DOT1L — the target where excellent chemistry met an indirect interaction

This is a prediction, not a measurement. No one has seen this protein in this shape. The colours say how much the software trusts itself, and that is worth reading before anything else — the orange stretches are places the model is effectively saying this part has no fixed shape.

• Indirect recruitment means buffering: DOT1L is reached through an AHD that four proteins compete for, so removing DOT1L may simply let another AHD ligand take its place.
• Delivery: the preclinical regimen that produced sustained regressions was continuous intravenous infusion. For an infant population that is a serious practical barrier independent of target biology.
• Partial response breeds resistance: because the recruitment-to-output relationship is graded rather than switch-like, achievable inhibition gives partial suppression of the leukemic programme, which is the selection pressure under which resistance arises.
• H3K79 methylation has roles outside the fusion programme, including in DNA damage response; combining a DOT1L inhibitor with genotoxic chemotherapy is not obviously safe.
• Compensation through the second axis: KMT2A fusions also drive their programme through menin-LEDGF chromatin tethering and SEC-mediated elongation, so removing the H3K79 mark leaves the fusion bound and the elongation machinery in place.

1. Why did an 80 pM, 37,000-fold selective, non-common-essential target underdeliver clinically? Separating pharmacokinetics (continuous infusion needed), indirectness of recruitment (through a competitive AHD) and gradedness of the phenotype would tell the menin programme which of its own assumptions to stress-test.
2. Is DOT1L inhibition synergistic with menin inhibition, and is the synergy additive on fusion output or genuinely mechanistic? The gradedness result predicts additivity, and a matrix in SEM and MV4-11 with H3K79me2 and HOXA9 readouts would test it.
3. For KMT2A::AFF1 specifically, which AHD-bearing partner delivers DOT1L - ENL or AF9? The DepMap split between MLLT1 (selectivity -0.132) and MLLT3 (+0.059) says ENL, but nobody has shown that DOT1L recruitment in a KMT2A::AFF1 line runs through ENL rather than AF9.
4. If AFF1, DOT1L, BCOR and CBX8 occupy the AHD mutually exclusively, how does a single fusion complex use more than one of them? Either multiple ENL/AF9 copies are present per complex or the partners exchange dynamically, and the two models predict different drug behaviour.
5. Is the HPA 'not detected in immune cells' call real at the protein level in haematopoietic stem and progenitor cells? If so it materially improves the predicted toxicity profile and nobody has checked it directly.
6. This entry summarises DOT1L recruitment as the AHD of ENL/AF9-family partners or the AF10/MLLT10 coiled coil. Both are correct and neither is present in KMT2A::AFF1, which leaves the index fusion with no stated route to DOT1L. Reported by the Q3b beat as an omission rather than an error - no citation settles it - and the rule it implies is worth holding: AHD binds AF9/ENL, the OM-LZ octamer binds AF10, and KMT2A::AFF1 has neither.

1. E1established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.database

   DOT1L structural coverage includes the catalytic domain with SAM (1NW3 2.5 A, 3QOW 2.1 A), with SAH (3QOX 2.3 A) and with a selective inhibitor (3SR4 2.5 A); active- and poised-state cryo-EM complexes on H2B-ubiquitinated nucleosomes (6NJ9 2.96 A, 6NQA 3.54 A, 6NOG 3.90 A, 7XCR 2.57 A, 7XCT 2.72 A, 6J99 4.1 A, 6O96 3.5 A, 6NN6 3.9 A); the DOT1L 877-900 complex with the AF9/MLLT3 AHD 500-568 (2MV7, NMR); and the DOT1L coiled-coil complex with AF10/MLLT10 699-782 (6JN2, 3.6 A) plus 7EDP (2.2 A).

   RCSB PDB entries 1NW3, 3QOW, 3QOX, 3SR4, 6NJ9, 6NQA, 6NOG, 7XCR, 7XCT, 6J99, 6O96, 6NN6, 2MV7, 6JN2 and 7EDP, with per-entity UniProt accessions, methods, resolutions and modelled residue ranges resolved against the RCSB search and data GraphQL APIs · RCSB Protein Data Bank · RCSB PDB 2026
   ▸What this evidence cannot show

   Identifiers, methods, resolutions and residue ranges were read from the RCSB API in this session rather than recalled, but residue ranges are deposited construct ranges annotated against UniProt, not ordered regions in the maps. None of these structures contains KMT2A or AFF1, which is the point: there is no structure of DOT1L with a KMT2A fusion protein.

   ▸Evidence that argues against this (1)

   • Counting PDB entries measures how tractable a protein was to CRYSTALLISE, which correlates with being small, ordered and well-behaved rather than with being druggable. What decides design value is whether the deposited density covers the site that matters, at a resolution that supports modelling, in the relevant state — and this graph has repeatedly found structures described in the direction that overstates what is resolved. DOT1L's problem was never structural. The compound reached patients, demethylated on target, and produced 2 complete remissions in 51 — so a dossier built on structural coverage is measuring the axis that did not fail. Blood 2018
2. E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

   The degree of DOT1L recruitment to MLL-AF9 determines the level of H3K79 di- and tri-methylation on target genes and the transformation potential, with graded mutants giving graded loss of both; the NMR structure of a DOT1L-AF9 AHD complex identifies three AF9 binding sites on DOT1L.

   Degree of recruitment of DOT1L to MLL-AF9 defines level of H3K79 di- and tri-methylation on target genes and transformation potential · Kuntimaddi A, Achille NJ, Thorpe J, et al. · Cell Reports 2015
   PMID 25921540DOI
   ▸What this evidence cannot show

   The system is MLL-AF9, not KMT2A::AFF1, and the transformation readout is a murine model. The abstract reports no numeric affinity, so no Kd should be attributed. Graded recruitment by mutation is not identical to graded occupancy by a drug, which also competes with substrate.

   ▸Evidence that argues against this (1)

   • Graded recruitment giving graded methylation and graded transformation is an elegant dose-response and it is measured in MLL-AF9, a murine myeloid model, with engineered mutants rather than pharmacological occupancy. Non-canonical H3K79me2-dependent pathways promote survival of KMT2A-rearranged leukaemia, so removing the mark and removing the requirement are not the same operation. eLife 2021
3. E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

   The AF10/MLLT10 leucine zipper binds the DOT1L coiled-coil domain, and the higher-order configuration of these heterodimeric coiled coils drives tetramerisation into a DOT1L-AF10 octamer that is required for MLL-AF10 leukemogenic activity.

   A higher-order configuration of the heterodimeric DOT1L-AF10 coiled-coil domains potentiates their leukemogenenic activity · Song X, Yang L, Wang M, et al. · PNAS 2019
   PMID 31527241DOI
   ▸What this evidence cannot show

   This is MLL-AF10 biology and does not transfer directly to KMT2A::AFF1, where AF10 is not part of the fusion. The oligomerisation requirement is shown for leukemogenic activity of the AF10 fusion, and whether the same octamer is needed for DOT1L function recruited via ENL or AF9 is not established here. No affinity is reported in the abstract.

   ▸Evidence that argues against this (1)

   • Octamerisation of DOT1L-AF10 is required in MLL-AF10, where AF10 is the partner. KMT2A::AFF1 does not contain AF10, so the tetramerisation requirement is a property of one fusion rather than of DOT1L, and importing it into an AFF1 dossier crosses a partner boundary. Leukemia 2023
4. E4strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

   AFF1/AF4 binds the AF9/MLLT3 AHD (structure 2LM0, AFF1 738-779 with MLLT3 490-568), and binding of AF4-family proteins, DOT1L, BCOR and CBX8 to the AHD is mutually exclusive, so the fusion cannot engage AFF1 and DOT1L on the same AHD simultaneously.

   BCOR binding to MLL-AF9 is essential for leukemia via altered EYA1, SIX, and MYC activity · Schmidt CR, Achille NJ, Kuntimaddi A, et al. · Blood Cancer Discovery 2020
   PMID 32954361DOI
   ▸What this evidence cannot show

   Mutual exclusivity is demonstrated for the isolated AHD with isolated partner peptides; in a cell, multiple ENL and AF9 molecules are present and different copies could hold different partners, so exclusivity at the level of one domain does not mean exclusivity at the level of the complex. The structural work is on MLL-AF9, not KMT2A::AFF1.

   ▸Evidence that argues against this (1)

   • Mutual exclusivity of AFF1, DOT1L, BCOR and CBX8 on the AF9 AHD was established in vitro with isolated domains, where competition reflects the concentrations the experimenter supplied. In a nucleus the same surfaces can be occupied by different partners on different molecules at one locus, so the constraint applies to a single AF9 chain and not to the complex at a gene. Nature 2012
5. E5strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.database

   DOT1L is not DepMap common-essential (mean Chronos -0.148 across 1,258 lines, 27.4% of lines depleted) and falls to -0.565 across 10 KMT2A-rearranged lines and -0.546 across 4 KMT2A::AFF1 lines, giving a lineage-controlled selectivity of -0.166 (rank 9 of 41) against a naive selectivity of -0.417; gnomAD gives pLI 1.0, LOEUF 0.288, 33 observed versus 153.3 expected loss-of-function variants; HPA classifies DOT1L as tissue enhanced, detected in all tissues, and not detected in immune cells.

   DepMap CRISPR (Chronos) gene effect via the Open Targets Platform GraphQL API; loss-of-function constraint via the gnomAD GraphQL API; normal-tissue expression via the Human Protein Atlas API · Broad Institute DepMap; gnomAD; Human Protein Atlas; Open Targets Platform · Databases 2026
   ▸What this evidence cannot show

   Means were computed by us from per-line API values, not read from a DepMap release table, and the release version is not stated by the API. Only 10 KMT2A-rearranged lines (4 KMT2A::AFF1) contribute. The HPA 'not detected in immune cells' classification is an RNA-level blood-cell call and should not be read as absence of DOT1L protein from haematopoietic progenitors, which is where toxicity would arise.

   ▸Evidence that argues against this (1)

   • Cell-line gene effect cannot see the in vivo requirement. The in vivo statement that MLL-rearranged leukemia is Dot1l-dependent, with little global effect of Dot1l inactivation outside direct target loci, comes from mouse genetics rather than from DepMap. Bernt KM et al. 2011
6. E6strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

   Pinometostat (EPZ-5676) inhibits DOT1L histone methyltransferase activity with a measured inhibition constant Ki of 80 pM and 37,000-fold selectivity over all other methyltransferases tested, and continuous intravenous infusion produced complete, sustained tumour regressions in a rat KMT2A-rearranged xenograft.

   Potent inhibition of DOT1L as treatment of MLL-fusion leukemia · Daigle SR, Olhava EJ, Therkelsen CA, et al. · Blood 2013
   PMID 23801631DOI
   ▸What this evidence cannot show

   This is the only numeric affinity we verified anywhere in this beat, and it is a biochemical methyltransferase assay, not a cellular or clinical potency. The requirement for continuous intravenous infusion to achieve sustained regressions is itself the important limitation: a target needing continuous infusion is a different clinical proposition from one addressable orally, independent of molecular quality.

   ▸Evidence that argues against this (1)

   • An 80 pM Ki with 37,000-fold selectivity is the strongest chemistry in this entire interactome and it is the clearest demonstration that potency and selectivity are not the limiting variables. The same compound's trial did not reach its maximum tolerated dose, so its modest activity is consistent with inadequate exposure as well as with a non-limiting pathway. Blood 2018
7. E7strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.database

   ChEMBL v34 target CHEMBL1795117 (DOT1L) carries 193 activity rows at pChEMBL >= 6 with a best Ki of 0.46 nM (CHEMBL2171169), dominated by one SAM/SAH-mimetic series, and carries no curated mechanism-of-action record for pinometostat.

   ChEMBL database version 34, target CHEMBL1795117 (histone-lysine N-methyltransferase, H3 lysine-79 specific): target_search, get_bioactivity and get_mechanism queries · EMBL-EBI ChEMBL · ChEMBL 2026
   ▸What this evidence cannot show

   The absence of a pinometostat mechanism record is a curation gap, not a biological statement; mechanism records in ChEMBL frequently sit on family or protein-complex targets rather than single-protein ones. The 193 rows and the 'best Ki' are from a page-local sample of 25 rows returned in activity-id order, so the best value is the best in a sampled page dominated by one publication, not a potency-ranked view of the whole target.

   ▸Evidence that argues against this (1)

   • A ChEMBL activity-row count measures how much medicinal chemistry has been DONE, not how tractable the target is, and the best reported affinity belongs to the most optimised compound in whichever series someone chose to pursue. Zero rows means nobody tried; thousands of rows means a target had a decade of industrial attention. 193 rows dominated by one SAM/SAH-mimetic series is a single chemical idea counted many times, which is the failure mode a row count is most prone to. ChEMBL, EMBL-EBI 2026

• ← related toExperiment: map what stays on chromatin when the menin inhibitor has done everything it can do

  competing mechanism to include as an arm: combined DOT1L and menin inhibition releases MLL::AF9 from chromatin globally whereas single agents have modest effects on occupancy

• ← related toMLLT10 / AF10 — domain architecture and residue coordinates

  Counter-evidence bearing on DOT1L-AF10 druggability: the functional unit is not the 1:1 heterodimer. PDB 6JN2 shows the AF10 leucine zipper plus OM domain driving tetramerisation of the binary complex into a DOT1L-AF10 OCTAMER, and that oligomerisation - not the heterodimer interface alone - is essential for MLL1-AF10 leukaemogenesis (Song 2019, PMID 31527241). Interface mutants are ambiguous between the two.

• ← bindsMLLT10 / AF10 — domain architecture and residue coordinates

  AF10 OM-LZ 704-779 binds the DOT1L coiled-coil domain (PDB 6CKN, 6CKO)

• ← depends onPICALM::MLLT10 (CALM-AF10), t(10;11)(p13;q14) - the DOT1L-dependent HOXA fusion that contains no KMT2A

  Dot1l knockout abolishes initiation and maintenance in mice

• ← supportsMEN1, DOT1L and LEDGF dependency in the only CALM-AF10 cell line DepMap has screened

  DOT1L dependency preserved at the KMT2A-rearranged level in a KMT2A-wild-type line

• ← recruitsCALM-AF10 reaches DOT1L through exactly the same OM-LZ interface as KMT2A::MLLT10 - the recruitment transfers, the addressing does not

  AF10 OM-LZ binds the DOT1L coiled coil; same interface in KMT2A::MLLT10

• ← regulatesCALM-AF10 strips H3K79 methylation from the rest of the genome while adding it at HOXA - one fusion, two opposite effects on the same mark

  sequesters DOT1L away from bulk chromatin

• ← recruitsSET::NUP214 is the second KMT2A-free HOXA fusion, and it converges on the same two proteins CALM-AF10 uses: CRM1 and DOT1L - and so does NPM1c, which IS menin-dependent (corrected 2026-09-21)

  SET-NUP214 interacts with DOT1L at HOXA promoters; interface unmapped

• ← depends onThe monocytic confound is worse than recorded: two of the six rearranged lines are one patient, and the four-line control group contains a HOXA-driven fusion and two NPM1c/myelomonocytic lines

  CALM-AF10 transformation is DOT1L-dependent, as is MLL-AF10

• ← related toAF10/MLLT10 fusions drive aberrant self-renewal of non-stem progenitors — found only by opening the paper

  the MOZ/ENL complex recruits the AF10 fusion/DOT1L complex

• ← related toHypothesis (first clause REFUTED 2026-09-21): the fusion's core dependencies may be cell-type-invariant, but the cell type itself is not fixed

  DOT1L moves -0.143 across the same-fusion cell-type contrast

• regulates →The differentiation block — what it actually is, operationally, in KMT2A::AFF1 leukaemia

  in murine MLL-AF9 leukaemia, Dot1l loss releases the differentiation block and downregulates the direct fusion target programme

• regulates →The stem-like self-renewal state — a limited stem-cell module grafted onto a committed cell

  DOT1L inhibitors block abnormal self-renewal induced by cohesin loss in murine HSPCs - not confined to the KMT2A fusion context

• related to →Granulocyte-monocyte progenitor (GMP) — the committed cell an MLL fusion can turn back into a stem cell

  required in KMT2A-rearranged lines of both lineages (-0.559 myeloid, -0.683 B-lineage); no lineage restriction

• ← related toExport or docking? A locus-resolved H3K79me2 ChIP under an XPO1 inhibitor separates the two CALM-AF10 models, and it has never been run

  DOT1L occupancy is a parallel readout because the dissenting model routes through it

• ← bindsSET / TAF-I beta - a histone chaperone that binds the retained N-terminus of MLL, co-activates HOXA with it, and is itself a HOXA-activating fusion partner that recruits DOT1L

  SET-NUP214 interacts with DOT1L and CRM1 at HOXA promoters

• part of →The clearance clock has machinery, and the machinery says the clock does NOT stop in a non-dividing cell - a postmitotic heart replaces its histones with a half-life of about two weeks

  yeast Dot1p has intrinsic histone chaperone activity at long transcribed genes

• ← related toThe 3.7% was mostly the query: DOT1L recall is 19.6% once you ask about leukaemia

  measures how much of the recent DOT1L literature this graph has read

• causes →H3K79me1 - the first methyl group, the one H2B ubiquitination is not needed for, and the state HOX loci fall back to as blood cells mature

  sole mammalian H3K79 methyltransferase; deposits the mono-methyl state

• causes →H3K79me2 - the di-methyl state the leukaemia actually runs on, and the one menin turns out to govern only half of

  sole H3K79 methyltransferase; deposits the di-methyl state at fusion targets

• causes →H3K79me3 - the third methyl group, the one that needs the most enzyme flux and the one human DOT1L makes least of

  terminal state of a distributive methylation reaction

• part of →DOT1L methylates H3K79 non-processively - so the three states are one distribution, not three signals

  the enzyme whose kinetics this entry describes

• part of →DOT1L does things that have nothing to do with methylation - which is why blocking its active site was never going to remove all of it

  functions of the protein that survive catalytic inhibition

• ← inhibitsDOT1L808 - a 5 nM DOT1L degrader that cleared an orthotopic leukaemia and still works after menin inhibitors fail

  PROTAC degradation, DC50 5 nM

• ← inhibitsMS2133 - the first molecule to degrade DOT1L rather than inhibit it

  first-in-class VHL-recruiting DOT1L PROTAC

• ← inhibitsBlocking where DOT1L binds instead of what it does - the modality that removes the mark only at fusion targets

  blocks recruitment rather than catalysis

• ← recruitsThe DOT1L-AF10 octamer - the second route to H3K79 methylation, required in MLL-AF10 and absent from KMT2A::AFF1

  AF10 LZ binds the DOT1L coiled coils; the complex tetramerises into an octamer

• causes →Normal blood needs DOT1L too - lose it in an adult mouse and the marrow fails within weeks

  adult marrow failure within weeks of Dot1l inactivation

• ← supportsEvery DOT1L dependency number this project has computed, in one place, lineage-split and in raw Chronos units

  lineage-split raw Chronos synthesis: -0.310 lymphoid, -0.186 myeloid, rank 61/8269 on effect

• ← regulatesHow much sugar the cell has decides how much DOT1L there is - a metabolic dial on the leukaemia's mark

  O-GlcNAc at S1511 blocks UBE3C and stabilises the protein

• ← bindsA long non-coding RNA binds DOT1L's catalytic domain and turns its activity up - and blocking it makes the fusion protein itself get eaten

  lncRNA binds the lysine-rich region of the DOT1L catalytic domain and raises activity

• ← regulatesH2BK120ub (H2Bub1) — the ubiquitin tag on histone H2B that licenses H3K79 methylation, and the mark this graph was missing

  direct intranucleosomal stimulation of DOT1L catalysis

• ← regulatesH2BK34ub — a second ubiquitination site on H2B that also stimulates DOT1L, written by MSL1/MSL2, and important at HOXA9 and MEIS1

  positions Dot1L for the productive conformation by nucleosome distortion

• ← part ofThe DOT1L–ubiquitin and DOT1L–acidic-patch interfaces: two non-catalytic surfaces that MV4;11 cells cannot do without

  two non-catalytic surfaces of DOT1L: the ubiquitin contact and the acidic-patch contact

• regulates →PROM1 / CD133 - a direct MLL-AF4 target switched on by an intragenic H3K79me2/3 enhancer, and the one surface target in this disease with a published human-relevant kill

  H3K79me2/3 at the intragenic PROM1 enhancer is the activating mark, making surface CD133 a candidate DOT1L pharmacodynamic readout

• ← depends onBCL2 — the one co-dependency in this beat that already has an approved drug pointed at it

  BCL2 upregulation in t(4;11) requires DOT1L-mediated H3K79 methylation

• ← part ofMLLT6 (AF17) — the fourth KMT2A fusion partner inside DotCom, and the only one no cell needs

  AF17 is a named DotCom subunit

• ← part ofCTNNB1 (beta-catenin) — the Wnt link inside DotCom, and the one piece of KMT2A biology that is not chromatin

  beta-catenin co-purifies with DOT1L in DotCom; Dot1 is required for a subset of Wingless targets in flies

• ← part ofTRRAP — a 3,859-residue DotCom subunit that every cell needs and no KMT2A cell needs more

  TRRAP co-purifies with DOT1L in DotCom

• ← depends onRBM15B — the bridge from the DOT1L mark to the RNA layer: it reads H3K79me2 and steers m6A to start codons

  H3K79me2, the DOT1L mark, guides RBM15B-directed m6A deposition at start codons

• ← related toTwo-thirds of this graph's core-complex interaction evidence comes from experiments where our protein was not the bait, and LEDGF has one bait-supported partner out of thirty-six

  THE HEALTHIEST OF THE SIX at 14 of 26 bait-supported, which fits a protein with a drug programme behind it.

• ← related toRunning the HOXA programme is not enough to need menin

  DOT1L dependency is retained in the menin-independent programme line, which is what makes the dissociation interpretable.

• ← bindsDAXX - the strongest of five selective dependencies among 254 unwritten interactors but it FAILS Bonferroni correction (p=0.0128, rank 4 of 254), and not one paper connects it to this disease

  one of four baits recovering DAXX, all on one platform

• proposed by →Kathrin Bernt (University of Pennsylvania / Children's Hospital of Philadelphia) — DOT1L and H3K79 methylation in KMT2A-rearranged leukemia

  established the H3K79 methylation dependency

• ← inhibitsPinometostat (EPZ-5676) — the DOT1L inhibitor, and the most instructive failure in this field

  the most instructive failure in the field

• ← testsCo-dependency scan: what else does a menin-dependent cell need? (run, not planned)
• ← supportsDoes the menin co-dependency survive lineage adjustment? (run)

  menin and DOT1L co-depend at r=0.68 within leukemia

• ← related toIs the drug target always in the retained half? An audit of the project's own framing
• ← supportsDOT1L sits in a B-lineage proximity map, and we nearly did not look
• ← related toMeasured against a technical floor of 4-10% from two independent input pairs, PCGF1 loss lowers baseline fusion occupancy at the top-FLAG sites by 16.3% and changes the DOT1L inhibitor's effect on that occupancy by 22.8% - while leaving the menin inhibitor's effect unchanged at 1.3%. In the knockout SGC0946 displaces more fusion than VTP-50469 does, which is the opposite of the control

  SGC0946s effect on fusion occupancy is genotype-dependent by 22.8%, outside the technical floor

• ← related toThe first measurement of any histone mark outside the top 0.1% of fusion sites: by ratio the drug's mark response is confined to the peaks as pre-registered, while in ABSOLUTE share the movement at the decile below is 5 to 33 times larger - though that is partly guaranteed by the stratum being 100x bigger. H3K79me2 falls at the peaks (0.8065) and is not measurably changed at D1, and H3K4me3 and H3K79me2 both break the floor at the lowest decile

  H3K79me2 falls at the peaks and is not measurably changed in the bulk

• ← bindsBRINP1 (DBC1/DBCCR1/FAM5A) - the last bait-supported partner of the six core proteins without an entry, a neural-specific BMP/retinoic-acid-inducible protein pulled down with DOT1L as bait in one quantitative AP-MS experiment and nowhere else

  one IntAct record, DOT1L (Q8TEK3) as bait and BRINP1 isoform 3 (O60477-3) as prey, anti-tag co-IP, interaction type association, from PMID 26496610 alone - a neural-specific protein recovered once in a non-neural tagged-bait screen, which is the canonical shape of a low-stoichiometry co-purifier

• ← supportsMenin dependence varies 2.83x across KMT2A::AFF1 lines and the spread survives at three donors - but RS4;11 is last on KMT2A itself, so the heterogeneity is largely fusion-dependence heterogeneity, and DAXX is the one gene decoupled from it

  DOT1L tracks KMT2A at Spearman +1.00 - the positive control that shows the method can see a fusion-axis relationship at n=4

• binds →KMT2A::AFF1 (MLL-AF4), t(4;11)(q21;q23)

  recruited through the partner side, deposits H3K79 methylation

• ← related toAll 56 core-complex partners that anyone deliberately fished for already have an entry: the unwritten interactome is 100% by-product

  FOURTEEN OF TWENTY-SIX, the best covered, which fits the protein with a drug programme.

• ← related to49 of 91 proteins in this graph have never had a drug-like molecule bound to them

  23 drug-like ligands at 37% coverage, but the count is flattered by SAM-competitive chemistry being easy to crystallise.

• ← related toDOT1L carries 52 in-gene genome-wide-significant SNPs at the 97.8th length-matched percentile, led by height, pulse pressure and blood-pressure traits - a speculative cardiovascular lead for DOT1L-directed drugs

  germline signal at DOT1L

• ← related toDOT1L is widely framed as a transcription-elongation factor, but the one direct test found in erythroleukaemic cells reported a role in initiation - in a non-KMT2A-rearranged line, so it is a counter-lead for the elongation model, not a refutation

  elongation versus initiation role of the DOT1L complex

• ← related toThe histone/ribosomal shape in the read queue is three classes, not one - H2BC21 is distinguishable AND essential at -0.70, so neither existing rejection ground reaches it, and HMGA1 is a harder dependency than MEN1 or KMT2A

  DOT1L -0.147 at 12.1%, the mildest of the three complex anchors in this pull, measured as a within-pull control rather than quoted from another cycle

• ← related toNothing beats menin after all, and the statistic that said otherwise ranks narrowness
• ← related toWe have read a tenth of the recent field, and the gap found a real interactor
• ← related toThomas Milne (MRC WIMM, Oxford) — MLL-AF4 target biology, enhancer architecture and PROM1/CD133

  H3K79me2/3 enhancer element (KEE) controls PROM1/CD133 activation; also co-authored the pinometostat resistance model

• ← related toKathrin Bernt (University of Pennsylvania / Children's Hospital of Philadelphia) — DOT1L and H3K79 methylation in KMT2A-rearranged leukemia

  established the H3K79me2 selectivity of the DOT1L dependency in KMT2A-rearranged leukaemia

• ← related toDOT1L's localization record is a mouse kidney paper wearing a human label - and the one image of it in a leukaemia cell is a supporting figure a title search cannot see

  subcellular and subnuclear localisation record for DOT1L, with evidence codes read

• ← related toH4K16ac - the one mark rule 80 actually rescued in this beat: elevated at MLL-AF9 targets in a supplementary figure, and the reason MLL1's own enzyme turns out to be dispensable

  trans-histone input to DOT1L, established in yeast and untested in human leukaemia

• ← recruitsH3K9ac - the mark KMT2A fusions pile up on their own targets, the one the AF9 reader actually binds, and the one a DOT1L inhibitor strips off first

  H3K9ac reading by AF9 YEATS is important for DOT1L chromatin recruitment

• ← related toSIRT1 - not a fusion partner, the least required gene in KMT2A-rearranged lines, and the one target here where the direction of the drug is genuinely disputed

  Direction dispute recorded 2026-09-19: the DOT1L-epistasis model (PMID 25822366) calls for a SIRT1 ACTIVATOR; SIRT1 inhibition kills primary paediatric ALL cells (PMID 25884180) and SIRT1 knockout suppresses BCR-ABL leukaemogenesis (PMID 22207735). No head-to-head comparison exists in a KMT2A-rearranged model, and tenovin-6 is not SIRT1-selective (PMID 23322738).

• ← co-occurs withMAT2A - the enzyme that makes the methyl donor, and the one metabolic dependency in this disease with two independent papers and a lymphoid PDX result

  MAT2A inhibition synergises with DOT1L inhibition; both depend on SAM

• ← related toSAM supply sustains the KMT2A-rearranged hypermethylated state - the metabolic requirement that IS a consequence of the chromatin programme, and can be attacked as one

  DOT1L is a SAM-consuming methyltransferase and a reported combination partner

• ← depends onKOPN-8 (CVCL_1866): the field's default KMT2A::MLLT1 line, and its fusion protein has no YEATS domain

  EPZ004777 IC50 0.62 uM

• ← bindsDOT1L and H3K79 in KMT2A::MLLT1: a direct partner contact, mutually exclusive with the elongation complex, and the only fusion with a DOT1L-inhibitor complete remission

  ENL AHD contacts DOT1L directly; mutually exclusive with the AF4-family contact

• ← bindsThe retained AF9 surface in residue detail: one disordered 70-mer, three DOT1L sites, two of them high-affinity and simultaneously occupiable

  three DOT1L motifs, two high-affinity, PDB 2MV7

• ← recruitsDOT1L and H3K79 in KMT2A::MLLT3: recruitment is a graded dial that sets methylation and transformation - and the fusion it was discovered in produced no remission in the DOT1L trial

  graded recruitment via the retained AHD

• ← related toIKK/NF-kB retains MLL on chromatin — BOTH the fusion and wild-type MLL, at two promoters, in mouse myeloid AML, and it is occupancy loss rather than protein loss

  H3K79me2 loss follows fusion eviction, consistent with loss of recruited DOT1L

• ← refutesHypothesis — the elongation hijack is how one partner class satisfies a more general requirement, and the general requirement is what should be drugged

  predicts DOT1L dependence is confined to the elongation partner class

• ← regulatesHow much of this graph's mechanism is somebody else's fusion? A counted answer: 54% of fusion-bearing evidence, and 0% from a dedicated human KMT2A::AFF1 experiment

  the DOT1L literature is where the import fraction is highest

• ← recruitsKMT2A::AFF1 has no mapped interface to DOT1L: it lacks both structurally defined recruitment modules, and the one complex-level route claimed for it is contested by reconstitution

  no documented interface; the recruitment step is unexplained in this fusion

• ← depends onImport class 1: murine MLL-AF9 myeloid transformation, the field's default assay and this graph's largest single import

  the founding DOT1L dependency result belongs to this class

• ← recruitsImport class 3: the DOT1L-AF10 coiled-coil axis, an MLLT10 mechanism with no counterpart in the index fusion

  the second documented DOT1L recruitment interface

• ← inhibitsAFF1 AF4_int motif to AF9/ENL AHD: the index fusion's only documented elongation interface, and it competes with DOT1L

  AF4 occupancy of the AHD excludes DOT1L in reconstitution

• ← testsDoes KMT2A::AFF1 deliver DOT1L to the AHD or displace it? A competition experiment in t(4;11) cells

  measures DOT1L occupancy on AHD-bearing proteins

• ← related toKMT2A::MLLT6 (MLL-AF17), t(11;17)(q23;q12-q21) — the bridge case: a leucine zipper on a DOT1L-complex subunit

  wild-type MLLT6 is a DotCom subunit with DOT1L; NOT shown for the fusion

• ← related toPhase-2 dossier: DOT1L - perfect chemistry, a completed paediatric trial, and a target that has already had its answer

  readiness layer on top of the DOT1L biology entry

• ← related toKMT2A-rearranged AML is two diseases on the phosphorylation layer, and DOT1L phosphorylation is one of the things that separates them

  increased DOT1L phosphorylation in MLLGA; residues and kinase unnamed

• ← related toThe standing priority asks for localization on every interactor; the graph has it on 14% of genes and 24% of its most central ones, and DOT1L has none at all

  HIGHEST-DEGREE GENE WITH NO LOCALIZATION AT ALL: degree 85, zero localization content of any kind in the entry.

• ← related toUSP7 - the enzyme that holds PRC1.1 together, and the only pharmacological handle on this complex that exists today

  USP7 named as a potential direct DOT1L target gene in GCB-DLBCL

• ← related to'Transcriptional memory' means two different things in this literature, and both of them are used about MLL leukaemia

  'DOT1L provides transcriptional memory' is an operational claim requiring a prime-washout-rechallenge experiment that has not been run here

• ← related toA second disease finds the same DOT1L-to-non-canonical-PRC1 axis in the same year - and the mark moves the opposite way

  DOT1L shapes chromatin-bound ncPRC1 composition in GCB-DLBCL as well as in MLL-fusion leukaemia

• ← co-occurs withZRANB2 - the only bait in any published proximity experiment that reaches AFF1, AFF4, ELL, MLLT1, CCNT1, KMT2A and DOT1L at once

  DOT1L recovered as prey

• ← co-occurs withAFF1-RBM15 proximity: the m6A-complex adaptor, itself a paediatric leukaemia fusion partner, has AFF1 in its BioID neighbourhood

  DOT1L co-recovered in the same neighbourhood

• ← related toA 109-transcription-factor BioID network puts KMT2A next to MYC, YY1 and the ETS and KLF families - and AF10 next to ERG and KLF5

  DOT1L recovered by GATA3 and TBXT baits

• ← related toPRMT5 - the type II arginine methyltransferase this graph has reached for sixteen times, whose mark on H3R2 RECRUITS WDR5 to chromatin

  PRMT5 and DOT1L inhibitors are synergistic in MLL-AF4/AF9 cord-blood cells

• regulates →H3K9me2 - named sixty times in this graph with no entry of its own, the mark that defines the lamina-associated domains the fusion's targets sit beside, and the only H3K9 methylation state ever measured at a KMT2A fusion's own binding sites

  DOT1L inhibition raises H3K9me2 at MLL-AF9-bound TSSs via SIRT1/SUV39H1 in murine myeloid leukaemia

• ← related toH3C1 stays rejected, and now with the two numbers the registry row lacked: its accession carries TEN gene names, and removing every substrate and ribosome symbol moves interactome coverage 43.8% -> 46.1%

  the cleanest demonstration that the census counts substrates: H3C13 enters the union as a DOT1L partner by METHYLTRANSFERASE ASSAY - the H3K79 enzyme acting on its own substrate

• ← related toThe interactome coverage figure is computed on 651 of the 1,015 IntAct rows available - 64% - and the gap is worst for menin and LEDGF, the two baits the menin-interface hypothesis rests on

  the SIXTH bait, omitted from this beat's dispatch brief: 51 rows, 24 human partners, 17 with entries (70.8%) - the best-covered bait of the six

• ← related toThe ATF4/ISR collapse under menin inhibitors reports loss of the KMT2A-fusion transcriptional programme by ANY route, not engagement of the menin interface - and the loss is of ATF4 activity, not ATF4

  a DOT1L inhibitor reproduces the ISR arm to within 0.02-0.24 log2, which is the basis for attributing the programme to H3K79-methylation-dependent elongation output rather than to the menin interface

• ← related toRe-bound at full text: the H3K79me2 decay half-life is still unmeasured - one paper in 12.4 million full texts contains the phrase 'half-life of H3K79me2', it is an unnumbered assertion citing a 2010 review, and the MLL::AF9 degron time-course that could have measured it uses the word zero times

  the unmeasured kinetic parameter that sets how long a DOT1L inhibitor must be held to clear the mark

• ← related toWhat fills the space: RING1B and BCOR arrive at the fusion's sites under BOTH drugs, but the DOT1L inhibitor puts them everywhere on the fusion's territory while the menin inhibitor puts them only in the quartile where the fusion is most completely evicted - and at Meis1 RING1B rises 16-fold

  the DOT1L inhibitor SGC0946 produces a displacement-independent PRC1 in-fill, consistent with H3K79me2 being the direct antagonist

• ← inhibitsA DOT1L inhibitor, which does not touch the menin-KMT2A interface, removes 70% of the fusion's enrichment from its own strongest sites - two thirds of what a menin inhibitor removes - so fusion chromatin residency is partly downstream of H3K79 methylation rather than only of menin

  SGC0946 inhibits DOT1L; at 5 uM for 48 h it removes about 70 percent of the fusion's enrichment from its own top sites

• ← related toHOXA9 is where menin leaves most completely (share ratio 0.14-0.23 over floor) and MLL1-N-reactive material stays most completely (0.74-1.02 over floor), in all three MLL-rearranged lines - so the menin inhibitor decouples menin from the HOXA9 locus without clearing the MLL1 N-terminus from it

  DOT1L leaves HOXA9 almost completely in RS4;11 (0.053 over floor) and stays in MOLM13 (0.992) and ML-2 (0.756)

• ← related toML-2 (KMT2A::AFDN, a non-elongation partner) is the line where menin eviction is weakest (retains 37.2% of its own top windows against 20.5% and 13.0%) and where the DOT1L track moves most of any track in the beat (retains 31.6%, 13.69 percentile points below a rank-matched null at p = 3e-10) - the opposite pattern to what an AF9/ENL-free fusion would predict

  ML-2's DOT1L track shows the largest rank effect in the beat (13.69 points below a rank-matched null, p = 3.2e-10) from the weakest library in the deposited table

• ← related toAt the 692 RS4;11 bins where an MLL1 N-terminus and menin co-occupy, VTP-50469 takes menin to 0.20 and DOT1L to 0.20, drops H3K79me2 to 0.66, and leaves MLL1-N-reactive material at 0.83 - four factors, one drug, one chromatin, and a clean ordering

  DOT1L occupancy at the MLL1-N held set falls to 0.2455 (0.299 over floor) and to 0.2003 at the co-occupied set - indistinguishable from menin's 0.2005

• ← related toThe four-way ordering given a second cell line: menin, DOT1L and the MLL1-N channel reproduce between KMT2A::AFF1 and KMT2A::MLLT3 to within 0.04 of a share ratio, and H3K79me2 - the one channel the ordering was built around - diverges by 0.21 to 0.30 and changes places with MLL1-N

  DOT1L occupancy falls to 0.2219 at the co-occupied MOLM13 bins while the mark it writes does not move, so enzyme eviction and mark persistence are separable at 3 days

• ← related toTwo instruments on the same cell line give opposite answers about DOT1L under a menin inhibitor: the depositors' own TSS-window table puts DOT1L at 0.6638 beside menin's 0.2513, and 2 kb genome-wide bins put it at 0.2219 beside menin's 0.1657 - and only the genome-wide instrument can see the 86% of the co-occupied set that is not at a TSS

  DOT1L leaves with menin genome-wide in MOLM13 (0.2219 against 0.1657) and appears to stay in the depositors' TSS table (0.6638 against 0.2513)

• ← depends onIn MOLM13 a menin inhibitor removes three quarters of DOT1L from the fusion's own sites and leaves H3K79me2 there completely intact for three days - the writer and its mark are separable on this timescale, and in RS4;11 they are not

  the dissociation is defined by DOT1L occupancy at the same bins falling to 0.2219 while the mark it writes does not move

• ← related toZMYND11::MBTD1 is the KMT2A-fusion architecture assembled from different parts - and the part it throws away is a direct physical brake on KMT2A itself

  the effector this architecture does NOT use - an acetyltransferase stands where the methyltransferase stands in a KMT2A fusion

• ← related toIn murine MLL-AF9, H3K79me2's response to a menin inhibitor runs 0.586 / 0.819 / 0.928 / 1.187 across the four eviction-depth quartiles of the fusion's own top-0.1% sites - Spearman -1.000, span 0.6005, the largest of any mark measured in these bins except H3K27me3 - while the aggregate over the same 1,228 bins is a single unremarkable 0.8156

  H3K79me2 is DOT1L's product; the gradient is in DOT1L's mark

• ← related toThe menin inhibitor's effect on H3K79me2 is a REDISTRIBUTION within the fusion's own site set, not a loss from it: the share falls 41.4% at the quartile the fusion is most evicted from and RISES 18.7% at the quartile it is least evicted from, in murine MLL-AF9

  redistribution of DOT1L's own mark

• ← inhibitsSGC0946 removes 86% of H3K79me2's share at the fusion's own sites and the four eviction quartiles are 0.1396 / 0.1453 / 0.1452 / 0.1360 - span 0.0036, Spearman +0.400, a bootstrap interval spanning zero - so a very large effect on this mark can carry no eviction-depth structure whatever

  SGC0946 inhibits DOT1L; its mark collapses to 0.1415 of vehicle at these bins

• ← related toStratifying by eviction depth does NOT rescue H3K79me2 as a resistance-detecting readout: at the most-evicted quartile the PCGF1-knockout and the control are 0.6123 against 0.5860, a difference of 0.0263, while the whole genotype effect sits at the LEAST-evicted quartile (-0.1401) - the opposite of eviction-graded

  the readout is DOT1L's mark

• ← related toIn MOLM13, human KMT2A::MLLT3, from the VTP-50469 founding paper's own series GSE127507, H3K79me2's aggregate response at MLL1-N's top sites is 0.998 - and the same sites split by eviction depth run 0.7746 / 1.0601 / 1.0995 / 1.1421 at Spearman -1.000, with the INPUT stratified identically staying flat

  DOT1L occupancy and its mark measured in the same cells

• ← depends onIn human MOLM13, DOT1L's own occupancy under a menin inhibitor runs 0.1364 / 0.3083 / 0.3555 / 0.3913 across MLL1-N eviction-depth quartiles at Spearman -1.000 - the writer is lost in proportion to eviction depth, and its mark follows with a 2.9-fold smaller span

  DOT1L occupancy falls in proportion to fusion eviction

• ← regulatesOGT - the O-GlcNAc transferase, a Polycomb-complexome bait that pulls down KMT2A, and already the metabolic dial on DOT1L in this graph

  O-GlcNAcylates DOT1L S1511, blocking UBE3C-mediated degradation and setting DOT1L abundance from hexosamine flux

• ← co-occurs withGATA3 - one of only two transcription-factor baits in a 109-bait BioID screen that recover DOT1L

  GATA3 bait / DOT1L prey, one spoke-expanded BioID row, IntAct IM-28767 - one of only two of 109 TF baits recovering DOT1L

• ← related toThe first same-condition H3K79me2 noise floor measured anywhere in this thread: 11.17% at D1 and 26.44% at the TOP 0.1% stratum, against 45.72% and 62.63% for H2AK119ub in the same arm, the same condition and the same pairing shape - 4.1x tighter. A mark floor transferred from H2AK119ub does not describe H3K79me2.

  H3K79me2 is DOT1L's product; this is the floor for measuring it

• ← related toTBXT / brachyury - the notochord master regulator, and one of only two of 102 transcription-factor baits whose neighbourhood contained DOT1L

  TBXT bait / DOT1L prey, one MI:1314 spoke-expanded BioID row, IM-28767, HEK293 Flp-In; one of only 2 of 102 TF baits reaching DOT1L

• ← testsIn the same deposit, the MENIN inhibitor removes DOT1L from all five DMSO DOT1L peaks at canonical target loci (p = 0.0432) while all three DOT1L inhibitors keep 5/5 - and the VTP DOT1L library still calls ACTB, GAPDH and EEF1A1, which is the internal positive control the MEN1 arm lacks

  DOT1L ChIP occupancy at the same 15 loci under the same four drugs

• ← related toUnder all three DOT1L inhibitors, DOT1L's fold-enrichment at canonical target loci is HIGHER than DMSO (PBX3 4.85 to 13.92, SENP6 3.37 to 9.68) - either catalytically dead DOT1L is retained or trapped on chromatin, or the reading is an artefact of a 1.35-fold spread in MACS fragment-size estimates, and this deposit cannot tell which

  predicts that a DOT1L degrader and a DOT1L catalytic inhibitor are not interchangeable

• ← inhibitsNovartis 'compound 10' - a DOT1L methyltransferase inhibitor from Perner et al. (Blood 2020), active at 100 nM, whose chemical structure is not given in the GEO deposit and whose target engagement at that dose IS demonstrated in the same series by a 5.6-fold spike-in-normalised H3K79me2 collapse by day 3

  DOT1L methyltransferase inhibitor, active at 100 nM, engagement shown by a 5.57-fold spike-in-normalised H3K79me2 collapse at day 3

• ← inhibitsNovartis 'compound 11' - a DOT1L methyltransferase inhibitor from Perner et al. (Blood 2020), active at 100 nM, whose chemical structure is not given in the GEO deposit and whose target engagement is not measured anywhere in the series

  DOT1L methyltransferase inhibitor by depositor labelling; target engagement is not measured anywhere in the series

• ← related toThe GSE149183 per-gene columns are RAW, not spike-in scaled: every one of 1,040,292 values in both files sits exactly on a 0.04 grid, which no per-library scale factor survives - so two entries in this graph called an unscaled number absolute, and the true collapse is 37-91x, not 5.8x

  the collapse being rescaled is the loss of DOT1L's own product under its inhibitors

• ← related toOn an absolute scale, H3K79me2 in the index fusion falls 52-fold (EPZ5676) to 91-fold (compound 10) by day 9 and NOT ONE of 17,341 measurable genes holds its signal - the first absolute-scale chromatin statement this project has been able to make about KMT2A::AFF1

  inhibiting DOT1L removes its product essentially completely and uniformly

• ← related toThe same baseline-matched test that kills the target effect under EPZ5676 fires hard under compound 10 - at d6 and d9, in both cell lines, 4 of 4 contrasts (p = 0.0012 to <0.0001) - and in MOLM13 HOXA9 holds 24.5% of its H3K79me2 where the genome median holds 4.5%

  two inhibitors of the same catalytic site leave different residual landscapes

• ← related toTwo inhibitors of the same DOT1L active site leave different residual H3K79me2 landscapes, and the difference sits at the KMT2A-fusion target loci - so 'how much H3K79me2 is left' is a compound-specific readout, not a generic measure of DOT1L inhibition

  two inhibitors of one active site, two residual landscapes

• ← testsThree hours after the fusion is degraded to 2.5-7.1% of its own sites, NOT ONE of the eight complex components profiled beside it falls below 68% retention genome-wide - menin 80.0%, DOT1L 68.7%, CDK9 83.6%, AFF4 92.7%, RNA Pol II 99.6%, pS2 98.3%, pS5 86.8% - so nothing in the complex is held on chromatin by the fusion at genome scale

  DOT1L retains 68.7% and is the MOST displaced component of the eight profiled - loss relative to the fusion 0.33 - consistent with the authors' report that DOT1L dissociates faster than menin.

• ← depends onDOT1L leaves chromatin before menin does when the fusion is degraded, and the ordering survives a change of instrument that destroys the magnitudes - ChIP-qPCR at selected loci, target-restricted read counts and genome-wide peak presence all rank DOT1L ahead of menin while disagreeing about every number

  DOT1L's chromatin tether at fusion sites is the direct AF9 contact, which is destroyed with the fusion - the mechanistic reason it leaves before menin, whose second anchorage is not.

• ← related toExperiment: a 6 / 24 / 72 hour / 7 day VTP-50469 time course in SEM reading menin occupancy and H3K4me3 together, to decide whether the 23% is a partial effect or a slow one

  The DOT1L arm's unverified target engagement is one reason the comparator needs an H3K79me2 readout

• ← related toMenin loses half its promoter signal where the fusion sat, and nothing where it did not

  DOT1L 0.401 at the same target genes, against MENIN 0.503

• ← related toThe DOT1L inhibitor removes menin from fusion promoters too, which a pure interface model does not predict

  DOT1L catalytic inhibition removes menin from fusion promoters

• ← related toENL is the only elongation cofactor that separates fusion-occupied promoters from wild-type KMT2A promoters by more than the H3K4me3 negative control does, in primary infant KMT2A::AFF1

  DOT1L clears the H3K4me3 benchmark in 1 of 3 primary states

• ← related toIn drug-naive primary infant KMT2A::MLLT1 B-ALL, menin is as enriched as the fusion N-terminus at the top fusion promoters (2.73x vs 3.12x over an accessibility reference) while the wild-type C-terminal reporter sits at background (1.10x)

  DOT1L is the most enriched channel at these promoters (16.10x)

• ← related toIn the HOXA-dead infant states IRX1 carries H3K4me3 and ENL while HOXA9/HOXA10 carry neither

  DOT1L measured at IRX1 alongside ENL, and is much weaker (0.53-4.61x)

• ← related toThe lymphoid-restricted DAXX dependency survives three nulls and two comparator restrictions - and ATRX, MEN1 and DOT1L do not survive the same test in the same lines

  DOT1L is flat in the B-lineage stratum (+0.061, p 0.78), consistent with its dependency being myeloid-weighted

• ← related to"Seen in MLL-AF4 and no other bait" excludes AFF1, AFF4 and ELL — the three most AFF1-specific proteins in the experiment — and keeps ACADM and GLUD1

  DOT1L under MLL-AF9 (p 0.00172, 24 spectra) is the only non-trivial true positive inside the singleton class and is used here as its calibrator

• ← related toCALM-AF10 strips H3K79 methylation from the rest of the genome while adding it at HOXA - one fusion, two opposite effects on the same mark

  the cytoplasmic-sequestration account of global H3K79 loss fails in leukaemia cells, where the fusion is nuclear and still suppresses the mark regardless of clathrin

• ← related toMLLT6 (AF17) — the fourth KMT2A fusion partner inside DotCom, and the only one no cell needs

  the one tissue where Af17 loss has a measured phenotype - renal collecting duct - shows INCREASED H3K79 dimethylation, i.e. AF17 constrains the DOT1L mark in vivo outside blood (PMID 21546577)

• expressed in →Cardiomyocyte - the non-haematopoietic cell that two of this graph's drug targets are required in, which makes it the graph's clearest bystander-organ liability

  RESIDENCY: DOT1L performs its function in cardiomyocytes by regulating Dystrophin transcription, established by cardiac-specific knockout with miniDmd rescue; DOT1L is also downregulated in idiopathic dilated cardiomyopathy patient samples

• regulates →Cardiomyocyte - the non-haematopoietic cell that two of this graph's drug targets are required in, which makes it the graph's clearest bystander-organ liability

  REQUIREMENT: cardiac-specific Dot1L knockout causes chamber dilation, cardiomyocyte death, systolic dysfunction and conduction abnormalities phenocopying dilated cardiomyopathy. GENETIC KNOCKOUT, not catalytic inhibition - the distinction is load-bearing

• ← testsThe six unwritten DOT1L partners are not six findings - they are nine spokes of ONE GFP-DOT1L pulldown, and the source paper already graded them

  Audits the entire IntAct record for DOT1L (Q8TEK3), 49 rows from 7 publications, and grades the nine spokes of the Hein 2015 pulldown

• ← bindsMAPK7 (ERK5) - a DOT1L partner on one 1:365 spoke of one atlas pulldown, and a kinase with real leukaemia literature that has never been connected to DOT1L

  ONE spoke of ONE atlas pulldown (PMID 26496610, IntAct EBI-11060394, spoke-expanded); see the entry for the stoichiometry and the source's own confidence class

• ← bindsEBNA1BP2 (EBP2) - the DOT1L spoke with the HIGHEST stoichiometry and the WEAKEST evidence, and the case that shows why abundance is the third dimension

  ONE spoke of ONE atlas pulldown (PMID 26496610, IntAct EBI-11060394, spoke-expanded); see the entry for the stoichiometry and the source's own confidence class

• ← bindsPARP16 - graded A- in the DOT1L pulldown, the best of the six, and a tail-anchored ER membrane protein that cannot be in the nucleoplasm

  ONE spoke of ONE atlas pulldown (PMID 26496610, IntAct EBI-11060394, spoke-expanded); see the entry for the stoichiometry and the source's own confidence class

• ← bindsIBTK - the only one of the six DOT1L spokes with a reciprocal test, and its own pulldown in the same study does not find DOT1L

  ONE spoke of ONE atlas pulldown (PMID 26496610, IntAct EBI-11060394, spoke-expanded); see the entry for the stoichiometry and the source's own confidence class

• ← bindsNIN (ninein) - a centrosomal scaffold recorded as a DOT1L partner under a UniProt isoform that UniProt itself calls dubious

  ONE spoke of ONE atlas pulldown (PMID 26496610, IntAct EBI-11060394, spoke-expanded); see the entry for the stoichiometry and the source's own confidence class

• ← related toIBTK - the only one of the six DOT1L spokes with a reciprocal test, and its own pulldown in the same study does not find DOT1L

  RECIPROCAL NEGATIVE: GFP-IBTK in the same study recovered 23 preys and DOT1L was not among them

• ← related toRegress prey abundance against interactor confidence class across all 1,330 baits of the Hein atlas - the data are already downloaded and the run is an afternoon

  Uses the DOT1L pulldown's A+ DotCom subunits as the internal spike control

• ← related toAF9's nuclear residence is chaperone-dependent, the Hsp90 site is the SAME C-terminal region the fusion keeps, and UniProt records no cytoplasmic pool at all

  The Hsp90 contact is on AF9 residues 475-568, the same AHD surface DOT1L binds, so chaperone engagement competes with the DOT1L contact

• ← related toTwo thirds of the curated interactome of this project's six core proteins is spoke-expanded - and the pairs this graph chose to write are significantly better than the ones it did not

  78.6% spoke-only, the highest of the six, with exactly one partner carrying an MI:0407 direct-interaction record

• ← related toIntAct curates SEVEN MLL fusion proteins as first-class interactors - ENL, AFF4, AF9, GAS7, ELL, AF6 and CALM - and not the index one

  the MLL-AF9 entity's DOT1L record is MI:0915 physical association and NOT spoke-expanded - the observed fusion-partner contact the index fusion has no equivalent of

• ← bindsBAG6 / BAT3 - a DOT1L partner that is about H3K79me2 itself, reciprocally co-immunoprecipitated, and not a spoke

  reciprocal non-spoke co-IP in U2OS; BAG6 UBL motif to DOT1L UIM

• ← bindsTCF7L2 - the protein IntAct's 'DOT1L-TCF4' row is actually about, and the Wnt effector that DOT1L and AF10 coactivate

  spoke-expanded rows only; the functional claim is beta-catenin-dependent coactivation, not a binary contact

• ← related toThe DOT1L partner IntAct calls TCF4 is TCF7L2 - the second UTP18/WDR5-class symbol mis-assignment found in this interactome, and gene-tcf4 is declined

  removes one partner from DOT1L's curated interactome

• ← co-occurs withSETD2 (Q9BYW2) - the H3K36 trimethyltransferase that binds all seven MLL fusions, and the only one of them with a published KMT2A::AFF1 dependency test

  SETD2 knockdown potentiates the DOT1L inhibitor EPZ5676 in MV4-11 (KMT2A::AFF1); SETD2 loss also elevates H3K79me2

• ← related toNobody has ever asked whether a KMT2A-rearranged sarcoma needs menin: zero records, and the reason is that no model exists

  DOT1L[tiab] AND sarcoma[tiab] returns 3 records, none in this entity

• ← related toAFF1 / AF4 — the 3' half of the fusion, and a target with almost no structural footing

  The two sides of the AFF1-DOT1L question, held in one place: a positive complex-level claim in mouse (PMID 17135274) against a reconstitution negative (PMID 21896721). No residue-level contact is mapped in either direction.

• ← related toIGF2BP3 — an RNA-binding protein required for MLL-Af4 leukaemogenesis in mice that CRISPR barely touches in human lines

  Via the methyl-donor pool only: methionine/SAM disruption in MLL-AF4 cells lowers H3K79 methylation AND lowers DOT1L mRNA and protein while raising H3K27me3 at the DOT1L promoter - so the enzyme and its substrate both fall, which the entry must not conflate. PMID 31717699 was cited in zero entries of this corpus before 2026-09-21.

• ← related toELL2 — a SEC subunit that almost no cell needs, next to a paralogue almost every cell needs

  ELL2 is required for DOT1L OCCUPANCY at a locus: Dot1L association with the IgH gene is impaired in the absence of ELL2, in mouse plasma cells. The experiment does not separate this from ELL2's effect on the marks themselves.

• ← related toSMYD3 - a lysine methyltransferase with three claimed histone marks and one robust non-histone substrate, sitting on an HOXA9 feed-forward loop in MLL-AF9 AML and a fatty-acid-oxidation axis in CML stem cells, with zero interventional trials

  combined disruption of the SMYD3-HOXA9 loop and DOT1L inhibition enhanced anti-leukaemia activity in MLL-AF9 AML in vitro and in vivo; SMYD3 activates HOXA9 independently of DOT1L

• ← recruitsWhat the reciprocal product assembles: P-TEFb, an H3K4 methyltransferase, an H3K79 methyltransferase, an arginine methyltransferase and an acetyltransferase

  An H3K79(me3) methyltransferase activity is reported among the 16 constituents.

• ← bindsSET::NUP214 recruits DOT1L by a different surface from CALM-AF10 and KMT2A::MLLT10: the founding paper tested for AF10 in the complex and did not find it

  anti-DOT1L IP recovers SET-NUP214 in LOUCY; one paper, 2008, never repeated

• regulates →The KMT2A-fusion causal chain: six links, one measured ordering, and two arms that are parallel rather than sequential

  link 4: H3K79me2/3 deposition, an arm parallel to the SEC arm

• ← depends onMLLT10 (AF10): the discarded PZP reader — the one partner where loss is proven to be the oncogenic event

  the OM-LZ/DOT1L interface argument is an H3K79 argument: conditional Dot1l knockout and EPZ004777 abolish MLL-AF10 and CALM-AF10 transformation (Chen 2013)

• ← regulatesMLLT10 (AF10): the discarded PZP reader — the one partner where loss is proven to be the oncogenic event

  discarded PZP normally licenses where the retained OM-LZ takes DOT1L

• ← bindsMLLT10 (AF10) — DOT1L cofactor and KMT2A fusion partner

  AF10 OM-LZ (aa 704-779) binds the DOT1L coiled coil

• ← recruitsMLLT3 (AF9): the YEATS acetyl-lysine reader is discarded in ~92% of MLL-AF9 patients — same shape as PZP, but the causal test has never been run

  intact AF9 YEATS couples H3K9ac to DOT1L chromatin recruitment

• ← related toCycle 104's M1 - 'no scoreable mark moves at D1, so the menin inhibitor's chromatin effect is proportionally a peak phenomenon' - DOES NOT SURVIVE. The same-condition floor at D1 is 2.4%, not the 10% borrowed from the top stratum, and all three scoreable marks exceed it (H3K4me3 9.8%, H3K79me2 7.8%, H3K27me3 4.8%). Two of the three exceed even GSE260742's OWN stratum-matched input floor, which was on disk the whole time. Cycle 104's alternative M2 fires instead
• ← related toA ratio-of-ratios combines four independent tracks and a pair ratio combines two, so its noise floor is wider by a factor of root two in log space - and cycles 101 and 102 scored two ratio-of-ratios against a pair floor. Applying the correction to the measured 9.4% peak floor gives 13.5%: VTP's 1.3% stays comfortably inside, and SGC0946's 22.8% stays outside but its margin falls from '2-6x outside' to 1.7x
• ← inhibitsPinometostat (EPZ-5676) — the DOT1L inhibitor, and the most instructive failure in this field

  SAM-competitive inhibition of the H3K79 methyltransferase

• ← related toA caveat that weakened the index fusion, wrongly: the MLL-AF4 spreading deposition is 30 SEM samples plus six other lines, all human

  the deposition carries an EPZ-5676 dose series in SEM

• ← related toThe AHD inhibitor's deposited arm is MOLM-13 and its in vivo arm is nowhere: a half-resolution that names the limit exactly

  an AF9/ENL-DOT1L interaction inhibitor tested in a MLLT3 fusion

• ← related toThree unnamed things at once: the DOT1L-PRC1.1 deposition names the fusion, the cell types and both inhibitors with concentrations

  SGC0946 at 3-5 uM across mouse MLLAF9 and human myeloid, erythroid and pluripotent cells

• ← related toThe unnamed Cre is Mb1-Cre, which acts at the pro-B stage: the one resolution in this beat that lands on the right cell

  conditional Dot1l loss from the pro-B stage onward

• ← related toBounded failure: 'menin inhibitor-resistant cells' is a drug entry's central claim and neither the paper nor any repository defines it

  a degrader removes DOT1L scaffolding as well as catalysis

• ← related toIf inhibiting one methyltransferase failed, constraining the methyl donor may not