# The emerging role of Twist proteins in hematopoietic cells and hematological malignancies

N. Merindol, Aurélien Riquet, Vanessa Szablewski, Jean-François Eliaou, A.

Puisieux, Nathalie Bonnefoy

► To cite this version:

N. Merindol, Aurélien Riquet, Vanessa Szablewski, Jean-François Eliaou, A. Puisieux, et al. The emerging role of Twist proteins in hematopoietic cells and hematological malignancies. Blood Cancer Journal, 2014, 4, pp.e206. 10.1038/bcj.2014.22. hal-01911065

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REVIEW

# The emerging role of Twist proteins in hematopoietic cells and hematological malignancies

N Merindol<fnref n="1" />, A Riquet<fnref n="1" />, V Szablewski²,³, J-F Eliaou²,⁴, A Puisieux⁵ and N Bonnefoy²

Twist1 and Twist2 (Twist1-2) are two transcription factors, members of the basic helix-loop-helix family, that have been well established as master transcriptional regulators of embryogenesis and developmental programs of mesenchymal cell lineages. Their role in oncogenesis in epithelium-derived cancer and in epithelial-to-mesenchymal transition has also been thoroughly characterized. Recently, emerging evidence also suggests a key role for Twist1-2 in the function and development of hematopoietic cells, as well as in survival and development of numerous hematological malignancies. In this review, we summarize the latest data that depict the role of Twist1-2 in monocytes, T cells and B lymphocyte activation, and in associated hematological malignancies.

Blood Cancer Journal (2014) **4**, e206; doi:10.1038/bcj.2014.22; published online 25 April 2014

INTRODUCTION

Twist1-2 main functions

Twist1 and Twist2 (Twist1-2) are two distinct tissue-restricted
transcription factor members of the basic helix-loop-helix (bHLH)
class B family that display high sequence similarity with each
other. They play a critical role in embryogenesis, particularly in
the inhibition of mesenchymal cell development. In humans,
mutations in *twist1* cause the Saethre-Chotzen syndrome, an
autosomal dominant inheritance disease principally characterized
by craniosynostosis (premature closure of clavicular suture).<sup>1,2</sup>
Heterozygous *twist1<sup>+/-</sup>* mice show craniofacial and limb
abnormalities, whereas *twist1<sup>ko</sup>* mice die early during
embryogenesis. Twist2 is expressed after Twist1 in mesodermal
tissues during embryogenesis. Twist2 inhibits terminal
differentiation of mesoderm-derived cells, such as myocytes,
osteoblasts and adipocytes.<sup>3-6</sup> In humans, mutations in *twist2* are
associated with the Setleis Syndrome, an inherited developmental
disorder characterized by bilateral temporal marks and other facial
features.<sup>7</sup> *Twist2<sup>ko</sup>* mice exhibit normal embryogenesis but die 2-3
days after birth of generalized cachexia caused by high levels of
proinflammatory cytokines.<sup>8</sup>

A complex regulation

The expression of Twist1-2 is triggered by diverse signaling
pathways linked to inflammatory-related cytokines via nuclear
factor (NF)-κB<sup>8,9</sup> and signal transducer and activator of
transcription 3 (STAT3),<sup>10</sup> and to stress conditions via WNT/β-
catenin<sup>11</sup> and hypoxia-inducible factor-1.<sup>12,13</sup> As described
throughout this review, their expression is tightly regulated,
context dependent and multifactorial.

To confuse the situation, once expressed, Twist1-2 may
function either as transcriptional activators or repressors, through
both direct and indirect mechanisms.<sup>14-16</sup> They act through direct
mechanisms when they bind to conserved DNA E-box sequences

(5'-NCANNTGN-3') present in the regulatory region of many
lineage-specific genes¹⁷ and function as transcription factors
(Figure 1); through indirect mechanisms when they activate/
repress gene expression via recruitment of co-activators/repressors
at the E-boxes they are bound to, or when they associate with
other transcription factors such as MEF2, RUNX1, RUNX2, CEBP-α
and NF-κB to modulate gene transcription<sup>5,8,18,19</sup> (Figure 1).

Mechanisms that influence transcription regulation by Twist1-2
include (1) their spatial and temporal regulation, (2) their post-
translational modifications (acetylation and phosphorylation),
(3) their dimer choice, (4) their affinity with potential dimers and
(5) protein-protein interactions (Figure 1).

The most characterized post-translational modification of Twist1
is its phosphorylation on serine 68 (S68) by mitogen-activated
protein kinases such as p38, c-Jun N-terminal kinase and
extracellular signal-regulated kinase-1/2.<sup>20,21</sup> S68 phosphorylation
inhibits ubiquitylation-mediated degradation of Twist1 and thus
regulates its half-life and availability. PKA (protein kinase A), PKC,
PKB/Akt and PP2 can all modulate Twist1 phosphorylation levels
at several other amino acid positions.<sup>20,22-24</sup>

Twist1-2 can also influence gene transcription through
epigenetic modulation of the promoter region. Indeed, Twist1-2
were reported to interact with and recruit histone acetyl
transferases/histone deacetylases to the promoter regulatory
region that results in transcription repression through histone
modification<sup>4,14,15</sup> (Figure 1).

Twist1-2 may form homodimers and heterodimers with class A
bHLH<sup>5,25</sup> E protein transcription factors or E2A that contain both
E12 and E47-Tcf3 and are implicated in immune cell
development.<sup>26</sup> Twist1-2 partner choice depends mostly on its
accessibility and on Twist1-2 phosphorylation acetylation
state,<sup>23,27</sup> and this choice of partner will determine which genes
are transcriptionally targeted (Figure 1). Availability of E proteins is
not only determined by their expression but also by the presence

<footnote n="1">¹Université de Lyon and INSERM U1111, Lyon, France; ²IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U896, Université Montpellier 1, Montpellier, France; ³Département de Biopathologie, Centre Hospitalier Régional Universitaire de Montpellier et Faculté de Médecine, Université Montpellier 1, Montpellier, France; ⁴Département d'Immunologie, Centre Hospitalier Régional Universitaire de Montpellier et Faculté de Médecine, Université Montpellier 1, Montpellier, France and ⁵Centre de Rececherche en Cancérologie de Lyon, INSERM UMR-S1052, Centre Léon Bérard, Lyon, France. Correspondence: Dr N Merindol, 380 rue Pie XII, Trois-Rivières, QC G8W1S8, Canada. or Dr N Bonnefoy, Institut de Recherche en Cancérologie de Montpellier, 208 rue des Apothicaires, Montpellier, F-34298, France.</footnote>

<footnote>E-mail: natachamerindol@yahoo.ca or nathalie.bonnefoy@inserm.fr</footnote>

<footnote>Received 25 February 2014; accepted 17 March 2014</footnote>
<figure><img src="image_3.png" /></figure>

**Figure 1.** Transcriptional regulation by Twist proteins. (a) The bHLH partner competition. Twist1-2 might act directly as transcription factors through binding to E-boxes (5'-NCANNTGN-3') in the promoter regulatory region of multiple lineage-specific genes. They may form homodimers and heterodimers with class A bHLH transcription factor E proteins (E12 and E47). Twist1-2 partner choice depends mostly on their accessibility and on Twist1-2 phosphorylation status. Availability of E proteins is determined by their level of expression and by the presence of Id (inhibition of DNA binding) proteins (Id1, Id2, Id3 and Id4) that lack a DNA-binding domain. The relative stoichiometry of each participant will define partner choice that, in turn, will determine the scheme of gene targeting. E-prt, E proteins; Twist; Twist1 or Twist2. (b) Pathways of transcriptional regulation. Twist1-2 might also activate gene expression through direct interaction with transcription factors (such as MEF2, RUNX1, RUNX2, CEBP-α and NF-κB) and co-activators of transcription factors. They can inhibit transcription through direct interaction and sequestration of transcription factors, their repressors or through epigenetic modulation by recruitment of histone acetyltransferase and histone deacetylase at the promoter region. CoA, co-activator; HAT, histone acetyltransferase; HDAC, histone deacetylase; Rep, repressor; TF, transcription factors.

of a third class of bHLH proteins that lack the DNA-binding domain: namely Id (inhibition of DNA binding) proteins (Id1, Id2, Id3 and Id4). They display great affinity for E2A proteins, in particular for E12, and therefore limit its availability, alter the possible dimerization pools, prevent its DNA binding and compete against Twist1-2<sup>27,28</sup> (Figure 1). The formation of Twist1/E47 heterodimers seems to stabilize Twist1, whereas detection of Twist1/Id1 heterodimers has been associated with destabilization and degradation of Twist1.<sup>29</sup> Twist1-2 also modulate the expression of E protein-dependent genes by sequestrating E-protein modulators. Ultimately, the relative stoichiometry of each E protein, Id repressors and Twist1-2 will determine the expression profile and cell fate, that is, homodimers of Twist will be preferentially found when Id proteins sequestrate E12 (Figure 1). Finally, in 2010, Danciu and Whitman<sup>30</sup> proposed that the redox state of the cell is also involved in transcriptional outcome by stabilizing disulphide bonds between Twist1-2 homodimers and thus preventing heterodimer formation.

### A major role for Twist1-2 in regulating inflammation

The perinatal death due to high levels of proinflammatory cytokines of mice with a *twist2*<sup>ko</sup> genotype or carrying *twist1* and *twist2* haploinsufficiency hints at a key role for Twist1-2 in the control of proinflammatory response, and illustrates the complexity of the transcriptional regulation mechanisms by and of Twist1-2. On one hand, they both regulate proinflammatory cytokine expression by interfering with NF-κB-dependent gene transcription. They interact physically with NF-κB (as demonstrated for Twist1 in COS cells), and bind to E-boxes in the regulatory regions of NF-κB-regulated cytokines, such as tumor necrosis factor-α (TNFα) and interleukin (IL)-1β, as shown in

murine macrophages<sup>8</sup> (Figure 1). Furthermore, Twist2 is able to inhibit IL12 and interferon-γ (IFNγ) expression and to activate the production of the anti-inflammatory cytokine IL-10,<sup>31</sup> whereas upon phosphorylation on S42 by PKB/Akt, Twist1 is able to enhance anti-inflammatory TGFβ receptor signaling.<sup>21</sup>

On the other hand, Twist1-2 expression itself is activated by NF-κB, suggesting the existence of a negative feedback loop where NF-κB pro- and anti-apoptotic pathways are activated by cytokines (such as TNFα), leading to downstream activation of other cytokines and of Twist1-2. In turn, Twist1-2 interact with p65/RelA subunit of NF-κB, causing the repression of NF-κB-mediated transactivation of cytokines.<sup>8</sup> Their activation and interaction by and with NF-κB is conserved across species. This negative feedback loop sets Twist1-2 as central modulators of the NF-κB proinflammatory pathway.

Finally, their role in the proinflammatory response incidentally implicates Twist1-2 in the modulation of the immune system response. Indeed, recent studies show their considerable role in immune cell function. Moreover, dysregulation of Twist1 or Twist2 are implicated in the pathogenesis of various hematological malignancies.<sup>31-33</sup>

## FUNCTIONS OF TWIST1-2 IN HEMATOPOIETIC LINEAGES

### Myeloid lineage

In the hematopoietic system, the expression of Twist1 is largely observed in CD34<sup>+</sup> hematopoietic stem cells,<sup>34-36</sup> whereas Twist2 is mostly expressed in the myeloid lineage.<sup>31</sup> Twist2 is a major negative modulator of both the development of myeloid cells and their proinflammatory responses. It is expressed in granulocyte-macrophage progenitors, and inhibits their proliferation and differentiation into macrophages, neutrophils and basophils,
through direct interaction and inhibition of RUNX1 and C/EBPα transcription factors.³¹ In mature myeloid cells, Twist2 negatively regulates the proinflammatory responses by inhibiting the expression of proinflammatory cytokines such as IL12, IFNγ, IL1, TNFα, IL6, monococyte chemoattractant protein-1 and macrophage inflammatory protein-1α through inactivation of C/EBPα and NF-κB, while activating anti-inflammatory IL10 expression³¹; (Table 1).

In murine mature macrophages, Twist1–2 are also implicated in an anti-inflammatory feedback loop triggered by IFNα to counteract production of TNFα.³³ Indeed, IFN type I strongly induced (1) mRNA and protein expression of Twist1–2 through induction of the receptor tyrosine kinase Axl, (2) responsiveness to Axl ligands and (3) concomitant reduction of TNFα expression. Sharif *et al*.³³ showed evidence that suppression of TNFα expression was mediated in part by Twist1–2 binding to E-boxes in the TNFα promoter region. Twist1–2 also modulate the function of several other transcription factors expressed in macrophages such as E-proteins, myocyte enhancer factor 2 (MEF2) and sterol regulatory element-binding protein-1 (SREBP1), positioning them as central in the regulation of macrophage priming and anti-inflammatory response.⁴,⁵,¹⁸,³⁷

Although its precise role is still unknown, Twist1 is also expressed in follicular dendritic cells that are specialized mesenchymal-derived stromal cells found in the germinal centers of lymphoid tissues that trap and present immune complexes to B cells, enabling their maturation.³⁷

## Lymphoid lineage

**T lymphocytes.** The T lymphocyte family encompasses CD8⁺ T lymphocytes that are cytolytic effector cells, CD4⁺ helper T cells whose role is auxiliary to other immune cells, and CD4⁺ regulatory T cells. T cells are further differentiated into naive, effector memory (EM), central memory (CM) and terminally

differentiated effector memory phenotypes depending on their exposure to and activation by their antigen (reviewed in Appay *et al*³⁸). CD4⁺ helper T lymphocytes are also divided in different subsets that display different functions. The best characterized includes T helper 1 (T<sub>H1</sub>) and T helper 2 (T<sub>H2</sub>), and other subsets such as T helper 17 (T<sub>H17</sub>), follicular helper T cells (TF<sub>H</sub>) and the more recently described T helper 9 (T<sub>H9</sub>) (reviewed in Wan *et al*³⁹).

There are ever-increasing data aiming to decipher the role of Twist1–2 in the T-cell response. These studies seem to suggest that, in this context, Twist1–2 display different and nonredundant functions.

**Twist1 and T lymphocytes.** In 2008, Niesner *et al*.⁴⁰ were the first to identify Twist1 as a key modulator of T<sub>H1</sub> lymphocyte proinflammatory response in mice. They observed that Twist1 expression was predominant in differentiated T<sub>H1</sub> effector memory cells. Accordingly, Twist1 expression was transiently induced following T cell receptor stimulation through NFAT (nuclear factor of activated T cells) and NF-κB, then enhanced and constantly detected upon repeated T cell receptor stimulations. Its expression was also activated by IL12 via STAT4. Following activation of naive T<sub>H1</sub> cells, NFAT, NF-κB and STAT4 had to be bound to their respective binding sites in the promoter region of *twist1* to induce its transcription, whereas in activated T<sub>H1</sub> cells, NFAT and NF-κB binding to the *twist1* promoter region was sufficient to trigger its expression. *Ex vivo*, Twist1 expression was mostly observed in memory CD4⁺ T cells and was especially high in chronically activated CD4⁺ T cells. Furthermore, the authors described that ectopic overexpression of Twist1 in 6-day-old T<sub>H1</sub> cells drastically reduced their pathogenic contribution in inflammation, whereas Twist1 silencing enhanced their potential to induce chronic inflammation. These results imply a key role for Twist1 in limiting inflammation in T<sub>H1</sub> cells. In T cells, as in macrophages, Twist1-mediated repression of NF-κB-activated pathways seems to act through binding to E-boxes in regulatory regions of NF-κB target genes. In particular, Twist1

<table><caption>**Table 1.** Functions of Twist1 and Twist2 in hematopoietic cells</caption><thead><tr><th></th><th>Twist1</th><th>Twist2</th></tr></thead><tbody><tr><td>Myeloid lineage:</td><td>- Key regulators of priming and of the anti-inflammatory response of macrophages<br>Mediators of an anti-inflammatory feedback loop triggered in response to IFNα through Axl to counteract TNFα production.³³<br>Modulation of E-proteins, MEF2 and SREBP1<sup>4,5,19,37</sup></td><td>- Expressed in myeloid progenitors.<br>Inhibition of proliferation and differentiation into macrophages, neutrophils, basophils through inhibition of RUNX1 and C/EBPα.³¹<br>- Expressed in mature myeloid cells.<br>Negative regulation of the proinflammatory response: activation of IL-10, inhibition of IL12, IFNγ, IL1, TNFα, IL6, MCP1 and MIP1α through inactivation of C/EBPα and NF-κB.³¹</td></tr><tr><td>T lymphocytes</td><td>- Expressed mainly in T<sub>H1</sub> lymphocytes<br>Induction by TCR stimulation through NFAT and NF-κB, by IL12 via STAT4<sup>40</sup><br>Prevention of chronic inflammation<sup>40,42,43</sup><br>Inhibition of the proinflammatory response.<sup>40</sup><br>Repression of NF-κB activated pathways, TNFα, IL2, SOCS1, SOCS2, IL1R decoy and Jak2.<sup>40</sup><br>Inhibition of IFN-γ through modulation of T-bet, STAT4 and RUNX3.<sup>41</sup><br>- Inhibition of T<sub>H17</sub> polarization and T follicular helper development.<sup>43</sup><br>Induced by STAT3 to control IL6 signaling</td><td>- Expressed in thymocytes and mature T lymphocytes<br>Prevention of galectin-1 mediated apoptosis after NF-κB activation during negative selection.<sup>19,32</sup></td></tr><tr><td>B lymphocytes</td><td>NA</td><td>Expressed in B cells (unknown function).<sup>45</sup></td></tr></tbody></table>

Abbreviations: C/EBP1, CCAAT/enhancer binding protein; IFN, interferon; IL, interleukin; MCP, monococyte chemoattractant protein; MEF2, myocyte enhancer factor 2; MIP, macrophage inflammatory protein; NA, not available; NF-κB, nuclear factor κ-light-chain-enhancer of activated B cells; NFAT, nuclear factor of activated T cells; RUNX1, runt-related transcription factor-1; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; SREBP1, sterol regulatory element-binding protein-1; TCR, T cell receptor; T<sub>H</sub>, T helper; TNF, tumor necrosis factor.
appeared to dampen inflammation triggered by T<sub>H</sub>1 memory cells in mice experiencing delayed-type hypersensitivity and antigen-induced arthritis through the specific targeting of TNFα, IL2, IFNγ, SOCS1, SOCS2, IL1R decoy and Jak2. Interestingly, and in contrast to the well-known properties of Twist1 in the induction of the epithelial-to-mesenchymal transition (EMT),<sup>41</sup> Twist1 expression did not interfere with the homing capacity of T<sub>H</sub>1 cells to the inflamed tissue.

The authors further validated their results in humans, showing that expression of Twist1 was low in naive CD4⁺ T cells, enhanced in activated effector memory CD4⁺ T cells and even higher in terminally differentiated effector memory CD4⁺ T cells.⁴⁰ Twist1-expressing CD4⁺ T cells exhibited the phenotype of activated T<sub>H</sub>1 effector memory cells (CD45RA⁻ CCR7⁻ CD27⁺/- and CCR5⁺). Interestingly, they also show that Twist1 expression was upregulated and persistent in CD4⁺ T cells recovered from inflamed tissues of patients with chronic inflammation (that is, rheumatic diseases, Crohn's disease and ulcerative colitis), despite therapeutic treatment. However, they did not definitely validate whether Twist1 was a hallmark of T<sub>H</sub>1 rather than of T<sub>H</sub>2 or of T<sub>H</sub>17 polarization in humans (Table 1).

In summary, Twist1 expression in T cells considerably lowered their
contribution to inflammation and tissue destruction in mice.
Expression of Twist1 in T<sub>H</sub>1 cells acts as an endogenous regulator
limiting the proinflammatory potential of T<sub>H</sub>1 cells in the continuous
presence of antigen with repeated activation of NFAT and NF-κB. In
humans, the specificity of Twist1 upregulation in CD4<sup>+</sup> T cells
recovered under a chronic inflammation context lead the authors to
propose that Twist1 could be used as a biomarker of chronic
inflammation.

Pham et al.⁴² further defined the role of Twist1 in T<sub>H</sub>1 cell-mediated inflammation. They showed that Twist1 modulated inflammation, as measured by inhibition of IFNγ production, by decreasing the expression of T-bet, STAT4 and RUNX3 and their binding to the IFNγ locus. Of note, only RUNX3 overexpression was able to compensate for Twist1-mediated inhibition of IFNγ, and this was achieved through direct physical interaction with Twist1. The authors did not observe direct binding of Twist1 to the promoter regions of IFNγ, TNFα and IL12Rβ2. Interestingly, they showed that Twist1 expression was not restricted to T<sub>H</sub>1 cells, but could also be measured in T<sub>H</sub>2 cells, although at a lower level.

In a subsequent study, the same group identified Twist1 as a key factor that hindered T<sub>H</sub>17 polarization.<sup>43</sup> They showed that Twist1 was a key component of a STAT3-induced feedback loop that controls IL6 signaling through inhibition of IL6Rα to block T<sub>H</sub>17 and TF<sub>H</sub> cell development differentiation. Phenotypically, mice with a T cell-specific deletion of Twist1 also exhibited early-onset experimental autoimmune encephalomyelitis, and increased antigen-specific antibody responses, demonstrating a significant role for Twist1 in limiting both cellular and humoral immunity.

*Twist2 and T lymphocytes.* The function of Twist2 in T lympho-cytes and immature thymocytes seems to focus mainly around the prevention of galectin-1-induced apoptosis, a process normally occurring after NF-κB activation during the negative selection in the thymus. This has been explored in two publications by Koh *et al*<sup>19,32</sup> in 2008 and 2009 (Table 1). CD7, a galectin-1 receptor, plays a crucial role in galectin-1-mediated apoptosis of activated T cells. E12 and Twist2 prevent NF-κB-mediated activation of the CD7 promoter. By doing so, they inhibit galectin-1-induced apoptosis in immature T cells. This targeted inhibition appears to be mediated by direct binding to the E-box in the CD7 promoter or through direct interaction with NF-κB. In any case, both bHLH transcription factors significantly reduce binding of NF-κB to the CD7 promoter.<sup>19,32</sup>

These studies characterize Twist2 as a dangerous inhibitor of
negative selection-mediated apoptosis. In this context, it is easily
conceivable that Twist2 uncontrolled expression may lead to
autoimmunity and/or T-cell oncogenesis.

B lymphocytes. The expression of Twist1–2 in normal B lymphocytes has been poorly described so far. Early evidence of their potential expression came from a study published in 2004 by Raval *et al*.⁴⁴ that focused on the *twist2* 5' upstream region and its consequence on expression in chronic lymphocytic leukemia (CLL) patients (see below). DNA methylation occurs in regions rich in CpG islands, typically in the 5' upstream region of genes and results in condensation of the chromatin and downregulation of transcription.⁴⁵ They showed that the *twist2* promoter was not methylated in CD19⁺ B cells purified from healthy controls and that Twist2 mRNA was indeed detected in these cells. In our hands, Twist1–2 could be detected in B cells in response to several proinflammatory signals (N Merindol, unpublished results). However, further studies are definitely needed to confirm the expression and possible functions of Twist1–2 in B cells.

TWIST1–2 AS EMERGING RISK FACTORS IN HEMATOLOGICAL MALIGNANCIES

The overexpression of Twist1 has been described as a poor prognostic factor in numerous epithelium-derived malignancies such as breast cancer, prostate cancer, colorectal cancer, bladder cancer, melanoma, hepatocellular carcinoma and neck carcinoma (reviewed in Puisieux *et al*<sup>46</sup>). Twist1–2 deregulations have also been more recently detected in hematological malignancies. Their precise roles are ill-defined in this latter context, and probable mechanisms of their oncogenic properties must be extrapolated from results obtained in solid tumors that we briefly summarize below.

The central role of Twist1–2 in embryogenesis and mesodermal
development and their targeting of multiple genes coding
for cell-fate proteins inevitably links them to cancer and
oncogenesis. In brief, Twist1 has been implicated in cancer
initiation by counteracting both senescence and apoptosis
programs and in cancer progression by increasing resistance to
treatment.

In cancer cells where Twist1 or Twist2 expression is increased, it
has been demonstrated that they neutralize senescence and cell
death through inhibition of both p53 and Rb tumor suppressor
pathways<sup>47</sup> (Figure 2). Interestingly, Twist1–2 E-boxes are found in
the regulatory region of the cyclin-dependent kinase inhibitors
p21, P15 (INK4B) and p16 that can induce cell cycle arrest upon
DNA damage. Both Twist1–2 repress senescence by inhibiting the
transcription of p53 target p21 and of p16.<sup>47</sup> Furthermore, they
inhibit the expression of p14ARF and perturb the p14ARF–MDM2–
p53 axis.<sup>48</sup> In fact, Twist1–2 are able to repress the p53
proapoptotic response at each step that may lead to its activation,
through: (1) inhibition of transcriptional co-activators of p53, such
as acetyltransferase p300,(2) prevention of p53 phosphorylation
and (3) preclusion of p53 DNA-binding activity.<sup>14,49–51</sup>

Twist1 also offsets the apoptosis response induced by other
pathways in addition to the DNA damage response. For example,
Twist1 is able to block the c-Myc-induced apoptotic response
through modulation of the p14ARF-MDM2-p53 pathway in
*in vitro* experiments on mouse embryonic fibroblasts.⁴⁸ N-Myc
expression is amplified in neuroblastomas accompanied by a
systematic overexpression of Twist1. Prevention of apoptosis by
Twist1 in this context could be partly attributable to impaired
p14ARF activity, once again through hindering of the p14ARF–
MDM2–p53 axis.⁵¹ As a direct consequence of their capacity to
override oncogene-induced senescence and apoptosis, Twist1–2
cooperate *in vitro* and *in vivo* with mitogenic oncoproteins for
malignant transformation⁴⁶,⁴⁹–⁵¹ (Figure 2).

In addition, Twist1–2 were recently shown to be involved in the
direct regulation of microRNAs (such as miR-199a/214 and miR-
10b involved in breast cancer invasiveness)⁵² and of genes linked
to cancer progression.⁵³,⁵⁴ Namely, Twist1 is able to bind to the
promoter of the proto-oncogene AKT2 to upregulate its
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Targets of Twist proteins in the DNA damage response pathway. In healthy conditions, DNA damage triggers the activation of ataxia telangiectasia mutated (ATM) and ATR kinases that usually results in activation of p53, of its transcriptional targets cyclin-dependent kinase inhibitor p21 (CIP/WAF1, CDKN1A) and/or Bax, and ultimately leads to senescence or apoptosis. DNA damage can also induce the cyclin-dependent kinase inhibitor p16 (INK4A) and the tumor suppressor retinoblastoma (Rb) that will further stabilize senescence cell cycle arrest. p14ARF might also come into play during the DNA damage response and is additionally induced by cellular stresses such as oncogene signaling and replication stress. p14ARF is an upstream regulator of p53 that acts through repression of p53-inhibitor MDM2. Myc transcription factor family members promote cell growth and proliferation, but might at the same time induce p53-dependent (through p14ARF) and -independent apoptosis. In cancer cells where Twist1 or Twist2 expression is increased, they neutralize senescence and cell death through inhibition of both p53 and Rb tumor-suppressor pathways. Twist1-2 repress senescence through binding to E-boxes in the regulatory region of p21 and p16. They might also inhibit the expression of p14ARF. Furthermore, Twist1 is able to block the Myc-induced apoptotic response through modulation of the p14ARF-MDM2-p53 pathway. Conversely, they increase cellular proliferation and trigger resistance to chemotherapy through the upregulation of AKT2 and YB-1 and the activation of the antiapoptotic NF-κB response.</figcaption></figure>

expression, and to activate the transcription factor Y-box-
binding protein 1 (YB-1) associated with cellular proliferation.
Overexpression of YB-1 and AKT2 have been observed in several
malignancies and linked to poor prognosis.⁵³,⁵⁴ In breast cancers,
Twist1 oncogenic functions that promote EMT are controlled by its
phosphorylation mediated by PKB/Akt, leading to modulation of
TGFβ2 transcription and enhanced TGFβ receptor signaling.²¹

Twist1 expression is also connected to cancer cell resistance to chemotherapeutic agents, such as taxol and vincristine that target microtubules, and that are used in nasopharyngeal, bladder, ovarian and prostate cancers.⁵³,⁵⁵⁻⁵⁷ This acquired resistance to apoptosis induced by chemotherapeutic drugs triggered by Twist1 is partly because of activation of the antiapoptotic NF-κB response.⁵⁸

Finally, Twist1-2 have also been implicated in metastasis
formation through EMT, thereby facilitating cancer cell inva-
sion.⁴¹,⁵⁹ Twist1 induction of metastasis through EMT plays a

central role in epithelium-based cancers but may not be the
most relevant pathological effect in hematological malignancies,
because they derive from cells with intrinsic motility capacity.

Twist 1–2 in malignancies derived from the myeloid lineage
*Myelodysplastic syndromes (MDS)*. MDS are clonal disorders of
hematopoietic stem/precursor cells characterized by abnormal
maturation and differentiation, an increasing resistance to
proapoptotic signals and a high risk for MDS patients to progress
to acute myeloid leukemia (AML).

Upregulation of TNFα is typically observed in MDS bone marrow (BM) and is associated with a decreased expression of Twist1 in stromal cells.⁶⁰ In contrast, Twist1 expression is augmented in the CD34⁺ hematopoietic stem cell compartment from MDS BMs.³⁴ This increase in Twist1 expression appears to be induced in hematopoietic precursors by stroma contact and more frequent in
advanced-stage compared with low-grade MDS. A physical interaction between endogenous Twist1 and p53 has been detected and associated with resistance to apoptosis through inhibition of proapoptotic protein Bax expression.³⁴ Furthermore, forced inhibition of Twist1 in myeloid cell lines and primary CD34⁺ MDS cells resulted in increased expression of p53 and Bax, and enhanced TNFα-triggered apoptosis (Table 2). Li *et al*.³⁴ also observed an inverse correlation between the protein levels of Twist1 and p53 phosphorylated at serine 46. In 2013, the same group established that interactions between Twist1-dependent miR-10 family member and p53 played a central role in regulating TNFα-mediated apoptosis in MDS clonal cells.⁶¹ They showed that miRs10a/b facilitated apoptotic responses and decreased cell proliferation through direct modulation of the p53 promoter and of downstream targets of NF-κB after transcriptional activation by Twist1. Of note, Twist1 inhibition also increased apoptosis in cell lines displaying mutated p53, suggesting that Twist1 contributes to additional pathways in the apoptotic response to stroma in MDS that have yet to be defined.

**Acute myeloid leukemia.** AML is characterized by a rapid growth and an increased number of myeloid-derived blast cells that

accumulate in the BM, paired with an arrest in their maturation. This process interferes with hematopoiesis and frequently results in hematopoietic insufficiency.

N-Myc overexpression is frequently detected in AML.⁶² Similar to observations made in neuroblastoma, experiments in mice showed that Twist1 is upregulated in AML-expressing N-myc, therefore suggesting that Twist1-mediated disruption of the apoptotic response through the ARF/p53/MDM2 pathway could be involved in N-Myc-positive AML⁶² (Table 2). Furthermore, studies on the myeloid leukemic cell line U937 showed that Twist1 expression activated by the PKB/Akt/mTor (mammalian target of rapamycin) pathway through the neutrophin receptor TrkC (tropomyosin-related kinase C) molecular network (implicated in leukemogenesis)⁶³ was involved in resistance to apoptosis,⁶⁴ whereas when activated by Axl, Twist1 expression was implicated in resistance to treatment.⁶⁵

Finally, plasma levels of TNFα, IL6 and IL10 are increased in AML patients.⁶⁶ IL6 level is inversely correlated to patient survival. IL6 signalization acts through STAT3/5 activation to promote survival in AML⁶⁷ and, in theory, this may lead to the activation of Twist1–2 expression. Importantly, deregulation of Twist1–2 expression in AML patients remains to be demonstrated.

<table><caption>**Table 2.** Functions of Twist1 and Twist2 in hematological malignancies</caption><thead><tr><th></th><th>Twist1</th><th>Twist2</th></tr></thead><tbody><tr><td><strong>Myeloid malignancies</strong></td><td></td><td></td></tr><tr><td>MDS</td><td>- Expressed in CD34<sup>+</sup> hematopoietic stem cells<sup>34</sup>;<br>Expression associated with disease stage<br>- Inhibition of apoptosis<br>Interaction with p53, and Bax inhibition<br>Regulation of TNFα-mediated apoptosis<sup>34,61</sup> through miR-10.<br>Modulator of unknown p53-independent pathway of<br>apoptosis</td><td>NA</td></tr><tr><td>AML</td><td>- Upregulated in N-Myc<sup>+</sup> AML<sup>62</sup>;<br>- Possible implication in resistance to apoptosis<sup>63</sup> and to<br>treatment<sup>65</sup></td><td>- Hypermethylation of twist2 promoter region in some<br>cases of childhood AML<sup>104</sup>;<br>Function unknown</td></tr><tr><td>CML</td><td>- Increased expression<sup>35,68</sup>;<br>- Associated with stage disease and treatment resistance.</td><td>- No hypermethylation of the twist2 promoter<sup>104</sup></td></tr><tr><td><strong>T-cell lymphoma</strong></td><td></td><td></td></tr><tr><td>CTCL</td><td>- Highly expressed in Sz, MF and CD30<sup>+</sup> CTCL.<br>Correlated with MF and Sz disease stage<sup>77,78</sup>;<br>- Probable inhibition of apoptosis through association with<br>c-Myc and p53.<sup>79</sup></td><td>- Highly expressed<br>- Antiapoptotic role<sup>81,82</sup><br>Downregulation of CD7, galectin-1 receptor.</td></tr><tr><td>ALCL</td><td>- Highly expressed<br>Upregulated via STAT3<br>- Probable role in invasiveness and chemoresistance linked to<br>phospho-Akt and Bmi-1<sup>89</sup></td><td>NA</td></tr><tr><td><strong>B-cell lymphoma</strong></td><td></td><td></td></tr><tr><td>DLBCL, MzBCL,<br>FL</td><td>- Overexpressed in DLBCL,<sup>99</sup> MzBCL and FL (unknown<br>function) (Merindol et al., this study, Figure 3a).</td><td>- Not overexpressed (Merindol et al., this study, Figure 3b).</td></tr><tr><td><strong>Leukemia</strong></td><td></td><td></td></tr><tr><td>ALL</td><td>NA</td><td>- Hypermethylation of twist2 promoter region frequent in<br>childhood ALL.<sup>105</sup><br>Associated with loss of Twist2 expression<br>Loss associated with disease stage/relapse<br>- Tumor-suppressor properties<br>Loss of Twist2 linked to cell growth and to resistance to<br>apoptosis and chemotherapeutic agents.</td></tr><tr><td>CLL</td><td>NA</td><td>Frequent promoter region methylation in mutated Ig V<sub>H</sub><br>CLL subtypes<br>Associated with favorable prognosis.<sup>44</sup></td></tr></tbody></table>

Abbreviations: ALCL, anaplastic large cell lymphoma; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; Ig, immunoglobulin; CML, chronic myeloid leukemia; CTCL, cutaneous T-cell lymphoma; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; MDS, myelodysplastic syndrome; MF, mycosis fungoides; MzBCL, marginal zone B-cell lymphoma; NA, not available; STAT, signal transducer and activator of transcription; Sz, Sézary syndrome; TNF, tumor necrosis factor; V<sub>H</sub>, variable heavy chain.
<figure><img src="image_5.png" /></figure>

Chronic myelogenous or myeloid leukemia (CML). CML is a clonal stem cell disorder characterized by an increased number and unregulated growth of myeloid cells in the BM and accumulation of these cells in the blood. This disease is associated with a characteristic chromosomal translocation called the Philadelphia chromosome, consisting of a Bcr-Abl tyrosine kinase fusion.

In 2003, Tipping *et al.*<sup>68</sup> detected the expression of Twist1 in KCL22 CML cell lines in a genome-wide microarray analysis. Twist1 expression was further amplified in KCL22 clones that were resistant to Imatinib, a tyrosine kinase inhibitor (TKI). In 2010, Cosset *et al.*<sup>35</sup> confirmed that Twist1 expression increased in CD34<sup>+</sup> hematopoietic stem cells and CD34<sup>-</sup> cells throughout CML disease progression. Its expression significantly decreased upon complete remission and increased again after relapse. Furthermore, they observed a 100-fold increase of Twist1 expression in TKI nonresponder compared with TKI responder samples (Table 2). The increase in Twist1 expression was specific to TKI-resistant patients in the CD34<sup>+</sup> cells compartment. Silencing of Twist1 by RNA interference experiments in cell lines decreased cell number and viability in response to Imatinib, whereas overexpression of Twist1 led to improved Imatinib resistance of previously sensitive cell lines. Therefore, Twist1 seems to be implicated in resistance to TKI in cell lines and the authors suggest that Twist1 may be used as a novel predictive marker of TKI resistance in CML.

A recent study proposed a new role for Twist1 in CML. Xin *et al.*<sup>69</sup> studied differences between the CML cell lines K562 and the multidrug-resistant K562/A02 derived by enrichment through treatment with doxorubicin. They showed that Twist1 was highly expressed in the multidrug-resistant cell lines derived from K562. Moreover, twist1 knockdown decreased the expression of stem cell markers such as Oct4, Sox2 and Nanog in this multidrug-resistant cell line, suggesting that, in addition to participating in chemoresistance, Twist1 is involved in maintaining tumor-initiating or cancer stemness properties.

## Twist1-2 in lymphoid lineage-derived malignancies

### T-cell lymphoma

Cutaneous T-cell lymphoma (CTCL). CTCLs are a group of lymphoproliferative disorders characterized by localization of neoplastic T cells in the skin. Classical forms of CTCLs are plaque-type mycosis fungoides (MF) and Sézary syndrome (Sz). The link between MF and Sz remains controversial. Sz was described by Albert Sézary, it can arise *de novo* or evolve from MF and is considered a rare leukemic variant. Sz is characterized by erythroderma, peripheral lymphadenopathy and the presence of malignant T cells that are CD4<sup>+</sup>CD45RO<sup>+</sup> and CD7<sup>-</sup>CD26<sup>-</sup> in the skin, lymph nodes and peripheral blood, chronically activated and skewed toward T<sub>H</sub>2 differentiation.<sup>70</sup> These T cells are derived from the same T-cell clone.<sup>71</sup> MF is a slowly progressive disease (87% 5-year survival), whereas Sz is more aggressive with a poor prognosis (11% 5-year survival). In both diseases, chemotherapy and BM transplantation are not curative and do not significantly improve survival.

Twist1. Sz and MF are defined by aberrations in four pathways: (1) evasion of activation-induced cell death, (2) T<sub>H</sub>2-biased lymphocyte differentiation, (3) TGFβ receptor expression and (4) modulation of TNF receptor ligands (reviewed in Dulmage *et al*<sup>70</sup>). Molecularly, typical observations of these diseases include the constitutive activation of STAT3<sup>72,73</sup> and NK-κB,<sup>74</sup> decreased expression of TGFβ receptor II, of downstream modulators such as SMAD3/5 and of its ligand TGFβ1,<sup>75</sup> diminished detection of p15 and p16<sup>76</sup> and dysregulation of the c-Myc and of TNF-associated pathways.

All these molecular alterations point toward a possible implication of Twist1. In 2004, van Doorn *et al.*<sup>77</sup> first identified high overexpression of the tyrosine kinase receptor EphA4 and of

the transcription factor Twist1 in Sz<sup>78</sup> (Table 2). Similar results were observed in a subset of patients with MF and with CD30-negative primary CTCL. Although they did not perform any mechanistic experiments, the authors proposed that Twist1 could block c-Myc-induced apoptosis by antagonizing the p53 pathway. In 2012, a study by Goswami *et al.*<sup>79</sup> showed that Twist1 expression was indeed correlated with MF and Sz stages. Moreover, they observed an association between increased Twist1 and c-Myc expression and abnormal p53 expression. The precise role of Twist1 and of other Sz- and MF-specific upregulated proteins (that is, PLS3, KIR3DL2, NKp36) in CTCL remains to be characterized.<sup>80</sup>

Twist2. Similar to conclusions from experiments in primary T lymphocytes, studies by SH Koh *et al.*<sup>19,32</sup> suggested that Twist2, like Twist1, plays an antiapoptotic role in T-cell lymphoma but through a distinct pathway (Table 2). Indeed, they showed that Twist2 was overexpressed in mature T-cell lymphoma cells and specifically highly expressed in Sz cells. Ectopic expression of Twist2 was able to downregulate the expression of the galectin-1 receptor CD7 through induction of chromatin deacetylation. This led to a decrease in galectin-1-mediated apoptosis in activated T cells that promoted malignant T-cell accumulation and progression of T-cell lymphoma. Accordingly, CD7 expression is dramatically downregulated in Sz and galectin-1-induced apoptosis by CD7 is inhibited.<sup>81,82</sup>

Of note, downregulation of CD7 is also related to the progression of adult T-cell lymphoma/leukemia where galectin-3-induced apoptosis is inhibited,<sup>83</sup> although Twist2 expression has not yet been detected in this context.

Anaplastic large cell lymphoma (ALCL). ALK<sup>+</sup> ALCL represents a small subset of non-Hodgkin's T-cell lymphoma characterized by a specific chromosomal translocation and the fusion of nucleophosmin (NPM) and anaplastic large cell lymphoma kinase (ALK) genes in the majority of cases. This leads to ubiquitous expression of ALK, whose expression is normally restricted to the brain and nervous system. NPM-ALK promotes oncogenesis by activating cellular signaling proteins, including STAT3, MEK/ERK, mTOR and (PI3K)/Akt (reviewed in Amin *et al*<sup>84</sup> and Chiarle *et al*<sup>85</sup>). The activation of these pathways has been linked to Twist1 expression in other malignancies as well as to increased cell proliferation and resistance to apoptosis.

NPM-ALK promotes tyrosine phosphorylation of STAT2 on Y705, essential for dimerization and activation of STAT3.<sup>86</sup> Inhibition of STAT3 leads to apoptosis and cell cycle arrest in ALK<sup>+</sup> ALCL cell lines and is required for its lymphomagenesis.<sup>87,88</sup> In 2012, Zhang *et al.*<sup>89</sup> reported that STAT3 directly upregulated Twist1 and that Twist1 was highly expressed in three ALK<sup>+</sup> ALCL cell lines and *ex vivo* in three ALK<sup>+</sup> ALCL tumor-derived samples (Table 2). They performed functional assays that suggested that Twist1 contributes to the invasiveness of ALK<sup>+</sup> ALCL. In two out of three cell lines, Twist1 was linked to expression of phospho-Akt and Bmi-1 and downregulation of p66Shc (a member of Src homology and collagen homology family). Twist1 knockdown resulted in increased sensitivity to the chemotherapeutic agent ALK inhibitor Crizotinib in those two cell lines. These results need to be confirmed on a larger population of patients. Nevertheless, they suggest a key role for Twist1 as a regulator of apoptosis and resistance to treatment in this T-cell malignancy.

In addition, an *in vitro* study by Degerman *et al.*<sup>90</sup> hints at a role for Twist1 during spontaneous oncogenesis of two Nijmegen breakage syndrome T-cell cultures established following mitogenic stimulation in the presence of IL-2. Twist1 expression is strongly increased in cell lines undergoing immortalization and appears to be of significance for senescence bypass in T cells.

### B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL), marginal zone B-cell lymphoma and follicular lymphoma. DLBCL is the most common subtype of
non-Hodgkin's lymphoma among adults with an overall survival at 5 years of 60%. Marginal zone B-cell lymphoma and follicular lymphoma are two other types of B-cell lymphoma, the latter being the second most common non-Hodgkin's lymphoma.

DLBCL arises from normal B cells or from the transformation of other types of lymphoma and leukemia. The disease is characterized by an aggressive and rapidly growing tumor that can arise in any part of the body. DLBCL is heterogeneous and encompasses a diverse set of clinical presentations and diagnosis. With the help of gene expression profiling, DLBCL can be divided into germinal centre B-cell-like, activated B-cell-like and primary mediastinal B cell-like subgroups.⁹¹,⁹²

The first suggestions of a possible Twist1 dysregulation came from genetic studies performed by Davis *et al*.⁹³ showing that the NF-κB pathway was constitutively activated in activated B-cell-like and primary mediastinal B cell-like DLBCL. In addition, the expression of STAT3 and IL6 is frequently triggered in activated B-cell-like subtypes.⁹⁴ c-Myc overexpression because of chromosomal rearrangement or translocation is observed in ~10% of DLBCL (regardless of the subtype) and associated with poor prognosis.⁹⁵⁻⁹⁸

In 2012, Lemma *et al*.⁹⁹ detected the expression of Twist1 in the nucleus of the majority of DLBCL samples (83/99) by immunohistochemical staining. Twist1 expression correlated with Slug and Zeb1 expression, two other transcription factors associated with EMT⁹⁹ (Table 2). Nevertheless, the expression of Twist1 could not be associated with any prognostic value. Interestingly, overexpression of YB-1, a known target of Twist1 implicated in cellular proliferation, has also been described and linked to poor prognosis and rituximab resistance in DLBCL samples.¹⁰⁰,¹⁰¹

With the aim of investigating a possible role of Twist1–2 in B-cell lymphoma, we performed real-time Q-PCR on samples derived from Origene (Lymphoma cDNA Array II LYRT102, OriGene Technologies, Inc., Rockville, MD, USA) (Figure 3). A description of each sample is found at www.origene.com/assets/documents/TissueScan/LYRT102.xls. As a positive control, an analysis of Twist1 expression from a sample of a patient with MF was included. Reference values used as healthy controls were obtained from six

samples of lymph nodes and spleen containing 100% normal cells, originally part of the mRNA samples present on the LYRT102 plate. We detected an increased expression of Twist1 in DLBCL samples and also in follicular and marginal zone B-cell lymphomas samples (although results were statistically significant only for follicular and marginal zone B-cell lymphoma compared with spleen and lymph node controls of normal appearance; Figure 3a). Twist2 expression was positively correlated with Twist1 expression. Although a high expression of Twist2 was detected in some patients in all three types of lymphoma, no significant overexpression of Twist2 could be measured compared with controls (Figure 3b).

In conclusion, Twist1 seems to be overexpressed in several types of B-cell lymphomas. Given its potential as a therapeutic target in other types of cancer, further investigations are needed to characterize its function and its regulation.

*Leukemia.* Although there are considerable data showing Twist1–2 oncogenic properties in T-cell lymphoma and in myeloid leukemia, publications on their implication in lymphoid leukemia remain sporadic and results remain to be confirmed.

Acute lymphoblastic leukemia (ALL). ALL is the most common type of leukemia in childhood and still accounts for 25% of all deaths from pediatric cancers. In adults, it is even more difficult to treat with a 5-year survival of 40%. It can derive from both B lymphocytes and T cells.

In several types of cancer such as leukemia, differences in biology and survival between patients may be due, in part, to inappropriate epigenetic regulation, such as silencing of tumor suppressor genes through DNA methylation.¹⁰²,¹⁰³

Preliminary evidence suggesting that Twist2 might play a role in ALL came from observations of specific dysregulated pathways of transcription factors that are known targets of Twist2, namely RUNX1 (fused with ETV6 in 25% of childhood ALL) and NF-κB.³¹ In 2012, Thathia *et al*.¹⁰⁴ identified that hypermethylation of the *twist2* promoter is frequent in both childhood (30 of 54 including 28 of 43 B-ALL and 2 of 7 T-ALL) and adult ALL (52 of 77 patients including 27 of 39 B-ALL and 13 of 15 T-ALL), whereas it was only moderately observed in childhood AML (4 of 28 cases) and rarely

<figure><img src="image_6.png" /></figure>

**Figure 3.** Twist1–2 mRNA levels in several types of non-Hodgkin’s B-cell lymphomas. The mRNA levels of *twist1* and *twist2* were measured by real-time PCR using the Applied Biosystems StepOnePlus Realtime PCR systems (Life Technologies, Carlsbad, CA, USA) for 35 amplification rounds in Fast runs using the Fast SYBR Green Master mix (FastStart Universal SYBR Green Master, Roche, Basel, Switzerland). CTRL (*n* = 6), healthy control samples; DLBCL (*n* = 16), diffuse large B-cell lymphoma samples; follicular (*n* = 10), follicular lymphoma samples; MCL (*n* = 4), mantle cell lymphoma samples; MF (*n* = 1), mycosis fungoides sample; MZBC (*n* = 5), marginal zone B-cell lymphoma samples. Gus (β-Glucuronidase) was the endogeneous control gene used to normalize DNA quantity. Relative expression was calculated as 2^(-log10 Twist1/2 - log10 Gus)!. Statistics were performed independently between groups and control samples using the one-tailed Mann–Whitney *t*-test. (a) *twist1* relative expression. *twist1* expression was significantly higher in follicular lymphoma and in marginal zone B-cell lymphoma. It was also higher in DLBCL, although it did not reach statistical significance. Of note, when comparisons were further separated in tissue-specific DLBCL samples, that is, that came either from spleen or from lymph nodes and compared with tissue-matched controls, results reached statistical significance (*P* = 0.0286 between healthy and DLBCL samples derived from lymph nodes and *P* = 0.0357 between healthy and DLBCL samples derived from spleen, data not shown). As *twist1* expression is known to be upregulated in MF, one sample derived from a patient with MF was included as a positive reference value in the figure. (b) *twist2* relative expression. *twist2* expression was measured in the same samples as in Figure 1a. No statistically significant difference could be observed between groups. Of note, high variability was observed in DLBCL- and MZBL-derived samples.
in CLL (5 of 110 cases). Noteworthy, *Twist2* hypermethylation was not observed in CML and adult AML. *Twist2* 5' upstream regulatory region hypermethylation was more frequent in ETV6-RUNX1-positive childhood ALL than in other childhood ALL cases (Table 2). Moreover, *twist2* hypermethylation was especially frequent in relapsed adult ALL (20 of 22 cases), and the corresponding frequencies of samples exhibiting hypermethylated *twist2* was higher following relapse than at the time of diagnosis.¹⁰⁴

Hypermethylation resulted in loss of Twist2 expression in patient samples and ALL cell lines. Interestingly, restoration of Twist2 expression through transfection of Nalm6 and Reh ALL cell lines resulted in a dramatic inhibition of cell growth (aggravated by decamethasone treatment), associated with induction of apoptosis in ETV6-RUNX1⁺ Reh cell lines.¹⁰⁴ The authors propose that Twist2 inhibition of cell growth could be mediated in some cases through inactivation of RUNX1 because the latter has been shown to increase proliferation and enhance B-cell survival¹⁰⁵; although no experiments were conducted to test this. Finally, re-expression of Twist2 leads to an increased sensitivity to the chemotherapeutic agents etoposide and daunorubicin. The authors hypothesize that chemotherapeutic sensitivity modulation by Twist2 is mediated through NF-κB inhibition, because increased expression of the latter is correlated with chemoresistance.

In summary, in opposition to its role in T-cell lymphoma and in myeloid leukemia, Twist2 appears to exhibit tumor-suppressor properties in ALL.

Chronic lymphocytic leukemia. CLL is the most common type of leukemia, displaying a great variability with regard to clinical course and survival. It is characterized by slow and uncontrolled proliferation with disrupted apoptosis of a clonal B cell that results in the accumulation of cells and disruption of normal immune function. Prognostic factors associated with worsened survival include expression of ZAP70 and CD38, chromosomal aberrations (reviewed in Stilgenbauer *et al*¹⁰⁶) and chromosomal deletions,¹⁰⁷,¹⁰⁸ p53 mutations¹⁰⁹ and absence of somatic mutations in genes coding for immunoglobulin variable heavy chain (Ig V<sub>H</sub>).¹¹⁰,¹¹¹ In addition, a high frequency of patients with unmutated Ig V<sub>H</sub> CLL exhibit p53 dysfunction.

DNA methylation is also implicated in the biology of CLL.¹¹² In 2005, Raval *et al*.⁴⁴ demonstrated that the Twist2 promoter was frequently methylated in patients exhibiting mutated Ig V<sub>H</sub> subtypes of CLL (associated with a favorable prognosis) but not in patients that exhibited unmutated Ig V<sub>H</sub> subtypes (associated with a poor prognosis) (Table 2). Methylation resulted in *twist2* silencing in mutated subtypes, whereas Twist2 was expressed in unmutated subtypes. The authors suggest that the expression of Twist2 in unmutated Ig V<sub>H</sub> patients (with unmethylated *twist2*) could lead to disruption of the p53 pathway as observed in several other types of cancer, although they did not confirm this experimentally. In conclusion, parallel to what is seen in T-cell lymphoma but in opposition to observations in ALL, the expression of Twist2 in CLL could be associated with a poor prognosis.

## CONCLUSION AND PERSPECTIVES: OTHER BHLH PROTEINS IN HEMATOLOGICAL MALIGNANCIES

Twist1-2-mediated gene expression modulation is intrinsically linked to the abundance of other bHLH members such as E2A and Id proteins. Interestingly, deregulation of expression of these proteins has been observed in various hematological malignancies. For instance, E2A<sup>ko</sup> mice display a high risk of developing T-cell tumors, as do E2A<sup>ko</sup>Id1<sup>ko</sup> mice.²⁶ E47 knockdown experiments in T-cell lymphoma cell lines linked E47 deficiencies to the development of T-cell malignancy.¹¹³ Loss of expression of E2A, or its sequestration by Id2, has been linked to resistance to apoptosis in primary effusion lymphoma,¹¹⁴ to loss of B-cell phenotype and reprogramming of neoplastic B cells in Hodgkin's

lymphoma.¹¹⁵ Id1 is frequently overexpressed in various malignancies and its deregulated expression in mice has been associated with MDS development and immortalization of myeloid progenitors.¹¹⁶ In AML, high expression of Id1 is implicated in leukemic cell proliferation and has been associated with poor prognosis.¹¹⁷ Id4 silencing is systematic in CLL and MDS and plays a role in its pathogenesis.¹¹⁸,¹¹⁹ In contrast, Id4 seems to act as an oncogene in B cell-derived malignancies such as ALL.¹²⁰,¹²¹ Unfortunately, Twist1-2 function was not investigated in the studies mentioned herein.

In conclusion, numerous questions remain to be addressed concerning the role of Twist1-2 in hematological malignancies. There is still a disturbing paucity of data on Twist2 implication in blood malignancies and on the role of Twist1-2 in non-T-cell lymphoma/leukemia. As potential therapeutic targets and as central regulators of apoptosis and senescence, large efforts should be undertaken to promptly clarify the role of Twist1-2 and their partners in these types of cancer.

## CONFLICT OF INTEREST

The authors declare no conflict of interest.

## ACKNOWLEDGEMENTS

NM was supported by a fellowship from the ARC foundation (fondation ARC pour la recherche sur le cancer) and AR by a fellowship from the Ligue Nationale contre le Cancer. This work was supported by institutional grants from INSERM, Université de Lyon (to NB) and by a specific grant from the Cancéropole Grand Sud-Ouest (to NB).

## REFERENCES

1. el Ghouzzi V, Le Merrer M, Perrin-Schmitt F, Lajeunie E, Benit P, Renier D *et al*. Mutations of the TWIST gene in the Saethre-Chotzen syndrome. *Nat Genet* 1997; **15**: 42–46.

2. Howard TD, Paznekas WA, Green ED, Chiang LC, Ma N, Ortiz de Luna RI *et al*. Mutations in TWIST, a basic helix-loop-helix transcription factor, in Saethre-Chotzen syndrome. *Nat Genet* 1997; **15**: 36–41.

3. Hebrok M, Wertz K, Füchtbauer EM. M-twist is an inhibitor of muscle differentiation. *Dev Biol* 1994; **165**: 537–544.

4. Lee YS, Lee HH, Park J, Yoo EJ, Glackin CA, Choi YI *et al*. Twist2, a novel ADD1/SREBP1c interacting protein, represses the transcriptional activity of ADD1/SREBP1c. *Nucleic Acids Res* 2003; **31**: 7165–7174.

5. Spicer DB, Rhee J, Cheung WL, Lassar AB. Inhibition of myogenic bHLH and MEF2 transcription factors by the bHLH protein Twist. *Science* 1996; **272**: 1476–1480.

6. Murray SS, Glackin CA, Winters KA, Gazit D, Kahn AJ, Murray EJ. Expression of helix-loop-helix regulatory genes during differentiation of mouse osteoblastic cells. *J Bone Miner Res* 1992; **7**: 1131–1138.

7. Tukel T, Šošić D, Al-Gazali LI, Erazo M, Casasnovas J, Franco HL *et al*. Homozygous nonsense mutations in TWIST2 cause Setleis syndrome. *Am J Hum Genet* 2010; **87**: 289–296.

8. Sosic D, Richardson JA, Yu K, Ornitz DM, Olson EN. Twist regulates cytokine gene expression through a negative feedback loop that represses NF-kappaB activity. *Cell* 2003; **112**: 169–180.

9. Thisse C, Perrin-Schmitt F, Stoetzel C, Thisse B. Sequence-specific transactivation of the Drosophila twist gene by the dorsal gene product. *Cell* 1991; **65**: 1191–1201.

10. Cheng GZ, Zhang WZ, Sun M, Wang Q, Coppola D, Mansour M *et al*. Twist is transcriptionally induced by activation of STAT3 and mediates STAT3 oncogenic function. *J Biol Chem* 2008; **283**: 14665–14673.

11. Howe LR, Watanabe O, Leonard J, Brown AM. Twist is up-regulated in response to Wnt1 and inhibits mouse mammary cell differentiation. *Cancer Res* 2003; **63**: 1906–1913.

12. Yang MH, Wu MZ, Chiou SH, Chen PM, Chang SY, Liu CJ *et al*. Direct regulation of TWIST by HIF-1alpha promotes metastasis. *Nat Cell Biol* 2008; **10**: 295–305.

13. Gort EH, van Haaften G, Verlaan I, Groot AJ, Plasterk RH, Shvarts A *et al*. The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha. *Oncogene* 2008; **27**: 1501–1510.

14. Hamamori Y, Sartorelli V, Ogryzko V, Puri PL, Wu HY, Wang JY *et al*. Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein twist and adenoviral oncoprotein E1A. *Cell* 1999; **96**: 405–413.
15 Gong XQ, Li L. Dermo-1, a multifunctional basic helix-loop-helix protein, represses MyoD transactivation via the HLH domain, MEF2 interaction, and chromatin deacetylation. *J Biol Chem* 2002; **277**: 12310–12317.

16 Pan D, Fujimoto M, Lopes A, Wang YX. Twist-1 is a PPARdelta-inducible, negative-feedback regulator of PGC-1alpha in brown fat metabolism. *Cell* 2009; **137**: 73–86.

17 Murre C, McCaw PS, Baltimore D. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. *Cell* 1989; **56**: 777–783.

18 Bialek P, Kern B, Yang X, Schrock M, Sosic D, Hong N *et al*. A twist code determines the onset of osteoblast differentiation. *Dev Cell* 2004; **6**: 423–435.

19 Koh HS, Lee C, Lee KS, Ham CS, Seong RH, Kim SS *et al*. CD7 expression and galectin-1-induced apoptosis of immature thymocytes are directly regulated by NF-kappaB upon T-cell activation. *Biochem Biophys Res Commun* 2008; **370**: 149–153.

20 Hong J, Zhou J, Fu J, He T, Qin J, Wang L *et al*. Phosphorylation of serine 68 of Twist1 by MAPKs stabilizes Twist1 protein and promotes breast cancer cell invasiveness. *Cancer Res* 2011; **71**: 3980–3990.

21 Xue G, Restuccia DF, Lan Q, Hynx D, Dirhofer S, Hess D *et al*. Akt/PKB-mediated phosphorylation of Twist1 promotes tumor metastasis via mediating cross-talk between PI3K/Akt and TGF-β signaling axes. *Cancer Discov* 2012; **2**: 248–259.

22 Firulli BA, Howard MJ, McDaid JR, McIlreavey L, Dionne KM, Centonze VE *et al*. PKA, PKC, and the protein phosphatase 2A influence HAND factor function: a mechanism for tissue-specific transcriptional regulation. *Mol Cell* 2003; **12**: 1225–1237.

23 Firulli AB, Conway SJ. Phosphoregulation of Twist1 provides a mechanism of cell fate control. *Curr Med Chem* 2008; **15**: 2641–2647. Review.

24 Vichalkovski A, Gresko E, Hess D, Restuccia DF, Hemmings BA. PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage. *Oncogene* 2010; **29**: 3554–3565.

25 Castanon I, Von Stetina S, Kass J, Baylies MK. Dimerization partners determine the activity of the Twist bHLH protein during Drosophila mesoderm development. *Development* 2001; **128**: 3145–3159.

26 Yan W, Young AZ, Soares VC, Kelley R, Benezra R, Zhuang Y. High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice. *Mol Cell Biol* 1997; **17**: 7317–7327.

27 Connerney J, Andreeva V, Leshem Y, Mercado MA, Dowell K, Yang X *et al*. Twist1 homodimers enhance FGF responsiveness of the cranial sutures and promote suture closure. *Dev Biol* 2008; **318**: 323–334.

28 Benezra R, Davis RL, Lockshon D, Turner DL, Weintraub H. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. *Cell* 1990; **61**: 49–59.

29 Hayashi M, Nimura K, Kashiwagi K, Harada T, Takaoka K, Kato H *et al*. Comparative roles of Twist-1 and Id1 in transcriptional regulation by BMP signaling. *J Cell Sci* 2007; **120**: 1350–1357.

30 Danciu TE, Whitman M. Oxidative stress drives disulfide bond formation between basic helix-loop-helix transcription factors. *J Cell Biochem* 2010; **109**: 417–424.

31 Sharabi AB, Aldrich M, Sosic D, Olson EN, Friedman AD, Lee SH *et al*. Twist-2 controls myeloid lineage development and function. *PLoS Biol* 2008; **6**: e316.

32 Koh HS, Lee C, Lee KS, Park EJ, Seong RH, Hong S *et al*. Twist2 regulates CD7 expression and galectin-1-induced apoptosis in mature T-cells. *Mol Cells* 2009; **28**: 553–558.

33 Sharif MN, Sosic D, Rothlin CV, Kelly E, Lemke G, Olson EN *et al*. Twist mediates suppression of inflammation by type I IFNs and Axl. *J Exp Med* 2006; **203**: 1891–1901.

34 Li X, Marcondes AM, Gooley TA, Deeg HJ. The helix-loop-helix transcription factor TWIST is dysregulated in myelodysplastic syndromes. *Blood* 2010; **116**: 2304–2314.

35 Cosset E, Hamdan G, Jeanpierre S, Voeltzel T, Sagorny K, Hayette S *et al*. Deregulation of TWIST-1 in the CD34 + compartment represents a novel prognostic factor in chronic myeloid leukemia. *Blood* 2011; **117**: 1673–1676.

36 Wang SM, Coljee VW, Pignolo RJ, Rotenberg MO, Cristofalo VJ, Sierra F. Cloning of the human twist gene: its expression is retained in adult mesodermally-derived tissues. *Gene* 1997; **187**: 83–92.

37 Mabbott NA, Kenneth Baillie J, Kobayashi A, Donaldson DS, Ohmori H, Yoon SO *et al*. Expression of mesenchyme-specific gene signatures by follicular dendritic cells: insights from the meta-analysis of microarray data from multiple mouse cell populations. *Immunology* 2011; **133**: 482–498.

38 Appay V, van Lier RA, Sallusto F, Roederer M. Phenotype and function of human T lymphocyte subsets: consensus and issues. *Cytometry A* 2008; **73**: 975–983. Review.

39 Wan YY. Multi-tasking of helper T cells. *Immunology* 2010; **130**: 166–171. Review.

40 Niesner U, Albrecht I, Janke M, Doebis C, Loddenkemper C, Lexberg MH *et al*. Autoregulation of Th1-mediated inflammation by twist1. *J Exp Med* 2008; **205**: 1889–1901.

41 Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C *et al*. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. *Cell* 2004; **117**: 927–939.

42 Pham D, Vincentz JW, Firulli AB, Kaplan MH. Twist1 regulates Ifng expression in Th1 cells by interfering with Runx3 function. *J Immunol* 2012; **189**: 832–840.

43 Pham D, Walline CC, Hollister K, Dent AL, Blum JS, Firulli AB *et al*. The transcription factor Twist1 limits T helper 17 and T follicular helper cell development by repressing the gene encoding the interleukin-6 receptor α chain. *J Biol Chem* 2013; **288**: 27423–27433.

44 Raval A, Lucas DM, Matkovic JJ, Bennett KL, Liyanarachchi S, Young DC *et al*. TWIST2 demonstrates differential methylation in immunoglobulin variable heavy chain mutated and unmutated chronic lymphocytic leukemia. *J Clin Oncol* 2005; **23**: 3877–3885.

45 Nguyen CT, Gonzales FA, Jones PA. Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation. *Nucleic Acids Res* 2001; **29**: 4598–4606.

46 Puisieux A, Valsesia-Wittmann S, Ansieau S. A twist for survival and cancer progression. *Br J Cancer* 2006; **94**: 13–17. Review.

47 Ansieau S, Bastid J, Doreau A, Morel AP, Bouchet BP, Thomas C *et al*. Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence. *Cancer Cell* 2008; **14**: 79–89.

48 Maestro R, Dei Tos AP, Hamamori Y, Krasnokutsky S, Sartorelli V, Kedes L *et al*. Twist is a potential oncogene that inhibits apoptosis. *Genes Dev* 1999; **13**: 2207–2217.

49 Stasinopoulos IA, Mironchik Y, Raman A, Wildes F, Winnard Jr P, Raman V. HOXA5-twist interaction alters p53 homeostasis in breast cancer cells. *J Biol Chem* 2005; **280**: 2294–2299.

50 Shiota M, Izumi H, Onitsuka T, Miyamoto N, Kashiwagi E, Kidani A *et al*. Twist and p53 reciprocally regulate target genes via direct interaction. *Oncogene* 2008; **27**: 5543–5553.

51 Valsesia-Wittmann S, Magdeleine M, Dupasquier S, Garin E, Jallas AC, Combaret V *et al*. Oncogenic cooperation between H-Twist and N-Myc overrides failsafe programs in cancer cells. *Cancer Cell* 2004; **6**: 625–630.

52 Haque I, Banerjee S, Mehta S, De A, Majumder M, Mayo MS *et al*. Cysteine-rich 61-connective tissue growth factor-nephroblastoma-overexpressed 5 (CCN5)/Wnt-1-induced signaling protein-2 (WISP-2) regulates microRNA-10b via hypoxia-inducible factor-1α-TWIST signaling networks in human breast cancer cells. *J Biol Chem* 2011; **286**: 43475–43485.

53 Cheng GZ, Chan J, Wang Q, Zhang W, Sun CD, Wang LH. Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel. *Cancer Res* 2007; **67**: 1979–1987.

54 Shiota M, Izumi H, Onitsuka T, Miyamoto N, Kashiwagi E, Kidani A *et al*. Twist promotes tumor cell growth through YB-1 expression. *Cancer Res* 2008; **68**: 98–105.

55 Wang X, Ling MT, Guan XY, Tsao SW, Cheung HW, Lee DT *et al*. Identification of a novel function of TWIST, a bHLH protein, in the development of acquired taxol resistance in human cancer cells. *Oncogene* 2004; **23**: 474–482.

56 Zhang X, Wang Q, Ling MT, Wong YC, Leung SC, Wang X. Anti-apoptotic role of TWIST and its association with Akt pathway in mediating taxol resistance in nasopharyngeal carcinoma cells. *Int J Cancer* 2007; **120**: 1891–1898.

57 Zhan X, Feng X, Kong Y, Chen Y, Tan W. JNK signaling maintains the mesenchymal properties of multi-drug resistant human epidermoid carcinoma KB cells through snail and twist1. *BMC Cancer* 2013; **13**: 180.

58 Pham CG, Bubici C, Zazzeroni F, Knabb JR, Papa S, Kuntzen C *et al*. Upregulation of Twist-1 by NF-kappaB blocks cytotoxicity induced by chemotherapeutic drugs. *Mol Cell Biol* 2007; **27**: 3920–3935.

59 Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. *Cell* 2009; **139**: 871–890. Review.

60 Stirewalt DL, Mhyre AJ, Marcondes M, Pogosova-Agadjanyan E, Abbasi N, Radich JP *et al*. Tumour necrosis factor-induced gene expression in human marrow stroma: clues to the pathophysiology of MDS? *Br J Haematol* 2008; **140**: 444–453.

61 Li X, Xu F, Chang C, Byon J, Papayannopoulou T, Deeg HJ *et al*. Transcriptional regulation of miR-10a/b by TWIST-1 in myelodysplastic syndromes. *Haematologica* 2013; **98**: 414–419.

62 Kawagoe H, Kandilci A, Kranenburg TA, Grosveld GC. Overexpression of N-Myc rapidly causes acute myeloid leukemia in mice. *Cancer Res* 2007; **67**: 10677–10685.

63 Eguchi M, Eguchi-Ishimae M, Tojo A, Morishita K, Suzuki K, Sato Y *et al*. Fusion of ETV6 to neurotrophin-3 receptor TRKC in acute myeloid leukemia with t(12;15)(p13;q25). *Blood* 1999; **93**: 1355–1363.
<figure><img src="image_7.png" /></figure>

64 Kim MS, Kim GM, Choi YJ, Kim HJ, Kim YJ, Jin W. TrkC promotes survival and growth of leukemia cells through Akt-mTOR-dependent up-regulation of PLK-1 and Twist-1. *Mol Cells* 2013; **36**: 177-184.

65 Hong CC, Lay JD, Huang JS, Cheng AL, Tang JL, Lin MT *et al*. Receptor tyrosine kinase AXL is induced by chemotherapy drugs and overexpression of AXL confers drug resistance in acute myeloid leukemia. *Cancer Lett* 2008; **268**: 314-324.

66 Sanchez-Correa B, Bergua JM, Campos C, Gayoso I, Arcos MJ, Bañas H *et al*. Cytokine profiles in acute myeloid leukemia patients at diagnosis: survival is inversely correlated with IL-6 and directly correlated with IL-10 levels. *Cytokine* 2013; **61**: 885-891.

67 Redell MS, Ruiz MJ, Gerbing RB, Alonzo TA, Lange BJ, Tweardy DJ *et al*. FACS analysis of Stat3/5 signaling reveals sensitivity to G-CSF and IL-6 as a significant prognostic factor in pediatric AML: a Children's Oncology Group report. *Blood* 2013; **121**: 1083-1093.

68 Tipping AJ, Deininger MW, Goldman JM, Melo JV. Comparative gene expression profile of chronic myeloid leukemia cells innately resistant to imatinib mesylate. *Exp Hematol* 2003; **31**: 1073-1080.

69 Xin H, Kong Y, Jiang X, Wang K, Qin X, Miao ZH *et al*. Multi-drug-resistant cells enriched from chronic myeloid leukemia cells by Doxorubicin possess tumor-initiating-cell properties. *J Pharmacol Sci* 2013; **122**: 299-304.

70 Dulmage BO, Geskin LJ. Lessons learned from gene expression profiling of cutaneous T-cell lymphoma. *Br J Dermatol* 2013; **169**: 1188-1197. Review.

71 Dippel E, Assaf C, Hummel M, Schrag HJ, Stein H, Goerdt S *et al*. Clonal T-cell receptor gamma-chain gene rearrangement by PCR-based GeneScan analysis in advanced cutaneous T-cell lymphoma: a critical evaluation. *J Pathol* 1999; **188**: 146-154.

72 Eriksen KW, Kaltoft K, Mikkelsen G, Nielsen M, Zhang Q, Geisler C *et al*. Constitutive STAT3-activation in Sezary syndrome: tyrphostin AG490 inhibits STAT3-activation, interleukin-2 receptor expression and growth of leukemic Sezary cells. *Leukemia* 2001; **15**: 787-793.

73 Zhang Q, Nowak I, Vonderheid EC, Rook AH, Kadin ME, Nowell PC *et al*. Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome. *Proc Natl Acad Sci USA* 1996; **93**: 9148-9153.

74 Izban KF, Ergin M, Qin JZ, Martinez RL, Pooley RJ JR, Saeed S *et al*. Constitutive expression of NF-kappa B is a characteristic feature of mycosis fungoides: implications for apoptosis resistance and pathogenesis. *Hum Pathol* 2000; **31**: 1482-1490.

75 Kadin ME, Cavaille-Coll MW, Gertz R, Massagué J, Cheifetz S, George D. Loss of receptors for transforming growth factor beta in human T-cell malignancies. *Proc Natl Acad Sci USA* 1994; **91**: 6002-6006.

76 Scarisbrick JJ, Woolford AJ, Calonje E, Photiou A, Ferreira S, Orchard G *et al*. Frequent abnormalities of the p15 and p16 genes in mycosis fungoides and Sezary syndrome. *J Invest Dermatol* 2002; **118**: 493-499.

77 van Doorn R, Dijkman R, Vermeer MH, Out-Luiting JJ, van der Raaij-Helmer EM, Willemze R *et al*. Aberrant expression of the tyrosine kinase receptor EphA4 and the transcription factor twist in Sézary syndrome identified by gene expression analysis. *Cancer Res* 2004; **64**: 5578-5586.

78 Ni X, Richmond HM, Liao XM, Decker WK, Shiu LH, Shpall EJ *et al*. Induction of T-cell responses against cutaneous T-cell lymphomas ex vivo by autologous dendritic cells transfected with amplified tumor mRNA. *J Invest Dermatol* 2008; **128**: 2631-2639.

79 Goswami M, Duvic M, Dougherty A, Ni X. Increased Twist expression in advanced stage of mycosis fungoides and Sézary syndrome. *J Cutan Pathol* 2012; **39**: 500-507.

80 Michel L, Jean-Louis F, Begue E, Bensussan A, Bagot M. Use of PLS3, Twist, CD158k/KIR3DL2, and NKp46 gene expression combination for reliable Sézary syndrome diagnosis. *Blood* 2013; **121**: 1477-1478.

81 Pace KE, Hahn HP, Pang M, Nguyen JT, Baum LG. CD7 delivers a pro-apoptotic signal during galectin-1-induced T cell death. *J Immunol* 2000; **165**: 2331-2334.

82 Rappl G, Abken H, Muche JM, Sterry W, Tilgen W, André S *et al*. CD4+CD7- leukemic T cells from patients with Sézary syndrome are protected from galectin-1-triggered T cell death. *Leukemia* 2002; **16**: 840-845.

83 Liu TY, Chen CY, Tien HF, Lin CW. Loss of CD7, independent of galectin-3 expression, implies a worse prognosis in adult T-cell leukaemia/lymphoma. *Histopathology* 2009; **54**: 214-220.

84 Amin HM, Lai R. Pathobiology of ALK + anaplastic large-cell lymphoma. *Blood* 2007; **110**: 2259-2267. Review.

85 Chiarle R, Voena C, Ambrogio C, Piva R, Inghirami G. The anaplastic lymphoma kinase in the pathogenesis of cancer. *Nat Rev Cancer* 2008; **8**: 11-23. Review.

86 Zamo A, Chiarle R, Piva R, Howes J, Fan Y, Chilosi M *et al*. Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death. *Oncogene* 2002; **21**: 1038-1047.

87 Amin HM, McDonnell TJ, Ma Y, Lin Q, Fujio Y, Kunisada K *et al*. Selective inhibition of STAT3 induces apoptosis and G(1) cell cycle arrest in ALK-positive anaplastic large cell lymphoma. *Oncogene* 2004; **23**: 5426-5434.

88 Chiarle R, Simmons WJ, Cai H, Dhall G, Zamo A, Raz R *et al*. Stat3 is required for ALK-mediated lymphomagenesis and provides a possible therapeutic target. *Nat Med* 2005; **11**: 623-629.

89 Zhang J, Wang P, Wu F, Li M, Sharon D, Ingham RJ *et al*. Aberrant expression of the transcriptional factor Twist1 promotes invasiveness in ALK-positive anaplastic large cell lymphoma. *Cell Signal*. 2012; **24**: 852-858.

90 Degerman S, Siwicki JK, Osterman P, Lafferty-Whyte K, Keith WN, Roos G. Telomerase upregulation is a postcrisis event during senescence bypass and immortalization of two Nijmegen breakage syndrome T cell cultures. *Aging Cell* 2010; **9**: 220-235.

91 Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A *et al*. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. *Nature* 2000; **403**: 503-511.

92 Wright G, Tan B, Rosenwald A, Hurt EH, Wiestner A, Staudt LM. A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma. *Proc Natl Acad Sci USA* 2003; **100**: 9991-9996.

93 Davis RE, Brown KD, Siebenlist U, Staudt LM. Constitutive nuclear factor kappaB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells. *J Exp Med* 2001; **194**: 1861-1874.

94 Lam LT, Wright G, Davis RE, Lenz G, Farinha P, Dang L *et al*. Cooperative signaling through the signal transducer and activator of transcription 3 and nuclear factor-kappaB pathways in subtypes of diffuse large B-cell lymphoma. *Blood* 2008; **111**: 3701-3713.

95 Barrans S, Crouch S, Smith A, Turner K, Owen R, Patmore R *et al*. Rearrangement of MYC is associated with poor prognosis in patients with diffuse large B-cell lymphoma treated in the era of rituximab. *J Clin Oncol* 2010; **28**: 3360-3365.

96 Klapper W, Stoecklein H, Zeynalova S, Ott G, Kosari F, Rosenwald A *et al*. Structural aberrations affecting the MYC locus indicate a poor prognosis independent of clinical risk factors in diffuse large B-cell lymphomas treated within randomized trials of the German High-Grade Non-Hodgkin's Lymphoma Study Group (DSHNHL). *Leukemia* 2008; **22**: 2226-2229.

97 Niitsu N, Okamoto M, Miura I, Hirano M. Clinical significance of 8q24/c-MYC translocation in diffuse large B-cell lymphoma. *Cancer Sci* 2008; **100**: 233-237.

98 Savage KJ, Johnson NA, Ben-Neriah S, Connors JM, Sehn LH, Farinha P *et al*. MYC gene rearrangements are associated with a poor prognosis in diffuse large B-cell lymphoma patients treated with R-CHOP chemotherapy. *Blood* 2009; **114**: 3533-3537.

99 Lemma S, Karihtala P, Haapasaari KM, Jantunen E, Soini Y, Bloigu R *et al*. Biological roles and prognostic values of the epithelial-mesenchymal transition-mediated transcription factors Twist, ZEB1 and Slug in diffuse large B-cell lymphoma. *Histopathology* 2013; **62**: 326-333.

100 Hanzawa K, Momose S, Higashi M, Tokuhira M, Watanabe R, Kajino K *et al*. Y-box binding protein-1 expression in diffuse large B-cell lymphoma: an impact on prognosis in the rituximab era. *Leuk Lymphoma* 2010; **51**: 2054-2062.

101 Xu W, Zhou L, Qin R, Tang H, Shen H. Nuclear expression of YB-1 in diffuse large B-cell lymphoma: correlation with disease activity and patient outcome. *Eur J Haematol* 2009; **83**: 313-319.

102 Cameron EE, Baylin SB, Herman JG. p15(INK4B) CpG island methylation in primary acute leukemia is heterogeneous and suggests density as a critical factor for transcriptional silencing. *Blood* 1999; **94**: 2445-2451.

103 Melki JR, Vincent PC, Brown RD, Clark SJ. Hypermethylation of E-cadherin in leukemia. *Blood* 2000; **95**: 3208-3213.

104 Thathia SH, Ferguson S, Gautrey HE, van Otterdijk SD, Hili M, Rand V *et al*. Epigenetic inactivation of TWIST2 in acute lymphoblastic leukemia modulates proliferation, cell survival and chemosensitivity. *Haematologica* 2012; **97**: 371-378.

105 Blyth K, Slater N, Hanlon L, Bell M, Mackay N, Stewart M *et al*. Runx1 promotes B-cell survival and lymphoma development. *Blood Cells Mol Dis* 2009; **43**: 12-19.

106 Stilgenbauer S, Bullinger L, Lichter P, Döhner H. German CLL Study Group (GCLLSG) Chronic lymphocytic leukemia. Genetics of chronic lymphocytic leukemia: genomic aberrations and V(H) gene mutation status in pathogenesis and clinical course. *Leukemia* 2002; **16**: 993-1007. Review.

107 Döhner H, Fischer K, Bentz M, Hansen K, Benner A, Cabot G *et al*. p53 gene deletion predicts for poor survival and non-response to therapy with purine analogs in chronic B-cell leukemias. *Blood* 1995; **85**: 1580-1589.

108 Stankovic T, Weber P, Stewart G, Bedenham T, Murray J, Byrd PJ *et al*. Inactivation of ataxia telangiectasia mutated gene in B-cell chronic lymphocytic leukaemia. *Lancet* 1999; **353**: 26-29.
109 Lin K, Manocha S, Harris RJ, Matrai Z, Sherrington PD, Pettitt AR. High frequency of p53 dysfunction and low level of VH mutation in chronic lymphocytic leukemia patients using the VH3-21 gene segment. *Blood* 2003; **102**: 1145–1146.

110 Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL *et al*. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. *Blood* 1999; **94**: 1840–1847.

111 Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. *Blood* 1999; **94**: 1848–1854.

112 Rush LJ, Raval A, Funchain P, Johnson AJ, Smith L, Lucas DM *et al*. Epigenetic profiling in chronic lymphocytic leukemia reveals novel methylation targets. *Cancer Res* 2004; **64**: 2424–2433.

113 Schwartz R, Engel I, Fallahi-Sichani M, Petrie HT, Murre C. Gene expression patterns define novel roles for E47 in cell cycle progression, cytokine-mediated signaling, and T lineage development. *Proc Natl Acad Sci USA* 2006; **103**: 9976–9981.

114 Lietz A, Janz M, Sigvardsson M, Jundt F, Dörken B, Mathas S. Loss of bHLH transcription factor E2A activity in primary effusion lymphoma confers resistance to apoptosis. *Br J Haematol* 2007; **137**: 342–348.

115 Mathas S, Janz M, Hummel F, Hummel M, Wollert-Wulf B, Lusatis S *et al*. Intrinsic inhibition of transcription factor E2A by HLH proteins ABF-1 and Id2 mediates reprogramming of neoplastic B cells in Hodgkin lymphoma. *Nat Immunol* 2006; **7**: 207–215.

116 Suh HC, Leeanaksiri W, Ji M, Klarmann KD, Renn K, Gooya J *et al*. Id1 immortalizes hematopoietic progenitors in vitro and promotes a myeloproliferative disease in vivo. *Oncogene* 2008; **27**: 5612–5623.

117 Yu WP, Scott SA, Dong WF. Induction of ID1 expression and apoptosis by the histone deacetylase inhibitor (trichostatin A) in human acute myeloid leukaemic cells. *Cell Prolif* 2008; **41**: 86–97.

118 Chen SS, Claus R, Lucas DM, Yu L, Qian J, Ruppert AS *et al*. Silencing of the inhibitor of DNA binding protein 4 (ID4) contributes to the pathogenesis of mouse and human CLL. *Blood* 2011; **117**: 862–871.

119 Wang H, Wang XQ, Xu XP, Lin GW. ID4 methylation predicts high risk of leukemic transformation in patients with myelodysplastic syndrome. *Leuk Res* 2010; **34**: 598–604.

120 Bellido M, Aventin A, Lasa A, Estivill C, Carnicer MJ, Pons C *et al*. Id4 is deregulated by a t(6;14)(p22;q32) chromosomal translocation in a B-cell lineage acute lymphoblastic leukemia. *Haematologica* 2003; **88**: 994–1001.

121 Yu L, Liu C, Vandeusen J, Becknell B, Dai Z, Wu YZ *et al*. Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia. *Nat Genet* 2005; **37**: 265–274.

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