The genetics and molecular biology of T-ALL

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy caused by the accumulation of genomic lesions that affect the development of T-cells. Since many years, it has been established that deregulated expression of transcription factors, impairment of the CDKN2A/2B cell cycle regulators and hyperactive NOTCH1 signaling play prominent roles in the pathogenesis of this leukemia. In the past decade, systematic screening of T-ALL genomes by high resolution copy number arrays and next- generation sequencing technologies has revealed that T-cell progenitors accumulate additional mutations affecting JAK/STAT signaling, protein translation and epigenetic control, providing novel attractive targets for therapy. In this review, we provide an update on our knowledge on T-ALL pathogenesis, on the opportunities for the introduction of targeted therapy and on the challenges that are still ahead.

Introduction

The characterization of chromosomal abnormalities, such as 9p deletions resulting in inactivation of CDKN2A (p16) and CDKN2B (p15) and translocations affecting the T-cell receptor genes, have been fundamental in providing initial insights in the genetic defects present in T-ALL. The incorporation of gene expression profiling has provided an additional view on the subgroups present in T-ALL, each characterized by the presence of specific chromosomal aberrations leading to ectopic expression of one particular transcription factor such as TAL1, TLX1, TLX3 or others. Sequencing approaches focusing on candidate oncogenes or more recently genome wide sequencing (exome sequencing, whole genome sequencing or transcriptome sequencing) have identified more than 100 genes that can be mutated in T-ALL. Only two of these genes, NOTCH1 and CDKN2A/2B are mutated in more than 50% of T-ALL cases, and a large variety of genes is mutated at lower frequency (Table 1). Based on all available genomic data, we can conclude that each T-ALL case contains probably more than 10 biologically relevant genomic lesions, each contributing to the transformation of normal T-cells into an aggressive leukemia cell with impaired differentiation, improved survival and proliferation characteristics, and altered metabolism, cell cycle and homing properties. These changes help the T-ALL cells to proliferate and survive by changing their own behavior as well as to interact effectively with normal cells, which further supports their stem cell characteristics and the survival of these high numbers of abnormal T-cells.

Table 1. Mutation frequencies in adult versus pediatric T-ALL.

Gene		Type of genetic aberration			Frequency (%)
					Pediatric	Adult
NOTCH1 signaling pathway
FBXW7		Inactivating mutations			14	14
NOTCH1		Chromosomal rearrangements/Activating mutations			50	57
Cell cycle
CDKN2A		9p21 deletion			61	55
CDKN2B		9p21 deletion			58	46
RB1		Deletions			12
Transcription factors
BCL11B		Inactivating mutations/deletions			10	9
ETV6		Inactivating mutations/deletions			8	14
GATA3		Inactivating mutations/deletions			5	3
HOXA (CALM-AF10, MLL-ENL and SET-NUP214)		Chromosomal rearrangements/inversions/expression			5	8
LEF1		Inactivating mutations/deletions			10	2
LMO2		Chromosomal rearrangements/deletions/expression			13	21
MYB		Chromosomal rearrangements/duplications			7	17
NKX2.1/NKX2.2		Chromosomal rearrangements/expression			8
RUNX1		Inactivating mutations/deletions			8	10
TAL1		Chromosomal rearrangements/5′ super-enhancer mutations/deletions/expression			30	34
TLX1		Chromosomal rearrangements/deletions/expression			8	20
TLX3		Chromosomal rearrangements/expression			19	9
WT1		Inactivating mutation/deletion			19	11
Signaling
AKT		Activating mutations			2	2
DNM2		Inactivating mutations			13	13
FLT3		Activating mutations			6	4
JAK1		Activating mutations			5	7
JAK3		Activating mutations			8	12
IL7R		Activating mutations			10	12
NF1		Deletions			4	4
KRAS		Activating mutations			6	0
NRAS		Activating mutations			14	9
NUP214-ABL1/ABL1 gain		Chromosomal rearrangement/duplication			8
PI3KCA		Activating mutations			1	5
PTEN		Inactivating mutations/deletion			19	11
PTPN2		Inactivating mutations/deletion			3	7
STAT5B		Activating mutations			6	6
Epigenetic factors
DNMT3A		Inactivating mutations			1	14
EED		Inactivating mutations/deletions			5	5
EZH2		Inactivating mutations/deletions			12	12
KDM6A/UTX		Inactivating mutations/deletions			6	7
PHF6		Inactivating mutations/deletions			19	30
SUZ12		Inactivating mutations/deletions			11	5
Translation and RNA stability
CNOT3		Missense mutations			3	8
mTOR		Activating mutations			5
RPL5		Inactivating mutations			2	2
RPL10		Missense mutations			8	1
RPL22		Inactivating mutations/deletion			4	0
%<5	5≤%<10	10≤ %<15	25>%>50	%>50

Genes targeted by genetic alterations in more than 2% of T-ALL cases are shown in the table. Calculation of the different frequencies is based on several previously published independent studies that analyzed pediatric and/or adult T-ALL patient cohorts. For frequency calculations, chromosomal rearrangements, copy number variations and mutations were considered. For HOXA, TLX1, TLX3, TAL1, NKX2.1, NKX2.2 and LMO2, also gene expression was considered. The HOXA group includes cases that are positive for CALM-AF10, SET-NUP214 or MLL-ELN chromosomal rearrangements. Frequencies are indicated in gray when it was not possible to have separate numbers for pediatric and adult cases.

Transcription factors

Careful gene expression profiling of T-ALL cases has led to the identification of subgroups of T-ALL, each characterized by a specific transcriptional profile and the ectopic expression of one particular transcription factor, often as a consequence of a chromosomal defect. The largest subgroup is defined by ectopic TAL1 expression (in some cases together with LMO1/LMO2), while other major subgroups show mutual exclusive expression of TLX1, TLX3, HOXA9/10, LMO2 or NKX2-1 (Table 2).^1–3 Furthermore, the early T-cell precursor subgroup (ETP-ALL) corresponds to immature T-ALLs expressing ETP/stem cell genes, is characterized by aberrant expression of LYL1,² and hematopoietic transcription factors such as RUNX1 and ETV6 are frequently mutated in this genetically heterogeneous subgroup.^1,4

Table 2. Chromosomal rearrangements resulting in ectopic expression of transcription factors or the generation of fusion genes with transcriptional/epigenetic activity.

chromosomal rearrangement or mutation	partner gene 1 (oncogene)	chromosome location	partner gene 2	chromosome location	consequence of the rearrangement/mutation
Major transcription factors
del(1)(p32p32)	TAL1	1p32	STIL	1p32	Ectopic TAL1 expression driven by STIL promoter
t(1;14)(p32;q11)			T-cell receptor α	14q11	Ectopic TAL1 expression driven by TCR enhancer
t(1;14)(p32;q11)			T-cell receptor δ	14q11	Ectopic TAL1 expression driven by TCR enhancer
small insertion			–	-	Ectopic TAL1 expression driven by de novo enhancer
t(10;14)(q24;q11)	TLX1	10q24	T-cell receptor α	14q11	Ectopic TLX1 expression driven by TCR enhancer
t(7;10)(q34;q24)			T-cell receptor β	7q34	Ectopic TLX1 expression driven by TCR enhancer
t(10;14)(q24;q11)			T-cell receptor δ	14q11	Ectopic TLX1 expression driven by TCR enhancer
t(5;14)(q35;q11)	TLX3	5q35	T-cell receptor δ	14q11	Ectopic TLX3 expression driven by TCR enhancer
t(5;14)(q35;q32)			BCL11B	14q32	Ectopic TLX3 expression driven by BCL11B
inv(7)(p15;q34)	HOXA9/HOXA10	7p15	T-cell receptor β	7q34	Ectopic expression of HOXA genes, predominantly HOXA9 and HOXA10, driven by TCR enhancer
t(10;11)(p13;q14)	PICALM (CALM)	11q14	MLLT10 (AF10)	10p13	PICALM-MLLT10 fusion transcript, resulting in upregulation of HOXA genes
del(9)(q34;q34)	SET	9q34	NUP214	9q34	SET-NUP214 fusion transcript, resulting in upregulation of HOXA genes
inv(14)(q11;q13)	NKX2-1	14q13	T-cell receptor α	14q11	Ectopic NKX2-1 expression driven by TCR enhancer
T(14;20)(q11;p11)	NKX2-2	20p11	T-cell receptor δ	14q11	Ectopic NKX2-2 expression driven by TCR enhancer
t(11;14)(p15;q11)	LMO1	11p15	T-cell receptor α	14q11	Ectopic LMO1 expression, mostly together with LYL1 or TAL1 expression.
			T-cell receptor δ	14q11
t(11;14)(p13;q11)	LMO2	11p13	T-cell receptor α	14q11	Ectopic LMO2 expression, mostly together with LYL1 or TAL1 expression.
			T-cell receptor δ	14q11
del(5)(q14;q14)	MEF2C	5q14	-	-	Small deletions close to MEF2C leading to upregulation of MEF2C expression.
t(7;19)(q34;p13)	LYL1	19p13	T-cell receptorβ	7q34	Ectopic expression of LYL1 driven by TCR enhancer
t(11;14)(p11;q32)	SPI1	11p11	BCL11B	14q32	Ectopic expression of SPI1 driven by BCL11B
Additional transcription factor aberrations
dup(6)(q23;q23)	MYB	6q23	-	-	Increased MYB expression due to extra copy of MYB
t(6;7)(q23;q34)			T-cell receptor β	7q34	Ectopic expression of MYB driven by TCR enhancer
Various translocations	KMT2A (MLL)	11q23	many different partners		KMT2A fusion genes
Mutation/deletion	BCL11B	14q32	-	-	Inactivation of BCL11B
Mutation/deletion	ETV6	12p13	-	-	Inactivation of ETV6
Mutation/deletion	RUNX1	21q22	-	-	Inactivation of RUNX1
Mutation/deletion	LEF1	4q25	-	-	Inactivation of LEF1
Mutation/deletion	WT1	11p13	-	-	Inactivation of WT1

TCR: T-cell receptor gene.

Almost all subgroups of T-ALL are characterized by the clear ectopic expression of one of these transcription factors, except for some immature T-ALL cases where MEF2C expression and other sporadic translocations where SPI1 could be important.¹ The oncogenic roles of TAL1 and TLX1 have been nicely illustrated by studies in the mouse, in cell lines and in patient samples. Overexpression of TAL1 or TLX1 in developing thymocytes in the mouse results in the development of T-ALL with long latency.^5–8 For TAL1, co-expression of LMO1 and ICN1 dramatically decreased disease latency, in part by increasing the number of leukemia initiating cells.⁹ Mouse leukemias that eventually developed in the context of TLX1 expression often harbored mutations in Bcl11b or Notch1, as also observed in human T-ALL.^7,8 These data confirm that ectopic expression of transcription factors such as TAL1 or TLX1 alone can be an initiating step in T-ALL development, but that additional mutations are required to fully transform normal T-cells to leukemia cells. The ectopic expression of these transcription factors could have a strong effect on the differentiation of the cells, as suggested by the correlation with immunophenotype. Moreover, for TLX1 it was recently shown that TLX1 can bind the T-cell receptor enhancer and in this way can suppress TCRalpha expression and T-cell differentiation.¹⁰

There are now four major mechanisms known to cause aberrant expression of transcription factors in T-ALL: (1) chromosomal translocations involving one of the T-cell receptor genes, (2) chromosomal rearrangements with other regulatory sequences, (3) duplication/amplification of the transcription factor, (4) mutations or small insertions generating novel regulatory sequences acting as enhancers (Table 2). The latter mechanism was only recently identified by studies that revealed changes in chromatin structure close to the TAL1 gene, indicative for the presence of a new enhancer region. Detailed analysis of this region led to the identification of mutations that created a de novo binding site for MYB, thereby resulting in recruitment of additional transcriptional regulators and activation of TAL1 expression in cis.^11,12 These data illustrate how non-coding mutations can have a strong effect on leukemia development and we can expect that more of these mutations will be identified in the future as more information comes available on the non-coding part of the genome.

Oncogenic NOTCH1 signaling

The NOTCH1 signaling pathway is essential for the commitment of multipotent hematopoietic progenitors to the T-cell lineage and for further development of thymocites.^13,14 Activation of NOTCH1 signaling constitutes the most predominant oncogenic event involved in the pathogenesis of T-ALL with activating mutations in more than half of T-ALL cases (Table 1).¹⁵ Loss-of-function of negative regulators of NOTCH1 is an alternative mechanism leading to aberrant activation of the pathway. Mutations of FBXW7 are present in 10–15% of T-ALL cases and lead to increased NOTCH1 protein stability.^16,17 The role of the NOTCH1 cascade in the context of T-ALL is discussed in more detail in a companion review article.¹⁸

Increased kinase signaling

Interleukin 7 (IL7) signaling is essential for normal T-cell development and is triggered by the interaction of IL7 with the heterodimeric IL7 receptor. This interaction induces reciprocal Janus kinases 1 (JAK1) and JAK3 phosphorylation and subsequent recruitment and activation of the signal transducer and activator of transcription factor 5 (STAT5). Upon phosphorylation, STAT5 dimerizes and translocates to the nucleus where it regulates the transcription of many target genes, including the anti- apoptotic B-cell lymphoma 2 (BCL-2) family member proteins.^19,20 In addition to the JAK/STAT pathway also the RAS-MAPK and PI3 kinase pathways are activated by IL2, IL7 and SCF that act on the developing T-cells (Figure 1).

Figure 1. Deregulation of the JAK-STAT signaling cascade in T-ALL.

Representation of the different oncogenic mechanisms that lead to aberrant activation of the IL7 signaling in T-ALL. Interaction of IL7 with the heterodimeric IL7 receptor induces reciprocal JAK1 and JAK3 phosphorylation and subsequent recruitment of STAT5. STAT5 dimerizes and translocates to the nucleus where it induces transcription of the pro-survival factor BCL2. IL7 also activates the RAS-MAPK and PI3K kinase pathways. IL7 signaling can indirectly be enhanced by abnormal NOTCH1 signaling, constitutive expression of ZEB2 or by increased presentation of IL7R on the cell surface of thymocytes due to impaired clathrin-dependent endocytosis caused by DNM2 mutations. Proteins that are mutated in T-ALL are indicated with an asterisk. Promising therapeutic agents targeting the oncogenic IL7-JAK-STAT cascade are indicated in red.

Activating mutations in IL7R, JAK1, JAK3 and/or STAT5 are present in 20-30% of T-ALL cases (Table 1),^4,21,22 with a higher representation within TLX3-positive, HOXA-positive and ETP-ALL patient subgroups.^4,23 A small percentage of cases also show aberrations in phosphatases like PTPN2 and PTPRC, which, amongst other substrates, dephosphorylate and inactivate JAK kinases.^24,25 Interestingly, the IL7R signaling cascade can also be hyperactivated in patients that do not carry any genetic aberrations in the IL7R, JAK or STAT5 genes, indicating that still other mechanisms exist to activate this pathway.^26,27 Indeed, only recently it was discovered that loss-of-function mutations in DNM2 lead to increased presentation of IL7R on the cell surface of thymocytes due to impaired clathrin-dependent endocytosis.²⁶ In addition, a rare translocation targeting the zinc finger E-box-binding homeobox 2 locus (ZEB2) has been recently described in T-ALL, and Zeb2 overexpression was shown to increase Il7r expression and Stat5 activation, promoting T-ALL cell survival in a mouse model.²⁷ While most mutations seem to result in activation of JAK1 and JAK3, rare fusion transcripts involving JAK2 have also been described, and wild type TYK2 may have an important role in activating survival pathways of T-ALL cells.^{28 29}

Aberrant activation of the PI3K-AKT pathway results in enhanced cell metabolism, proliferation and impaired apoptosis.^30,31 Hyperactivation of the oncogenic PI3K-AKT pathway in T-ALL is mainly caused by inactivating mutations or deletions of the phosphatase and tensin homologue (PTEN), the main negative regulator of the pathway.^22,32–36 In addition, some T-ALL cases show gain-of-function mutations in the regulatory and catalytic subunits of PI3K, respectively p85 and p110; or in the downstream effectors of the cascade such as AKT and mTOR (Table 1).^22,35,37 PI3K-AKT signaling activation is also achieved through IL7 stimulation or RAS activation.^22,31,38 A recent study on 146 pediatric T-ALL cases described that almost 50% of the patients harbored at least one mutation in the JAK-STAT, PI3K-AKT or RAS-MAPK pathways, underscoring the importance of activation of those cascades for the leukemic cells.²²

In addition to the activation of the JAK kinases or the PI3K pathway, activation of the ABL1 kinase is observed in up to 8% of T-ALL cases, with 6% of patients expressing the NUP214-ABL1 fusion protein.³⁹ NUP214-ABL1 is a constitutively active tyrosine kinase that activates STAT5 and the RAS-MAPK pathway, but much weaker as compared to BCR-ABL1.^39,40 Interestingly, the kinase activity of NUP214-ABL1 is dependent on its location to the nuclear pore^39,41 and its oncogenic properties rely on its interaction with MAD2L1, NUP155 and SMC4 and on the activity of the LCK, a member of the SRC family kinase.⁴² All data collected to date suggest that the NUP214-ABL1 fusion kinase is a weak oncogene that cooperates with additional oncogenic events to drive leukemia development. In that sense, it is of interest to note that the NUP214-ABL1 fusion is always found together with TLX1 or TLX3 expression and is often associated with loss of PTPN2, a negative regulator of NUP214-ABL1.^25,39

RAS-activating mutations were recently described in 44% of diagnosis - relapse sample pairs, indicating an overrepresentation of these defects in high-risk ALL. Interestingly, KRAS mutations rendered lymphoblasts resistant towards methotrexate, while sensitizing them to vincristine.⁴³

Epigenetic factors

T-ALL is one of the pediatric tumor types with the highest incidence of epigenetic lesions, and 56% of samples from children with T-ALL contain mutations in this gene class.⁴⁴ Also in adult T-ALL, mutations in epigenetic factors are highly common (Table 1).^45,46 For a complete overview of all epigenetic lesions identified in T-ALL, we refer to recent reviews on this topic.^47,48

The plant homeodomain protein 6 (PHF6) protein was postulated as an epigenetic factor because it contains 2 atypical plant homeodomain (PHD)-like zinc fingers. Canonical PHD domains typically bind post-translationally modified histones. However, the C-terminal PHD-like domain in PHF6 binds double stranded DNA. Besides DNA, PHF6 binds to a growing list of transcriptional regulators such as the Nucleosome Remodeling Deacetylase (NuRD) complex, the RNA Polymerase II Associated Factor 1 (PAF1) transcriptional elongation complex and Upstream Binding Transcription Factor, RNA Polymerase I (UBTF1), a key transcriptional activator of rRNA. The latter brings us to the nucleolar role of PHF6 and its proposed role in ribosome biogenesis. Besides interacting with UBTF1, PHF6 binds the rDNA promotor and rDNA-coding sequences and modulation of PHF6 levels alters rRNA synthesis rates. Todd and colleagues propose that the N-terminal PHD-like domain may mediate binding to rRNA while the C-terminal domain may bind rDNA. As such, PHF6 may act as a scaffold bridging rDNA transcriptional elongation with early processing of produced rRNA. Finally, PHF6 seems involved in the DNA damage response and cell cycle regulation: PHF6 is a substrate of the DNA damage checkpoint kinase ATM and loss of PHF6 results in accumulation of phosphorylated γH2AX, a mark of DNA double-strand breaks, and in G2/M cell cycle arrest.⁴⁹ Further studies are however required to investigate how inactivation of PHF6 in T-ALL promotes leukemia.

Enzymes involved in regulating methylation of histone 3 lysine 27 (H3K27) are also implicated in T-ALL. Loss-of-function defects in the polycomb repressive complex 2 (PRC2) components EZH2, SUZ12 and EED suggest a tumor suppressor role for this complex in T-ALL,^4,46,50 which is supported by accelerated leukemia onset in mice upon EZH2 downregulation.⁴⁶ PRC2 catalyzes H3K27 trimethylation (H3K27me3) and its tumor suppressor role may be explained by antagonism with NOTCH1: NOTCH1 activation causes eviction of PRC2 and recruitment of the Lysine Demethylase 6B (KDM6B, also known as JMJD3) at NOTCH1 target gene promoters, resulting in loss of the repressive H3K27me3 chromatin modification and activation of these genes.^46,51 Also the Lysine Demethylase 6A (KDM6A, also called UTX) that demethylates H3K27me3 shows inactivating lesions in T-ALL,^50–52 and its downregulation accelerates NOTCH1-driven leukemia in mice.^51,52 Interestingly, UTX binds TAL1, and it is recruited to TAL1 target genes to remove repressive H3K27me3 marks and activate genes. In TAL1-positive leukemias, UTX behaves as a proto-oncogene and is required for leukemia maintenance, which may explain why UTX inactivation so far has not been detected in TAL1 positive cases.⁵³ The mechanism by which UTX plays a tumor suppressor role in TAL1 negative T-ALL is currently not understood. The KDM6B and KDM6A inhibitor GSKJ4 shows promising preclinical results in T-ALL. Whereas the Brand group reported specificity of this drug for the TAL1 positive T-ALL subgroup, the Aifantis group observed sensitivity in all tested T-ALL cell lines and samples.^51,53

RNA metabolism and translation

A few new oncogenes and tumor suppressors recently emerged in T-ALL. Somatic mutations in ribosomal protein genes RPL5, RPL10, and RPL22 have been described in ~20% of T-ALL cases.^50,54 Whereas RPL5 and RPL22 show heterozygous inactivating mutations and deletions, RPL10 contains an intriguing mutational hotspot at residue arginine 98 (R98), with 8% of pediatric T-ALL patients harboring an R98S RPL10 missense mutation. Inactivation of Rpl22 accelerates myristoylated Akt-driven T-cell lymphoma in mice.^54,55 Mechanistically, heterozygous Rpl22 deletion activates NF-κB and induces stemness factor Lin28B.⁵⁴ The roles of the RPL5 and RPL10 R98S defects in T-ALL development remain to be determined. Characterization of RPL10 R98S in yeast revealed that it impairs ribosome formation and translation fidelity. Over time, compensatory mutations are acquired, restoring ribosome biogenesis but not translation fidelity, which may drive expression of an oncogenic protein profile.^56,57 Translational deregulation in T-ALL may however go far beyond mutations in ribosomal proteins: PHF6 has been linked to ribosome biogenesis, major T-ALL oncogenes and tumor suppressors such as PTEN, NOTCH1 and FBXW7 regulate translation, and T-ALL cells are sensitive to translation inhibitors 4EGI-1 and silvestrol.^58–60

Another intriguing novel tumor suppressor that is inactivated in 8% of adult T-ALL samples is CCR4-NOT Transcription Complex Subunit 3 (CNOT3).⁵⁰ The CCR4-NOT complex catalyzes mRNA deadenylation and may also link again to control of protein translation. In addition, roles for CNOT3 as transcriptional regulator have been proposed.⁶¹ Using studies in a fruitfly eye cancer model that is driven by Notch activation, we confirmed that NOT3 (CNOT3 homolog in the fruitfly) is a tumor suppressor gene, but more studies are required to determine how mutations in CNOT3 contribute to leukemia development. It is interesting to note that somatic mutations in CNOT3 have now also been observed in chronic lymphocytic leukemia (CLL) and in some solid tumors.⁶²

Micro RNAs (miRNAs) and long non-coding RNAs (lncRNAs)

Inactivation of Dicer1, an essential component of the miRNA processing machinery, impairs NOTCH1 driven T-ALL development in mice and can induce regression of established NOTCH1 driven tumors, indicating a role for one or more miRNAs in (at least NOTCH1 driven) T-ALL.⁶³ Indeed, many miRNAs are misexpressed in T-ALL, either due to a translocation, as key downstream target of T-ALL oncogenes such as NOTCH1 or TAL1, or via unknown mechanisms.^64–67 Oncogenic miRNAs (onco-miRs) have been identified downregulating the expression of known T-ALL associated tumor suppressor genes. Examples are miR-19b, miR-20a, miR-26a, miR-92 and miR-223 that cooperatively downregulate IKZF1 (or IKAROS), PTEN, BIM, PHF6, NF1 and FBXW7 transcripts.⁶⁸ Other miRNAs are underexpressed in T-ALL, leading to overexpression of T-ALL associated oncogenes. An example here is miR-193b, that regulates the expression of MYB and the anti-apoptotic factor MCL1.⁶⁹ Many additional onco-miRs and tumor suppressive miRNAs have been described in T-ALL. For a partial overview, we refer to reference⁶⁶.

A related field of interest that recently emerged in T-ALL are lncRNAs, a heterogeneous class of transcripts defined by a minimum length of 200 nucleotides and an apparent lack of protein-coding potential. Diverse molecular mechanisms have been described by which lncNRAs control a wide variety of cellular functions, developmental processes and promote disease pathogenesis, including cancer.⁷⁰ Interestingly, different T-ALL subgroups are characterized by distinct lncRNA expression profiles.⁷¹ In addition, a series of NOTCH1 regulated lncRNAs were identified.^72,73 The best characterized one is leukemia-induced noncoding activator RNA 1 (LUNAR1), which is a NOTCH1 regulated pro-oncogenic lncRNA that is required for efficient growth of T-ALL cells by maintaining high expression of insulin-like growth factor 1 receptor (IGF1R).⁷³

Cooperation of oncogenic events

Recent sequencing studies have suggested that on average 10 to 20 protein-altering mutations are present in T-ALL cells.^4,50,74 This accumulation of mutations does not occur randomly, and specific combinations of mutations are often found, suggesting that those activated oncogenes and inactivated tumor suppressor genes are physiologically interconnected and cooperate during the development and progression of the leukemia.⁷⁵ Initial founder genomic lesions initiate a pre-malignant process by interacting with the existing machinery of the physiological cell state⁷⁶ and additional mutations are necessary to drive transformation^75,77 and lead to the development of sub-clonal variegation.

This has been very nicely observed in TAL1/LMO2 positive T-ALL cases, where PTEN inactivation occurs most frequently. Interestingly, transplantation of TAL1 rearranged leukemia cells into immune deficient mice allowed development of leukemic subclones with newly acquired PTEN microdeletions.³² These data suggest that there is an enormous pressure for TAL1 expressing T-ALL cells to inactivate PTEN, and that TAL1 expression and PTEN inactivation cooperate during T-cell transformation. In contrast, components of the IL7R-JAK signaling pathway are frequently mutated in immature T-ALL cases or in TLX/HOXA-positive cases, but are underrepresented in TAL1/LMO2-positive cases. In addition, we observed that mutations of IL7R-JAK are positively associated with mutations and deletions of members of the PRC2 complex (EZH2, SUZ12 and EED).²¹ Similarly, WT1 mutations are most prevalent in TLX3 positive cases.⁷⁸ These and other genomic data (Figure 2) suggest that initiating lesions leading to ectopic transcription factor expression sensitize the cells to alterations of very specific pathways.

Figure 2. Representation of the cooperation of oncogenic events.

The major subclasses of T-ALL are shown based on the expression of the transcription factors TAL1, TLX1, TLX3, HOXA genes, NKX2-1 or LMO2/LYL1. For each subclass, additional genes are shown that are most frequently mutated in that subclass. The PRC2 complex contains EZH2, SUZ12 and EED.

T-ALL Treatment

Current treatment of T-ALL consists of high intensity combination chemotherapy and results in a very high overall survival for pediatric patients.⁷⁹ Unfortunately, this treatment comes with significant short term and long term side effects. Especially for young children, the effects of this high dose chemotherapy on bone development, central nervous system and fertility should not be underestimated.⁸⁰ A complete overview of the treatment options was recently described.^81,82

In addition to the side effects, the occurrence of relapse is another important challenge as it is observed in up to 20% of pediatric and 40% of adult T-ALL.⁸³ Relapsed T-ALL cases are often refractory to chemotherapeutics and are associated with a poor prognosis.^84,85 From comparative genetic studies conducted on T-ALL samples taken at diagnosis and relapse, and studies with xenotransplantation in mice, it is clear that relapse is determined by the clonal evolution of either a minor genetic subclone present in the primary leukemia, or from the clonal expansion of an ancestral cell (pre-diagnosis).^86–89 However, some rare T-ALL late-relapsed cases are considered to be secondary malignancies rather than originated by the initial disease.⁹⁰

The use of whole-exome sequencing has provided a way to take a detailed view on the genomes of relapsed T-ALL cases and led to the identification of activating mutations in cytosolic 5’-nucleotidase II (NT5C2) as one of the causes of relapse.^86,87 NT5C2 is an enzyme responsible for the inactivation of nucleoside-analog chemotherapy drugs.⁹¹ When NT5C2 mutant proteins were expressed in T-ALL lymphoblast, an increased nucleotidase activity was observed and the cells became resistant to 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), two chemotherapeutic drugs used in maintenance treatment of T-ALL, suggesting the important role of the identified NT5C2 activating mutations in therapy resistance.⁸⁶

To further improve the treatment of T-ALL and to reduce the toxicity of current treatment, the introduction of newer targeted agents is awaited. Recent drug development in both oncology and immunology has generated a spectrum of new drugs that could be useful for the treatment of specific T-ALL subsets.

With NOTCH1 being the major oncogene in T-ALL and with drugs available that can interfere with the activation (cleavage) of NOTCH1 by the gamma-secretase complex, a number of clinical trials have been initiated with gamma-secretase inhibitors (GSIs). However, these trials led to disappointing clinical results due to the dose-limiting toxicity and low response rates. It remains to be determined how second generation GSIs will perform, but at least some promising anecdotal responses have already been reported and with milder toxicities.^92–95 Besides GSIs, other therapeutic approaches for the inhibition of NOTCH1 have been developed. Monoclonal antibodies against the NOTCH1 receptor have anti-tumor effects in vitro and in vivo with limited gastrointestinal toxicities.^96,97 Inhibition of ADAM10 may also facilitate effective inhibition of wild-type and mutant NOTCH receptors.⁹⁸ An antibody against the gamma-secretase complex (A5226A) showed pre-clinical activity against T-ALL.⁹⁹ Mastermind inhibiting peptides that mimic the NOTCH1 interaction with MAML (SAMH1 peptides) are also being tested.^100,101

Since both JAK and ABL1 kinases are often activated in T-ALL, available JAK inhibitors (ruxolitinib and tofacitinib) and ABL1 inhibitors (imatinib, dasatinib, nilotinib) could be repurposed for the treatment of T-ALL cases with documented mutations leading to JAK/STAT or ABL1 activation. Several pre-clinical studies have demonstrated activity of ruxolitinib or tofacitinib for the inhibition of T-ALL cells with IL7R or JAK1/JAK3 mutations, while case reports have shown some activity of imatinib or dasatinib for the treatment of NUP214-ABL1 positive T-ALL.^4,23,102,103 The therapeutic potential of targeting the IL7Rα signaling and the use of JAK inhibitors in ALL has been recently reviewed.^104,105

Similarly, recently approved hedgehog pathway inhibitors could show activity in T-ALL. The hedgehog signaling pathway plays an important role in normal T-cell development, which is steered by the secretion of the sonic hedgehog ligand (SHH) by certain thymic epithelial cells.¹⁰⁶ In T-ALL, the hedgehog pathway was reported to be aberrantly activated by rare mutations or ectopic expression of hedgehog pathway genes, and those cases showed response to hedgehog inhibitor treatment.^50,107,108 Finally, some other attractive novel therapies have recently been described in T-ALL. Selective Inhibitors of Nuclear Export (SINE) were shown to be highly toxic to T-ALL and AML cells in mouse xenograft models, while having little toxic effects on normal mouse hematopoietic cells.¹⁰⁹ In addition, it was demonstrated that TCR-positive T-ALL cells can be targeted by mimicking thymic negative selection as obtained by TCR stimulation via antigen/MHC presentation or via an antibody against CD3.¹¹⁰ The advantage of these types of therapies is that they seem independent of genetic subtypes, avoiding extensive genetic characterization before therapeutic choices are to be made.

Challenges ahead

In the past decades, enormous progress was made in our understanding of the genetics and biology of T-ALL, but there are still significant gaps in our knowledge. To date, almost all attention has gone to defects affecting protein coding genes. The recent identification of mutations in non-coding regions of the genome that result in aberrant transcription factor expression and the discovery of several miRNAs and lncRNAs with a pathogenic role in T-ALL underscore that the ‘non-coding genome’ should not be neglected and that novel classes of oncogenes and tumor suppressor genes may remain to be discovered.

Genomic screens have identified recurrent novel oncogenes and tumor suppressors, such as PHF6, RPL10 and CNOT3, for which the exact role in the pathogenesis of T-ALL remains poorly understood. Moreover, genome wide screening has mainly been limited to diagnostic cases and such screens of large patient cohorts are lacking for relapse T-ALL. A better understanding of the biology of relapse is an absolute requirement to improve the dismal prognosis these patients currently are facing.

Analysis of mutational patterns in large patient cohorts revealed that the mutational landscape of T-ALL is not random and that particular defects often co-occur or are mutually exclusive.²¹ More research is needed to understand the biology behind particular mutational patterns and to characterize the specific clinical behaviors and distinct prognosis associated with these patterns. Related to this issue, also the order in which mutations are acquired in T-ALL development is most likely not random. In addition, the cellular context and exact hematopoietic developmental stage of the cell in which a mutation arises might be very relevant for the oncogenic action of the defect. At this point, not much is known on the order in which mutations are acquired and on the cell of origin in which they need to arise. Extensive modeling in mice and single cell sequencing will be required to answer these questions.

Finally, also the interaction of the leukemia cells with their microenvironment and characterization of the T-ALL niche are essential to fully capture T-ALL pathogenesis. In this context, recently developed advanced microscopy techniques allow in vivo imaging of interactions of leukemia cells with their environment and an essential role in T-ALL maintenance has been discovered for the CXCL12 chemokine and its receptor CXCR4.^111–113

The ultimate goal of the T-ALL research community is to translate the knowledge into highly efficient and low toxicity targeted therapies. Whereas the current chemotherapy-based regimens in pediatric T-ALL are associated with high survival rates, the toxicities of these therapies should not be underestimated. With many new targeted agents under development for cancer and auto-immune diseases, it should be possible to effectively repurpose the best agents for the treatment of T-ALL, and to design effective combination therapies directed against the specific sets of cooperating mutations in T-ALL.

Table 3. Promising targeted agents in T-ALL and their stage in clinical testing.

Compound	Specificity	Clinical Phase	Disease
NOTCH1 Signaling
MK-0752	γ-secretase inhibitor (GSI)	Phase 1	T-ALL and Lymphoma
PF 03084014	γ-secretase inhibitor (GSI)	Phase 1	Advanced-stage cancers, T ALL, Lymphoblastic Lymphoma
BMS 906024 (with dexamethasone)	γ-secretase inhibitor (GSI)	Phase 1	T-ALL or T cell lymphoblastic lymphoma
OMP 52M51	Notch1 inhibitory antibody	Phase 1	Dose-escalation study in lymphoid malignancy
LY3039478	Notch1 Inhibitor	Phase 1	Dose-escalation study in advanced-stage cancers
BMS-536924	ATP-competitive IGF-1R/IR inhibitor	Pre-clinical
JAK-STAT inhibitors
Ruxolitinib	JAK1/2 inhibitor	Phase 1	Acute myeloid leukemia (AML)
		Phase 2	B-ALL
		FDA Approved	Myelofibrosis
		Phase 1/2	Fallopian Tube cancer, Ovarian Cancer, Primary Peritoneal Cancer
Tofacitinib	JAK3 inhibitor	FDA Approved	Rheumatoid arthritis
		Phase 1	Systemic Lupus Erythematosus
		Phase 3	Juvenile Idiopathic Arthritis
Pimozide	STAT5 inhibitor	FDA Approved	Schizophrenia, Psychotic Disorders
		Phase 2	Amyotrophic Lateral Sclerosis (ALS)
17-AAG	HSP90 inhibitor	Phase 3	Multiple Myeloma (MM)
		Phase 2	Pancreatic Cancer
		Phase 2	Advanced Malignancies
		Phase 2	Ovarian Cancer
PU-H71	HSP90 inhibitor	Phase 1	Solid Tumor, Lymphoma
		Phase 1	Metastatic Solid Tumor, Lymphoma, Myeloproliferative Neoplasms
Inducers of apoptosis
ABT-263 (Navitoclax)	inhibitor of Bcl-xL, Bcl-2 and Bcl-w	Phase 1	Non-Small Cell Lung
		Phase 2	Platinum-resistant or Refractory Ovarian Cancer
		Phase 1	Hepatocellular Carcinoma
		Phase 1/2	Melanoma
ABT-199 (Venetoclax)	Bcl-2-selective inhibitor	FDA Approved	Chronic Lymphoblastic Leukemia (CLL)
		Phase 3	Relapsed/Refractory Multiple Myeloma
PI3K inhibitors
CAL-130	inhibits p110γ and p110δ catalytic domains	Pre-clinical
Ly294002	inhibit PI3Kα/δ/β	Phase 1	Neuroblastoma
Pictilisib (GDC-0941)	inhibitor of PI3Kα/δ	Phase 2	Breast Cancer
		Phase 2	Non-Squamous Non-Small Cell Lung Cancer
Apitolisib (GDC-0980, RG7422)	inhibitor for PI3Kα/β/δ/γ	Phase 2	Endometrial Carcinoma
		Phase 1/2	Prostate Cancer
		Phase 2	Renal Cell Carcinoma
MEK inhibitors
CI-1040	ATP non-competitive MEK1/2 inhibitor	Phase 2	Breast Cancer, Colorectal Cancer, Lung Cancer, Pancreatic Cancer
Selumetinib (AZD6244)	MEK1 inhibitor	Phase 2	Triple Negative Breast Cancer
		Phase 1	Lung Cancer, Melanoma, Head and Neck Carcinoma, Gastroesophageal Cancer, Breast Cancer, Pancreatic Adenocarcinoma, Colorectal Cancer
Trametinib (GSK1120212)	MEK1/2 inhibitor	Phase 2	Gastrointestinal Stromal Tumors
		Phase 1	Melanoma
		Phase 1	Neuroblastoma
		Phase 2	Non-Small-Cell Lung Cancer
ABL inhibitors
Imatinib	v-Abl, c-Kit and PDGFR inhibitor	FDA Approved	Chronic Myeloid Leukemia (CML)
		Phase 2	B-ALL, B Lymphoblastic Lymphoma, T-ALL, T Lymphoblastic Lymphoma
		Phase 3	Philadelphia Chromosome Positive Adult ALL
Dasatinib	Abl, Src and c-Kit inhibitor	FDA Approved	CML
		Phase 2	ALL
		Phase 1	Chronic Kidney Disease
Nilotinib	Abl, Src and c-Kit inhibitor	Phase 2	Glioma
		Phase 3	Philadelphia Chromosome Positive Adult Acute Lymphoblastic Leukemia
		FDA Approved	CML
Hedgehog inhibitors
Vismodegib (GDC-0449)	SMO inhibitor	FDA approved	Basal cell carcinoma
		Phase 2	Breast Cancer
		Phase 2	Non-Hodgkin Lymphoma, Non-Hodgkin, MM, Advanced Solid Tumors
GANT61	GLI1 inhibitor	Pre-clinical
Translation inhibitors
Rapamycin	specific mTOR inhibitor	Phase 2	Myelodysplastic Syndrome, CLL, ALL, T Lymphoblastic Lymphoma, Acute Myelogenous Leukemia, Acute Biphenotypic Leukemia, Acute Undifferentiated Leukemia
		Phase 1	Fallopian Tube Carcinoma, Ovarian Carcinoma, Primary Peritoneal Carcinoma
4EGI-1	competitive eIF4E/eIF4G interaction inhibitor	Pre-clinical