Epigenetic Rewiring of Protein Kinase Signalling in T-Cell Acute Lymphoblastic Leukaemia

Abstract

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive neoplastic malignancy characterised by the accumulation of multiple oncogenic and epigenetic alterations in haematopoietic T-cell precursors leading to their uncontrolled proliferation and accumulation in the bone marrow. For many years it has been established that the occurrence of activating mutations, alterations in transcription factors expression, impairment in cell cycle regulators, and hyperactivation of NOTCH1 signalling play prominent roles in the pathogenesis of this disease. Recently, the introduction of high-resolution screening and next-generation sequencing platforms revealed that T-cell progenitors accumulate additional mutations, affecting protein kinase signalling, protein translation, and epigenetic control mechanisms, providing novel attractive targets for therapy. While the contributions of direct genomic events are well understood as causative agents of hyperactive kinase signalling pathways, the epigenetic rewiring of kinase signalling cascades via DNA methylation, histone post-translational modifications, and non-coding miRNAs remains less well explored. In this review, we provide novel perspectives on epigenetic regulatory aspects of kinase signalling heterogeneity in T-ALL pathogenesis and therapeutic outcomes.

1. Introduction

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive malignant neoplasm accounting for 10–15% of paediatric and around 25% of adult ALL cases [1]. Clinically, T-ALL originates from lymphoid progenitor T cells that undergo malignant transformation into preleukaemic or leukaemic-initiating cells (LICs) due to the accumulation of multiple oncogenic events [1]. This accumulation results in the uncontrolled clonal expansion of immature T-lymphoblasts, which spread via the peripheral blood circulation and infiltrate several tissues, including the liver, spleen, lymph nodes, and central nervous system [2].

The molecular basis of T-ALL lies in aberrant genetic modifications leading to alterations in cell growth, survival, and differentiation processes during the development of thymocytes into T cells [1]. Unlike other leukaemias such as chronic myeloid leukaemia (CML) and Philadelphia-positive ALL, which are merely kinase-driven malignancies, T-ALL is driven by oncogenic transcription factors that act along with secondary acquired mutations. These lesions, together with active signalling pathways, may be targeted by therapeutic agents [3]. Notch1 signalling activation is T-ALL’s most predominant tumour driver in this context. Activating mutations in the NOTCH1 gene itself and in negative regulators such as FBXW7 and VAV-1 are found in nearly 60% of T-ALL patients [4,5,6]. Additionally, chromosomal rearrangements and genetic lesions leading to aberrant expression of TF oncogenes have been associated with different molecular groups of T-ALL [1]. Importantly, these genetic modifications can also be combined with recurrent cytogenetic and molecular alterations resulting in impaired regulation of cell cycle, cell growth/proliferation, chromatin remodelling, T-cell differentiation, and T-cell self-renewal pathways [1].

Recently, several novel treatment strategies, including risk-based treatment assignment, multi-agent chemotherapy, and prophylactic central nervous system therapy, have significantly improved the overall survival of childhood T-ALL patients to about 80–90% [7]. The risk-based therapeutic regimen consists of steroids, microtubule-destabilising agents (vincristine), alkylating agents (cyclophosphamide), anthracyclines (doxorubicin or daunorubicin), antimetabolites (methotrexate, MTX), nucleoside analogues (6-mercaptopurine, thioguanine, or cytarabine), and hydrolysing enzymes (L-asparaginase), and in some cases, it is followed by stem cell transplantation. However, the prognosis for adults who relapse is poor, with a complete remission rate of 27–40% [7]; this is mainly due to the severe side effects of intensive chemotherapy treatments, often leading to the discontinuation of therapy [7]. Novel insights into the molecular mechanisms in T-ALL have been crucial to develop more targeted treatment approaches of the disease [3]. Briefly, oncogenic NOTCH1 signalling can be inhibited via monoclonal antibodies (brontictuzumab), ADAM10 metalloprotease, gamma-secretase inhibitors, SERCA inhibitors, and CXCR4 antagonists (plerixafor and BL8040). Immunotherapy approaches for T-ALL include monoclonal antibodies against surface CD38 (daratumumab or isatuximab), as well as CAR T cells directed towards surface CD1, CD5, CD7, and CD38. The increased expression of anti-apoptotic BH3 proteins such as BCL2 and BCLXL can be counteracted by the use of BH3 mimetics (venetoclax, navitoclax, and AZD-5991). APR-246 can bind mutant p53 and restore its wild-type, tumour-suppressing function, whereas MDM2 inhibitors (idasanutlin and NVP-HDM201) can prevent wild-type p53 ubiquitination and consequent degradation via the proteasome. Alternatively, tumour suppressor protein degradation can be prevented by proteasome inhibitors (bortezomib). The increased activity of cell cycle regulators such as CDK4/6 can be blocked by CDK inhibitors (ribociclib or palbociclib), whereas aberrant transcription induced by BRD4 can be targeted by BET inhibitors (OTX015). The nuclear trafficking of oncogenic mRNA and proteins can be targeted via XPO1 inhibitors (selinexor). Alternatively, molecularly targeted protein kinase inhibitors have gained an important place in the therapeutic portfolio for T-ALL, used separately or in combination therapies with traditional treatments [8,9,10,11] (Table 1).

Protein kinases are phosphotransferases responsible for transferring the γ-phosphate from ATP to the specific amino acid residues of the substrate proteins. This process is known as protein phosphorylation. Phosphorylation is a critical mechanism that regulates many cellular functions, such as cell proliferation, cell cycle, apoptosis, motility, growth, and differentiation [12]. Imbalance in protein kinase expression and activity play a central role in the survival and spread of cancer cells [12]. There are several ways for kinases to become involved in carcinogenesis: either via imbalanced expression, aberrant phosphorylation, or genetic changes, including gene mutations, chromosomal translocations, and alterations in epigenetic regulation [13,14]. Consequently, kinase inhibitors have become a cornerstone of personalised cancer therapy. This therapeutic approach has expanded substantially, with over 70 kinase inhibitors currently approved for clinical use and more than twice that number undergoing clinical evaluation [15].

Table 1. Epigenetic regulation of kinase expression and therapeutic kinase inhibitors in T-ALL.

Kinase Classification	Kinase Targets	Epigenetic Signalling Involved and Biological Relevance	Organism and/or Cell Type	Inhibitors	Article(s)
Serine/threonine kinases	CDKN2B/p15INK4b	- 5′CpG island hypermethylation of CDKN2B - Blocked cell division G1/S	Human (paediatric and adult T-ALL)	/	[16,17,18,19]
	DAPK	- DAPK promotor hypermethylation - Apoptosis resistance	Human—Burkitt lymphoma and B-ALL	/	[20,21,22]
	MAP2K7	- KLF4 (TF) deficiency - Aberrant activation of MAP2K7 - Biological relevance unknown	Mouse and human (paediatric) T-ALL—lymphoblasts	/	[23]
	CDK2	- Deacetylation of CDK2 - Increased cell proliferation - Decreased apoptosis	Mouse human T-ALL—T6E, 8946, and KOPT-K1 cells	/	[24]
	IRAK1	- hsa-miR-204 promotor hypermethylation - Activates NF-κB pathway - Cytokine secretion - Promotes metastasis	Human T-ALL—Jurkat cells	/	[25,26,27,28]
	ERK	- Expression of miR-181 - Biological relevance unknown	Mouse—lymph node cells	Indirect through MEK inhibitors: Selumetinib Trametinib	[29]
Tyrosine kinases	EphB6	- EPHB6 promotor hypomethylation - Altered lymphokine secretion - Inhibition of cell proliferation	Human—Jurkat cells	/	[30,31]
		- EPHB6 promotor hypomethylation - Fas-mediated apoptosis of T cells - Suppression of the JNK/GTPase/Rac1 pathway - Downregulation of TCR-mediated apoptosis	Mouse—thymocytes		[32,33]
	EphB1	- EPHB1 promotor hypomethylation - Anti-apoptotic signals to neighbouring thymocytes	Mouse—embryonic and adult thymic T cells	/	[34]
	EphB4	- EPHB4 promotor hypermethylation - Reduced apoptosis - Increased cell growth	Human—cell lines and bone marrow samples	/	[35]
		- EPHB4 promotor hypermethylation - Reduced apoptosis - Increased cell growth	Human (paediatric)—newly diagnosed patients		[36]
	EphA3	- No to low methylation in 5′ upstream region of EPHA3 - Biological relevance unknown	Human—HSB2, Jurkat, MOLT4 cell lines and patients with high blast counts	/	[37]
	SYK	- SYK promotor hypermethylation - Biological relevance unknown	Human—T-ALL patients and pro-B-lineage ALL	/	[38,39]
	LCK	- Expression of miR-181 - Biological relevance unknown	Mouse—lymph node cells	Indirect through ABL/SRC inhibitors: Imatinib Dasatinib	[29]
	ZAP70	- Expression of miR-181 - Biological relevance unknown	Mouse—lymph node cells	/	[29]
Nucleotide kinase	dCK	- H3 and H4 deacetylation at dCK promotor - Replication stress - Cell growth arrest - DNA damage - Impaired T-cell signalling	Human T-ALL—cell lines	TRE-515	[40,41,42]
RNA polymerase II kinase CTD Ser kinase	BRD4	- BRD4 activation - c-Myc upregulation - Apoptotic arrest	Human (paediatric)—primary cells T-ALL	/	[43,44,45]
		- BRD4 inhibition - Depletion of LIC development - Delayed leukaemia development	Mouse T-ALL		[46]
Lipide kinase Ser/Thr kinase	PI3K/AKT(/mTOR)	- Overexpression of miR-363-3p - Suppression of PTEN and BIM - Anti-apoptotic - Pro-proliferative	Human (paediatric) T-ALL—patients and cells	PI3K: Buparlisib AKT: MK-2206 mTOR: Sirolimus Everolimus Temsirolimus	[47,48,49,50]
		- Inactivation of Circ-PRKDC - Inhibition of cell proliferation - Induced autophagy - Induced apoptosis	Human T-ALL—tissue		[51]
		- Upregulation of mTOR - Decreased miR-150 expression - Accumulation of immature/abnormal T cells - Increased cell proliferation - Increased cell differentiation - Increased cell survival	Human T-ALL—cell lines		[52]
		- miR-26 inhibition - Cell proliferation	Mouse—Pten-deficient T-ALL		[53]
Non-receptor tyrosine kinases	JAK/STAT	- Overexpression of miR-363-3p - Suppression of PTPRC and SOCS2	Human (paediatric) T-ALL—patients and DND-41, ALL-SIL cells	JAK: Ruxolitinib	[48]
Lipide kinase/phosphatase	PI3K/PTEN	- Expression of miR-19 - Degradation of PTEN - Enhanced proliferation - Enhanced cytokine secretion - Inhibition of apoptosis	Mouse and Human T-ALL	PI3K: Buparlisib	[54,55]

Table 1. Epigenetic regulation of kinase expression and therapeutic kinase inhibitors in T-ALL.

Kinase Classification	Kinase Targets	Epigenetic Signalling Involved and Biological Relevance	Organism and/or Cell Type	Inhibitors	Article(s)
Serine/threonine kinases	CDKN2B/p15INK4b	- 5′CpG island hypermethylation of CDKN2B - Blocked cell division G1/S	Human (paediatric and adult T-ALL)	/	[16,17,18,19]
	DAPK	- DAPK promotor hypermethylation - Apoptosis resistance	Human—Burkitt lymphoma and B-ALL	/	[20,21,22]
	MAP2K7	- KLF4 (TF) deficiency - Aberrant activation of MAP2K7 - Biological relevance unknown	Mouse and human (paediatric) T-ALL—lymphoblasts	/	[23]
	CDK2	- Deacetylation of CDK2 - Increased cell proliferation - Decreased apoptosis	Mouse human T-ALL—T6E, 8946, and KOPT-K1 cells	/	[24]
	IRAK1	- hsa-miR-204 promotor hypermethylation - Activates NF-κB pathway - Cytokine secretion - Promotes metastasis	Human T-ALL—Jurkat cells	/	[25,26,27,28]
	ERK	- Expression of miR-181 - Biological relevance unknown	Mouse—lymph node cells	Indirect through MEK inhibitors: Selumetinib Trametinib	[29]
Tyrosine kinases	EphB6	- EPHB6 promotor hypomethylation - Altered lymphokine secretion - Inhibition of cell proliferation	Human—Jurkat cells	/	[30,31]
		- EPHB6 promotor hypomethylation - Fas-mediated apoptosis of T cells - Suppression of the JNK/GTPase/Rac1 pathway - Downregulation of TCR-mediated apoptosis	Mouse—thymocytes		[32,33]
	EphB1	- EPHB1 promotor hypomethylation - Anti-apoptotic signals to neighbouring thymocytes	Mouse—embryonic and adult thymic T cells	/	[34]
	EphB4	- EPHB4 promotor hypermethylation - Reduced apoptosis - Increased cell growth	Human—cell lines and bone marrow samples	/	[35]
		- EPHB4 promotor hypermethylation - Reduced apoptosis - Increased cell growth	Human (paediatric)—newly diagnosed patients		[36]
	EphA3	- No to low methylation in 5′ upstream region of EPHA3 - Biological relevance unknown	Human—HSB2, Jurkat, MOLT4 cell lines and patients with high blast counts	/	[37]
	SYK	- SYK promotor hypermethylation - Biological relevance unknown	Human—T-ALL patients and pro-B-lineage ALL	/	[38,39]
	LCK	- Expression of miR-181 - Biological relevance unknown	Mouse—lymph node cells	Indirect through ABL/SRC inhibitors: Imatinib Dasatinib	[29]
	ZAP70	- Expression of miR-181 - Biological relevance unknown	Mouse—lymph node cells	/	[29]
Nucleotide kinase	dCK	- H3 and H4 deacetylation at dCK promotor - Replication stress - Cell growth arrest - DNA damage - Impaired T-cell signalling	Human T-ALL—cell lines	TRE-515	[40,41,42]
RNA polymerase II kinase CTD Ser kinase	BRD4	- BRD4 activation - c-Myc upregulation - Apoptotic arrest	Human (paediatric)—primary cells T-ALL	/	[43,44,45]
		- BRD4 inhibition - Depletion of LIC development - Delayed leukaemia development	Mouse T-ALL		[46]
Lipide kinase Ser/Thr kinase	PI3K/AKT(/mTOR)	- Overexpression of miR-363-3p - Suppression of PTEN and BIM - Anti-apoptotic - Pro-proliferative	Human (paediatric) T-ALL—patients and cells	PI3K: Buparlisib AKT: MK-2206 mTOR: Sirolimus Everolimus Temsirolimus	[47,48,49,50]
		- Inactivation of Circ-PRKDC - Inhibition of cell proliferation - Induced autophagy - Induced apoptosis	Human T-ALL—tissue		[51]
		- Upregulation of mTOR - Decreased miR-150 expression - Accumulation of immature/abnormal T cells - Increased cell proliferation - Increased cell differentiation - Increased cell survival	Human T-ALL—cell lines		[52]
		- miR-26 inhibition - Cell proliferation	Mouse—Pten-deficient T-ALL		[53]
Non-receptor tyrosine kinases	JAK/STAT	- Overexpression of miR-363-3p - Suppression of PTPRC and SOCS2	Human (paediatric) T-ALL—patients and DND-41, ALL-SIL cells	JAK: Ruxolitinib	[48]
Lipide kinase/phosphatase	PI3K/PTEN	- Expression of miR-19 - Degradation of PTEN - Enhanced proliferation - Enhanced cytokine secretion - Inhibition of apoptosis	Mouse and Human T-ALL	PI3K: Buparlisib	[54,55]

Epigenetic modifications involve changes in chromatin structure through chemical DNA alteration [56], post-translational modifications of DNA-bound histone proteins [56], gene expression regulation through non-coding RNAs [56], and changes in the higher-order 3D chromatin structure [57,58,59]. All of these contribute to tumorigenesis and metastatic predisposition [56]. The epigenetic reshaping of the T-ALL genome has shown the occurrence of genetic lesions in genes involved in epigenetic homeostasis [60,61]. These highlight the role of key epi-modulators such as the histone lysine N-methyltransferase enzyme EZH2, the transcriptional repressor EED, the lysine histone demethylase UTX encoded by the KDM6A gene, and the transcriptional regulator PHF6, suggesting their role in T-ALL development and expansion [60,62,63]. A genome-wide sequencing study covering thousands of paediatric cancer genomes reported by Huether et al. [61] found that T-ALL was amongst the paediatric tumours with the highest frequency of mutations (56%, 46% gliomas, 43% medulloblastomas) in epigenetic regulator genes [61]. In this review, we will focus on the current state of knowledge on the role of protein kinases in T-ALL pathogenesis and how epigenetic mechanisms can influence the signalling cascade orchestrated by these enzymes in T-ALL pathology.

2. Role of Protein Kinases in T-ALL

Every living cell relies on efficient communication pathways for its survival. Millions of different signalling molecules reach the cell at any given moment, and these must be processed to generate the appropriate response. This requires the presence of an accurate and fast network that can distinguish and transmit these signals to the correct cellular compartment [64]. Protein kinases are amongst the most important players in this vast signalling transduction network [65]. This “kinome” family of proteins consists of 538 different kinases, which play a role in many vital cellular processes, including differentiation, cell death, cell mobility, cell cycle proliferation, and many more [65,66]. This functional diversity is also reflected at the gene level, where almost 2% of all eukaryotic genes are translated into kinases, making them one of the largest eukaryotic gene families [67].

Chromosomal mapping has provided evidence indicating that more than 40% of protein kinase genes map either disease loci or cancer amplicons [67] implicated in the pathogenesis and drug resistance mechanisms of practically all types of leukaemia [68]. Around 65% of T-ALL cases have mutations that activate kinase signalling pathways, including PI3K/AKT, JAK/STAT, and RAS signalling, as reported by Liu et al. [69]. In addition, genomic rearrangements involving multiple kinase-coding genes have also been identified in T-ALL pathophysiology. These rearrangements include the episomal amplification of NUP214-ABL1 and activating mutations in FLT3 genes [69,70,71]. Given the importance of these kinases in cancer biology, many “small molecule inhibitor drugs” have already been developed and are widely used in managing acute leukaemia [8]. In T-ALL, the JAK1/2 inhibitor ruxolitinib, when combined with dexamethasone, showed a strong synergistic pro-apoptotic effect on T-ALL patient-derived xenograft models independent of the occurrence of JAK/STAT mutations [72]. Moreover, BEZ235 and PKI-587, both dual PI3K/AKT/mTOR inhibitors, have been demonstrated to exert tumour suppressor effects on T-ALL [73]. BEZ235 potentiated dexamethasone cytotoxicity in cell lines established from newly diagnosed and relapsed T-ALL paediatric patients by regulating the expression of apoptosis signalling-related molecules such as BIM, MCL-1, and AKT1 [73]. PKI-587, in turn, inhibited cell growth and in vitro colony formation and delayed tumour formation in T-ALL cell lines and mouse xenograft models [74]. Additionally, other potentially druggable kinases, including the JAK family member tyrosine kinase 2 (TYK2), activated by gain-of-function mutations, or the Polo-like kinases (PLKs), Aurora kinases (AURKs), and PIM1 kinase, the latter being regulated by the glucocorticoid–IL7R pathway, have been linked to the development of T-ALL [3,75,76,77]. An overview of the role of these protein kinases in T-ALL is presented below.

2.1. PI3K/AKT/mTOR Signalling in T-ALL

The PI3K/AKT signalling network is one of the most frequently altered pathways in cancer. It is a master regulator of signalling cascades that control cell proliferation, differentiation, self-renewal, and survival and contributes to tumorigenesis and cancer therapy resistance [78]. Upon activation through, e.g., growth factor stimulation, PI3K phosphorylates PIP2 to PIP3, leading to the phosphorylation of AKT ending in the subsequent phosphorylation of target genes and proteins with both tumorigenic effects or tumour suppression activity, such as MDM2, GSK-3β, FOXO1, BAD, mTOR, and NK-κB [78]. During normal T-cell development, the PI3/AKT/mTOR pathway is activated by NOTCH1, IL7R, and pre-TCR signalling to promote cell survival [79], as demonstrated in knockout mouse models where the inhibition of PI3K/AKT/mTOR pathway resulted in impaired T-cell differentiation [80].

Aberrant activation of the PI3K/AKT/mTOR pathway is a common feature in T-ALL, being detectable in 50–80% of the patients [81] and associated with worsened disease outcomes [78]. Several studies have demonstrated the constitutive and induced hyperactivation of the PI3K/Akt/mTOR pathway in T-ALL [82,83,84], which is mostly linked to PTEN and PTPN2 deletions and AKT1 mutations [69]. PTEN is a lipid phosphatase with tumour suppressor activity that acts as the main negative regulator of the PI3K/AKT/mTOR pathway [85]. The inactivation of PTEN in T-ALL has been linked to the occurrence of non-sense mutations and deletions in the PTEN gene [85], resulting in a significant decrease in the levels of this protein. A lack of PTEN expression leads to the hyperactivation of PI3K and its downstream effectors, enhancing a pro-tumoural environment favouring the proliferation and survival of cancerous cells [86].

However, the regulation of the PTEN–PI3K axis in T-ALL remains controversial in T-ALL. Proof of this is the multicentre study performed by Silva et al. [87] involving paediatric and adult T-ALL patients showing that PI3K/Akt activation may occur in the presence of high levels of PTEN protein [87]. This study demonstrated that PTEN phosphorylation (by CK2) and oxidation (by ROS) in T-ALL cells downregulated PTEN activity without decreasing its expression and consequently promoted PI3K/Akt/mTOR constitutive hyperactivation [87]. In line with this, it was previously reported that the phosphorylation of PTEN prevents proteasome-mediated degradation, resulting in a more stable but less active protein, ultimately leading to PI3K/Akt pathway constitutive activation [88]. In the study of Silva et al., T-ALL cells had constitutively high levels of ROS [87]. It has been recently reported that ROS, produced through IL-7 signalling, might induce PI3K/AKT/mTOR activation, promoting the proliferation and survival of T-ALL cells [89]. These mechanisms were confirmed when CK2 inhibitors and ROS scavengers restored PTEN activity and impaired PI3K/Akt signalling in T-ALL cells [87]. And the inhibition of PI3K and/or CK2 promoted T-ALL cell death without affecting normal T-cell precursors [87].

Besides PTEN inactivation, other mechanisms, including stimulation with cytokines and growth factors, can lead to the hyperactivation of the PI3K/Akt/mTOR pathway in T-ALL. One of them is the overexpression of the IGF-1/IGF-1R and Notch-1 signalling pathways, where the pharmacologic inhibition or genetic deletion of IGF-1R reduces T-ALL cell proliferation and survival [90]. In addition, cytokines produced by thymic epithelial cells and the bone marrow microenvironment, including the interleukins IL-4 and IL-7, as well as CXC chemokine ligand 12 (CXCL12), constitute important sources for the PI3K/AKT/mTOR signalling activation in this disease. Cardoso et al. demonstrated that IL-4 induces the proliferation, cell cycle progression, and growth of T-ALL cells by mediating phosphorylation and activating the PI3K/Akt/mTOR pathway [91]. Additionally, increased signalling downstream of IL-7Rα has been described in about 9% of paediatric T-ALL [92] patients. CXCL12, another cytokine produced by bone marrow stromal cells from T-ALL patients, is also an activator of the PI3K/Akt/mTOR signalling and is implicated in drug resistance mechanisms in T-ALL cells [93]. Finally, IL-7 signalling has also been implicated in activating the MAPK/ERK pathway and driving MAPK/ERK-induced steroid resistance in T-ALL [50].

2.2. MAPK/ERK Signalling in T-ALL

The MAPK/ERK pathway regulates a wide variety of basic cellular processes, including proliferation, differentiation, apoptosis, stress responses, and tissue remodelling [94]. Several stimuli can activate the MAPK/ERK pathway, including growth factors, cytokines, viruses, Ras kinase, PKC, and GPCR activation [95]. ERK is a potent serine/threonine kinase with a diverse range of nuclear and cytoplasmic substrates. ERK1/2 is located in the cell cytoplasm, and when activated, it is transferred to the nucleus, where it regulates (through phosphorylation) the activity of various transcription factors and proto-oncogenes such as c-Fos, c-Jun, the ETS domain-containing protein Elk-1, the c-Myc proto-oncogene, and the transcription factor ATF2 [50]. Cytoplasmic ERK1/2, in turn, can also phosphorylate cytoskeletal components such as microtubule-associated proteins MAP1, MAP2, and MAP4, regulating cell morphology processes and cytoskeletal redistribution [50].

The MAPK/ERK pathway is critically involved in normal T-cell development, and it has been shown that ERK1/2-deficient thymocytes may alter the transition of double-negative to double-positive and positive selection of T-cell precursor cells during T-cell differentiation [96]. RAS/MEK/ERK signalling is frequently hyperactivated in T-ALL patients through gain-of-function mutations in the NRAS, KRAS, and BRAF genes [69]. On the other hand, inactivating mutations targeting NF1; activating mutations in FLT3, EGFR, and PTPN11; and chromosomal translocations, including those of BCR-ABM and TEL-PDGFR, have been identified as alternative pathways for downstream MEK/ERK pathway activation [69].

In a recent study, Van der Zwet et al. showed that IL-7-induced steroid resistance in paediatric T-ALL patient-derived xenografts was mediated by the activation of the MAPK/ERK pathway [50]. Activated ERK induced the phosphorylation of the pro-apoptotic BIM (Bcl-2 interacting mediator of cell death) molecule [50]. BIM is an important transcriptional target gene of the glucocorticoid receptor (NR3C1) [97], and its downregulation has been linked to steroid resistance in paediatric ALL patients [97]. In the study by Van der Zwet et al., the phosphorylation of BIM resulted in decreased binding of this molecule to the anti-apoptotic molecules BCLXL, BCL2, MCL1, and BMF, leading to steroid resistance [50]. Of interest, the inhibition of the MAP/ERK pathway by an MEK inhibitor had a synergistic interaction with steroids, reverting the resistance mechanism. These findings illustrate the importance of MAPK/ERK signalling in T-ALL and its potential in future steroid-combined therapies.

2.3. IL7R/JAK/STAT Pathway Activation in T-ALL

The JAK/STAT pathway includes serine/threonine Janus kinase (JAK) and signal transducer and activator of transcription (STAT) molecules. This pathway is activated in response to the binding of cytokines, chemokines, and growth factors through several transmembrane receptor families resulting in changes in gene expression [98]. These signal transduction events control embryonic development as well as processes such as stem cell maintenance, haematopoiesis, and the inflammatory response [98]. Many of these receptors transmit anti-apoptotic, proliferative, and differentiation signals, and their expression and functions are critical in the pathogenesis of lymphoid and myeloid malignancies [98].

In humans, IL7 is crucial for T-cell development and the homeostasis of mature T cells. Loss-of-function mutations in IL7R lead to severe T-cell lymphopaenia, while hyperactive IL7R signalling is correlated with the occurrence of different lymphoid malignancies, including T-ALL [92,99]. Gain-of-function mutations in IL7R are found in 10% of childhood T-ALL patients, leading to constitutive IL7Rα activation [92]. However, mutations are seen not only in IL7R but at all levels of the IL7/IL7Rα signalling cascade, including cytokine receptors, JAKs, STATs, and associated regulatory proteins [92]. Gain-of-function mutations in JAK1, JAK3, and STAT5 have been identified in a 6–27% of T-ALL patients. However, JAK mutations do not always occur in an isolated manner but most of the time are co-expressed with activating mutations in TYK2 and IL-7R and deactivating mutations in PTPN2 and PTPRC genes, all resulting in constitutive activation of the JAK/STAT pathway [100,101]. In general, the regulation of the JAK/STAT pathway is extremely complex and covers interactions with broad immune and cell cycle-dependent signalling mechanisms, irrespective of intrinsic mutations.

2.4. Activation of ABL1 Kinase in T-ALL

The Abl family of non-receptor tyrosine kinases in vertebrates consists of two proteins, Abl (Abl1) and Arg (Abl2), which are generated by the alternative splicing of the first exon of the ABL1 gene [102,103]. The ABL1 gene (v-abl Abelson murine leukaemia viral oncogene homologue 1) was initially mapped in chromosome 9 by Heisterkamp et al. [104]. Later, De Klein et al. [105] showed the translocation of the gene from chromosome 9 to chromosome 22, resulting in an abnormal ABL1 protein with tyrosine kinase activity [106]. This non-receptor tyrosine protein kinase plays an important role in many key processes linked to cell growth and survival, receptor endocytosis, autophagy, and DNA repair processes [107]. More importantly, ABL kinases are activated by TCR engagement and are required for maximal TCR signalling [108]. They play a crucial role in the modulation of T-cell development and in the control of TCR signalling, as well as in the regulation of T-cell cytoskeletal remodelling processes, which, in turn, influence immunological, migration, and adhesion responses [108].

Episomal ABL1 gene rearrangements are observed in 8% of T-ALL patients [109]. Amongst these rearrangements, NUP214-ABL1 fusion is the most frequent and highly specific for T-lineage cells and has been reported in 5% of T-ALL patients [110]. In contrast, other gene fusions such as EML1-ABL1, BCR-ABL1, and ETV6-ABL1 are rare in T-ALL and most often associated with other haematologic malignancies like CML or precursor B-ALL [111]. NUP214-ABL1 gene fusion results in an ABL1 product displaying constitutive tyrosine kinase activity and activates survival and proliferation pathways that contribute to tumour progression and expansion [71,110,112]. Further, ABL1 rearrangement-associated leukaemia has been associated with poor prognosis. And although the discovery of tyrosine kinase inhibitors has improved the outcome, the sensitivity to these drugs is quite variable [110]. Of interest, NUP214-ABL1 was shown to confer experimental susceptibility to the ATP-ABL competitor imatinib treatment in T-ALL patients [113]. However, clinical studies addressing this field are limited and no conclusive answer can be made.

2.5. Role of PIM Kinases in T-ALL

The serine/threonine proviral integration sites for Moloney murine leukaemia virus (PIM) serine/threonine kinases have been implicated in promoting growth and survival in multiple cell types. The PIM kinase family consists of three family members, PIM1, PIM2, and PIM3, which, unlike most other kinases, are constitutively active, containing a kinase domain but not a regulatory domain [114]. Therefore, their activity is not dependent on post-translational modifications but their expression is rather tightly regulated at the transcriptional and translational level [115]. PIM kinase transcription is rapidly upregulated upon stimulation with growth factors, interleukins (e.g., IL4 and IL7), interferons, and GM-CSF [116,117], which transduce signals mainly through the JAK/STAT pathway [118]. In the context of T-cell homeostasis, PIM kinases are required for efficient T-cell activation, proliferation, differentiation, and cytokine secretion [118,119,120].

PIM kinases were originally identified as drivers of haematological malignancies [121,122], and PIM1 is considered a potential molecular target in human T-ALL [123]. Two independent studies reported the aberrant activation of PIM1 linked to TCRβ-PIM1 translocation in T-ALL patients [123,124].

The overexpression of PIM kinase family members has been associated with poor prognosis in T-ALL [125]. A study performed by Padi et al. [126] using T-ALL model cell lines and bioinformatics analysis of clinical datasets demonstrated that pan-PIM inhibitors (AZD1208 and LGB32) could block the growth of human T-ALL cell lines. Importantly, the sensitivity of T-ALL cell lines to the inhibitors was positively correlated with high PIM levels, the activation of the JAK/STAT pathway, reduced MYC expression, and a mature (CD4⁺/CD8⁺) disease phenotype [126]. Later, it was shown that cell-intrinsic PIM1 activation in primary T-ALL cells, rendering them susceptible to pan-PIM inhibitors (AZD1208 and TP3654), could occur through TCR-driven translocations [123,124] or activating mutations in IL7RA, JAK1, JAK3, and STAT5B or loss-of-function alterations in PTPN2 [123,124]. In addition, De Smedt et al. [75,125,126] focused on the study of the “non-cell-autonomous” activation of specific oncogenic pathways as an alternative to increasing the success of PIM inhibitors in T-ALL. This study demonstrated that increasing endogenous IL-7 levels via glucocorticoid administration upregulated PMI1 levels in CD127+ T-ALL/T-LBL PDX models, rendering them sensitive to the PIM inhibitor PIM447 and improving leukaemia survival when combined with induction chemotherapy [75,125,126]. To conclude, there is no doubt that PIM1 plays a crucial role in T-ALL pathogenesis and represents a valuable molecule for the management of the disease. However, more research needs to be conducted to address the several mechanisms behind chemotherapy and PIM inhibitors in the clinic.

2.6. Src Family of Kinases

The function of the Src-family kinases (SFKs) Lck and Fyn in T cells has been intensively studied over the past 20 years. There is considerable evidence that these tyrosine kinases play a critical role in T-cell development, activation, and T-cell receptor (TCR) signalling pathways. TCR signalling affects the selection and survival of T cells at different stages of their development and, therefore, has an important role in T-cell leukaemogenesis.

T-cell glucocorticoid-induced apoptosis is mediated by the activation of glucocorticoid receptor during the T-lymphocyte selection process in the thymus [127]. However, activation of the TCR blocks glucocorticoid-induced apoptosis, implying functional crosstalk between these two distinct signalling processes [127]. A recent phospho-proteome profiling study aiming to identify targetable kinases in T-ALL identified a group of highly activated tyrosine kinases that includes Src-family kinases such as LCK, SRC, FYN, YES1, LYN, INSR, and IGF-1R and serine/threonine kinases such as CDK1/2, AKT, and PAK1/2 [9]. This study also evaluated possible combinatorial treatments using several kinase inhibitors. The results obtained indicated that the inhibition of the INSR/IGF-1R axis sensitised cancer cells to the SRC/ABL kinase inhibitor dasatinib, suggesting that this combination is a promising therapeutic option for T-ALL [9].

Recently, Laukkanen et al. showed that the combination of an mTORC1 inhibitor (temsirolimus) and the general tyrosine kinase inhibitor dasatinib effectively repressed the growth of human cell lines and primary human T-ALL and leukaemia proliferation in patient-derived xenograft mouse models [128]. The effect of these drugs was mediated by the inhibition of LCK phosphorylation and activation and by a reduction in MCL-1 protein expression, resulting in TCR signalling inhibition and the apoptosis of leukaemia cells [128].

2.7. c-Jun NH₂ Kinase (JNK) Signalling

JNK is a member of the mitogen-activated protein kinase (MAPK) superfamily, which also includes extracellular signal-regulated kinase (ERK) and the p38 family of kinases [129]. JNK has two ubiquitously expressed isoforms, JNK1 and JNK2, and a tissue-specific isoform, JNK3 [129]. Activation of JNK is induced by sequential protein phosphorylation through the MAPK pathway in response to a variety of extracellular stimuli [130]. Once activated, JNK phosphorylates and regulates the activity of several transcription factors, such as c-Jun and c-Myc, as well as other molecules, including the Bcl-2 family proteins, playing a critical role in tumorigenesis, cell cycle control, and apoptosis [130,131].

The role of JNK kinases in T-ALL was addressed by Cui et al. [132], showing that the inhibition of JNK1 but not JNK2 resulted in a decrease in growth and pro-apoptotic effects on T-ALL cells [132]. Further, the authors observed the aberrant nuclear accumulation of JNK protein and basal JNK activity in T-ALL cells, in contrast to primary T cells, which exhibited exclusive cytoplasm localisation [132]. These findings suggested that mechanisms other than JNK hyperactivation may underlie the pro-tumorigenic role of JNK in T-ALL.

Moreover, interesting work from Liu and colleagues showed that the inhibition of JNK signalling when using NF-κB inhibitor treatment in Jurkat (T-ALL) cell lines resulted in a synergistic anti-leukaemic effect [133]. Of importance, this effect was exclusive to leukaemic stem/progenitor cells and not healthy haematopoietic stem/progenitor cells, suggesting that this combinatorial approach might provide better treatment for T-ALL [133]. In addition, Liou et al. observed that the resistance of T-ALL Jurkat cells to tetrandrine (a potentially anti-aleukaemic and adjuvant drug for T-ALL) was mediated by the activation of the JNK/AP-1 pathway, as the inhibition of JNK but not ERK suppressed AP-1 activity and tetrandrine resistance [134]. Altogether, these results suggest that targeting JNK may be an important therapeutic strategy in T-ALL management; however, more in vivo studies are needed before it can be used in a clinical set-up.

2.8. Aurora B Kinase

Aurora kinases belong to a family of serine/threonine kinases comprising Aurora A (AURKA), Aurora B (AURKB), and Aurora C (AURKC), well known to have integral roles in the regulation of cell division and for which overexpression or gene amplification has been demonstrated in several cancer pathologies [135,136]. Within the Aurora kinase family, AURKB has been shown to play a crucial role in cell cycle regulation by suppressing p53 activity and stabilising MYC protein, leading to cell cycle progression and survival in cancer cells [135]. The MYC proto-oncogene encodes the c-Myc transcription factor that controls cell growth, proliferation, and survival and is overexpressed in various human malignancies [137]. Levels of c-Myc protein are overexpressed in patients with T-ALL [138]. This results from the phosphorylation of c-Myc at Serine 67 by AURKB, promoting c-Myc protein stability [138]. Of interest, the stabilisation of c-Myc activates, in turn, AURKB transcription, resulting in a feedback mechanism which sustains their promotion of deregulation and malignant T-cell transformation [138]. Further, it was shown that the inhibition of c-Myc leads to T-ALL remission by inhibiting LIC activity [138,139]. This suggests that AURKB inhibitors hold great promise for MYC-targeted therapy in T-ALL [140].

3. Epigenetic DNA Methylation Regulation of Kinase Expression in T-ALL

T-ALL is amongst the paediatric cancer subtypes with the highest frequency of somatic mutations in genes regulating DNA methylation or histone modifier enzymes [61]. Accumulating evidence suggests that epigenetic changes are an important driving force behind the acquisition of drug resistance in cancer [9,14,20,101,102,103,104,105,141,142]. This is because of the dynamic alterations in gene expression due to chemotherapy-induced chromatin remodelling changes, independent of genetic mutations [143]. This is why it is pivotal to understand the biological meaning of methylation in T-ALL. In this second part of this review, we will discuss the epigenetic regulation of kinases in T-ALL, as some of the pathways and kinases affected by these epigenetic changes might have relevance as novel therapeutic targets in this disease [14,144]. Of special note, since epigenetic enzyme activity is also susceptible phosphosite regulation by kinases, targeted kinase inhibitors may also be effective for reversing the epigenetic remodelling of signalling networks [145].

3.1. DNA Methylation Programming of Kinase Signalling Pathways in T-ALL

DNA methylation refers to the transfer of a methyl group to a cytosine nucleotide by DNA methyltransferases such as DNMT1 and DNMT3B [146]. In humans and most other animals, methylation is almost exclusively observed on cytosine nucleotides, followed by guanine nucleotides (the so-called CpG sites). In general, DNA hypermethylation at gene promoter regions leads to the repression of gene expression by preventing TFs from binding and/or by binding methyl-binding domain (MBD) proteins to recruit other transcriptional repressors complexes [147]. However, a more complex relationship between methylation and gene expression may emerge depending on the genomic context [147]. It has been demonstrated that differential DNA methylation is related to T-ALL patient prognosis [148,149]. Global genome hypomethylation together with region-specific hypermethylation, the so-called CpG island methylator phenotype (CIMP), can be considered a hallmark of several cancer pathologies, including T-ALL [149,150,151,152]. Recently, it was observed that CIMP⁺ T-ALL patients have a better prognosis than CIMP⁻ T-ALL patients, although some discrepancies have been reported in this respect [153]. An overview of protein kinase signalling changes by epigenetic dysregulation in T-ALL is presented below.

3.1.1. Hypermethylation of Cyclin-Dependent Kinase Inhibitor Gene Promoters

The cell cycle process is extensively controlled by a family of cyclin-dependent kinases (CDKs), which consist of serine/threonine kinases that become catalytically active when bound to cyclins and control the progression of the cell cycle through the different checkpoints [154]. CDKN2A and CDKN2B are tumour suppressor genes that encode for p16^INK4a/p14ARF and p15^INK4b, respectively, which inhibit cyclin/CDK-4/6 complexes, resulting in the blockage of cell division during the G1/S phase of the cell cycle [16,154]. Tsellou et al. demonstrated that the inactivation of CDKN2B but not CDKN2A was associated with 5′ CpG island hypermethylation in childhood T-ALL [17].

These findings were later confirmed by Batova et al., who found hypermethylation at the 5′ CpG island of the promoter of CDKN2B in 38% of T-ALL patients at diagnosis and in 22% of patients at relapse [18]. CDKN2A hypermethylation, however, was less frequent, as it was observed in only 4% of T-ALL patients at diagnosis and in none at relapse [18]. In addition, Jang et al. observed that T-ALL patients (within his study cohort) with more than 45% methylation in the CDKN2B promoter region exhibited a poor prognosis, with an overall survival rate of 43%, in contrast to those patients with low methylation (<45%), who showed an overall survival of 85% [19]. In the same study, the authors suggested that mutations in DNMT3A observed in the highly methylated group may be one of the mechanisms inducing this increase in CDKN2B promoter methylation leading to T-ALL pathology, as suggested before (Figure 1) [155,156,157,158,159,160].

3.1.2. Differential Methylation Status of Ephrin Tyrosine Kinase Genes

Eph tyrosine kinases comprise the largest family of surface receptors, represented by sixteen Eph receptors, of which fourteen are expressed in human cells [67]. Eph receptors are divided into EphA or EphB subclasses depending on their structural properties and ligand-binding (ephrinA and ephrinB) preferences [161]. Eph signalling controls a wide range of physiological and pathological processes, including immune cell development, survival, migration, and activation, by modifying the organisation of the cellular actin cytoskeleton [162,163]. A particular hallmark of Eph receptors is their capacity to bind to ephrin ligands expressed on the plasma membrane of a neighbouring cell, often forming multimeric structures [164]. They can initiate signal transduction events in each “receptor- and ligand-presenting cell” upon cell–cell contact, triggering forward (through ephrin-ligated Eph receptors) and/or reverse (through Eph-bound ephrins) responses [165].

Several studies have suggested the role of Eph receptors and their ligands in T-cell development. In a study of Shimoyama and colleagues, the T-cell lineage-specific expression of EphB6 was strictly conserved in human leukaemia/lymphoma cells and decreased with T-cell maturation, suggesting its role in T-cell development [31]. Moreover, Luo et al. demonstrated that EphB6 protein is expressed at high levels in Jurkat leukaemic T cells, and cross linking with CD3 protein complex resulted in altered lymphokine secretion, the inhibition of proliferation, and the induction of Fas-mediated apoptosis in these cells [30]. Later, Freywald and colleagues demonstrated that the stimulation of mouse thymocytes with Ephrin B1 (ligand of EphB6) or the overexpression of the EphB6 receptor itself resulted in a reduction in IL-2 secretion and the upregulation of IL-2Ra, resulting in the downregulation of TCR-mediated apoptotic cell death, needed for the removal of self-reacting double-positive thymocytes [32,33]. Of interest, this response was mediated by the suppression of JNK/GTPase Rac1 pathway activation [33].

Importantly, this complex signalling mechanism was evidenced to play a role in T-cell development, as suggested by Yu et al. [34]. This study showed that EphB1 and Ephrin B1 receptors were expressed at high levels in CD4⁺, CD8⁺, and CD4⁺CD8⁺ mouse embryonic and adult thymic T cells [34]. Of interest, soluble Ephrin B1 seems to block the physiological role of cell surface EphB1 in promoting the survival of neighbouring Ephrin B1-bearing thymocytes by delivering anti-apoptotic signals [34].

Eph receptors are expressed in many cancer types, such as lung, breast, and prostate cancer, as well as in leukaemia [166,167]. Their expression is not limited to cancer cells but also to cells in the tumour microenvironment, and they may have either suppressive or tumour-promoting activity, depending on the expression pattern [168,169,170]. In the leukaemia context, EphB6 is overexpressed in T-ALL cell lines and in paediatric patient samples and may suppress T-ALL resistance to doxorubicin by interfering with Akt activity [171]. Moreover, Ephrin B1 was also strongly expressed by paediatric T-ALL cell lines and patients and suppressed the adhesive responses of T-ALL cell lines, resulting in an increase in invasive behaviour [172].

Aberrant DNA methylation has been reported to be an important mechanism in the inactivation of Eph/Ephrin signalling, leading to acute lymphocytic leukaemia progression [173]. Kuang et al. [173] demonstrated in 2010 that fifteen Eph/ephrin family genes were hypermethylated in T- and B-ALL primary cells. This hypermethylation was correlated with the transcriptional silencing of EPHB4, EFNB2, and EFNA5 [35]. Moreover, EPHB4 promoter hypermethylation resulted in its transcriptional silencing in T- and B-ALL cell lines and bone marrow samples [35]. This translated into a reduced apoptotic and increased cell growth capacity of the tumour cells and was potentially associated with poor outcomes in ALL.

Years later, Li et al. studied the effect of EPHB4 methylation on leukaemia pathogenesis and prognosis in a two-year disease-free survival study including forty newly diagnosed cases of childhood ALL (including both T-ALL and B-ALL) [36]. The study supported previous findings by demonstrating that patients with a methylated EPHB4 gene exhibited a shorter disease-free survival time than patients with low or no promoter methylation. This suggests that the methylation status of the EPHB4 gene may serve as a potential prognostic marker for childhood ALL [36].

Another study performed by Dottori et al. demonstrated that the EphA3 receptor is differentially expressed in various human haematopoietic leukaemia cell lines [37]. In the study, the authors showed a high expression of this Eph receptor in HSB2, JM, and MOLT4 cell lines and in T-ALL patients with high blast counts. To investigate the regulation of this gene expression, they analysed the DNA methylation in each cell line and patient sample. Of interest, methylation percentages in the 5′ upstream region did not correlate with the gene and protein expression levels in normal cells but correlated well in leukaemia cell lines and patient samples [37]. T-ALL samples showed low or no methylation in the genomic region analysed, corresponding to the high levels of expression observed [37]. Although the function of the overexpression of EphA3 in T-ALL was not analysed in the study, given the role of EphA3 in controlling cell movement and expansion, these results may be related to tumour cell development and expansion in the disease [37]. However, further research is needed to fully understand the functional consequences of EphA3 methylation in T-ALL.

3.1.3. Hypermethylation of Death-Associated Protein Kinase Gene Promoter

The death-associated protein kinase (DAPK) family of proteins comprises Ca²⁺/Calmodulin-regulated serine/threonine kinases associated with the cytoskeleton that play important roles in a broad range of signal transduction pathways, such as apoptosis, autophagy, and immune responses [174]. DAPK family proteins include DAPK1, DAPK2, DAPK3, and DAPK-related apoptosis-inducing protein kinases (DRAK-1 and DRAK-2) [175].

A lack of expression of DAPK has been associated with resistance to apoptosis in B-cell lymphoma and leukaemia cell lines subjected to INF-γ stimulation, as described by Cohen and Kimchi [20]. However, this altered expression seems not to be exclusively a result of gene promoter hypermethylation. Gene loss or inactivation may also occur due to mutations, deletions, or other DNA rearrangements [176]. In paediatric B-cell malignancies (Burkitt lymphoma and B-cell ALL), the DAPK promoter region has been observed to be methylated in 80–100% of patients, leading to the loss of gene expression and resulting in an increase in malignant cell survival [21,22]. However, the studies reporting DAPK methylation changes in T-ALL are rather limited and controversial. Whereas the gene promoter was observed to be hypermethylated in 17% and 10% of adult and childhood T-ALL patients, respectively, in a Spanish population [177], no methylation was observed in adult T-ALL patients from a German cohort [22,178]. Another comparative study carried out by Gutierrez et al. demonstrated that the degree of methylation of the DAPK gene promoter was significantly lower in T-ALL (13%) than in B-ALL (35%) samples [179]. The above results indicate that a lack of expression of this kinase due to promoter hypermethylation may play a vital role in B-ALL; however, no clear correlation has been found for T-ALL patients.

3.1.4. Hypermethylation of Spleen Tyrosine Kinase (SYK) Gene Promoter

The SYK protein belongs to the cytoplasmic SYK/ZAP tyrosine kinase family, which plays a critical role in early T-cell development in the thymus by regulating key signalling events involved in lineage commitment, pre-TCR signalling, and the β-selection checkpoint. Its activity is essential for the proper differentiation, survival, and maturation of early thymocyte progenitors into functional T cells, paving the way for the subsequent stages of T-cell development [180]. It is involved in several signal transduction pathways that regulate T-cell development, activation, and differentiation, as well as the haematopoiesis of T-cell receptor engagement [180]. SYK is highly expressed in the thymus but downregulated in the periphery [181]. Differential expression levels of SYK may play an important role in the functioning of the immune system and, therefore, may also be involved in oncogenesis.

Several studies have demonstrated that the hypermethylation of CpG islands in the promoter region of the SYK gene plays a crucial role in the pathogenesis of different cancer types [182,183,184]. Goodman et al. demonstrated that T cells and bone marrow of a subset of T-ALL patients showed low or no expression of SYK protein when compared to non-leukaemic cells and that this downregulation directly depended on the hypermethylation of the CpG island in the promoter region of the SYK gene [38]. These findings suggest that SYK may act as a tumour suppressor molecule in T-ALL, as already described in breast cancer [182] and pro-B-lineage ALL [39]. However, further studies are needed to clarify the exact clinical relevance of this SYK promoter methylation in T-ALL.

3.1.5. Epigenetic Silencing of Krüppel-like Factor 4 and MAP2K7 Signalling

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor playing an important role in the regulation of cell growth, proliferation, and differentiation [185].

KLF4 protein has dual functions and may act as either an activator or repressor of gene transcription via several mechanisms. The first mechanism is based on its capacity to both methylate and de-methylate GC-rich sequences or exhibit protein-to-protein interactions with proteins bound to gene regulatory elements, resulting in the recruitment of coactivators or corepressors depending on the cell context or environment [186,187]. The second and third mechanisms involve binding to proteins that impact DNA methylation status and epigenetic marks [188] and the formation of higher-order chromatin structures, especially in embryonic and induced pluripotent stem cells [188,189]. As such, KLF4 may induce or suppress tumorigenesis in a tissue-dependent manner [188,189]. In contrast to that in solid tumours, the role of KLF4 in haematological malignancies has not been well-studied.

This transcription factor directly represses NOTCH1 expression in T-ALL as a negative regulator [190]. It has also been shown that KLF4 is inactivated in Jurkat (T-ALL) cell lines and in paediatric T-ALL patients due to the hypermethylation of this gene promoter region, resulting in transcriptional repression [191].

Further mechanistic studies performed in T-ALL cell-derived xenograft mouse models revealed that a lack of KLF4 expression resulted in the development of a NOTCH1-induced T-ALL phenotype by affecting transition in leukaemic cells promoting LIC expansion [191]. Moreover, KLF4 deficiency leads to the aberrant activation (by phosphorylation) of MAP2K7 and its downstream effectors JNK, c-JUN, and ATF2, as observed in LICs from KLF4-deficient T-ALL mice and lymphoblasts from children with T-ALL [192]. MAP2K7 is a kinase activated in response to cellular stress, leading to the downstream activation of JNK, c-Jun, and ATF2, which regulate the expression of downstream targets involved in the proliferation and self-renewal of leukaemic cells and LICs [192]. However, further studies should be performed to elucidate how MAP2K7 is activated in normal and leukaemic T cells and whether KLF4 epigenetic silencing is associated with patient outcomes.

4. Histone Mark Regulation of Kinase Expression in T-ALL

Epigenetic histone modification marks have been suggested as valuable diagnostic biomarkers (prognosis, therapy response) in T-ALL [193]. However, little is known about how global changes in histone modifications are translated to kinase network signalling, therapy response, or disease stage. High expression of H3K27me3, H3K4me2, and H3K4me3 was observed in bone marrow lymphoid blasts of T-ALL patients [193]. Of interest, although no specific kinase was linked to these histone modifications, ALL patients with Philadelphia cytogenetic aberration [194] and resistance to tyrosine kinase inhibitors (TKIs) showed significantly reduced levels of histone methylation marks at diagnosis compared to long-term survivors [193]. In addition, in vitro and in vivo studies have demonstrated synergistic anti-leukaemic effect of HDAC on ALL upon combination with proteasome, DNMT, and kinase inhibitors (e.g., the Aurora kinase inhibitor MK-0457 and the BCR-ABL1 inhibitor KW-2449) [195,196,197].

4.1. Histone Chromatin Regulation of Deoxycytidine Kinase (dCK) Expression in T-ALL

As stated before, drug resistance represents a significant problem in acute leukaemia therapy, especially in the paediatric patient population [198]. In this context, the efficacy of purine nucleoside analogues such as nelarabine is currently being investigated in treating children with de novo and refractory T-ALL [199]. A study by Yoshida et al. revealed that resistance to nelarabine, observed in several T-ALL cell lines, was correlated with low gene expression of deoxycytidine kinase (dCK) [41]. Moreover, the reduced expression of the enzyme in nelarabine-resistant cells was due to histone H3 and H4 deacetylation at the dCK promoter region (Figure 2). dCK is the rate-limiting enzyme in the nucleoside salvage pathway and plays an important role in nelarabine metabolism [200]. A deficiency in dCK protein has been reported to induce replication stress, cell growth arrest, and DNA damage in erythroid, B-lymphoid, and T-lymphoid lineages. The link of histone deacetylation with nelarabine resistance and dCK expression was confirmed when the sensitive phenotype could be restored after treatment with the histone deacetylase inhibitor vorinostat [42].

4.2. (Histone) Acetylation Regulation of Cyclin-Dependent Kinase 2 (CDK2) in T-ALL

Although the role of CDKs in regulating the cell cycle in the pathogenesis of T-ALL is well established, new regulatory mechanisms have recently been discovered involving chromatin writer–eraser proteins in the last ten years (Figure 2). For example, in vitro experiments revealed that Notch signalling increases protein levels of the histone deacetylase SIRT1 via MYC regulation, which promotes the proliferation of T-ALL cells [24]. Furthermore, interactions between MYC, the histone SIRT1, and CDK2 in Notch-induced T-ALL were found to promote the deacetylation of CDK2 [24,201]. Acetylation has been reported to decrease CDK2-mediated phosphorylation, while deacetylation increases it [202]. The binding of SIRT1 to CDK2 results in increased CDK2 kinase activity. Furthermore, CDK2 deacetylation promotes ubiquitination and phosphorylation at Thr187 of the CDK inhibitor p27 (CDKN1B/KIP1) molecule, resulting in its degradation and increased cell proliferation. Altogether, these results suggest that targeting CDK2 kinase acetylation holds promise as a potential drug target for T-ALL therapy.

4.3. Dual Chromatin Acetylase–Kinase Bromodomain Protein 4 (Brd4) Enhances c-Myc Expression in T-ALL

Bromodomain protein 4 (BRD4) is a chromatin reader–writer protein known to coordinate chromatin organisation and transcription through its dual histone acetyltransferase (HAT) and atypical kinase activity [203]. BRD4 plays a crucial role in the maturation step of thymocyte differentiation [43], and hence, it is a crucial molecule in the pathogenesis of T-cell malignancies.

BRD4 acetylates histone tails and the globular domain of histone H3, resulting in nucleosome dissociation and chromatin remodelling at BRD4-targeted genes [203]. With its intrinsic kinase activity, the enzyme phosphorylates Ser2 in the carboxy terminal of RNA polymerase II, which results in early transcriptional elongation [203]. Wu and colleagues found that BRD4 was overexpressed in paediatric primary T-ALL cell lines, and this overexpression was associated with a poor prognosis due to the enhanced c-Myc expression [43,44]. Reciprocally, the inhibition of BRD4 led to the downregulation of c-Myc and the induction of apoptotic cell death, translating into a markedly reduced leukaemic burden and significantly increased survival rate in T-ALL [43,45].

More recently, Piya et al. demonstrated that the degradation of BRD4 substantially decreases the expression of key molecules involved in leukaemia pathogenesis, such as HES1 (direct target of NOTCH1), PI3K/AKT pathway proteins, and CD44, in PDX and PTEN T-ALL mouse models [46]. The same group also showed that the inhibition of BRD4 protein results in delayed leukaemia development by inhibiting LIC development and improving survival in T-ALL-engrafted mice [46]. Altogether, these results suggest that BRD4 degradation is a promising LIC-targeting therapeutical strategy for T-ALL treatment.

5. Epigenetic microRNA Regulation of Kinase Expression in T-ALL

MicroRNAs (miRNA) belong to a class of non-coding RNAs playing important regulatory roles in gene expression via epigenetic and posttranscriptional (translational) targeting mechanisms. Dysregulated miRNA expression can have oncogenic or tumour-suppressive regulatory functions in T-ALL. Together with protein kinases, miRNAs constitute an important control point in the pathogenesis and development of T-ALL. Moreover, a systematic meta-analysis published by Kyriakidis et al. identified significantly overexpressed members of the miR-128, miR-181, miR-130, and miR-17 families and significantly reduced expression of miR-30, miR-24-2 and miR143~145 clusters, miR-574, and miR-618 in paediatric T-ALL cases compared to that in control individuals [204]. Further, Almeida et al. compared the miRNA expression profiles of bone marrow mononuclear cells from paediatric patients with T-ALL and B-ALL before chemotherapy [205] and found thirty-three differentially expressed miRNAs and sixteen modulated miRNAs that may be used for distinguishing childhood T-ALL and B-ALL subtypes [205].

In this section, we will focus on miRNAs that regulate directly or indirectly the expression of protein kinases influencing T-ALL pathogenesis.

5.1. hsa-miR-363-3p Crosstalk with PI3K/AKT Signalling

MiR-363-3p belongs to the mir-106a-363 oncogenic cluster with an eminent role in many types of cancer, including T-ALL [206]. The overexpression of miR-363-3p together with miR-20b-5p was previously reported to target important tumour suppressor genes such as PTEN and BIM in a cohort of paediatric T-ALL patients [47]. MiR-363-3p directly targets these genes and downregulates their expression levels in vitro, resulting in an anti-apoptotic and pro-proliferative effect on T-ALL cells [48]. PTEN is a negative regulator of the PI3K/AKT pathway, inhibiting cell growth and proliferation by inducing apoptosis, as discussed above [86,97]. BIM has been demonstrated to be repressed by upregulated MYC and PI3K/AKT pathways, resulting in steroid resistance and enhanced survival in T-ALL cells [49,50]. In addition, the results of an integrated study combining mRNA, miRNA, methylation, and proteomic data from T-ALL patient cell lines also identified the PTPRC and SOCS2 genes as direct targets of miR-363-3p [206]. PTPRC (CD45) is a receptor tyrosine phosphatase known to inhibit the SRC and JAK families of kinases by dephosphorylation [207]. Similarly, suppressors of cytokine signalling (SOCSs) are a family of intracellular proteins that also negatively regulate JAK/STAT signal transduction by targeting and degrading cytokine receptors and signalling proteins [98]. In the aforementioned study, the overexpression of miR-363-3p resulted in the downregulation of both transcript and protein expression levels of PTPRC and SOCS2 [48]. These findings prove the oncogenic role of miR-363-3p, which activates JAK/STAT signalling by suppressing two negative regulators, PTPRC and SOCS2. In this respect, miR-363-3p may represent a new predictive diagnostic biomarker for personalised treatment options against T-ALL (Figure 3).

5.2. hsa-miR-204 Crosstalk with IRAK Signalling

Another miRNA, hsa-miR-204, is downregulated in T-ALL, leading to progression and cancer development [25]. One of the identified targets of this miRNA is interleukin-1 receptor-associated kinase (IRAK). This is a ubiquitously expressed serine/threonine kinase that acts as an upstream signalling component of the NF-κB signalling pathway and mediates signal transduction downstream of Toll-like (TLRs) and interleukin-1 receptors (IL1Rs) [26]. The activation of IRAK1 is associated with cytokine secretion from cancer-associated leucocytes, promoting tumour invasion and metastasis [27]. IRAK1 is overexpressed and functional in all T-ALL subtypes, and its inhibition reverses resistance to corticosteroids in Jurkat (T-ALL) cells, suggesting that it is a promising therapeutic target (Figure 3) [28].

Lin et al. observed that DNA methylation in the promoter region of miR-204 directly downregulated its expression during T-ALL development in the Jurkat cell line. The authors also demonstrated that hsa-miR-204 directly targets the 3′UTR of the IRAK1 gene, negatively regulating its expression at both the transcript and protein levels. Of interest, the inhibition of IRAK1 by hsa-miR-204 inhibited the proliferation and enhanced the apoptosis of T-ALL cells [27]. The study also showed that IRAK1 enhanced the expression of MMP-2/MMP-9 by activating the downstream NF-κB pathway, resulting in the proliferation of T-ALL cells [27]. These findings support previous statements that miR-204 targeting of IRAK1 may be a valid targeted therapy for T-ALL patients.

5.3. miR-653-5p Crosstalk with Circ-PRKDC and PI3K/AKT/mTOR Signalling Pathways

Circular RNAs have been suggested to participate in the regulation of lymphocyte differentiation and maturation in haematopoietic compartments via microRNAs such as Circ-PRKDC (Circ 0136666) and miR-653-5p [208]. Circ-PRKDC (Circ 0136666) has previously been associated with drug resistance, tumour cell invasion, and migration in colorectal cancer [51]. MiR-653-5p acts as a tumour suppressor in cervical cancer and melanoma [209,210]. The involvement of these RNAs in T-ALL was addressed by Ling et al., who showed increased expression levels of Circ-PRKDC and decreased levels of miR-653-5p in T-ALL tissues [51,209]. Moreover, the silencing of Circ-PRKDC in T-ALL inhibited cell proliferation, induced autophagy and apoptosis, and inactivated the PI3K/AKT/mTOR pathway. On the other hand, the experimental upregulation of miR-653-5p levels restored the effect of Circ-PRKDC. Finally, this study concluded that Circ-PRKDC upregulates miR-653-5p and increases the expression of RELN to inhibit the PI3K/AKT/mTOR signalling pathway, facilitating cell apoptosis and autophagy in T-ALL. This may represent another valid strategy for targeted treatment of patients with T-ALL.

5.4. hsa-miR-150 Crosstalk with mTOR Signalling

MicroRNA-150 is involved in various biological processes, including haematopoiesis and immune system development. This miRNA has been extensively studied in the context of T-ALL. Research has shown that miR-150 acts as a tumour suppressor in T-ALL, meaning it inhibits the development and progression of cancer. Its expression is frequently downregulated or lost in T-ALL cells, which contributes to the malignant transformation and aggressive behaviour of this disease. Several mechanisms have been identified through which miR-150 exerts its tumour-suppressive effects on T-ALL. For example, miR-150 directly targets and suppresses the expression of genes involved in cell proliferation, survival, and differentiation pathways. By doing so, it helps to maintain the normal balance of cell growth and prevents the uncontrolled expansion of leukaemic cells. Furthermore, miR-150 has been found to regulate the differentiation of T-cell precursors. Its reduced expression in T-ALL impairs the normal maturation of T cells, leading to the accumulation of immature and abnormal cells in the bone marrow and peripheral blood. Overall, the dysregulation of miR-150 in T-ALL contributes to disease progression and aggressiveness. Understanding the role of miR-150 in T-ALL may provide insights into potential therapeutic strategies, such as miRNA replacement therapy or the targeted modulation of its downstream targets, to restore normal cellular processes and inhibit leukaemic growth. A study performed by Podshivalova et al. addressed the role of miR-150 in the pathophysiology of T-ALL [52]. This research revealed decreased miR-150 expression in T-ALL cell lines as compared to that in normal peripheral blood cells. Of interest, the researchers proposed that normal expression of miR-150, for example, in resting T cells, is due to the inactivation of the mTOR pathway, which is constitutively activated in T-ALL. This was confirmed by activating mTOR signalling in non-malignant T cells, which resulted in a significant decrease in miR-150 expression [211]. Moreover, the in vitro treatment of T-ALL cell lines with rapamycin (pharmacological mTOR inhibitor) increased miR-150 expression in a dose-dependent manner. Analyses of the genes serving as targets for miR-150 included genes regulating the cell cycle, molecules in the ERK/MAPK pathway, and genes involved in regulating DNA replication and cell division. An important point in this work is that regulation of miR-150 by mTOR was only observed in T-ALL cells and not in epithelial cancer or B-ALL cells. These findings hold promise for developing new targeted treatment strategies for T-ALL.

5.5. miR-181 Crosstalk with LCK, ZAP70, and ERK Kinase Signalling Pathways

The miR-181 family is composed of six mature miRNAs: miR-181a-1, miR-181a-2, miR-181b-1, miR-181b-2, miR-181c, and miR-181d, playing important roles in cellular metabolism, growth, and development [211,212]. The expression of these miRNAs is dynamically regulated during T-cell development in the thymus and during T-cell differentiation in the periphery [213]. In the thymus, miR-181a facilitates the development of conventional and regulatory T cells, dampening TCR activation by repressing multiple phosphatases that lead to the inactivation of LCK, ZAP70 and ERK kinases [29]. On the other hand, at lower levels in the periphery, miR-181a increases TCR sensitivity to antigens promoting the activation of peripheral of T cells [29].

5.6. miR-19 Crosstalk with PI3K/PTEN Signalling

This miRNA belongs to the miR-17-92 cluster and has been demonstrated to be a potent regulator of Th1 cell responses by enhancing T-cell proliferation, facilitating IFN-γ production, and blocking iTreg differentiation [54]. In Th1 cells, miR-19 targets PTEN, resulting in enhanced proliferation, cytokine secretion, and apoptosis inhibition in T cells [54]. The involvement of miR-19 in T-ALL was addressed by Mavrakis et al. [55], who demonstrated that the miRNA is an oncogene that promotes leukaemogenesis by activating the PI3K pathway in NOTCH-induced T-ALL in vivo models [55]. In turn, the activation of PI3K directly targets key survival molecules such as BIM, PRKAAI, PTEN, and PP2A [55].

5.7. miR-26 Crosstalk with PI3K/AKT Signalling

miR-26 was identified by Mavrakis et al. as part of a group of five miRNAs responsible for the cooperative inhibition of several tumour suppressor genes [55]. Later, Yuan et al. [53] performed miRNA profiling in a T-ALL Pten-deficient mouse model, revealing low expression of miR-26 in T-ALL mice when compared to that in wild-type mouse thymocytes. In the search for mechanistic pathways, the authors demonstrated that miR-26 directly binds to the PIK3CD gene. It is already known that activation of the PI3K/AKT pathway inhibits T-ALL cell proliferation. In the same study, the authors observed that miR-26b expression is regulated by PTEN, which in turn controls the expression of the transcription factor Ikaros, a direct transcriptional regulator of miR-26b. The decreased expression of this miRNA in the T-ALL experimental models points to the role of this miRNA as a tumour suppressor in the development of the disease, and it may be promising for the development of new treatment options.

6. Conclusions

Despite the improvements achieved in integrative medicine using multiple chemotherapeutic agents in T-cell acute lymphoblastic leukaemia (T-ALL), relapse remains the leading cause of death in children. Resistance to conventional chemotherapy, including glucocorticoids (GCs), is a frequent feature of relapsed and refractory T-ALL, being a critical factor influencing treatment response and outcome [214,215]. One of the major causes of this phenomenon is the high clinical and molecular clonal heterogeneity of this disease and the extensive epigenetic plasticity of protein kinase signalling in T-ALL development, from progenitor T-cell differentiation and maturation to the formation of leukaemia-initiating cells, leading to different pathological phenotypes and therapeutic outcomes. Of particular interest, in addition to the multi-omic characterisation of kinase expression signatures, innovative phosphopeptidome-based kinome activity profiling may further allow researchers to identify pharmacologically targetable cell signalling states in the kinome space to overcome therapy resistance/relapse [216,217,218].

Unfortunately, kinase inhibitor therapies are frequently challenged by the emergence of resistance, often within one to two years of treatment initiation [219,220,221]. Resistance mechanisms include secondary mutations within the targeted kinase, alternative splicing events, and the activation of compensatory signalling pathways through epigenomic, transcriptomic, or kinomic feedback loops [14]. Additionally, a metabolically heterogeneous and hypoxic tumour microenvironment (TME) promotes the clonal heterogeneity of the disease, resulting in only the partial elimination of leukaemia cells upon therapy [222,223,224,225]. Therefore, resistant T-ALL clones may be selected and survive under the selective pressure of treatment [225,226,227]. Targeting supportive kinase signals within the TME has been proposed as a strategy to enhance existing therapies and mitigate disease relapse [223,224,225].

A major clinical challenge is that kinase inhibitors, when administered at maximum tolerated doses, impose strong selective pressures that accelerate tumour evolution, leading to the expansion of resistant clones [144,227,228]. High-dose monotherapy often triggers adaptive resistance by activating alternative signalling pathways, while high-dose combinatorial regimens are frequently limited by toxicity, which may necessitate treatment discontinuation [228]. Despite the extensive pipeline of targeted agents in preclinical and clinical testing, the optimal combination strategies to prevent resistance remain uncertain. Emerging evidence suggests that sustained antitumour efficacy with reduced toxicity may be achievable through low-dose, multitarget therapeutic approaches. Current pharmacological strategies aimed at overcoming resistance and re-sensitising tumours to targeted therapies include the development of second- and third-generation inhibitors that address resistance mutations, as well as novel drug combinations that concurrently inhibit primary oncogenic pathways and compensatory survival mechanisms. Examples include kinase inhibitors co-administered with agents targeting epigenetic regulators or hybrid kinase–epigenetic enzyme inhibitors [229,230,231,232]. A deeper understanding of signalling networks and the epigenetic adaptations induced by targeted therapies will be critical for designing effective treatment strategies that prevent disease relapse [227,228]. Overcoming resistance will require the multi-level targeting of tumour cells, either through single agents with polypharmacological activity or rational combinations of highly selective therapies [3,9,228,229,232,233,234]. Finally the unprecedented integrative multi-omic insights into kinase cancer cell signalling networks [234] may offer future opportunities to stratify personalised combination therapies of FDA-approved protein kinase inhibitors that offer the best immune–oncological clinical benefits.