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Molecular basis ofacute lymphoblastic leukemia 73

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heterogeneity of the iAMP21- chromosome revealed by single- cell analysis suggests progressive amplification of theiAMP21 over time. Although rare, constitutional structural chromosomal abnormalities, including Robertsonian translocation rob(15; 21), isodicentric chromosome 21 idic(21), or a germline ring chromosome 21, are associated with an elevated risk of iAMP21. Other common concurrent lesions include deletions of the regions of histone genes at 6p22.2, copy number neutral LOH of 12q, and JAK–STAT pathway gene (SH2B3, IL7R, P2RY8::CRLF2) alterations, suggesting ruxolitinib as the potential target (Table 5.1). Similar to high hyperdiploid ALL, UV- related signature is enriched in iAMP21; however, UV- induced mutations occur before most chromosomal gains in contrast to high hyperdiploid ALL.

ETV6::RUNX1 and ETV6::RUNX1- like ALL

ETV6::RUNX1 is the most common fusion, accounting for 20%–25% of pediatric B- ALL but is rare in adults (Figure5.2). Clinically, ETV6::RUNX1 ALL cases are CD27

low- neg

CD44

and show a favorable outcome. This fusion gene is caused by the t(12:21)(p13:q22) translocation but typically cryptic on cytogenetic analysis. The frequent incidence of ETV6::RUNX1 in unselected normal cord blood supports a prenatal origin; however, the existence of ETV6::RUNX1 alone is not sufficient to induce overt leukemia due to the following reasons: the substantial variation of leukemia onset in identical twins, long latency for the acquisition of secondary mutations, and heterogeneity in the subclonal composition. Indeed, a mouse transplantation model with retroviral ETV6::RUNX1 expression resulted in a low incidence of leukemia with a long latency, although transduction of ETV6::RUNX1 in human cord blood hematopoietic progenitors resulted in the generation of the aberrant CD34+CD38−CD19+ population, associated with an early stage of B-

cell lineage commitment. Secondary alterations, including deletion of normal allele of ETV6, PAX5, TBL1XR1, CDKN2A/B, and BTG1, are considered to drive leukemic transformation, many of which arise from aberrant RAGmediated genomic recombination. Among them, TBL1XR1 alteration is associated with inferior outcomes and is exclusively found in ETV6::RUNX1 ALL and not in ETV6::RUNX1- like ALL (Table5.1).

ETV6::RUNX1- like ALL is a subtype that phenocopies

ETV6::RUNX1 ALL with similar expression profile, immu-

nophenotype (CD27

pos

and CD44

low- neg

), and mutational signature (APOBEC), but lacks ETV6::RUNX1 fusion. Instead of ETV6::RUNX1 fusion, 40% of ETV6::RUNX1- like ALL has the deletions or rearrangements of transcription factors, including members of the ETS family (ETV6, ERG, FLI1), IKZF1 and TCF3. The germline ETV6 variants with addi-

pos

and

tional PAX5 and ETV6 (the remaining, normal allele) alterations are associated with ETV6::RUNX1- like ALL (Table5.1). In contrast to ETV6::RUNX1 ALL, ETV6::RUNX1- like ALL is associated with inferior outcomes with high levels of MRD.

TCF3- rearranged ALL (TCF3::PBX1 and TCF3::HLF)

The fusion gene TCF3::PBX1 is generated by the translocation t(1;19)(q23;p13) and is found in 5–6% of childhood and 1% of adult B- ALL patients (Figure5.2). TCF3::PBX1 ALL is associated with a pre- B immunophenotype expressing cytoplasmic μ heavy chain, indicating the presence of pre- B- cell receptor (BCR) signaling that could be targetable by SRC kinases inhibitors, including dasatinib and ponatinib, not by imatinib. ROR1inhibition may have a synergistic effect by inhibiting the compensatory upregulation of ROR1 expression by dasatinib (Table 5.1). Conditional activation of TCF3::PBX1 results in enhanced self- renewal and leukemia with somatic alterations in PAX5, JAK–STAT, and Ras pathways after a pre- leukemia phase. Although JAK–STAT pathway gene mutations are generally not found in human TCF3::PBX1 ALL, Ras pathway gene alterations and deletions in PAX5 and CDKN2A are observed. Importantly, CDXN2A deletions in TCF3::PBX1 ALL are associated with inferior outcomes. TCF3::PBX1- mediated leukemogenesis may arise through the interaction of PBX1with HOXB7. In addition, a recent study revealed an important role of TCF3::PBX1 fusion protein in leukemogenesis as a coactivator for RUNX1, resulting in the activation of RUNX1related gene programs through enhancement of the RUNX1 autoregulatory loop.

The translocation t(17;19)(q22;p13) generates a rare TCF3::HLF fusion associated with an extremely poor outcome. Despite common TCF3 rearrangements, TCF3::HLF ALL differs from TCF3::PBX1 ALL in gene expression profiles (Figure5.1A) with the enrichment of stem cell and myeloid features and in the mutational landscape with recurrent PAX5 and CDKN2A/B deletions and Ras signaling mutations. The TCF3::HLF fusion contains the transactivation domains in TCF3 and the DNA binding and dimerization domains in HLF, which drives lymphoid differentiation. The TCF3::HLF fusion protein works as a pioneer TF by cooperating with ERG and recruits EP300, activating MYC and promoting a stempromising results in a preclinical study by the inactivation of the TCF3::HLF binding enhancers. High expression of Aurora A kinase (AURKA) may have therapeutic potential in TCF3::HLF ALL, in addition to inhibiting BCL2 (venetoclax), pre- BCR signaling (Src family inhibitors), and BIRC5 (Table5.1).

like state. The EP300inhibitor A- 485 showed

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74 Molecular Hematology

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KMT2A- rearranged ALL

KMT2A- rearranged ALL is a subtype of high- risk B- ALL with both lymphoid and myeloid features, defined by the rearrangement of the KMT2A (MLL1) gene on chromosome 11q23 to more than 100 different partner genes. KMT2A rearrangements are known to occur in utero and thus associated with high frequency in infant ALL. The second peak of onset is in adults, accounting for more than 10% of adult ALL (Figure5.2). Although rare, exposure to topoisomerase II inhibitors induces KMT2A rearrangement, resulting in therapy- related leukemia. KMT2A rearranged ALL typically lacks CD10 expression but expresses some myeloid markers, consistent with lineage plasticity and an early B cell differentiation stage, including early lymphocyte precursor and pro- B state. The most frequent translocations in this subtype is t(4;11)(q21;q23), KMT2A::AFF1, following t(11;19) and t(9;11). Some translocations show lineage preference, including KMT2A::AFF1 in B-

ALL and KMT2A::MLLT4 in T- ALL. The difference in partner genes also associates with different outcomes, showing inferior outcomes for KMT2A::AFF1- rearranged leukemia. Furthermore, multi- omic analysis of infant ALL identified two main subtypes, IRX and HOXA subtype, depending on the partner genes. Although infant ALL has few genomic alterations, perturbations in PI3K and Ras pathways are the most frequent and often subclonal, indicating their cooperating role in leukemogenesis and potential targets. The interaction of KMT2A and its rearrangement partners leads to the assembling of a large multiprotein complex and recruiting excessive DOT1L (K3K79methyltransferase), resulting in epigenetic dysregulation and aberrant transcription. The targeted therapy against this complex, including inhibitors of DOT1L, Menin, bromodomain, and polycomb repressive complex, is promising (Table5.1). Although some CD19- targeted immunotherapies have produced disappointing results in KMT2A- rearranged ALL due to lineage plasticity and immune escape, the low incidence of relapse after the combined treatment with Interfant-

06 backbone and blinatumomab is promising. Furthermore, the development of new markers, including CD72, might have therapeutic potential for this high- risk subtype.

BCR::ABL1 (Ph+) and Ph- like ALL

BCR::ABL1 positive ALL is characterized by the Philadelphia chromosome (Ph), derivative chromosome 22, induced by the reciprocal translocation t(9;22)(q34;q11). The frequency of BCR::ABL1 ALL is rare in children (2–5%) but increases with age to be the most common genetic subgroup in adults with an incidence of 20–25% (Figure5.2). BCR::ABL1 ALL

is a high-

risk subgroup associated with poor prognosis; however, the use of TKIs has dramatically improved the outcome. Secondary cooperative mutations include alterations of IKZF1 and CDKN2A/B in more than 50% of cases that cooperate in lymphoid leukemogenesis. IKZF1 alterations are included in the criteria of “IKZF1

plus

,” a recently defined marker of high- risk ALL, and are associated with an inferior outcome irrespective of the TKI exposure. Alterations of IKZF1 resulted in stem cell- like features, including selfrenewal and increased stromal adhesion, which may be reversed with retinoid receptor agonists by introducing the wild- type IKZF1 expression or with focal adhesion kinase inhibitors by inhibiting downstream integrin signaling pathways (Table5.1). Importantly, BCR::ABL1 fusion gene is also a hallmark of chronic myeloid leukemia (CML) and its lymphoid blast crisis is often indistinguishable from BCR::ABL1 ALL, though there is a prevalence of the majorBCR::ABL1 fusion transcript (p210) for CML and the minor- BCR::ABL1 fusion transcripts (p190) for B- ALL. However, DNA- based MRD monitoring by combining Ig/T- cell receptor (TCR) and genomic BCR::ABL1 fusion can reveal “CML- like” BCR::ABL1 ALL resembling lymphoid blast crisis by detecting the discordance of MRD level between them due to the existence of BCR::ABL1 fusion in non- ALL hematopoietic cells. Their outcomes are similar, but the difference in the speed of treatment response might suggest that BCR::ABL1 ALL and CML- like ALL are biologically distinct.

Ph- like or BCR::ABL1- like ALL is a high- risk subgroup that phenocopies BCR::ABL1 ALL with a similar expression profile and poor outcome, but alternative kinase- activating alterations rather than BCR::ABL1 fusion (Figure 5.1A). Ph- like ALL is more common in adults, males, and individuals with Down syndrome, and is associated with inferior outcomes with persistent MRD positivity. However, there is no consensus approach to define this subtype in clinical practice, partly because Ph- like ALL is quite heterogeneous in terms of genomic alterations other than concomitant IKZF1 alterations that resulted in the acquisition of stem cell-

like features and poor TKI response. However, the diverse genomic alterations in Ph- like ALL are characterized by the activation of cytokine receptor and kinase signaling that can be targetable by available TKIs, and fall into three types (Figure5.4): (1) activation of JAK–STAT signaling by alterations of CRLF2, JAK1/2, IL7RA, or EPOR (70%); (2) ABL- class fusions involving ABL1/2, CSF1R, LYN, PDGFRA/B (10–15%); and (3) other rare fusions involving NTRK3, FLT3, BLNK, and FGFR1. In addition, activation of Ras signaling induced by alterations of NRAS, KRAS, and PTPN11, and copy number alterations in genes related to B- cell differentiation (IKZF1, PAX5 , EBF1, and

ETV6) and cell cycle regulators (CDKN2A/B, TP53, and RB1) are recurrently found. Among them, CRLF2 alterations

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Molecular basis ofacute lymphoblastic leukemia 75

mTOR inhibitors

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EPOR

JAK2

SH2B3

EPO

Truncated

EPOR

EBF1

PAX5

ATF7IP

etc.

TSLP

CRLF2

IL-7Rα

JAK1

JAK2

Fusion proteins

JAK2

SMARC4A

JAK-STAT Signaling

STAT

JAK-STAT inhibitors

BCL2 inhibitors

PI3K inhibitors

mTOR inhibitors

IL-7Rα

JAK1

IL-7Rα

insertion

MYB

ZNF430

etc.

PI3K/

mTOR

JAK3

TYK2

JAK2

CRLF2

ABL-class fusion proteins

ABL1/2

RCSD1

ETV6 NUP214 RANBP2

etc.

ATF7IP

EBF1

ETV6

etc.

CSF1R

ABL Signaling

STAT

CRKL

Kinase inhibitors

PI3K inhibitors

mTOR inhibitors

PDGFRA/B

LYN

PI3K/

mTOR

Fusion proteins

ETV6

NTRK3

ZMYM2

TMEM2 PTK2B

FLT3

DTT2

BLNK

FGFR1

Other Signaling

MAPK/

ERK

STAT

TRK inhibitors

FLT3 inhibitors

FGFR1 inhibitors

MEK inhibitors

FAK inhibitors

JAK-STAT inhibitors

PI3K inhibitors

FLT3 ITD

NRAS

KRAS

PI3K/

mTOR

Figure 5.4 Schema showing the activation of cytokine receptor and kinase signaling in Ph- like ALL and potential targets. Approximately 70% of Ph- like ALL cases are characterized by JAK–STAT signaling with alterations of CRLF2, JAK1/2, IL7RA, and EPOR. ABL- class fusions are observed in 10–15%, which can be druggable by kinase inhibitors including dasatinib. The remaining cases include rare fusions involving NTRK3, FLT3, BLNK, FGFR1, and Ras signaling.

are the most common, found in approximately 50% of Ph- like ALL, including cryptic rearrangements between CRLF2 and the immunoglobulin heavy chain locus (IGH), focal deletions in pseudoautosomal region 1 (PAR1) of chromosomes Xp22 and Yp11 resulting in P2RY8::CRLF2, and less commonly, the single nucleotide variant (SNV) CRLF2 F232C. Deletions at PAR1 region are commonly used as a surrogate marker of P2RY8::CRLF2 alterations, and one of the criteria of “IKZF1

plus

” defined by cooccurrence of the IKZF1 deletion with deletion of CDKN2A, CDKN2B, PAX5 , and/or PAR1 region in the absence of ERG deletion. However, there is a caveat in “IKZF1

plus

” definition in that the use of SNP or multiplex ligation- dependent probe amplification (MLPA) arrays do not detect IGH::CRLF2 and CRLF2 SNVs. Among Ph- like ALL, cases

with JAK2 and EPOR rearrangements/alterations are asso- ciated with inferior outcomes than cases with the other alterations.

Several clinical trials are testing the efficacy of FDAapproved TKIs in frontline studies, which include JAK inhibitors (ruxolitinib) for JAK–STAT activating alterations, imatinib/dasatinib/ponatinib for ABL- class fusions, tropomyosin receptor kinase (TRK) inhibitors (larotrectinib, PLX7486) for NTRK3 fusions, and FLT3inhibitors forFLT3 alterations (Table 5.1). In addition, several immunotherapeutic approaches show promising response, including blinatumomab (bispecific anti- CD3/CD19monoclonal antibody), inotuzumab (CD22 antibody drug- conjugate), and Chimeric antigen receptor (CAR) T cell therapy targeting CD19.

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76 Molecular Hematology

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DUX4- rearranged ALL

DUX4- rearranged ALL is a distinct subtype induced by the cryptic translocation of DUX4 on chromosomes 4q/10q to the IGH locus, resulting in the overexpression of a 3′ truncated DUX4 protein. DUX4- rearranged ALL comprises approximately 5% of B- ALL (both children and adults) with a slight peak in adolescents and young adults (AYAs), and shows a distinct immunophenotype (CD2 and CD371 positive) that may be used as a promising surrogate marker for this subtype (Figure5.2). In addition to DUX4 translocations, ERG and IKZF1 deletions and alterations of epigenetic modifiers are frequently observed. ERG deletions are widely used as a surrogate marker for this subtype, but are secondary events that are not present in all cases. The DUX4 rearrangements result in expression of elevated expression of an N- terminal fragment of DUX4, that binds to an intronic region of ERG, resulting in transcriptional deregulation of ERG, and commonly, intragenic ERG deletion and expression of a non- canonical C- terminal ERG fragment (ERGalt). ERGalt retains the DNA- binding and transactivating domain of ERG and shows a dominant negative effect, leading to lymphoid lineage shift and transforming. Overall, DUX4- rearranged ALL is associated with a favorable prognosis regardless of IKZF1 alterations despite high MRD positivity at the EOI. Further subclassification by gene expression profiles revealed two developmentally different subtypes in DUX4- rearranged ALL; one with the enrichment of ERG and TBL1XR1 alterations, high NFATC4 expression, and excellent outcomes; and the other one with

NRAS, IKZF1, and KMT2D alterations, high expression of CEBPA and FLT3, and higher incidence of relapse but can

be salvaged.

MEF2D- rearranged ALL

MEF2D- rearranged ALL is characterized by fusion genes of N- terminal MEF2D to several partner genes, including BCL9 and HNRNPUL1, and found in up to 5% of pediatric and less than 1% of adult B- ALL (Figure5.2). Deletions of CDKN2A/B are frequently observed in this subtype. MEF2D- rearranged ALL shows a distinct immunophenotype with CD38, CD5, and cytoplasmic μ chain positivity and low/absent CD10 expression. Although most cases are MRD negative at the EOI, MEF2D- rearranged ALL shows inferior outcomes except for MEF2D::HRNPUL1 cases which are associated with relatively high CD10 expression and late pre- B stage. MEF2D fusion protein enhances MEF2D transcriptional activity through an autoregulatory loop of core regulatory circuitry (CRC, a network of TFs and co- factors that function together to regulate key

biological processes within a cell) and evasion from miRNA- mediated degradation, resulting in impairment of B- cell differentiation and dysregulation of MEF2D targets including HDAC9which can be targeted by histone deacetylase inhibitors. MEF2D fusion- CRC integrates preBCR signaling that can be targetable by SRC inhibitors (Table5.1). In addition, SREBF1 is a critical component of CRC and could be a promising target.

ZNF384- rearranged leukemia

Rearrangements of ZNF384 define a subtype of lineage ambiguous leukemia with a distinct gene expression profile and characteristic immunophenotype (weak CD10 and aberrant myeloid markers of CD13 or CD33) (Figure 5.1A). ZNF384- rearranged leukemia may manifest as B- ALL or B/ myeloid mixed phenotype acute leukemia (MPAL) depending on the expression of myeloperoxidase at diagnosis. Moreover, ZNF384- rearranged leukemia is immunophenotypically multiclonal in many cases, with lineage plasticity during the course of disease, and transition between predominantly myeloid or lymphoid marker expression. This lineage plasticity was also shown in the mouse transplantation model by the reconstitution of the immunophenotypic diversity from the flow- sorted subpopulations. Secondary alterations in ZNF384- rearranged leukemia include Ras pathway mutations, ETV6 deletions, and CDKN2A/B deletions, but lack variability between immunophenotypic subclones. In addition to these findings, detection of ZNF384 rearrangements in a subset of hematopoietic progenitor cells (HPC) at diagnostic samples and the report of twin ZNF384- rearranged leukemia cases may indicate that ZNF384 rearrangements are founder alterations in HPC as a cell of origin. Thus, ZNF384- rearranged leukemia may be considered to be a single entity regardless of immunophenotype (B- ALL or MPAL) at diagnosis.

ZNF384 rearrangements induce fusion oncoproteins that retain almost entire coding regions of ZNF384. Nfusion partners include TFs (TAF15, TCF3) and chromatin modifiers (CREBBP, EP300). Ep300::Znf384 knock- in mouse model revealed ZNF384 fusion oncoproteins cause hematopoietic expansion, myeloid lineage skewing, and self- renewal, supporting ZNF384 rearrangements are founder alterations. Furthermore, ZNF384 fusion oncoproteins bind to intergenic enhancer elements at the FLT3 locus, leading to overexpression of FLT3 with sensitivity to FLT3inhibitors (Table5.1). Although lineage- directed conventional chemotherapy and recent immunotherapies, including CAR T- cell therapy and blinatumomab, may result in lineage switch due to the lineage plasticity in ZNF384- rearranged leukemia, FLT3inhibitors might be a potential targeted therapy.

terminus

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PAX5- driven ALL (PAX5alt and PAX5 P80R)

PAX5- driven ALL is classified into two subtypes, PAX5alt and PAX5 P80R, and is characterized by loss of function alterations in PAX5, an essential TF in B- cell development. PAX5alt and PAX5 P80R are defined by the type of PAX5 alterations and gene expression profiles (Figure5.1A), and both types of alterations are essential for leukemogenesis as initiating or cooperating lesions.

PAX5 alterations in PAX5alt include rearrangements (except for PAX5::JAK2 and PAX5::ZCCHC7 also observed in B-ALL), focal intragenic amplifications (PAX5amp), and sequence mutations. These sequence mutations generally do not include P80R except for heterozygous P80R mutation in rare cases. Despite the diversity of alterations, PAX5alt cases exhibit similar gene expression profiles in B- ALL, though there is some granularity within the PAX5alt subtype as PAX5::ETV6 and R38H/R140L cases appear as distinct groups. PAX5 rearrangements typically preserve the C- terminus DNA- binding domain and fuse to over 20 different partner genes, including PAX5::ETV6. Exceptions of PAX5 rearrangements are PAX5::JAK2 cases exclusively observed in Ph- like ALL and PAX5::ZCCHC7 found in sev- eral other subtypes. The lesions of PAX5amp are variable but always involve exon 5, leading to in- frame duplication of the DNA- binding domain, and frequently harbor CDKN2A/B alterations and trisomy 5. Non- silent PAX5 sequence mutations are generally compound heterozygous with multiple alterations, PAX5amp, PAX5 fusions, or structural variants. The most frequent compound heterozygosity in PAX5alt is R38H and R140L in the DNAbinding paired domain. The same concomitant alteration pattern was also reported in B- ALL cases with the germline R38H variant. In addition to PAX5 alterations, PAX5alt cases have co- lesions in cell- cycle regulation genes (CDKN2A, RB1, BTG1), B- cell development genes (IKZF1), and transcriptional/epigenetic regulators (KDM6A, KMT2A, ATRX).

The other subtype of PAX5defined by the non- silent PAX5 hot spot mutation in the paired domain. PAX5 P80R exhibits distinct gene expression profiles (Figure5.1A). Importantly, PAX5 P80R is generally hemizygous, concomitantly showing inactivation of wild- type PAX5 allele by deletion, loss- of- function mutation, or copy- neutral LOH. In addition, inactivation of the wild- type Pax5 allele is observed in the heterozygous

P80R/+

Pax5

knock- in mouse model, which develops transplantable B- ALL, suggesting the importance of biallelic PAX5 alterations in PAX5 P80R and that the PAX5 P80R mutation may be leukemia initiating events. Additional colesions include alterations of CDKN2A/B, Ras, and JAK– STAT pathway genes, and SETD2. In addition to some

driven ALL is PAX5 P80R,

PAX5alt fusion genes (PAX5::NOL4L, PAX5::ASXL1), the structural rearrangements of chromosomal arms 9p and 20q found in PAX5 P80R may be associated with dic(9:20). One PAX5 P80R specific feature might be the disruption of CD58 enhancer activity. Loss of CD58 expression results in impaired blinatumomab- induced T- cell activation, inducing CD19 positive resistance in blinatumomab treatment in vitro experimental model, though this should be examined in clinical settings.

CDX2/UBTF ALL

B- ALL with UBTF::ATXN7L3/PAN3,CDX2 is an uncom- mon B- ALL subtype characterized by unique gene expression profile (Figure5.1A) and harboring two alterations in all cases, UBTF::ATXN7L3 fusion and a deletion upstream of FLT3 that results in deregulation of CDX2. This entity is succinctly called “CDX2/UBTF ALL” to acknowledge these two distinct mutations. Most CDX2/UBTF ALL cases are adults, especially AYA females, and is in children (Figure5.2), has distinct immunophenotypes including partial/negative CD10 expression, CD20negativity, and positivity for CD34, CD38, and cytoplasmic IgM. CDX2/UBTF ALL patients are more likely to have positive MRD at the EOI and exhibit a higher relapse rate. CDX2/UBTF ALL cases commonly have dual micro- deletions in 13q12.2 (PAN3, FLT3, and CDX2 region) and 17q21.31 (UBTF and ATXN7L3 region) through aberrant RAG activity. Importantly, two types of deletions in 13q12.2 have been reported in B- ALL; Type I deletion with deletions sparing the FLT3 promoter, leading to high FLT3 expression (in non- CDX2/UBTF ALL), and Type II deletion with deletions including the FLT3 promoter, resulting in CDX2 overex- pression at the cost of FLT3 expression (in CDX2/UBTF ALL) (Figure5.5A). In both types of deletions, retargeting of the PAN3 enhancer induces aberrant expression of FLT3 or CDX2. The other micro- deletions on chromosome 17 generate in-

frame UBTF::ATXN7L3 fusion, which can be detectable by RT- PCR or genomic at diagnosis (Figure5.3). Additional genomic alterations include gains of 1q, PAX5 alterations, and CXCR4 truncating mutations. Detection of two distinct clonal, genomic subtype- defining driver events is a unique feature in CDX2/UBTF ALL. By quantitation of variant frequency, CDX2 dysregulation is estimated to arise. The mechanism of leukemogenesis mediated by these multiple alterations remains to be elucidated.

NUTM1- rearranged ALL

NUTM1- rearranged ALL is a rare subtype of pediatric B- ALL that is frequently found in infant ALL without KMT2A rearrangements, and not in adult ALL. Unlike

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(A) (B)

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Hg19 chromosome 13

28,500,000 28,600,000 28,700,000 28,800,000 28,900,000

CDX2/UBTF H3K27ac HiChIP

PDX1

PLUT

CDX2/UBTF (Type II deletion)

RNA-seq

H3K27ac HiChIP Sig. loops

B-other (Type I deletion)

RNA-seq

H3K27ac HiChIP Sig. loops

URAD PAN3

CDX2

FLT3 FLT1

Type II deletion

Type I deletion

raw read-pairs

039

[0–1523]

[0–774]

[0–1523]

[0–774]

t(8:14) H3K27ac HiChIP

Chr 14

H3K27ac HiChIP

Sig. loops

t(7:14) H3K27ac HiChIP

BCL11B SETD3 BENC

Breakpoint

BCL11B

Breakpoint

chr14

chr8

MYC BENC enhance

Chr 8

[0–299]

BCL11B

chr14

chr7

CDK6 enhancer

ETV6::RUNX1 (No deletion in this region)

RNA-seq

H3K27ac HiChIP Sig. loops

Figure 5.5 H3K27ac HiChIP showing enhancer retargeting in CDX2/UBTF (B- ALL) and BCL11B- driven ALL (T- ALL and T/M MPAL). Enhancer region is annotated in green and retargeted genes are colored in red. The heatmap shows raw read counts for all pairwise 5 kb genomicbins. Significant (false discovery rate, 0.01) loops are shown as blue arcs. (A) Representative cases of CDX2/UBTF (TypeII deletion), Type I deletion, and no deletion in chromosome 13q12.2 from the top to the bottom. Enhancer on PAN3 gene is retargeting CDX2 (inCDX2/UBTF) and FLT3 (in Type I deletion), respectively. (B) Representative cases of BCL11B- driven ALL mapped on customized reference genome based on translocations. The top case has translocation of chromosome 8 (right, brown) and 14 (left, purple), resulted in bringing BENC enhancer close proximity to BCL11B. The bottom case has translocation of chromosome 14 (right, purple) and 7 (left, brown), resulted in bringing CDK6 region proximity to BCL11B.

[0–1523]

KMT2A- rearranged infant ALL, NUTM1- rearranged ALL is associated with excellent outcome. NUTM1- rearranged ALL exhibits an aberrant expression of NUTM1 by fusing to several different partner genes including ACIN1, AFF1, ATAD5,

BRD9, CHD4, CUX1, IKZF1, RUNX1, SLC12A6, and ZNF618. Almost all the coding region of NUTM1 is retained

in fusion genes, including NUT domain, which is considered important to regulate histone acetylation. Especially for the cases with BRD9::NUTM1, histone deacetylase inhibitors or bromodomain inhibitors may be potentially druggable (Table5.1).

Chr 14

[0–603]

[0–774]

Chr 7

CDK6 BCL11B SETD3

H3K27ac HiChIP

Sig. loops

IKZF1N159Y

Heterozygous IKZF1N159Y mutations define a unique subtype of mutation- initiated ALL with distinct gene expression profiles (Figure 5.1A), characterized by upregulation of YAP1 and SALL1 that are not observed in other subtypes of ALL with other IKZF1 alterations as secondary events. The N159mutation is located at the critical DNA- binding domain of the second zinc finger of IKZF1, and this residue is highly conserved across species, suggesting an important role. Germline mutations of IKZF1 are known to cause

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combined immunodeficiency through haploinsufficiency or familial pediatric ALL; however, patients with germline mutations at N159 residue (N159S, N159T) behave in a dominant- negative fashion and exhibit more severe earlyonset clinical phenotypes. Furthermore, all IKZF1N159 germline mutations are de novo and no asymptomatic individuals have been detected. Thus, although IKZF1N159Y mutation induces nuclear mislocalization resulting in aberrant leukemic cell adhesion and drug resistance as with other IKZF1 alterations, IKZF1 N159Y might have additional unique mechanisms to drive leukemia.

Other subtypes of B- ALL

B- ALL cases with ZEB2 hotspot H1038R mutation and IGH::CEBPE alterations exhibit similar gene expression profiles based on unsupervised clustering and define a rare B- ALL subtype in AYA and adult ALL. However, unlike PAX5 P80R and IKZF1N159Y, ZEB2 H1038R might not act as a founder alteration because some B- ALL cases with ZEB2 H1038R mutations are found in other B- ALL subtypes, including DUX4- and ZNF384- rearranged ALL. In addition, ZEB2 H1038R and IGH::CEBPE do not co- occur and some cases with identical gene expression profile lack either. Enrichment of NRAS mutations and upregulation of LMO1 are characteristic of this group.

The translocations of IGH (less frequently light chain loci) and MYC, resulting in aberrant expression of MYC oncogene, are a hallmark of mature B cell malignancies including Burkitt lymphoma (BL) and diffuse large B- cell lymphomas, but also observed in immature B- ALL (B cell precursor ALL, BCP- ALL) though they are rare. In contrast to BL, IGH::MYC in BCP- ALL can be clonal or subclonal. BCP- ALL with IGH::MYC are classified into three categories: (1) cases with B- ALL specific drivers (e.g. KMT2A- , DUX4- rearranged) which represent similar expression profiles to concurrent driver genes; (2) cases with concurrent BCL2 and/or BCL6 rearrangements which exhibit “double/triple hit” lymphoma features (BCL2/MYC); and (3) cases without disease defining drivers. The clinical decision of treatment strategy (ALLdirected, BL- directed, or hybrid protocols) in BCP- ALL with IGH::MYC may be challenging because there is no consensus due to the lack of understanding of biology and appropriate clinical trials.

IDH1 R132C and IDH2 R140Q mutations define a rare B- ALL subtype with distinct gene expression profiles. These mutations may be initiating events to drive leukemia, though functional studies are required. Consistent with the roles of IDH1/2 in cellular metabolism and epigenetic regulation, IDH1 R132C and IDH2 R140Q ALL exhibit hypermethylated profiles that can distinguish from other subtypes of B- ALL.

T- cell precursor acute lymphoblastic leukemia(T- ALL)

Early T- cell precursor (ETP) ALL, immature T- ALL, and BCL11B-

T- ALL exhibiting an immature T- cell immunophenotype (cytoplasmic CD3+, CD7+, CD8−, CD1a−, CD5 expression of myeloid and/or stem- cell markers is clinically called ETP- ALL. ETP- ALL is one of the heterogeneous subsets in the immature T- ALL group and is composed of several different T- ALL subgroups defined by genomic alterations, including alterations and rearrangements of BCL11B (not to

TLX3), HOXA13, HOXA9, NUP98, ZFP36L2, ETV6, and MED12. Importantly, these immature T- ALL cases represent

similar gene expression profiles (Figure5.1B), and their gene expression profiles are more analogous to HPC than T cell precursor, suggesting that at least some parts of these immature T- ALL cases overlap with immature ALAL, and they might be in the same spectrum of immature leukemias. Thus, these immature leukemias should be classified by genomic/biological features ideally, not by immunophenotype, when we decide on treatment strategy.

One example is BCL11B- activated leukemia, which includes one- third of ETP- ALL, and T/Myeloid MPAL cases, and less commonly, AML and acute undifferentiated leukemia, and exhibits a distinct expression profile (Figure5.1B). BCL11B- activated leukemia is characterized by aberrant expression of BCL11B in the early hematopoietic stem and progenitor cells developmental stage of HPC. This is caused by interchromosomal rearrangements at 14q32, the region around BCL11B gene, and enhancers active at the HPC stage in 80% of cases (Figure5.5B). The remaining 20% of cases harbor focal amplification downstream of BCL11B, generating a neoenhancer. Approximately 80% of BCL11B- activated leukemia possess FLT3 activating mutations. Concurrent expression of BCL11B and FLT3- ITD on HPC showed syner- gistic effects toward T- cell- directed differentiation to express cytoplasmic CD3 while blocking myeloid differentiation. With high BCL2 expression in BCL11Bcombination of FLT3 and BCL2inhibitors exhibited efficacy in a patient- derived xenografts preclinical model.

activated leukemia

weak

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activated leukemia, a

TLX1 and TLX3- driven T- ALL

T- ALL with TLX1 or TLX3 rearrangements, are among the most common genomic events found in pediatric T- ALL, and generally juxtapose these genes to TCR genes or BCL11B regulatory regions, resulting in aberrant expression of TLX1 or TLX3. Although most translocations in TLX3- driven T- ALL involve BCL11B regulatory regions, they are totally different from above- mentioned BCL11B- activated leukemia in that TLX3- driven T- ALL places TLX3 gene under the control of

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BCL11B regulatory regions for TLX3 overexpression, while BCL11B- activated leukemia puts BCL11B gene under the

control of active regulatory elements in the early developmental stage for aberrant BCL11B expression (Figure5.5B). Therefore, TLX3- driven T- ALL exhibits distinct gene expression profiles that can be distinguished from the immature T- ALL group (Figure5.1B). TLX1- driven T- ALL also represents unique gene expression profiles, but its expression profiles are close to that of TLX3- driven T- ALL, consistent with their early developmental stages of cortical thymocyte maturation. In addition, TLX1 and TLX3- driven T- ALL share genomic and epigenomic features, including a high prevalence of NOTCH1 activating alterations, CDKN2A inactivation, mutations in JAK–STAT pathway and epigenomic regulator genes, and DNA methylation profiles. The presence of NUP214::ABL1 fusion oncogenes and alterations in JAK– STAT pathway genes are considered druggable by TKIs and ruxolitinib, and these inhibitors have already been incorporated into ongoing clinical trials.

TAL1- and class II basic helix- loophelix (bHLH) factor- deregulated T- ALL

Deregulation of class II bHLH (a common structural motif found in proteins involved in gene regulation that consists of two important regions: a basic region and a helix–loop–helix region) factors, including TAL1, TAL2, LYL1, and BHLHB1, is involved in leukemogenesis of T- ALL. Among them, aberrant expression of TAL1 is observed most commonly and accounts for approximately 40% of T- ALL. There are several mechanisms to deregulate TAL1 expression: (1) chromosomal translocations with TCRA/D loci; (2) RAG- mediated submicroscopic interstitial deletion (STIL::TAL1); (3) loss of CTCF binding sites (disruption of insulated neighborhoods); (4) somatic indels generating aberrant MYB binding site (MuTE) for aberrant super- enhancer activity; and (5) amplification of neighborhood enhancers. TAL1 plays a central role in forming a CRC for the positive autoGATA3, RUNX1, MYB, the ETS family genes, and the LIMonly domain factors (LMO1 and LMO2). In this regard, concurrent deregulation of LMO2 (or less frequently LMO1) with aberrant TAL1 expression is recurrently observed in human T- ALL, and transgenic mouse model with double Tal 1 and Lmo1/2 overexpression accelerates T- ALL development. In contrast, Ta l 1 single transgenic mouse model needs a latent period to develop T- ALL, suggesting the requirement of additional alterations for leukemogenesis. Mechanistically, overexpression of TAL1 (or LYL1) results in forming TAL1/ LYL1- E- protein heterodimers, leading to inhibition of the function of E- protein dimers, which have an essential role in T- cell differentiation. In addition to CRC genes, several genes and non- coding RNAs, including ARID5B, ARIEL, and MYC, cooperate with TAL1 to exhibit distinct T- ALL features.

regulatory loop with

By gene expression profiles, TAL1- and Class II bHLH factor- deregulated T- ALL cases are further sub- classified into two subtypes (Figure5.1B): (1) TAL1- RA subtype and (2) TAL1- RB subtype expressing PTCRA (pre- TCR) and RAG1/2 genes with LCK activation that could be druggable by dasatinib. Frequent co- lesions are LMO1/2 deregulating alterations, PI3K- AKT pathway gene alterations (PTEN, PIK3CD), USP7, and deletion of chromosome 6q. Among them, activation of the PI3K- AKT pathway is associated with glucocorticoid resistance in this subtype and can be reversed by inhibiting this pathway. TAL1 also regulates cell cycle regulators such as CDK6 and CCND3, which could be poten- tial targets.

Other subtypes of T- ALL

T- ALL cases with deregulated homeobox TF genes other than TLX1/3, including KMT2A- , HOXA9- , MLLT10- ,

NUP98- , NKX2- 1- , NKX2- 5- rearrangements, and SET::NUP214, each exhibit distinct gene expression profiles

(Figure5.1B). Some HOXA9- deregulated T- ALL share gene expression and DNA methylation features with immature T- ALL, including clinical ETP- ALL. Except for KMT2A- and MLLT10- rearrangements and SET::NUP214. Most homeobox TF gene deregulations are induced by alterations in non- coding regions or TCR loci that require whole genome sequencing for detection.

T- ALL with SPI1 fusion genes, encoding PU.1, exhibit a unique subtype in T- ALL (Figure 5.1B). This subtype is enriched in the Asian population with approximately 5% frequency. SPI1- rearranged T- ALL exhibits a distinct gene expression signature with LCK activation that might be targetable by dasatinib. Ras signaling mutations might be important secondary lesions for leukemogenesis. Clinically, this subtype is associated with poor outcomes, and recurrent cases of Langerhans cell histiocytosis are reported as secondary events of SPI1- rearranged T- ALL.

Summary

Recent advances in high- throughput sequencing technologies, including whole transcriptome and whole genome sequencing, have revolutionized the molecular basis of B- ALL and T- ALL, enabling us to define ALL subtypes based on deregulated driver events, not on conventional clinical features such as age and sex (Figure5.2). That being said, it is not the era yet to treat all ALL patients with personalized treatment strategies based on genomic events or classification, though some TKIs and JAK–STAT inhibitors have started to be incorporated in some frontline clinical trials. Now, we have defined genomic alterations in most ALL cases; however, we have already understood that ALL is not

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such a simple disease and cannot be explained only by genomic events, leading to ongoing study of additional areas of the molecular basis of ALL, including epigenome, proteome, metabolism, cell of origin, heterogeneity, and role of the microenvironment. Several developing technologies might be useful to leverage these issues. One example is High- throughput Chromosome Conformation Capture (HiC) or HiChIP (method to capture critical threedimensional genome organization and long- range genetic information in one assay) for examining three- dimensional chromosome structures and enhancer- promoter looping (Figure5.5). These technologies have aided the elucidation of several novel ALL subtypes, including CDX2/UBTF ALL and BCL11B- activated ALL, but they are also beneficial to explore critical deregulated genes or regions for the potential targets. In addition, the development of single- cell analysis will be essential for understanding cell of origin and heterogeneity of ALL. Furthermore, emerging evidence suggests the involvement of liquid–liquid phase separation in leukemia biology, especially for cases with fusion oncoproteins. Thus, in a post- genomic era, it is critical to explore these advanced issues in ALL biology for a deep understanding that will improve treatment strategies in the future.