Competing model 4: t(4;11) cells are addicted to the direct fusion and completely indifferent to the reciprocal one
also called addiction to MLL-AF4 but not AF4-MLL · reciprocal dispensable · Kumar knockdown
Somebody switched off the mirror-image fusion in leukaemia cells and nothing happened. Switching off the famous one killed them. That is the strongest argument that the mirror image does not matter once the disease exists.
The same thing, for a scientist
In the human t(4;11) lines RS4;11 and SEMK2-M1, siRNA knockdown of AF4-MLL had no effect on growth in either line and no effect on cell cycle or apoptosis in SEMK2-M1, whereas knockdown of MLL-AF4 significantly inhibited growth of both and caused G2/M arrest and increased apoptosis [E1]. The authors' conclusion is that the direct fusion is critical for proliferation and survival while the reciprocal product is dispensable. The paper drew a published comment and an authors' reply in the same journal [E2][E3], which is worth knowing because the disagreement is on the record rather than only in the field's folklore. Two things this result establishes incidentally are as valuable as its headline. **Both RS4;11 and SEMK2-M1 express both fusions** [E1] - which answers an open question standing in three entries of this graph, that asked whether the standard t(4;11) lines are reciprocal-positive. They are. And because they are, every mechanistic experiment ever run in those lines was run in a reciprocal-positive background, so the field has no reciprocal-negative cell-line comparator at all.
What the result does and does not exclude
Does: it excludes a requirement for the reciprocal product in the proliferation and survival of an established t(4;11) line in vitro. Does not: it says nothing about initiation, where the opposite result is published in mouse 4; nothing about in vivo or niche-dependent phenotypes; nothing about differentiation state; and nothing about protein, because no reagent can confirm that the reciprocal-derived protein was actually depleted. The knockdown is scored on transcript.
The incidental finding that should be propagated
This graph carries an open question in at least three entries - concept-an-mll-c-antibody-cannot-separate-wild-type-from-reciprocal-fusion, hyp-menin-primacy-was-argued-from-one-of-two-fusion-products and concept-reciprocal-aff1-kmt2a-retains-the-entire-wrad-interface - asking whether SEM, RS4;11 and MV4-11 are reciprocal-positive or reciprocal-negative, and noting that the answer determines how every published t(4;11) result should be read. 1 states plainly that RS4;11 and SEMK2-M1 (a subline of SEM) both express MLL-AF4 and AF4-MLL. The answer is positive for the two lymphoid lines. MV4-11 is not covered by this paper and remains open.
| effect of reciprocal knockdown | none on growth, cell cycle or apoptosis |
|---|---|
| effect of direct knockdown | growth inhibition in both; G2/M arrest and apoptosis in SEMK2-M1 |
| assay level | transcript knockdown; protein depletion unverifiable |
lines tested
[ "RS4;11", "SEMK2-M1" ]
reciprocal status of standard t411 lines
{
"RS4;11": "positive",
"SEMK2-M1 (SEM subline)": "positive",
"MV4-11": "not reported in this source"
}answers open question in
[ "concept-an-mll-c-antibody-cannot-separate-wild-type-from-reciprocal-fusion", "hyp-menin-primacy-was-argued-from-one-of-two-fusion-products", "concept-reciprocal-aff1-kmt2a-retains-the-entire-wrad-interface" ]
- If the reciprocal product is dispensable for maintenance, then the therapeutic case for targeting it collapses even though the biology remains interesting.
- Because every standard t(4;11) line is reciprocal-positive, any preclinical menin-inhibitor result from those lines was obtained in the presence of a menin-blind KMT2A oncoprotein - which is either reassuring (the drug works anyway) or an unexamined confound, and nobody has said which.
- Is MV4-11 reciprocal-positive? It is a t(4;11) line used throughout menin-inhibitor pharmacology and this beat could not find its reciprocal status stated.
- Does any reciprocal-negative t(4;11) cell line exist? If not, no cell-based experiment in this disease has ever had the comparator arm it needs.
- Would a stable knockout of the der(4) allele, rather than transient siRNA, give the same answer? CRISPR-engineered t(4;11) human HSPCs now make that feasible.
- Was the reciprocal protein actually depleted in this experiment? Nobody can say, because the assay to check it does not exist.
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
In the human leukaemia cell lines RS4;11 and SEMK2-M1, both of which express MLL-AF4 and AF4-MLL, siRNA knockdown of AF4-MLL had no effect on growth of either line and no change in cell cycle or apoptosis in SEMK2-M1, while knockdown of MLL-AF4 significantly inhibited growth of both and caused G2/M arrest and increased apoptosis in SEMK2-M1.
t(4;11) leukemias display addiction to MLL-AF4 but not to AF4-MLL. · Kumar AR, Yao Q, Li Q, et al. · Leuk Res 2011What this evidence cannot show
Two lines from two patients, transient siRNA, in vitro readouts only, no in vivo arm. Knockdown of the reciprocal product cannot be verified at protein level because no reagent distinguishes the reciprocal-derived MLL-C fragment from the wild-type one. A negative in a maintenance assay does not address initiation.
Evidence that argues against this (1)
- The same fusion expressed in murine Lin-/Sca1+ progenitors induced acute leukaemia where the direct fusion alone did not, so initiation and maintenance give opposite answers for the same protein. Bursen A et al. 2010
- E2moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper
The result prompted a published commentary in the same journal questioning the significance of the AF4-MLL reciprocal fusion in t(4;11) leukaemias.
Significance of AF4-MLL reciprocal fusion in t(4;11) leukemias? · Sanders DS, Muntean AG, Hess JL · Leuk Res 2011What this evidence cannot show
A commentary with no abstract in the PubMed record; this beat cites it as evidence that the disagreement is formally on the record, not for any scientific content, which it could not read from the record alone.
Evidence that argues against this (1)
- A commentary is not data; the underlying disagreement was not resolved by either the comment or the reply, and both predate every subsequent model system. Kumar AR et al. 2011
- E3moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper
The original authors published a reply in the same journal restating that t(4;11) leukaemias display addiction to MLL-AF4 but not to AF4-MLL.
Authors' comments: t(4;11) leukemias display addiction to MLL-AF4 but not to AF4-MLL. · Kumar AR, Yao Q, Li Q, et al. · Leuk Res 2011What this evidence cannot show
Authors' reply, no abstract in the PubMed record; cited for the existence and position of the reply only.
Evidence that argues against this (1)
- The opposing laboratory's position - that the reciprocal product is indispensable for leukaemia onset - was published in Blood and has not been withdrawn. Bursen A et al. 2010
- E4strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
AF4.MLL expressed in murine Lin-/Sca1+ progenitors induced pro-B ALL, B/T biphenotypic acute leukaemia or mixed-lineage leukaemia, whereas MLL.AF4 alone caused no disease over 13 months.
The AF4.MLL fusion protein is capable of inducing ALL in mice without requirement of MLL.AF4. · Bursen A, Schwabe K, Rüster B, et al. · Blood 2010What this evidence cannot show
Murine, retroviral, partially penetrant; a sufficiency experiment in a different species and a different cell state from the knockdown experiment it is set against.
Evidence that argues against this (1)
- In human cord-blood CD34+ cells the same fusion initiated no leukaemia within 36 weeks, so the murine sufficiency result does not transfer to human cells. Prieto C et al. 2017
The strongest published negative on this protein's role in established disease.
Answers that entry's first open question: RS4;11 and SEMK2-M1 both express the reciprocal fusion, so the three-way attribution problem applies to every experiment in them.
Answers that entry's second open question on the reciprocal status of the standard t(4;11) lines: both lymphoid lines are positive.
Maintenance and initiation give opposite answers for the same protein; this is the maintenance side.
Points the opposite way to the t(4;11) knockdown, in a symmetric design but in rare partners and a transformation surrogate.