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R. Marke et al.
which is even more evident in Btg1-/-;Btg2-/- mice.101 These studies have demonstrated that BTG1, together with BTG2, is required to suppress a T-lineage inappropriate expression program in progenitor B cells. Thus, mono- allelic gene deletions of IKZF1 in combination with EBF1, PAX5 or BTG1 may contribute to a more prominent block in B-cell development and increased proliferative expan- sion of precursor B cells. Indeed, intercrossing haplodefi- cient Ikzf1 animals with heterozygous Ebf1 or Pax5 knock- out mice promotes the onset of ALL, giving rise to both B- ALL and T-ALL.102 On the other hand, Btg1-deficiency specifically accelerates the development of T-ALL in Ikzf1+/- mice, which suggests that B-lineage-restricted mouse models will be required to establish their synergis- tic action in the pathogenesis of B-ALL.
Effector pathways downstream of IKZF1 involved in leukemia pathogenesis
Since lymphoid transcription factors are commonly deleted in BCP-ALL, the tumor suppressive functions of IKZF1 and other B-cell master regulators, such as EBF1 and PAX5, have been mostly linked to the suppression of their B-cell differentiation programs in these leukemic cells. However, this would not fully explain the predilection of IKZF1 alterations in BCR-ABL1-positive and BCR-ABL1- like leukemia, suggesting that IKZF1 also regulates other molecular pathways. Furthermore, loss of IKZF1 function probably affects different target genes in human leukemic cells as compared to mouse progenitor B cells, which could even be distinct from those deregulated by expres- sion of dominant-negative isoforms, such as IK6. Nonetheless, mouse studies performed over the past 5 years have been very instrumental in deciphering the tran- scriptional networks downstream of IKZF1. Thus, gene expression profiling in different Ikzf1 knockout mouse models combined with genome-wide chromatin immuno- precipitation studies has uncovered IKZF1-specific targets that are not only linked to lymphoid lineage commitment and B-cell differentiation, but also to leukemia develop- ment.
A large group of those Ikzf1-target genes can be classi- fied as signal transducers, some of which drive early lym- phoid differentiation, such as c-Kit, Flt3 and Il7r.12,32,37,53 Adult ALL samples harboring IKZF1 deletions display increased expression of IL7R together with reduced expression of SH2B3, which represents a defined subset of high-risk B-ALL.103 Other genes differentially expressed in Ikzf1-mutant mice are important for pre-B-cell receptor signaling, and several of these IKZF1 targets appear to be deregulated in BCR-ABL1-positive B-ALL, including IGLL1, SYK, and SLP65.104,105 Indeed, defective pre-B-cell receptor function is a hallmark of BCR-ABL1-positive ALL, and loss of IKZF1 function enhances SRC phosphorylation at the expense of the SYK/SLP65 pathway activation, which is required for pre-B-cell differentiation.104 Besides transcriptional regulation of signal transducers, Ikzf1 con- trols the expression of cell surface receptors, such as CD34 and CD43, and these molecules confer a leukemic growth advantage to IKZF1-mutated BCR-ABL1-positive B-ALL cells.55
Another group of IKZF1 target genes identified in mouse progenitor B cells seems to converge on a cellular network coupling cell surface protein expression with intracellular Wnt and Rho signaling as well as catenin-dri- ven gene regulation inside the nucleus.55,106 A critical target
gene within this subgroup includes Ctnnd1 encoding p120- catenin. This is a multifunctional protein that regulates cadherin stability at the cell membrane, activation of the Rho family of GTPases in the cytoplasm and Wnt/β- catenin target genes within the nucleus by interacting with Kaiso.107 Activation of CTNND1 expression is observed in samples from patients with IKZF1 deletions,108 and inactivation of p120-catenin reduces the proliferative capacity of BCR-ABL1-positive leukemic cells.55,106 A relat- ed downstream effector pathway of IKZF1 that plays an eminent role during mouse B-cell development is integrin- dependent survival signaling, which involves activation of focal adhesion kinase (FAK).12,56 In mouse models of BCR- ABL1-positive B-ALL, perturbation of Ikzf1, including loss- of-function deletions and expression of IK6, leads to acti- vation of an adhesive phenotype, which correlates with overexpression of FAK.63,109 FAK pathway upregulation is also observed in BCR-ABL1-positive BCP-ALL, especially in the context of IK6 expression.109 Moreover, FAK inhibi- tion potentiates the responsiveness to the ABL inhibitor dasatinib in a xenograft model system and improves sur- vival.109
Recently, it has been proposed that the B-lymphoid transcriptional program regulated by IKZF1, as well as PAX5, acts as a metabolic barrier against malignant trans- formation of B-cell precursor cells.110 Inducible reconstitu- tion of functional IKZF1 in patient-derived IKZF1-deleted B-ALL cells results in activation of the LKB1-AMPK ener- gy-stress-sensor pathway, and decreased protein levels of the insulin receptor, the glucose transporters GLUT1, GLUT3 and GLUT6, as well as the effectors of glucose metabolism, such as HK2, HK3, and G6PD. On the other hand, the expression of glucose-transport inhibitors, such as TXNIP and CNR2, are strongly induced by IKZF1. Consequently, these IKZF1-reconstituted B-ALL cells tran- sit into a state of chronic energy deficit. Thus, this ‘meta- bolic gatekeeper’ function of IKZF1 may force silent pre- leukemic clones that carry potentially oncogenic lesions to remain in a latent state.
Besides imposing a change on pre-B-cell receptor signal- ing, cell adhesion and metabolic state, IKZF1 alterations in combination with BCR-ABL1 expression also result in acquisition of stem cell-like features and enhanced self- renewal of progenitor B cells63,105 (Figure 4). Activation of THY1 expression has been linked to enhanced self-renew- al,63 and Ikzf1 has been shown to regulate expression of multiple genes involved in cell cycle regulation, including Cdkn1a, Cdkn2a, and Cdk6.53,55 In mouse progenitor B cells and human B-ALL, BCL6 and MYC have been identified as IKZF1 targets,32,111-113 and probably both contribute to enhanced cell proliferation of IKZF1-deleted B-ALL. However, it remains to be established whether targeting these pathways has therapeutic potential in high-risk B- ALL patients.
IKZF1 alterations mediate therapy resistance
The presence of IKZF1 gene lesions in BCR-ABL1-posi- tive B-ALL results in inferior treatment outcome and mouse xenograft models suggest that IKZF1 loss contributes to resistance to tyrosine kinase inhibitor-based therapy.63,80 Reactivation of cell adhesion pathways by perturbation of IKZF1 function leads to elevation of key adhesion mole- cules, such as integrins (ITGA5) and CD90, and adhesion regulators, such as FAK, as well as increased phosphoryla- tion of FAK itself, which permits relocalization of leukemic
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haematologica | 2018; 103(4)