Hematopoietic and Chronic Myeloid Leukemia Stem Cells: Multi-Stability versus Lineage Restriction

Abstract

There is compelling evidence to support the view that the cell-of-origin for chronic myeloid leukemia is a hematopoietic stem cell. Unlike normal hematopoietic stem cells, the progeny of the leukemia stem cells are predominantly neutrophils during the disease chronic phase and there is a mild anemia. The hallmark oncogene for chronic myeloid leukemia is the BCR-ABLp210 fusion gene. Various studies have excluded a role for BCR-ABLp210 expression in maintaining the population of leukemia stem cells. Studies of BCR-ABLp210 expression in embryonal stem cells that were differentiated into hematopoietic stem cells and of the expression in transgenic mice have revealed that BCR-ABLp210 is able to veer hematopoietic stem and progenitor cells towards a myeloid fate. For the transgenic mice, global changes to the epigenetic landscape were observed. In chronic myeloid leukemia, the ability of the leukemia stem cells to choose from the many fates that are available to normal hematopoietic stem cells appears to be deregulated by BCR-ABLp210 and changes to the epigenome are also important. Even so, we still do not have a precise picture as to why neutrophils are abundantly produced in chronic myeloid leukemia.

Full text

Citation: Brown, G. Hematopoietic
and Chronic Myeloid Leukemia Stem
Cells: Multi-Stability versus Lineage
Restriction. Int. J. Mol. Sci. 2022,23,
13570. https://doi.org/10.3390/
ijms232113570
Academic Editor: Haifa
Kathrin Al-Ali
Received: 19 October 2022
Accepted: 3 November 2022
Published: 5 November 2022
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International Journal of
Molecular Sciences
Review
Hematopoietic and Chronic Myeloid Leukemia Stem Cells:
Multi-Stability versus Lineage Restriction
Geoffrey Brown
Institute of Clinical Sciences, School of Biomedical Sciences, College of Medical and Dental Sciences, University of
Birmingham, Birmingham B15 2TT, UK; g.brown@bham.ac.uk; Tel.: +44-(0)12-1414-4082
Abstract:
There is compelling evidence to support the view that the cell-of-origin for chronic myeloid
leukemia is a hematopoietic stem cell. Unlike normal hematopoietic stem cells, the progeny of the
leukemia stem cells are predominantly neutrophils during the disease chronic phase and there is a
mild anemia. The hallmark oncogene for chronic myeloid leukemia is the BCR-ABLp210 fusion gene.
Various studies have excluded a role for BCR-ABLp210 expression in maintaining the population
of leukemia stem cells. Studies of BCR-ABLp210 expression in embryonal stem cells that were
differentiated into hematopoietic stem cells and of the expression in transgenic mice have revealed
that BCR-ABLp210 is able to veer hematopoietic stem and progenitor cells towards a myeloid fate. For
the transgenic mice, global changes to the epigenetic landscape were observed. In chronic myeloid
leukemia, the ability of the leukemia stem cells to choose from the many fates that are available to
normal hematopoietic stem cells appears to be deregulated by BCR-ABLp210 and changes to the
epigenome are also important. Even so, we still do not have a precise picture as to why neutrophils
are abundantly produced in chronic myeloid leukemia.
Keywords:
chronic myeloid leukemia; leukemia stem cells; hematopoietic stem cells; oncogenes;
epigenetics
1. Introduction
From findings in the 1950s, tumorigenesis is a multistep process [
1
] and a complex
sequence of events needs to be completed to transform a normal cell into a cancerous
one. The identification of oncogenes has been one of the major advances to unraveling the
biology of cancer. Oncogenic insults to a cell arise from cellular genes (proto-oncogenes)
that are mutated, and then typically dominant in nature, and chromosomal translocations,
that give rise to a constitutively active fusion protein, such as a signaling kinase, or that
place a strong promoter close to the involved genes leading to overexpression. The normal
gene products control cell survival, proliferation or differentiation, and the oncogene
products essentially deregulate the controls on these processes. Hence, oncogenes have an
intrinsic capacity to control cell survival, proliferation, and differentiation.
However, the induced expression of an oncogene within cells, even to a very high level,
does not lead invariably to cell transformation. The BCR-ABLp210 fusion gene (also termed
BCR-ABL1) arises from the t(9; 22) (q34; q11) reciprocal translocation, is characteristic of
chronic myeloid leukemia (CML), and the chimeric protein has constitutive kinase activity.
Expression of the BCR-ABLp210 protein at a high level did not transform NIH-3T3 cells,
but expression of BCR-ABLp210 with C terminal rearrangements transformed permissive
NIH-3T3 subclones [
2
,
3
]. Changes to the C terminal had activated the transformation
capacity of BCR-ABLp210, and a particular function domain(s) is/are required for trans-
formation. The transformation of particular subclones brings to attention that there is an
important dynamic relationship between the functionality of the BCR-ABLp210 protein
and constraints that prevail within the intracellular environment. Cell status also influences
the precise nature of the outcome from oncogene expression within cells. The oncogenes
Int. J. Mol. Sci. 2022,23, 13570. https://doi.org/10.3390/ijms232113570 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022,23, 13570 2 of 13
CTNNB1,TERT, and MYC are characteristic of liver cancer, but they induced senescence in
primary human hepatocytes and fibroblasts, and when fibroblasts were reprogrammed to a
liver progenitor cell (induced hepatocytes) they transformed these cells [
4
]. The environ-
ment a cell resides in is a further consideration to whether an oncogene transforms a cell
or fails to do so. Mutant KRAS-G12V induces pancreatic ductal adenocarcinoma in mice
when expressed in embryonic acinar lineage cells whereas chronic pancreatitis is needed
for KRAS-G12V to induce pancreatic tumors in adult mice [
5
]. For adult mice, changes to
the environment will have altered the intracellular status of cells. Barr has argued in favor
of cell context-specific mechanisms of transformation because there is also the need for
the avoidance of any oncogene-mediated toxic effects and a complex relationship exists
between a translocation event and the juxtaposed loci [
6
]. The oncogene’s nature, the target
cell’s status, and the environment a cell resides in are all part of the complex series of events
for cancer.
As target cell status is important to successful transformation, we need to be certain
about the cell-of-origin of a cancer to unravel its transformation process. This review focuses
on CML because it is well known to arise from a hematopoietic stem cell (HSC), and, as
mentioned above, its hallmark oncogene BCR-ABLp210 is well established. The course of
the disease is complex. Neutrophils are massively over produced at disease presentation
and during the chronic phase. At three to five years after onset, CML generally progresses
to a blast and accelerated phase. Two thirds of cases have blasts with a phenotype similar
to that of acute myeloid leukemia (AML) cells. The remaining cases have blasts with a
lymphoid morphology. Consideration is given to the influence of BCR-ABLp210 expression
on the behavior of CML leukemia stem cells (LSCs) that give rise to the chronic phase of
disease, and why the behavior of these cells is very different to that of HSCs.
2. CML Arises in an HSC
CML is a clonal disease arising from a pluripotent HSC, as revealed by studies in the
late 1960s of the expression of the glucose-6-phosphate dehydrogenase (G-6-PD) isoen-
zymes A and B within cells from three women with CML [7,8]. For normal cells, only one
G-6-PD gene is active in each female cell, due to X chromosome inactivation, and they
are a mixture of cells that express either the A or B isoenzyme. One enzyme isotype is
present in the blood granulocytes, erythrocytes, platelets, monocytes, macrophages, and B
lymphocytes of CML patients’ cells. A stem cell that is common to the development of the
above cell types is the origin of CML.
We can exclude a committed myeloid progenitor cell as the target for transforma-
tion in CML from consideration of the action of induced BCR-ABLp210 expression. LSCs
must self-renew to sustain leukemia. Common myeloid progenitors (CMP) and granulo-
cyte/monocyte progenitors (GMP) do not self-renew and the BCR-ABLp210 protein lacks
the capacity to confer self-renewal upon CMPs and GMPs. CMPs and GMPs were trans-
duced with BCR-ABLp210 and did not serially re-plate when cultured in methycellulose
supplemented with cytokines, and BCR-ABLp210 expression did not affect their survival
and differentiation. When BCR-ABLp210 transduced CMPs and GMPs were injected into
a lethally-irradiated congenic mouse, there was no evidence of leukemia at the time of
sacrifice between 113 and 240 days. In essence, BCR-ABLp210 had not conferred an essential
attribute of LSCs within committed murine myeloid progenitors [9].
An HSC origin for CML is not exceptional because some of the sub-types of AML
arise from an HSC. Just a small fraction (0.01%) of human AML cells was able to initiate
leukemia in nonobese diabetic-severe combined immunodeficient mice, their cell surface
phenotype corresponded to that of a normal HSC (CD34++, CD38-), and, like HSCs, the
cells that initiated leukemia were able to self-renew. These findings led to the cancer stem
cell model which states that rare cancer stem cells (CSCs) generate the hierarchy of cells that
sustains a cancer, with the progeny of CSCs differentiating either partially or fully [10,11].
A general view is that leukemias and other cancers, originate from a rare population of
cells. From early mapping of the various hematopoietic progenitor cells (HPC), these rare

Int. J. Mol. Sci. 2022,23, 13570 3 of 13
cells were included as ‘target’ cells for transformation for some of the human leukemias [
12
].
Acute promyelocytic leukemia (AML-M3) was seen as arising in a myeloid-committed
progenitor, as there is an excessive production of promyelocytic blasts [
13
,
14
]. Childhood
acute B-cell lymphoblastic leukemia (B-ALL) and T-cell acute lymphoblastic leukemia
were described as arising in a B-cell lineage and T-cell lineage committed progenitors,
respectively, as there is an excessive production of B-cell lineage blast cells and T-cell
lineage blast cells, respectively [
12
]. More recent analyses have revealed a more primitive
origin for acute promyelocytic leukemia [
15
], childhood B-ALL [
16
–
18
], and infant MLL-AT4
B-cell precursor ALL [19]. Chronic lymphocytic leukemia has long been seen as arising in
an antigen-experienced B-cell, whereas new evidence supports the view that this leukemia
arises from transformation of an HSC [
20
,
21
]. The origins of some leukemias, for example,
childhood B-ALL, is still a matter of debate [
22
]. Even so, it is conceivable that all of the
leukemias arise from an HSC, with cancers in general arising from a tissue-specific stem cell.
CSCs have been identified for bladder [
23
], head and neck squamous cell carcinoma [
24
],
lung [
25
], pancreatic [
26
], prostate [
27
], and sarcoma [
28
], but there is often uncertainty
regarding whether their normal counterpart is a tissue-specific stem cell or a progenitor
cell because of a lack of appropriate markers.
3. CML LSCS Are Restricted to Neutrophil Production during Chronic Phase
The most striking feature of CML LSCs is a massive over production of neutrophils
during the chronic phase of disease, and there is a mild anemia. This restriction of CML
LSCs to myeloid cell production during the chronic phase is supported by findings from
the induced expression of BCR-ABLp210 in transgenic mice. The targeting of BCR-ABLp210
to the bone marrow hematopoietic stem and progenitor cells, via stem cell antigen (Sca1)-
BCR-ABLp210, led to a leukemia resembling human chronic phase CML [
29
,
30
]. BCR-
ABLp210 expression was also targeted to the bone marrow stem cell compartment using
the tetracyclin (tet)-off system. The transgenic mice developed a human CML-like disease
upon the induction of BCR-ABLp210 expression (tet withdrawal), and the disease was
transplantable by the use of bone marrow cells that lacked lineage markers (lin-) and that
expressed the Sca-1 antigen and the c-kit receptor (c-kit+) for stem cell factor (termed
LSK), which is a classical signature for HSCs. The disease was fatal in the transgenics and
primary transplant recipients [
31
]. From these findings, the BCR-ABLp210 oncogene is able
to restrict very primitive bone marrow cells to a neutrophil fate. Perhaps cell status-related
factors, e.g., transcription factors and cell signaling, interact with BCR-ABLp210 leading to
a massive expansion of myeloid cells.
4. What Is the Role of BCR-ABLp210?
For many years, CML was seen as a “one-hit tumor”, and therapeutic approaches
focused on achieving complete inhibition of the kinase activity of the BCR-ABLp210 protein.
However, the kinase activity is not required for the survival and self-renewal of CML LSCs
because they are insensitive to the second-generation inhibitors dasatinib, nilotinib, and
bosutinib that are used to treat patients, and their persistence leads to minimal residual dis-
ease in patients [
32
,
33
]. For human CD34+ CML cells, BCR-ABLp210 knockdown achieves
partial inhibition of BCR-ABLp210 activity and the addition of dasatinib completely in-
hibited the phosphorylation of CrkL and STAT5. A substantial proportion of the CD34+
CML cells survived (~50%) when cultured [
31
]. For the tet-off BCR-ABLp210 transgenic
model and when BCR-ABLp210 expression was shut off, CML LSCs persisted
in vivo
and
upon re-expression of BCR-ABLp210 they were able to initiate leukemia in secondary
recipients [31].
We might exclude BCR-ABLp210 from having a role in the maintenance/proliferation
of CML LSCs, and therefore does induced BCR-ABLp210 expression influence their dif-
ferentiation? The engineered expression of BCR-ABLp210 in embryonal stem (ES) cells
allowed sustained expression when these cells were differentiated by using OP9 cell layers
to provide support. The BCR-ABLp210+ ES cells differentiated into hemangioblasts which

Int. J. Mol. Sci. 2022,23, 13570 4 of 13
produced HSCs, HPCs, and finally mature blood cells. The outcomes from the expression
of the BCR-ABLp210 gene were two-fold. Multipotent and myeloid HPCs were increased
and there was suppression of erythroid progenitor cell development. From
in vitro
colony
formation assays, there was a dominance of myeloid colonies, with the balance of myeloid
to erythroid colonies shifting from 1:2 to 4:1. Therefore, BCR-ABLp210 expression can
directly and acutely expand a very immature cell type with multilineage differentiation
capabilities (c-kit+, lin- or CD34+, lin- cells) and change the balance of lineage development
to favor myeloid development, despite the presence of erythropoietin (Epo) in cultures.
Tet-regulated expression of BCR-ABLp210 allowed investigation of whether the change to
the balance of erythroid versus myeloid colonies was reversable. Shutting of BCR-ABLp210
during the last phase of culture development led to a normal erythroid over myeloid
dominance [34].
Though BCR-ABLp210 can direct the lineage fate of HSCs, the presence of the BCR-
ABLp210 protein alone may not be sufficient to initiate the chronic phase of human CML.
BCR-ABLp210 mRNA, as encoded by the Philadelphia chromosome 22 (chromosome 22
with a piece of 9 attached), is present at a very low level in the cells of individuals who
do not succumb to CML [
35
]. In earlier studies, investigators followed myelodysplasia
patients who eventually developed CML by using X-chromosome linked G-6-PD A or B
isotype expression and observed a peripheral clonal dominance prior to the development
of CML. [
36
]. As mentioned above, CML clones, as identified by G-6-PD isotype expres-
sion, can differentiate into mature blood granulocytes, erythrocytes, platelets, monocytes/
macrophages, and B lymphocytes. The pathogenesis of CML may be, at least, a two-step
process. Another possibility to explain the above phenomenon is that the translocation
might be present in a more differentiated cell that is resistant to the oncogenic effect of the
fusion protein, instead of being in the right, HSC, target cell.
5. Is Another Event Needed for CML before or after the Philadelphia Chromosome?
How might other genetic and epigenetic events, facilitated by BCR–ABLp210 or
otherwise, lead to the onset of the chronic phase of CML? Cells that express BCR–ABLp210
accumulate genetic abnormalities, and this led to the proposal that BCR-ABLp210 is a
multifaceted promotor of DNA mutation [
37
]. Possibilities are that the expression of BCR-
ABLp210 leads to error cascades, and an accumulation of errors by influencing DNA repair
or making changes to the accumulation of DNA damage. Regarding DNA repair, the
BCR-ABLp210 tyrosine kinase facilitates the repair of DNA double-strand breaks, and
other investigators postulated that genetic instability within CML cells may be due to
unfaithful repair of double stranded breaks [
38
]. BCR-ABLp210 is known to enhance the
DNA damage that is provoked by endogenous reactive oxygen species and exogenous
genotoxic agents (reviewed in [
39
]). Presently, the precise involvement of BCR-ABLp210 in
DNA repair processes is unclear.
There is good evidence to support the view that alterations to the epigenome plays
a role in the development of the chronic phase of CML. The tet-inducible transgenic
model of CML was used to show that BCR-ABLp210 triggers DNA methylation changes.
DNA methylation patterns were examined for cells that were harvested as HSCs from
non-induced and control BCR-ABLp210 mice, induced and leukemic BCR-ABLp210 mice,
and repressed and rescued BCR-ABLp210 mice. Cells from the leukemic mice showed a
moderate increase of CpG islands’ DNA methylation levels. The investigators argued that
a single oncogenic protein can trigger changes to DNA methylation at several gene loci
and that the epigenetic abnormalities lead to the escape of the leukemic clone resulting in
disease [
40
]. A pathway to widespread DNA methylation changes involves a class of RNAs
called DNA (cytosine-5)-methytransferase 1 (DNMT1)-interacting RNAs. They originate
from transcriptionally active gene loci and bind with high affinity to DNMT1 to prevent
DNA methylation of the corresponding gene loci. BCR-ABLp210 might negatively regulate
the expression of DNMT1-interacting RNAs to cause the silencing of specific genes [
41
].
From analysis of the proportions of methylated and unmethylated genes in CML stages,

Int. J. Mol. Sci. 2022,23, 13570 5 of 13
other investigators proposed that DNA methylation is increased in advanced disease and
is associated with disease progression, resistance to imatinib, and shortened survival [42].
A significant loss of methylation at CpG islands that have a low-to-moderate level of
methylation in wild-type HSPCs was reported for cells harvested as HSCs/HPCs from the
Sca1-BCR-ABLp210 transgenic mice. This change was lasting within the mature leukemic
myeloid cells, despite an absence of oncogene expression in the mature leukemic cells.
DNMT1 was upregulated within HSCs/HPCs from the mice, and the expression of Dnmt1
in HSCs/HPCs, under control of the Sca1, led to malignancies that were mostly myeloid
with a marked expansion of granulocytes in the bone marrow and blood. DNA hypomethy-
lation was observed for cells from Sca1-DNMT1 transgenic mice, and the pattern was similar
to that observed for the cells from the Sca1-BCR-ABLp210 mice. The investigators concluded
that slight perturbations to the function of DNMTs are sufficient for the chronic phase of
CML because epigenetic reprogramming by itself was sufficient to drive leukemogene-
sis. Again, global changes are important and DNMT1, DNMT3A, and DNMT3B interact
with EZH2, the catalytic subunit of PRC2. Overexpression of DNMT1 may have sterically
hindered the association between DNMT3A and EZH2, and DNMT3A homo-tetramers
efficiently methylate cytosine leading to global hypomethylation [30].
Perturbation to the chromatin landscape has been shown for mutant RUNX1 oncopro-
teins that are able to guide HSC development. Expression of four types of RUNX1 was in-
duced in ES cells which were then differentiated towards hematopoietic cells. RUNX1-ETO
expression led to a bias towards a B cell identity by reducing the expression and binding
of transcription factors (TF) that regulate myeloid differentiation (PU.1 and C/EBP
α
). Ex-
pression of R201Q, which has a mutation in the DNA binding domain, led to a bias away
from megakaryocyte differentiation by reducing the interaction of wild-type RUNX1 with
CBF
β
(a master regulator of hematopoiesis) and increasing GATA1 binding (a TF for ery-
thropoiesis). Differentiation of myeloid and erythroid cells was reduced by RUNX1-EV11
expression. Expression of the mutant proteins perturbed chromatin priming of lineage-
specific sites [
43
], and RUNX1 plays a role in the organization of the chromatin landscape at
the onset of hematopoiesis in order to maintain accessibility [
44
,
45
], and shapes landscapes
via a cascade of direct and indirect targets [46].
Changes to the epigenetic landscape, which in turn control TF and signaling pathway
components, are required for LSCs to initiate the chronic phase of CML LSCs, and, in 1957,
Waddington proposed that an epigenetic landscape dictates stem-cell decision-making.
Developing stem and progenitor cells roll down valleys that branch towards an end-
fate with the ridges to the hills maintaining a chosen fate [
47
]. The valleys and hills are
the epigenetic landscape that controls the expression of key transcription factors. The
existing landscape has also been described as “the judge, jury, and executioner of stem
cell fate” [
48
]. From the ability of BCR-ABLp210 to increase myeloid progenitor cell
development and suppress erythroid progenitor cell development by HSCs (derived from
hemangioblasts), BCR-ABLp210, either alone or in combination with other factors, can
mis-shape the epigenetic landscape.
6. HSC Decision-Making for Lineage Fate
An understanding of how HSCs choose a particular pathway of development is
clearly important to unraveling the lineage restriction of CML LSCs. Therefore, what is the
underlying principle to HSC decision-making? A general view is that cells make binary
decisions that are irreversible such as all-or-nothing decisions. The cellular controls are
bi-stable, with cells switching from one steady state to another when there is a change to
external and/or internal systems. Well-studied examples are whether to undergo apoptosis
or not and to mature or not, with both based on information from the environment reaching
a threshold level which is processed to an outcome [
49
]. Binary decision-making, as
depicted as a tree-like process, also underpins models for the development of the different
cells of an entire organism and longstanding fate maps for hematopoiesis. In classic models,
HSCs first choose between the myeloid and lymphoid pathways of development. A series

Int. J. Mol. Sci. 2022,23, 13570 6 of 13
of stepwise decisions then progressively restrict lineage options to ultimately give rise
to single lineage-restricted HPCs. Routes to each cell type are via preferred intermediate
HPCs [50].
For HSCs and to add to the choice of a developmental pathway, there is the need to
integrate controls on various other cell states. They include the maintenance of survival
and choices between quiescence versus cell division and self-renewal versus differentiation.
Multi-stability allows cells to process more information and to regulate gene expression
for more than two mutually exclusive stable states. It exists for gene regulatory net-
works [
51
,
52
], signaling pathways [
53
,
54
], and metabolic networks [
55
]. Multi-stability
would allow HSCs to switch to an appropriate state to accord with the various changes to
external influences, and modelling has revealed that it is important to HSCs choosing a
cell lineage [
56
]. The TFs GATA1, GATA2, and PU-1 play essential roles in HSC and HPC
development. GATA2 is a driver of hematopoiesis; a high level of expression of GATA1
is likely to veer HSPCs towards megakaryocyte/erythroid development, and a high level
of expression of PU-1 veer HSPCs towards granulocyte/macrophage development. The
framework to the mathematical modeling of the three TFs was based on the embedding
of sub-systems with less stable states and the use of equations that took-into-account the
genes in the system, their regulation, and the various equilibria. Two bi-stable models
were embedded to achieve a tri-stable model which accorded with experimental data. The
tri-stable model was then modified to achieve four stable states. For GATA1 and GATA-2,
the modelling assumed that there is an exchange of GATA1 for GATA2 at the chromatin
site, which then controls GATA1 and GATA2 gene expression. The simulated four sta-
ble states were maintenance of the HSC state related to unsuccessful GATA2 to GATA1
switching when the displacement of GATA2 is not sufficient, a myeloid progenitor state
related to unsuccessful GATA-2 to GATA1 switching when the displacement of GATA2 is
not sufficient and there is a low expression of all three genes, a myeloid progenitor state
related to successful GATA switching and there is a high level of expression of PU.1, and a
megakaryocyte/erythroid progenitor state related to successful GATA switching and there
is a high level of expression of GATA-1.
A high order of multi-stability within HSCs is interesting because it fits with newer
continuum [
57
] and diffusion map models [
58
] for hematopoiesis. In these models, HSCs
can veer directly towards any of the lineage options that are available as a spectrum.
Affiliation to a cell lineage occurs much earlier than previous thought, and the finding
that supports this view is that HSCs are really a consortium of multipotent cells and
subtypes that have an intrinsic bias/affiliation towards a cell lineage. The subtypes include
megakaryocyte- [
59
], lymphoid-, myeloid- and dendritic cell-biased [
60
–
63
] HSCs and
erythroid- and macrophage-affiliated HSCs [64].
Embedding bi-stable models together for multi-stability is in keeping with continuum
and diffusion map models because developmental pathways are still placed close to one
another. The adjacent relationships in a continuum model are megakaryocytes
↔
erythro-
cytes
↔
basophils/mast cells
↔
eosinophils
↔
neutrophils
↔
monocytes
↔
dendritic
cells
↔
B cells
↔
innate lymphoid cells
↔
T cells [
57
]. They were inferred from close
relationships between particular cell lineages as seen for HPCs when assayed
in vitro
. In
addition, the differentiation of HSCs towards each of the various end cell types has long
been attributed to a complex network of TFs. For fates that are contiguous in the above con-
tinuum, there is shared usage of transcription factors [
57
]. For example, GATA-1, GATA-2
and Friend of GATA-1 (FOG-1) are important for the megakaryocyte
→
erythrocyte
→
basophil/mast cell
→
eosinophil span of the spectrum [
65
], and GATA-1 restricts mast cell
development [
66
], possibly by combining with FOG-1 to disrupt the association between
GATA-1 and PU-1 [
67
]. In essence, TFs that play a role in lineage commitment set up a new
gene expression pattern and extinguish others (Figure 1). The importance of the sharing of
TFs to the level of gene expression noise within cells is considered below.

Int. J. Mol. Sci. 2022,23, 13570 7 of 13
Int.J.Mol.Sci.2022,23,xFORPEERREVIEW7of13


setupanewgeneexpressionpatternandextinguishothers(Figure1).Theimportanceof
thesharingofTFstothelevelofgeneexpressionnoisewithincellsisconsideredbelow.

Figure1.SharedusageofTFsbycontiguousfates.ThecoordinatedactivityofmultipleTFsisim‐
portanttowhetheracelladoptsoravoidsaparticularfate.TFscanpromotethedevelopmentof
onecelltypeorsetofadjacentcelltypesandsuppressneighboringfates.Boxesthatareshaded
greenshowthatTFsmustbeactivetodirectthedevelopmentoftheparticularcelltype,andtheTF
inhibitstheadoptionofafateforboxesthatareshadedred(reviewedin[57]).Meg,megakaryocyte;
Ery,erythroid;Bas,basophil/mastcell;Eos,eosinophil;Neu,neutrophil;Mon,monocyte;DC,den‐
driticcell;B,Bcell;ILC,innatelymphoidcell;T,Tcell.
Morerecentmolecularstudiescapturedtheglobaltranscriptomeofdeveloping
HSCsfromsinglecellRNAsequencingandlinkedthisinformationtocellfate.Thenear‐
neighborpathwaysinacontinuumweretowardserythrocytes,basophils,neutrophils,
monocytes,dendriticcells,Bcells,andTcells[68],whichissimilartotheabove.Fromthe
singlecellRNAsequencing,HSCdevelopmentisaprogressiveprocesswithbroaddevel‐
opmentaltrajectoriesallowingcellstomovetotheleftorrightofaninitialchosenfateto
onethatisadjacentinthelandscape[58].Alternativefatesmightbeviewedasremaining
latentwithinHSCsandHPCs,andthisplasticityisimportanttoconsiderationofwhether
theprogenyofCMLLSCsarerestrictedtotheneutrophilpathwaybecausethecell‐of‐
originisanHSCwithanintrinsicbias/affiliationtowardsneutrophils.Thisisunlikely
becausedevelopingHSCsandHPCscan“changetheirmind”.
7.NoiseandBurstingofGeneExpression
Thereremainsanimportantquestion:HowdolineageoptionsarisewithinHSCsin
thefirstinstance?Thereisnaturalvariationtotheexpressionoflineage‐affiliatedgenes
withinHSCsbecause,asmentionedabove,sub‐setsofmouseHSCsexpressmRNAfor
thereceptorforEpoatalowlevelandthereceptorformacrophagecolony‐stimulating
factor(M‐CSF)attheirsurfaceatalowlevel[64].Amuchearlierfindingfromtheuseof
RT‐PCRtomeasuremRNAlevelswasthattherewaslowlevelexpressionofanumberof
lineage‐affiliatedgeneswithinthemultipotentmurineFDCP‐mixA4cellspriortocelllin‐
eagecommitment.Variableexpressionwasobservedforsinglecellsforthereceptorsfor
Epo,granulocytecolony‐stimulatingfactor,granulocyte/macrophagecolony‐stimulating
factorandM‐CSF.LowlevelmRNAexpressionwaspromiscuousbecauseβ‐globin
(erythroid)andmyeloperoxidase(myeloid)expressionoccurredwithinthesamecell,and
thismightrelatetotranscriptionalepisodesandmRNApersistenceorcellscycling
throughprograms[69].AstothenoisewithinHSCsregardingtheexpressionofgenes
thatencodelineage‐affiliatedreceptors,itisimportanttobearinmindthattheircytokines
Figure 1.
Shared usage of TFs by contiguous fates. The coordinated activity of multiple TFs is
important to whether a cell adopts or avoids a particular fate. TFs can promote the development of
one cell type or set of adjacent cell types and suppress neighboring fates. Boxes that are shaded green
show that TFs must be active to direct the development of the particular cell type, and the TF inhibits
the adoption of a fate for boxes that are shaded red (reviewed in [
57
]). Meg, megakaryocyte; Ery,
erythroid; Bas, basophil/mast cell; Eos, eosinophil; Neu, neutrophil; Mon, monocyte; DC, dendritic
cell; B, B cell; ILC, innate lymphoid cell; T, T cell.
More recent molecular studies captured the global transcriptome of developing HSCs
from single cell RNA sequencing and linked this information to cell fate. The near-neighbor
pathways in a continuum were towards erythrocytes, basophils, neutrophils, monocytes,
dendritic cells, B cells, and T cells [
68
], which is similar to the above. From the single cell
RNA sequencing, HSC development is a progressive process with broad developmental
trajectories allowing cells to move to the left or right of an initial chosen fate to one that
is adjacent in the landscape [
58
]. Alternative fates might be viewed as remaining latent
within HSCs and HPCs, and this plasticity is important to consideration of whether the
progeny of CML LSCs are restricted to the neutrophil pathway because the cell-of-origin
is an HSC with an intrinsic bias/affiliation towards neutrophils. This is unlikely because
developing HSCs and HPCs can “change their mind”.
7. Noise and Bursting of Gene Expression
There remains an important question: How do lineage options arise within HSCs in
the first instance? There is natural variation to the expression of lineage-affiliated genes
within HSCs because, as mentioned above, sub-sets of mouse HSCs express mRNA for
the receptor for Epo at a low level and the receptor for macrophage colony-stimulating
factor (M-CSF) at their surface at a low level [
64
]. A much earlier finding from the use of
RT-PCR to measure mRNA levels was that there was low level expression of a number
of lineage-affiliated genes within the multipotent murine FDCP-mixA4 cells prior to cell
lineage commitment. Variable expression was observed for single cells for the receptors for
Epo, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating
factor and M-CSF. Low level mRNA expression was promiscuous because
β
-globin (ery-
throid) and myeloperoxidase (myeloid) expression occurred within the same cell, and this
might relate to transcriptional episodes and mRNA persistence or cells cycling through
programs [
69
]. As to the noise within HSCs regarding the expression of genes that en-
code lineage-affiliated receptors, it is important to bear in mind that their cytokines can
instruct HSC fate. M-CSF instructs myeloid lineage fate within single HSCs
in vitro
, and
intravenous injection of recombinant M-CSF into mice increased activation of the myeloid-
associated TF PU.1 in long term reconstituting-HSCs and the proportion of myeloid-biased
HSCs [
70
]. Epo instructs an erythroid fate within multipotent HPCs and decreases myeloid

Int. J. Mol. Sci. 2022,23, 13570 8 of 13
output [
71
]. A low level of expression of cytokine receptors by HSCs cells may allow these
cells to explore various fates leading to an eventual survival dependency as they differen-
tiate further. For the soil bacterium Pseudomonas putida, it is interesting that noise within
metabolic regulatory networks allows cells to explore various nutritional landscapes [72].
Noise is widespread within cells and is caused by the complex dynamics to the TF reg-
ulation of gene expression together with protein turnover. The gene cis-regulatory elements
that are accessible to TFs, as described as chromatin nuclease hypersensitive sites, are scat-
tered throughout the nucleus. TFs complexed with chromatin remodelers/modifiers and
bound to cis-regulatory elements lead to the activation of gene expression. Gene expression
noise is drastically enhanced by spatial fluctuations in TF levels, due to diffusion [
73
], and
the sharing of TFs between genes [74]. As considered above, developing HSPCs share the
usage of TFs to promote or prevent the adoption of a fate [
57
]. The strength of signals that
a cell receives from its environment are integrated into the process of TF-mediated gene
regulation and also influence noise.
Hence, noise and the bursting of gene expression at various sites throughout the
genome is very likely to play a role in how lineage options are made available to HSCs
(Figure 2). It has been proposed that noise distorts the epigenetic landscape to shape cell
decision-making [
75
]. Furthermore, to take-into-account that the signals received by a cell
influence noise to distort the epigenome, its geometric landscape has been modelled for ES
cells directed by appropriate signals towards the neural and mesoderm fates [
76
]. However,
and by contrast to a focus on changes to the epigenetic landscape guiding decision-making,
the promyeloid cell line HL60 is able to differentiate into macrophages, neutrophils, mono-
cytes, and monocyte-derived macrophages, and when HL60 cells differentiated along these
pathways there were few differential changes in the chromatin landscape for up to 24 h.
Instead, changes occurred during the middle to late stages of differentiation [77].
Int.J.Mol.Sci.2022,23,xFORPEERREVIEW8of13


caninstructHSCfate.M‐CSFinstructsmyeloidlineagefatewithinsingleHSCsinvitro,
andintravenousinjectionofrecombinantM‐CSFintomiceincreasedactivationofthemy‐
eloid‐associatedTFPU.1inlongtermreconstituting‐HSCsandtheproportionofmyeloid‐
biasedHSCs[70].EpoinstructsanerythroidfatewithinmultipotentHPCsanddecreases
myeloidoutput[71].AlowlevelofexpressionofcytokinereceptorsbyHSCscellsmay
allowthesecellstoexplorevariousfatesleadingtoaneventualsurvivaldependencyas
theydifferentiatefurther.ForthesoilbacteriumPseudomonasputida,itisinterestingthat
noisewithinmetabolicregulatorynetworksallowscellstoexplorevariousnutritional
landscapes[72].
NoiseiswidespreadwithincellsandiscausedbythecomplexdynamicstotheTF
regulationofgeneexpressiontogetherwithproteinturnover.Thegenecis‐regulatoryel‐
ementsthatareaccessibletoTFs,asdescribedaschromatinnucleasehypersensitivesites,
arescatteredthroughoutthenucleus.TFscomplexedwithchromatinremodelers/modifi‐
ersandboundtocis‐regulatoryelementsleadtotheactivationofgeneexpression.Gene
expressionnoiseisdrasticallyenhancedbyspatialfluctuationsinTFlevels,duetodiffu‐
sion[73],andthesharingofTFsbetweengenes[74].Asconsideredabove,developing
HSPCssharetheusageofTFstopromoteorpreventtheadoptionofafate[57].The
strengthofsignalsthatacellreceivesfromitsenvironmentareintegratedintotheprocess
ofTF‐mediatedgeneregulationandalsoinfluencenoise.
Hence,noiseandtheburstingofgeneexpressionatvarioussitesthroughoutthege‐
nomeisverylikelytoplayaroleinhowlineageoptionsaremadeavailabletoHSCs(Fig‐
ure2).Ithasbeenproposedthatnoisedistortstheepigeneticlandscapetoshapecellde‐
cision‐making[75].Furthermore,totake‐into‐accountthatthesignalsreceivedbyacell
influencenoisetodistorttheepigenome,itsgeometriclandscapehasbeenmodelledfor
EScellsdirectedbyappropriatesignalstowardstheneuralandmesodermfates[76].
However,andbycontrasttoafocusonchangestotheepigeneticlandscapeguidingdeci‐
sion‐making,thepromyeloidcelllineHL60isabletodifferentiateintomacrophages,neu‐
trophils,monocytes,andmonocyte‐derivedmacrophages,andwhenHL60cellsdifferen‐
tiatedalongthesepathwaystherewerefewdifferentialchangesinthechromatinland‐
scapeforupto24h.Instead,changesoccurredduringthemiddletolatestagesofdiffer‐
entiation[77].

Figure2.LowlevelburstingofgeneexpressionwithinHSCs.HSCsareableto‘choose’directly
fromalloftheoptions.Theburstingofgeneexpressionatavarioussiteforaparticularfateisshown
bythebrightyellowhighlightedclouds.Thenoiseisnotexclusivetojustapathwayofdevelopment
becausedevelopmentaltrajectoriesarebroadandtherearenear‐neighborrelationshipsbetweenthe
Figure 2.
Low level bursting of gene expression within HSCs. HSCs are able to ‘choose’ directly from
all of the options. The bursting of gene expression at a various site for a particular fate is shown by
the bright yellow highlighted clouds. The noise is not exclusive to just a pathway of development
because developmental trajectories are broad and there are near-neighbor relationships between
the cell lineages. The close relationships shown between the pathways of development are as for
continuum and diffusion map models of hematopoiesis. Trajectories are broad and binary flips to an
adjacent landscape are shown by the clouds with a reduced yellow highlighting. The depiction is in
keeping with the mathematical embedding together of bi-stable models.
8. Waking up Old and Damaged Cells
As mentioned above, the BCR-ABLp210 oncogene occurs in the cells of individuals
who do not develop CML, and these cells are apparently normal. An entirely new view of
cancer has questioned whether oncogenes are the tipping point to the onset of cancer. Of

Int. J. Mol. Sci. 2022,23, 13570 9 of 13
interest were why non-smokers get lung cancer, if carcinogen-induced DNA damage is all
important, and how air pollutant particulate matter 2.5 (PPM2.5) causes cancer without
damaging DNA. As to the latter and for mice, PPM2.5 exposure led to the release of
interleukin-1
β
in the lung, which caused inflammation, whereby activated cells help to
repair lung damage. Blocking the action of interleukin-1
β
and inflammation prevented
the formation of lung cancers. Additionally, a surprising finding was that the risk of lung
cancer was cut when the action of interleukin-1
β
was blocked in a cardiovascular disease
trial. Thus, how might the release of interleukin-1
β
be the tipping point to lung cancer?
For a 50-year old person, lung cells with potentially cancer mutations are at a frequency of
around one in every 600,000 cells, due to damage to our cell’s DNA as we age. The new
postulate is that the released interleukin-1
β
had woken up damaged cells that appear to be
healthy but are normally inactive to give rise to lung cancer [78].
The waking up of pre-existing and damaged cells that appear to be normal may be an
element that is missing from our understanding of the onset of CML. There is damage to
the genome of stem cells as they divide to replenish themselves. Three mutations occur
every time they divide, resulting from random mistakes during DNA replication [
79
]. For
self-renewing tissues, more than half of the somatic mutations occur prior to the initiation
of tumors [
80
]. There is also a strong correlation between normal stem cell divisions and
cancer incidence as seen from studies of the risk of 17 types of cancer [
81
]. The lineage
capabilities of HSCs changes with age because there is a predominance of myeloid-biased
cells within the HSC compartment of aged mice [61,82]. The complex process of retention
of the availability of all of the lineage options might, therefore, be prone to error. As to
an increase in myeloid-biased of HSCs with age, it is interesting to note that around 50%
of CML patients are aged 66 and older [
83
]. The new view to the onset of lung cancer
may apply to many, if not all, cancers. Therefore, an as-yet-unseen interleukin or colony-
stimulating factor may play a role in the overproduction of neutrophils by CML LSCs as a
counterpart to age-related damage to the genome, BCR-ABLp210 expression and changes
to the epigenetic landscape (Figure 3).
Int.J.Mol.Sci.2022,23,xFORPEERREVIEW9of13


celllineages.Thecloserelationshipsshownbetweenthepathwaysofdevelopmentareasforcon‐
tinuumanddiffusionmapmodelsofhematopoiesis.Trajectoriesarebroadandbinaryflipstoan
adjacentlandscapeareshownbythecloudswithareducedyellowhighlighting.Thedepictionisin
keepingwiththemathematicalembeddingtogetherofbi‐stablemodels.
8.WakingupOldandDamagedCells
Asmentionedabove,theBCR‐ABLp210oncogeneoccursinthecellsofindividuals
whodonotdevelopCML,andthesecellsareapparentlynormal.Anentirelynewviewof
cancerhasquestionedwhetheroncogenesarethetippingpointtotheonsetofcancer.Of
interestwerewhynon‐smokersgetlungcancer,ifcarcinogen‐inducedDNAdamageis
allimportant,andhowairpollutantparticulatematter2.5(PPM2.5)causescancerwithout
damagingDNA.Astothelatterandformice,PPM2.5exposureledtothereleaseofinter‐
leukin‐1βinthelung,whichcausedinflammation,wherebyactivatedcellshelptorepair
lungdamage.Blockingtheactionofinterleukin‐1βandinflammationpreventedthefor‐
mationoflungcancers.Additionally,asurprisingfindingwasthattheriskoflungcancer
wascutwhentheactionofinterleukin‐1βwasblockedinacardiovasculardiseasetrial.
Thus,howmightthereleaseofinterleukin‐1βbethetippingpointtolungcancer?Fora
50‐yearoldperson,lungcellswithpotentiallycancermutationsareatafrequencyof
aroundoneinevery600,000cells,duetodamagetoourcell’sDNAasweage.Thenew
postulateisthatthereleasedinterleukin‐1βhadwokenupdamagedcellsthatappearto
behealthybutarenormallyinactivetogiverisetolungcancer[78].
Thewakingupofpre‐existinganddamagedcellsthatappeartobenormalmaybe
anelementthatismissingfromourunderstandingoftheonsetofCML.Thereisdamage
tothegenomeofstemcellsastheydividetoreplenishthemselves.Threemutationsoccur
everytimetheydivide,resultingfromrandommistakesduringDNAreplication[79].For
self‐renewingtissues,morethanhalfofthesomaticmutationsoccurpriortotheinitiation
oftumors[80].Thereisalsoastrongcorrelationbetweennormalstemcelldivisionsand
cancerincidenceasseenfromstudiesoftheriskof17typesofcancer[81].Thelineage
capabilitiesofHSCschangeswithagebecausethereisapredominanceofmyeloid‐biased
cellswithintheHSCcompartmentofagedmice[61,82].Thecomplexprocessofretention
oftheavailabilityofallofthelineageoptionsmight,therefore,bepronetoerror.Astoan
increaseinmyeloid‐biasedofHSCswithage,itisinterestingtonotethataround50%of
CMLpatientsareaged66andolder[83].Thenewviewtotheonsetoflungcancermay
applytomany,ifnotall,cancers.Therefore,anas‐yet‐unseeninterleukinorcolony‐stim‐
ulatingfactormayplayaroleintheoverproductionofneutrophilsbyCMLLSCsasa
counterparttoage‐relateddamagetothegenome,BCR‐ABLp210expressionandchanges
totheepigeneticlandscape(Figure3).

Figure 3.
Waking up of damaged cells that pre-exist. Damage to the normal HSC genome occurs
from random mistakes during DNA replication, and BCR-ABLp210 is known to enhance DNA
damage. The BCR-ABLp210 tyrosine kinase facilitates the repair of DNA double-strand breaks,
but it has been proposed that there is genetic instability within CML cells due to the unfaithful
repair of double stranded breaks. There are BCR-ABLp210-provoked changes to the epigenetic
landscape. The damaged cells are apparently normal but potentially cancerous. They are inactive and
woken up by signals received from the environment, perhaps from interleukins or colony stimulating
factors (CSFs).
9. Concluding Remarks
The behavior of CML LSCs is very different from that of HSCs. There is substantial
natural variation to lineage options within HSCs, and for CML LSCs there is an intrinsic

Int. J. Mol. Sci. 2022,23, 13570 10 of 13
stability regarding the neutrophil fate during the chronic phase of disease. Global changes
to the epigenome are important, as seen from the studies of transgenic mouse models of
CML, and they may influence the noise to/priming of lineage fates and bursting of gene
expression. CML might then be viewed as a perturbation to the epigenetic landscape to
normal stem cell development. During HSC cell fate specification there is likely to be the
need to buffer noise to ensure a ‘chosen’ outcome. For CML LSCs, we might speculate
that there is inappropriate/excessive buffering of fate options and that myeloid fate is
facilitated by BCR-ABLp210 or some other event. From the transgenic mouse studies
and as seen for the progeny of BCR-ABLp210+ hemangioblasts, BCR-ABLp210 can veer
HSC development towards a myeloid fate. For BCR-ABLp210-mediated transformation,
there may be a connection between BCR-ABLp210 expression and the bursting of myeloid
gene expression at this moment in time. The waking up of apparently healthy, inactive,
and damaged cells adds a further consideration to the onset and nature of CML, but the
importance of this new view on cancer to CML remains to be seen.
Presently, we do not have a clear explanation as to why the progeny of CML LSCs
are directed towards a neutrophil fate during the chronic phase. The way forward is to
develop a deeper understanding of how complex networks of cis-regulator elements and
TFs and changes to the epigenetic landscape cooperate to allow HSCs to affiliate to a cell
lineage. It seems that the epigenetic landscape is the judge, jury and executioner, but the
precise manner of this entire process is still unclear. Even so, perhaps CML disease and
other tissue-specific stem cell cancers arise from deregulation of the epigenome.
Funding:
G.B. received funding from the European Union’s Seventh Framework Programme for
research, technological development and demonstration under grant agreement no. 315902. G.B. was
the coordinator of the Marie Curie Initial Training Network DECIDE.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The author declares no conflict of interest.
References
1. Fould, L. The natural history of cancer. J. Chronic Dis. 1958,8, 2–37. [CrossRef]
2.
Shore, S.K.; La Cava, M.; Yendapalli, S.; Reddy, E.P. Structural alterations in the carboxyl-terminal domain of the BCRABL gene
product activate its fibroblastic transforming potential. J. Biol. Chem. 1994,269, 5413–5419. [CrossRef]
3.
Renshaw, M.W.; McWhirter, J.R.; Wang, J.Y.J. The human leukemia oncogene bcr-abl abrogates the anchorage requirement but not
the growth factor requirement for proliferation. Mol. Cell. Biol. 1995,15, 1286–1293. [CrossRef] [PubMed]
4. Barr, F.G. Translocations, cancer, and the puzzle of specificity. Nat. Genet. 1998,19, 121–124. [CrossRef] [PubMed]
5.
Sahu, B.; Pihlajama, P.; Zhang, K.; Palin, K.; Ahanan, S.; Cervera, A.; Ristimaki, R.; Aaltanen, L.A.; Hautaniemi, S.; Taipale, J.
Human cell transformation by combined lineage conversion and oncogene expression. Oncogene
2021
,40, 5533–5547. [CrossRef]
[PubMed]
6.
Guerra, C.; Schuhmacher, A.J.; Canamero, M.; Grippo, P.J.; Verdaguer, L.; Perez-Gallego, L.; Dubus, P.; Sandgren, E.P.; Barbacid,
M. Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice. Cancer
Cell 2007,11, 291–302. [CrossRef]
7.
Fialkow, P.J.; Gartler, S.M.; Yoshida, A. Clonal origin of chronic myelocytic leukaemia in man. Proc. Natl. Acad. Sci. USA
1967
,58,
1468–1471. [CrossRef]
8.
Fialkow, P.J.; Jacobson, R.J.; Papyannopoulou, T. Chronic myelocytic leukaemia: Clonal origin in a stem cell common to the
granulocyte erythrocyte, platelets and monocyte/macrophage. Am. J. Med. 1977,63, 125–130. [CrossRef]
9.
Huntly, B.J.P.; Shigematsu, H.; Deguchi, K.; Lee, B.H.; Mizuno, S.; Duclos, N. MOZ-TIF2, but not BCR-ABL, confers properties of
leukemic stem cells to committed murine hematopoietic progenitors. Cancer Cell 2004,6, 587–596. [CrossRef]
10.
Bonnet, D.; Dick, J.E. Human acute myeloid leukemia is organised as a hierarchy that originates from a primitive hematopoietic
cell. Nat. Med. 1997,7, 730–737. [CrossRef]
11. Dick, J.E. Stem cell concepts renew cancer research. Blood 2008,112, 4793–4807. [CrossRef]
12.
Greaves, M.F. Analysis of the clinical and biological significance of lymphoid phenotypes in acute leukemia. Cancer Res.
1981
,
41, 4752–4766.
13. Grimwade, D.; Enver, T. Acute promyelocytic leukemia: Where does it stem from? Leukaemia 2004,18, 375–384. [CrossRef]

Int. J. Mol. Sci. 2022,23, 13570 11 of 13
14.
Turhan, A.; Lemoine, F.M.; Debert, C.; Bonnet, M.L.; Baillou, C.; Picard, F.; Macintyre, E.A.; Varet, B. Highly purified primitive
hematopoietic stem cells are PML-RARA negative and generate nonclonal progenitors in acute promyelocytic leukemia. Blood
1995,85, 2154–2161. [CrossRef]
15.
Edwards, R.H.; Wasik, M.A.; Finan, J.; Rodriguez, R.; Moore, J.; Kamoun, M.; Rennert, H.; Bird, J.; Nowell, P.C.; Salhany, K.E.
Evidence for Early Hematopoietic Progenitor Cell Involvement in Acute Promyelocytic Leukemia. Am. J. Clin. Pathol.
1999
,112,
819–827. [CrossRef]
16.
Quijano, C.A.; Moore, D., II; Arthur, D.; Feusner, J.; Winter, S.S.; Pallavicini, M.C. Cytogenetically aberrant cells are present in the
CD34+ CD33
−
38
−
19
−
marrow compartment in children with acute lymphoblastic leukemia. Leukemia
1997
,11, 1508–1515.
[CrossRef]
17. Cox, C.V.; Blair, A. A primitive cell origin for B-cell precursor ALL. Stem Cell Rev. 2005,1, 189–196. [CrossRef]
18.
Hirt, A.; Schmid, A.-M.; Amman, R.A.; Leibundgut, K. In pediatric lymphoblastic leukemia of B cell origin, a small population of
primitive blast cells is noncycling, suggesting them to be leukemia stem candidates. Pediatr. Res. 2011,69, 194–199. [CrossRef]
19.
Malouf, C.; Ottersbach, K. The fetal liver lymphoid-primed multipotent progenitor provides the prerequisites for the initiation of
t(4;11) MLL-AT4 infant leukemia. Haematologica 2018,103, e573. [CrossRef]
20.
Alizadeh, A.A.; Majeti, R. Surprise! HSCs are aberrant in chronic lymphocytic leukemia. Cancer Cell
2011
,20, 135–136. [CrossRef]
21.
Kikushige, Y.; Iskikawa, F.; Miyamoto, T.; Shima, T.; Urata, S.; Yashimoto, T.; Mari, Y.; Iiono, T.; Yamauchi, T.; Eto, T.; et al.
Self-renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic lymphocytic leukemia. Cancer Cell
2011,20, 246–259. [CrossRef] [PubMed]
22.
Bernt, K.M.; Armstrong, S.A. Leukemia stem cells and acute lymphoblastic leukemia. Semin. Hematol.
2009
,46, 33–38. [CrossRef]
[PubMed]
23.
She, J.J.; Zhang, P.G.; Wang, Z.M.; Gan, W.M.; Che, X.M. Identification of side population cells from bladder cancer cells by
dyecycle violet staining. Cancer Biol. Ther. 2008,7, 1663–1668. [CrossRef] [PubMed]
24.
Prince, M.E.; Sivanandan, R.; Kaczorowski, A.; Wolf, G.T.; Kaplan, M.J.; Dalerba, P.; Weissman, I.L.; Clarke, M.F.; Ailles, L.E.
Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc. Natl.
Acad. Sci. USA 2007,104, 973–978. [CrossRef] [PubMed]
25.
Eramo, A.; Lotti, F.; Sette, G.; Pilozzi, E.; Biffoni, M.; Di Virgilio, A.; Contocello, C.; Roco, L.; Peschle, C.; De Maria, R. Identification
and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 2008,15, 504–514. [CrossRef]
26.
Hermann, P.C.; Huber, S.L.; Herrler, T.; Aicher, A.; Ellwart, J.W.; Guba, M.; Bruns, C.J.; Heeschen, C. Distinct populations of cancer
stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell
2007
,1, 313–323. [CrossRef]
27.
Collins, A.T.; Berry, P.A.; Hyde, C.; Stower, M.J.; Maitland, N.J. Prospective identification of tumorigenic prostate cancer stem
cells. Cancer Res. 2005,65, 10946–10951. [CrossRef]
28.
Wu, C.; Wei, Q.; Utomo, V.; Nadesan, P.; Whetstone, H.; Kandel, R.; Wunder, J.S.; Alman, B.A. Side population cells isolated from
mesenchymal neoplasms have tumor initiating potential. Cancer Res. 2007,67, 8216–8222. [CrossRef]
29.
Pérez-Caro, M.; Cobaleda, C.; González-Herrero, I.; Vicente-Dueñas, C.; Bermejo-Rodríguez, C.; Sánchez-Beato, M.; Orfao,
A.; Pintado, B.; Flores, T.; Sánchez-Martín, M.; et al. Cancer induction by restriction of oncogene expression to the stem cell
compartment. EMBO J. 2008,28, 8–20. [CrossRef]
30.
Vicente-Dueñas, C.; González-Herrero, I.; Sehgal, L.; García-Ramírez, I.; Rodríguez-Hernández, G.; Pintado, B.; Blanco, O.; Criado,
F.J.G.; Cenador, M.B.G.; Green, M.R.; et al. Dnmt1 links BCR-ABLp210 to epigenetic tumor stem cell priming in myeloid leukemia.
Leukemia 2018,33, 249–278. [CrossRef]
31.
Hamilton, A.; Helgason, G.V.; Schemionek, M.; Zhang, B.; Myssina, S.; Allan, E.K.; Nicolini, F.E.; Muller-Tidow, C.; Bhatia, R.;
Brunton, V.G.; et al. Chronic myeloid leukemia stem cells are not dependent on the bcr-abl kinase activity for their survival. Blood
2012,119, 1501–1510. [CrossRef]
32.
Graham, S.M.; Jorgensen, H.G.; Allan, E.; Pearson, C.; Alcorn, M.J.; Richmond, L.; Holyoake, T.L. Primitive, quiescent,
Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571
in vitro
.Blood
2002
,99,
319–325. [CrossRef]
33.
Corbin, A.S.; Agarwal, A.; Loriaux, M.; Cortes, J.; Deininger, M.W.; Druker, B.J. Human chronic myeloid leukemia stem cells are
insensitive to imatinib despite inhibition of BCR-ABL activity. J. Clin. Invest. 2011,121, 396–409. [CrossRef]
34.
Era, T.; Witte, O.N. Regulated expression of P210Bcr-Abl during embryonic stem cell differentiation stimulates multipotent
progenitor expansion and myeloid fate. Proc. Nat. Acad. Sci. USA 2000,97, 1737–1742. [CrossRef]
35.
Biernaux, C.; Loos, M.; Sels, A.; Huez, G.; Stryckmans, P. Detection of major bcr-abl gene expression at a very low level in blood
cells of some healthy individuals. Blood 1995,86, 3118–3122. [CrossRef]
36.
Fialkow, P.J.; Martin, P.J.; Najfeld, V.; Penfold, G.K.; Jacobson, R.J.; Hansen, J.A. Evidence for a multistep pathogenesis of chronic
myelogenous leukemia. Blood 1981,58, 158–163. [CrossRef]
37.
Burke, B.A.; Carroll, M. BCR-ABL: A multifaceted promoter of DNA mutation in chronic myelogenous leukemia. Leukemia
2010
,
246, 1105–1112. [CrossRef]
38.
Slupianek, A.; Nowicki, M.O.; Koptyra, M.; Shorski, T. BCR/ABL modifies the kinetics and fidelity of DNA double-strand break
repair in hematopoietic cells. DNA Repair 2006,5, 243–250. [CrossRef]
39. Skorski, T. BCR/ABL, DNA damage and DNA repair. Leuk. Lymphoma 2008,46, 610–614. [CrossRef]

Int. J. Mol. Sci. 2022,23, 13570 12 of 13
40.
Amabile, G.; Di Ruscio, A.; Muller, F.; Welner, R.S.; Yang, H.; Ebralidze, A.K.; Zhang, H.; Levantini, E.; Qu, L.; Martinelli, G.; et al.
Dissecting the role of aberrant DNA methylation in human leukaemia. Nat. Commun. 2015,6, 7091. [CrossRef]
41.
Ruscio, A.; Welner, R.S.; Tenen, D.G.; Amabile, G. The second hit of DNA methylation. Mol. Cell. Oncol.
2016
,3, e1093690.
[CrossRef] [PubMed]
42.
Jelinek, J.; Gharibyan, V.; Estecio, M.R.H.; Kondo, K.; He, R.; Chung, W.; Lu, Y.; Zhang, N.; Liang, S.; Kantarjian, H.M.; et al.
Aberrant DNA methylation is associated with disease progression, resistance to imatinib and shortened survival in chronic
myelogenous leukemia. PLoS ONE 2011,6, e22110. [CrossRef]
43.
Kellaway, S.G.; Keane, P.; Edgington-White, B.; Regha, K.; Kennett, E.; Bonifer, C. Different mutant RUNX1 oncoproteins program
alternative hematopoietic trajectories. Life Sci. Alliance 2021,4, e202000864. [CrossRef] [PubMed]
44.
Lichtinger, M.; Hoogenkamp, M.; Krysinska, H.; Ingram, R.; Bonifer, C. Chromatin regulation by RUNX1. Blood Cells Mol. Dis.
2010,44, 287–290. [CrossRef] [PubMed]
45.
Lichtinger, M.; Ingram, R.; Hannah, R.; Muller, D.; Clarke, D.; Assis, S.A.; Lie-A-Ling, M.; Noailles, L.; Vijayabasker, M.S.; Liu, M.;
et al. RUNX1 reshapes the epigenetic landscape at the onset of haematopoiesis. EMBO J. 2012,31, 4318–4333. [CrossRef]
46.
Hass, M.R.; Brissette, D.; Parameswaran, S.; Pujato, M.; Donmez, O.; Kottyan, L.C.; Weinrauch, M.T.; Kopan, R. Runx1 shapes the
chromatin landscape via a cascade of direct and indirect targets. PLoS Genet. 2012,17, e1009574. [CrossRef]
47.
Waddington, C.H. The Strategy of the Genes: A Discussion of Some Aspects of Theoretical Biology; Allen & Unwin: London, UK, 1957.
48. Tollervey, J.R.; Lunyak, V.V. Judge, jury and executioner of stem cell fate. Epigenetics 2012,7, 823–840. [CrossRef]
49.
Porter, J.R.; Andrews, B.W.; Iglesias, P.A. A framework for designing and analysing binary decision-making strategies in cellular
systems. Integr. Biol. 2012,4, 310–317. [CrossRef]
50.
Spangrude, G.J.; Heimfeld, S.; Weissman, I.L. Purification and characterization of mouse hematopoietic stem cells. Science
1988
,
241, 58–62. [CrossRef]
51.
Ozbudak, E.M.; Thattai, M.; Lim, H.N.; Shraiman, B.I.; Oudenaarden, A.V. Multistability in the lactose utilization network of
Escherichia coli. Nature 2004,427, 737–740. [CrossRef]
52.
Santos-Moreno, J.; Tasiudi, E.; Stelling, J.; Schaerli, Y. Multistable and dynamic CRISPRi-based synthetic circuits. Nat. Commun.
2020,11, 2746. [CrossRef]
53.
Angeli, D.; Ferrell, J.E.; Sontag, E.D. Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-
feedback systems. Proc. Natl. Acad. Sci. USA 2004,101, 1822–1827. [CrossRef]
54.
Harrington, H.A.; Feliu, E.; Wiuf, C.; Stumpf, M.P. Cellular compartments cause multistability and allow cells to process more
information. Biophys. J. 2013,104, 1824–1831. [CrossRef]
55.
Craciun, G.; Tang, Y.; Feinberg, M. Understanding bistability in complex enzyme-driven reaction networks. Proc. Natl. Acad. Sci.
USA 2006,103, 8697–8702. [CrossRef]
56.
Wu, S.; Zhou, T.; Tian, T. A robust method for designing mutistable systems by embedding bistable subsystems. Syst. Biol. Appl.
2022,8, 10. [CrossRef]
57.
Ceredig, R.; Rolink, A.G.; Brown, G. Models of haematopoiesis: Seeing the wood for the trees. Nat. Rev. Immunol.
2009
,9, 293–300.
[CrossRef]
58.
Nestorowa, S.; Hamey, F.K.; Pijuan Sala, B.; Diamanti, F.; Shepherd, M.; Laurenti, E.; Wilson, N.K.; Kent, D.G.; Gottens, B. A
single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation. Blood
2016
,128, e20–e31. [CrossRef]
59.
Sanjuan-Pla, A.; Macaulay, I.C.; Jensen, C.T.; Woll, P.S.; Luis, T.C.; Mead, A.; Moore, S.; Carella, C.; Matsuoka, S.; Bouriez Jones, T.;
et al. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature
2013
,502, 232–236. [CrossRef]
60.
Challen, G.A.; Boles, N.C.; Chambers, S.M.; Goodell, M.A. Distinct hematopoietic stem cell subtypes are differentially regulated
by TGF-beta1. Cell Stem Cell 2010,6, 265–278. [CrossRef]
61.
Gekas, C.; Graf, T. CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age. Blood
2013
,121,
4463–4472. [CrossRef]
62.
Shimazu, T.; Iida, R.; Zhang, Q.; Welner, R.S.; Medina, K.L.; Alberola-Lla, J.; Kincade, P.W. CD86 is expressed on murine
hematopoietic stem cells and denotes lymphopoietic potential. Blood 2012,119, 4889–4897. [CrossRef]
63.
Naik, S.H.; Perie, L.; Swart, E.; Gerlach, C.; van Rooij, N.; de Boer, R.J.; Schumaker, T.N. Diverse and heritable lineage imprinting
of early haematopoietic progenitors. Nature 2013,496, 229–232. [CrossRef] [PubMed]
64.
Mooney, C.J.; Cunningham, A.; Tsapogas, P.; Toellner, K.M.; Brown, G. Selective expression of flt3 within the mouse hematopoietic
stem cell compartment. Int. J. Mol. Sci. 2017,18, 1037. [CrossRef] [PubMed]
65. Graf, T. Differentiation plasticity of hematopoietic cells. Blood 2002,99, 3089. [CrossRef] [PubMed]
66.
Metcalf, D.; Majewski, I.; Mifsud, S.; Alexander, W.S. Clonogenic mast cells progenitors and their excess numbers in chimeric
BALB/c mice with inactivated GATA-1. Proc. Natl. Acad. Sci. USA 2007,104, 18642. [CrossRef]
67.
Sugiyama, D.; Tanaka, M.; Kitajima, K.; Zheng, J.; Yen, H.; Murotani, T.; Yamatodani, A.; Nakano, T. Differential context-dependent
effects of friend of GATA-1 (FOG1) on mast-cell development and differentiation. Blood 2008,111, 1924. [CrossRef]
68.
Weinreb, C.; Rodriguez-Fraticelli, A.; Camargo, F.D.; Klein, A.M. Lineage tracing on transcriptional landscapes links state to fate
during differentiation. Science 2020,367, aaw3381. [CrossRef]
69.
Hu, M.; Krause, D.; Greaves, M.; Sharkis, S.; Dexter, M.; Heyworth, C.; Enver, T. Multilineage gene expression precedes
commitment in the hematopoietic system. Genes Dev. 1997,11, 774–785. [CrossRef]

Int. J. Mol. Sci. 2022,23, 13570 13 of 13
70.
Mossedegh-Keller, N.; Sarrazins, S.; Kandella, P.K.; Espinosa, L.; Stanley, E.R.; Nutt, S.L.; Moore, L.; Siewke, M.H. M-CSF instructs
myeloid lineage fate in single haematopoietic stem cells. Nature 2013,497, 239–243. [CrossRef]
71.
Grover, R.; Mancini, I.C.; Moore, S.; Mead, A.J.; Atkinson, D.; Rasmussen, K.D.; O’Carrol, D.O.; Jacobsen, S.E.W.; Nerlov, C.
Erythropoietin guides multipotent progenitors towards an erythroid fate. J. Exp. Med. 2014,211, 181–188. [CrossRef]
72.
Silva-Rocha, R.; Perez-Pantoja, D.; de Lorenzo, V. Decoding the genetic networks of environmental bacteria: Regulatory
moonlihjting of the TOL system of Pseudomonas putida. ISME J. 2013,7, 229. [CrossRef]
73.
van Zon, J.; Morelli, M.J.; Tanase-Nicola, S.; Rein ten Wolde, P. Diffusion of transcriptional factors can drastically enhance the
noise in gene expression. Biophys. J. 2006,91, 4350–4367. [CrossRef]
74.
Das, D.; Day, S.; Brewster, R.C.; Choubey, S. Effect of transcription factor sharing on gene expression. PLoS Comput. Biol.
2017
,
13, e1005491. [CrossRef]
75.
Coomer, M.A.; Ham, L.; Stumpf, M.P.H. Noise distorts the epigenetic landscape within cells and shapes cell-fate decisions. Cell
Syst. 2022,13, 83–102. [CrossRef]
76.
Saez, M.; Brassberg, R.; Camacho-Aguillar, E.; Siggia, E.D.; Rand, D.A.; Briscoe, J. Statistically derived geometrical landscapes
capture principles of decision-making dynamics during cell fate transitions. Cell Syst. 2022,13, 12–28. [CrossRef]
77.
Ramirez, R.N.; El-Ali, N.C.; Mager, M.A.; Wyman, D.; Cenesa, A.; Mortazavi, A. Dynamic gene regulatory networks of human
myeloid differentiation. Cell Syst. 2017,4, 416–429. [CrossRef]
78.
Swanton, C.; Hill, W.; Lim, E.; Lee, C.; Weeden, C.E.; Augustine, M.; Chen, K.; Kuan, F.; Marongiu, F.; Rodrigues, F.; et al.
LBA1-Mechanism of action and an actionable inflammatory axis for air pollution induced non-small cell lung cancer: Towards
molecular cancer prevention. Ann. Oncol. 2022,33, S808–S869. [CrossRef]
79.
Lynch, M. Rate, molecular spectrum, and consequences of human mutation. Proc. Natl. Acad. Sci. USA
2010
,107, 961–968.
[CrossRef]
80.
Tomasetti, C.; Vogelstein, B.; Parmigiani, G. Half or more of the somatic mutations in cancers of self-renewing tissues originate
prior to tumor initiation. Proc. Natl. Acad. Sci. USA 2013,110, 1999–2004. [CrossRef]
81.
Tomasetti, C.; Li, L.; Vogelstein, B. Stem cell divisions, somatic mutations, cancer etiology, and cancer prevention. Science
2017
,
355, 1330–1334. [CrossRef]
82.
Beerman, I.; Bhattacharya, D.; Zandi, S.; Sigvardsson, M.; Weissman, I.L.; Bryder, D.; Rossi, D.J. Functionally distinct hematopoietic
stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion. Proc. Natl. Acad. Sci. USA
2010,107, 5465–5470. [CrossRef] [PubMed]
83.
Brunner, A.M.; Campigotto, F.; Sadrzadeh, H.; Drapin, B.J.; Chen, Y.-B.; Neuberg, D.S.; Fathi, A.T. Trends in all cause mortality
among patients with chronic myeloid leukemia: A surveillance epidemiology and end results data base analysis. Cancer
2013
,
119, 2620–2629. [CrossRef] [PubMed]