL-AF4 fusion oncoprotein. · Caslini C, Serna A, Rossi V, Introna M, Biondi A · Leukemia 2004What this evidence cannot show
Tetracycline-regulated overexpression of MLL-AF4 in U937, a myelomonocytic line with no KMT2A rearrangement of its own — an ectopic construct in a non-index cell, which is the construction this project's fusion-attribution audit rates weakest. High-level ectopic MLL fusion expression is independently known to arrest cells and induce apoptosis, so a dose-dependent G1 accumulation may be a transgene artefact rather than a property of the disease. The comparison of doubling times between t(4;11) and non-t(4;11) lines is uncontrolled for lineage and for time in culture. Read: abstract only.
Evidence that argues against this (1)
- This result and E1 are in tension about what kind of cycle abnormality is present. Caslini describes accumulation in G1 with a reduced S fraction, i.e. a cell that stalls at a recognisable position; Liu describes a cell that cannot be stalled at any position by any of three agents. A population that is intrinsically G1-skewed should be, if anything, easier to arrest in G1 with mimosine, not harder. Either the two are describing different cells — an ectopic construct in U937 versus endogenous fusion in SEM/THP-1 — or one of the two phenotypes is a property of the system rather than of the lesion. No published study has measured cycle distribution and synchronisability in the same cells. Read: abstract only. Liu H et al. 2007
SEM and RS4;11 could not be synchronised by mimosine, double thymidine or nocodazole, and endogenous MLL-AF4 protein did not fluctuate across elutriated cell-cycle fractions
THP-1, NOMO-1 and MOLM-13 named in the same sentence as refractory to all three synchronisation agents; note all three are monocytic, a lineage confound this project tracks separately
- ← regulatesKMT2A / MLL1 — the gene that breaks
wild-type KMT2A is an ATR-S516 S-phase checkpoint effector degraded biphasically by SCF-Skp2 and APC-Cdc20; fusions act as dominant negatives on that step
constrains the design: the proposed phase-stratified sensitivity assay must sort or elutriate, because a chemical block does not work in these lines
the hypothesis is testable but not by block-and-release; this entry names the method that did work (centrifugal elutriation)
- related to →The G1 state in KMT2A-rearranged leukaemia — where CDK6 does a job that is not about the cell cycle at all
the same cycle, two different descriptions: a CDK6-dependent G1 restriction point that is pharmacologically addressable, and a population that cannot be held at any phase chemically
the 2007 fusion measurement is anti-AF4-C-terminal IP blotted with anti-MLL aa 720-780 and discriminated by gel size — another instance of the size dimension doing the attribution work
three of the five refractory lines are monocytic AML lines, so the panel-level monocytic confound applies to this observation too
distinct states that are easy to conflate: quiescence is exit from the cycle, this is inability to be halted within it
the persister cells are smaller, and centrifugal elutriation separates on size and is the one method demonstrated to work in KMT2A-rearranged cells — an untried antibody-free enrichment route
step zero re-tests whether the five lines can be synchronised at all; arm A then measures phase-resolved fusion and menin occupancy by elutriation
records the four-route read of PMID 17908926 (fullTextXML HTTP 500, PMC HTML 91,692 stripped characters) that this entry rests on
NARROWS ITS ABSENCE CLAIM: occupancy and complex composition through the cycle HAVE been measured for wild-type MLL1, though not for the fusion.
the fusion's cell-cycle phenotype, against which this wild-type set-point is the comparator
the other time-domain anomaly of KMT2A-rearranged cells recorded in this graph
the mitotic-entry timer is set from the CENTROSOME: interphase centrosome-associated CHK1 prevents premature cyclin-B-CDK1 activation
SMC3 has four distributions within one cell cycle, so any bulk measurement in an asynchronous blast population averages them - the same problem this cell-state entry records (Q91 2026-09-22)
SBDS occupies four positions in one cell cycle - nucleolus in G1/G2, diffuse nuclear in S, mitotic spindle, MTOC in interphase - so a bulk measurement in an asynchronous culture averages them (Q91 2026-09-22)
KAT2A's centrosomal pool is phase-restricted to late G1 and G1/S (PMID 27796307), so any bulk immunofluorescence in an asynchronous blast population averages a nuclear majority with a phase-limited cytoplasmic minority - the measurement problem this node records (Q93 2026-09-21)
NASP is a CELL-CYCLE-REGULATED protein and tNASP overexpression alters cycle progression (PMIDs 10893414, 12509435), so the soluble H3-H4 reservoir this entry treats as a rate term oscillates - and a bulk measurement in an asynchronous blast population averages it (Q93 2026-09-21)
The EP400 pathway has a phase requirement nobody has connected to this disease: H2A.Z delivered by VPS72/YL1 is needed for NUCLEAR REASSEMBLY AFTER MITOSIS, and VPS72 knockdown extends telophase in HeLa (PMID 32708675). VPS72 serves both SRCAP and EP400, so the requirement is not assignable to EP400 (Q93 2026-09-21)