Received: 1 January 2020 Revised: 1 February 2020 Accepted: 8 February 2020

DOI: 10.1002/stem.3173

CANCER STEM CELLS

Selective killing of leukemia cells: Yamanaka factors’ new trick

Huafeng Xie1,2,3 | Thomas Graf4,5

1
Department of Hematology, Guangzhou First
People’s Hospital, Institutes for Life Sciences Abstract
and School of Medicine The four transcription factors of the Yamanaka cocktail (Oct4, Sox2, Klf4 and Myc,
2
National Engineering Research Center for
termed OSKM) are famously capable of reprogramming somatic cells into induced
Tissue Restoration and Reconstruction
3
Key Laboratory of Biomedical Engineering of pluripotent stem cells (iPSCs). In a paper recently published in Nature Communica-
Guangdong Province, South China University tions, Wang et al. now describe the unexpected discovery that short-term activation
of Technology, Guangzhou, P. R. China
4
of OSKM expression in acute myeloid leukemia cells in vivo induces apoptosis while
Centre for Genomic Regulation (CRG), The
Barcelona Institute of Science and Technology, negligibly affecting normal hematopoietic stem and progenitor cells.1 These findings
Dr. Aiguader 88, Barcelona, Spain
have potential implications for novel anti-cancer strategies.
5
Universitat Pompeu Fabra (UPF), Barcelona,
Spain

Correspondence
Thomas Graf
Email: thomas.graf@crg.eu

asked whether HSPCs from reprogrammable mice transduced with

A long-standing question in the reprogramming field is whether tumor
cells, like normal cells, can be induced to convert into iPSCs capable
of generating the diversity of normal tissues and even animals. The
overall picture is that, while some cancer cell lines, in particular leuke-
mias, can be reprogrammed (typically at very low efficiencies), many
others appear to be resistant (see for example,2,3). This could be
ascribed to differences in the cells’ genetic and epigenetic status, with
cells that are either insensitive to incoming signals or cells that are
receptive but activate a stress response resulting in cell death, in both
cases preventing rewiring of the cells’ genome required for the forma-
tion of a new and viable cell phenotype.
In the recent study by Tao Cheng and colleagues this question
was revisited, using a leukemia induction model developed earlier by
the authors. This consists of the retrovirus mediated transduction of
MLL-AF9 fusion protein into bone-marrow (BM) derived hematopoi-
etic stem and progenitor cells (HSPCs) followed by transplantation
into mice.2 The result is an acute myeloid leukemia (AML) dissemi-
nated in the BM, spleen and blood, causing the death of the mice
within 2 months. They now repeated these experiments but using
‘reprogrammable‘mice containing a doxycycline inducible OSKM cas-
sette. With such mice the group of Manuel Serrano had shown that
the formation of iPS cells can be induced in vivo.4 Wang et al. now
MLL-AF9-GFP in culture and then transplanted into sub-lethally irra-
diated mice would convert into iPSCs following activation of the
Yamanaka factors by doxycycline administration in the drinking water.
Surprisingly, they observed that in the treated animals the green fluo-
rescent MLL-AF9 cells disappeared and the mice were saved from
death by AML, whereas uninduced control animals succumbed within
a matter of months (Figure 1A). Strikingly, OSKM activation had
essentially no effects on uninfected hematopoietic progenitors in the
same animals, permitting the mice to survive long-term. The lack of
sensitivity of normal HSPCs to OSKM activation was further corrobo-
rated in a competitive repopulation experiment between cells from
reprogrammable mice and mice lacking OSKM, showing robust sur-
vival of the OSKM-induced cells after doxycycline treatment
(Figure 1B). This raised the question as to whether the immune sys-
tem is involved, which was not the case as the effects could also be
observed in immunodeficient mice and after depletion of macro-
phages. Instead, the relevant mechanism was found to consist in the
induction of apoptosis, with OSKM induction in MLL-AF9 cells leading
to increased levels of P53, Puma and cleaved caspase 3, resulting in
the selective increase of annexin V in these cells compared to normal
cKit+ progenitors.
To elucidate the molecular mechanism behind the activation of
apoptosis by OSKM in MLL-AF9 cells, Wang et al. performed chroma-
tin accessibility assays (ATAC-seq) after OSKM induction. They found
that MLL-AF9 cells re-organize global genome accessibility more

Stem Cells. ;9999:• •. wileyonlinelibrary.com/journal/stem ©AlphaMed Press 2020 1

2
extensively than wild type cKit+ cells. This included transitions from
open to closed and closed to open chromatin regions. When they ana-
lyzed the newly opened chromatin regions, likely representing sites
bound by incoming or newly activated transcription factors, they
found an enrichment for the sequence motifs Ets, Runt, Sox and Klf,
but strikingly not that of the well-defined Oct4 motif. This raised the
possibility that only a subset of the factors in the Yamanaka cocktail is
sufficient for the apoptosis-inducing effect in the leukemic cells and
that Oct4 is not among them. Indeed, when they tested the factors
individually, they discovered that Sox2 and Klf4 are sufficient to
induce the effect in the leukemic cells. To determine the mechanism
at the epigenetic level, they analyzed OSKM-induced changes in his-
tone modifications and found a reduction of global H3K9me3 levels in
MLL-AF9 cells not seen in wild-type cells. Searching for the dys-
regulation of enzymes known to be involved either in the deposition
or erasure of methyl residues in H3K9me3, they found the selective
downregulation of the methyltransferases Suv39h1/h2 and the
upregulation of the demethylase KDM3a. Consistent with a role of
these enzymes in the process, the apoptosis inducing capacity of
OSKM in MLL-AF9 cells could be impaired both with chaetocin, an
inhibitor of H3K9 methylation, and by knocking down the histone
demethylase Kdm3a. Moreover, overexpression of Kdm3a in normal
cKit+ progenitors caused cell death by apoptosis. Finally, they showed
that OSKM-induced apoptosis is not restricted to MLL-AF9 cells, as
three additional tumor types (mouse models of Notch1-ALL and
NRIP3-AML leukemia and cultured THP-1 monocytic leukemia cells)
exhibited a similar response. Table 1 summarizes the major differ-
ences in the response to OSKM activation between MLL-AF9 and
normal HSPCs.
An antitumor effect of transcription factors associated with cell
fate has been observed before, prominently by the activation of
endogenous RARa in acute promyelocytic leukemia cells treated with
all trans-retinoic acid. However, in this case the mechanism is not pri-
marily the induction of apoptosis but that of terminal differentiation.5
Likewise, the anti-tumorigenic effect of overexpressing C/EBPa in
human acute B cell leukemia cells is a consequence of their induced
transdifferentiation into non-malignant macrophages. However, mac-
rophages were never recovered in vivo, raising the possibility that
6
they were eliminated following apoptosis. In another study, B-ALL
cells carrying a BCR-ABL oncogene have been shown to spontane-
ously transdifferentiate into myeloid cells. In this scenario macro-
phages could be identified in patients carrying the same
immunoglobulin rearrangements as in the leukemic cells of the same
patient, showing that a malignant B cell to normal myeloid cell transi-
7
tion can also happen in vivo and with no apparent cell death.
In contrast, as shown by Wang et al.,1 overexpression of either
Sox2 or Klf4 in AML cells does not initiate a differentiation program
and their co-expression only caused a slight increase in apoptosis,
suggesting that they act redundantly through the same pathway.
However, what they have in common to set this machinery in motion
selectively in cancer cells remains unclear. An intriguing possibility is
that they somehow share the capacity to discriminate differences in
the heterochromatic landscape between cancer and normal cells.
Significance statement
The four transcription factors of the Yamanaka cocktail
(Oct4, Sox2, Klf4 and Myc, termed OSKM) are famously
capable of reprogramming somatic cells into induced plurip-
otent stem cells (iPSCs). In a paper published Dec. 6 in
Nature Communications, Wang et al. now describe the
unexpected discovery that short-term activation of OSKM
expression in acute myeloid leukemia cells in vivo induces
apoptosis while negligibly affecting normal hematopoietic
stem and progenitor cells1. These findings have potential
implications for novel anti-cancer strategies.
Studies by Kenneth Zaret and colleagues have described that a large
proportion of the mouse cell genome decorated with H3K9me3 is ini-
tially resistant to the binding of the OSKM pluripotency factors.8 They
recently showed that these regions are specifically associated with
almost two hundred proteins, and proteins tested individually were
found to impair reprogramming.9 An intriguing hypothesis therefore is
that AML differs from normal hematopoietic progenitors in the distri-
bution of heterochromatin sub-domains and that the interaction with
Sox2 and Klf4, but not Oct4, leads to H3K9me3 de-methylation that
activates apoptosis specifically in the cancer cells. However, things
may turn out to be more complex, as OSKM factors have been
described to not only induce reprogramming into iPSCs, but also
cause apoptosis in fibroblasts.8 In addition, Klf4 has been described to
behave as an anti-apoptotic oncogene in some cellular contexts and
as an apoptosis-inducing suppressor gene in others.10 Another exam-
ple for such a context dependency is the interaction of OSKM and
MLL-AF9 cells itself: earlier work by the Cheng lab has shown that
when the factors are activated in culture conditions that favor the
outgrowth of pluripotent stem cells, iPSC colonies can be obtained.2
In contrast, under the in vivo conditions described here, where MLL-
AF9 cells grow in the environment of normal hematopoietic cells, this
does not seem to occur. Together, these observations suggest that
both the cells’ epigenetic state and their environment influence the
outcome of the effect induced by cell fate-instructive transcription
factors.
In relationship to the possible clinical relevance of the described
phenomenon it is worth pointing out that a broad array of cancers are
enriched in mutations within genes that encode enzymes affecting
H3K9 methylation.11 The experiments described by Wang et al.,
pointing to a selective de-methylation by the Yamanaka factors of
H3K9 in malignant blood cells, therefore warrant searching for combi-
nations of compounds that mimic the effect of OSKM in cancer cells.
In this context it would be interesting to know if soluble factors or
chemical compounds that have been described to functionally replace
Sox2 or Klf4 during reprogramming (see for example references,12,13)
induce apoptosis of MLL-AF9 cells but not that of normal cells and
whether the combination with H3K9me3 inhibitors enhances the
effects. The search for compounds active in such screens could
XIE AND GRAF
represent an as yet unexplored therapeutic approach for leukemia
therapy that promises to yield drugs that exhibit lower tissue toxicity
than currently used chemotherapeutic regimens.
ACKNOWLEDGMENTS
H.X. was supported by funds form the National Key R&D Program of
China (2018YFA0108200) and the National Natural Science Founda-
tion of China (31970625). T.G. was supported by funds from MINECO
(Plan Estatal 2015, SAF2015-68740-P) and the European Research
Council (Synergy Grant 4D-Genome).
DIS CLOSURE OF POTENTI AL CONF LICTS OF INTERES T
The authors declared no potential conflict of interest.
AUTHOR CONTRIBUTION
“HX and TG wrote the piece”.
DATA AVAI LAB ILITY S TATEMENT
Data sharing is not applicable to this article as no new data were cre-
ated or analyzed in this study.
ORCID
Huafeng Xie https://orcid.org/0000-0002-8387-5368
RE FE R ENC E S
1. Wang Y, Lu T, Sun G, et al. Targeting of apoptosis gene loci by repro-
gramming factors leads to selective eradication of leukemia cells. Nat
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2. Liu Y, Cheng H, Gao S, et al. Reprogramming of MLL-AF9 leukemia
cells into pluripotent stem cells. Leukemia. 2014;28:1071-1080.
3. Hochedlinger K, Blelloch R, Brennan C, et al. Reprogramming of a
melanoma genome by nuclear transplantation. Genes Dev. 2004;18:
1875-1885.
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4. Abad M, Mosteiro L, Pantoja C, et al. Reprogramming in vivo pro-
duces teratomas and iPS cells with totipotency features. Nature.
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5. Lo-Coco F, Avvisati G, Vignetti M, et al. Retinoic Acid and Arsenic Tri-
oxide for Acute Promyelocytic Leukemia. N Engl J Med. 2013;369:
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6. Rapino F, Robles EF, Richter-Larrea JA, Kallin EM, Martinez-
Climent JA, Graf T. C/EBPα Induces Highly Efficient Macrophage
Transdifferentiation of B Lymphoma and Leukemia Cell Lines and
Impairs Their Tumorigenicity. Cell Rep. 2013;3:1153-1163.
7. McClellan JS, Dove C, Gentles AJ, Ryan CE, Majeti R. Reprogramming
of primary human Philadelphia chromosome-positive B cell acute lym-
phoblastic leukemia cells into nonleukemic macrophages. Proc Natl
Acad Sci U S A. 2015;112:4074-4079.
8. Soufi A, Garcia MF, Jaroszewicz A, Osman N, Pellegrini M, Zaret KS.
Pioneer transcription factors target partial DNA motifs on nucleo-
somes to initiate reprogramming. Cell. 2015;161:555-568.
9. Becker JS et al. Genomic and Proteomic Resolution of Heterochroma-
tin and Its Restriction of Alternate Fate Genes. Mol. Cell. 2017;68:
1023-1037.e15.
10. Ghaleb AM, Yang VW. Krüppel-like factor 4 (KLF4): What we cur-
rently know. Gene. 2017;611:27-37.
11. Tollefsbol TO, ed. Epigenetics in Human Disease. Vol 6. second ed.
Academic Press; 2018.
12. Ichida JK, Blanchard J, Lam K, et al. A Small-Molecule Inhibitor of Tgf-
β Signaling Replaces Sox2 in Reprogramming by Inducing Nanog. Cell
Stem Cell. 2009;5:491-503.
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How to cite this article: Xie H, Graf T. Selective killing of
leukemia cells: Yamanaka factors’ new trick. Stem Cells. 2020;
9999:n/a. https://doi.org/10.1002/stem.3173
4

T A B L E 1 Effects of OSKM activation

MLL-AF9 cells cKit positive HSPCs
in AML cells and normal hematopoietic
P53, Puma, activated Caspase 3 Increase No change progenitors.
% 7AAD/Annexin positive cells Increase No change
Global H3K9me3 levels Decrease No change
Suv39h1/h2 expression Strong decrease Decrease
Kdm3a expression Strong increase Increase
Chaetocin-induced apoptosis High Low
XIE AND GRAF 5

F I G U R E 1 A. Schematics of the experimental protocol used to demonstrate the selective anti-tumor effect of the OSKM factors in vivo.
Briefly, HSPCs from reprogrammable mice were isolated, infected in culture for 2 days with a retrovirus containing MLL-AF9-GFP and
transplanted into sub-lethally irradiated mice. One week later the percentage of GFP+ cells in the bone marrow and blood was determined and
animals treated with doxycycline (doxy) for 1 week to activate OSKM in the donor cells or left untreated (no doxy). The mice were then
monitored for several months for the onset of leukemia and death. While untreated animals died within 20 days, most of the mice with transient
OSKM activation survived for at least one year. B. MLL-AF9 infected HSPCs from reprogrammable mice were competitively repopulated with
control HSPCs from reprogrammable mice and the recipients treated with doxy. This showed that the MLL-AF9 cells died while the control
HSPCs survived

(A)
Mice with
Dox-inducible OSKM
different
MLL-AF9-GFP cells
GFP levels

No doxycycline

Doxycycline
induction

(B)

MLL-AF9 cells Cell death HSPCs Cell survival

+doxycycline +doxycycline
 Graphical Abstract

The contents of this page will be used as part of the graphical abstract of html only.
It will not be published as part of main.

(A)
Mice with
Dox-inducible OSKM
different
MLL-AF9-GFP cells
GFP levels

No doxycycline

Doxycycline
induction

(B)

MLL-AF9 cells Cell death HSPCs Cell survival

+doxycycline +doxycycline

A. Schematics of the experimental protocol used to demonstrate the selective anti-tumor effect of the OSKM factors in vivo. Briefly, HSPCs from
reprogrammable mice were isolated, infected in culture for 2 days with a retrovirus containing MLL-AF9-GFP and transplanted into sub-lethally
irradiated mice. One week later the percentage of GFP+ cells in the bone marrow and blood was determined and animals treated with doxycycline
(doxy) for 1 week to activate OSKM in the donor cells or left untreated (no doxy). The mice were then monitored for several months for the onset
of leukemia and death. While untreated animals died within 20 days, most of the mice with transient OSKM activation survived for at least one
year. B. MLL-AF9 infected HSPCs from reprogrammable mice were competitively repopulated with control HSPCs from reprogrammable mice
and the recipients treated with doxy. This showed that the MLL-AF9 cells died while the control HSPCs survived.