ZNF384-Related Fusion Genes in Acute Lymphoblastic Leukemia

Abstract

Zinc finger protein 384 (ZNF384) encodes a C2H2-type zinc finger protein that can function as a transcription factor. ZNF384 rearrangement in acute lymphoblastic leukemia (ALL) was first reported in 2002. More than 19 different ZNF384 fusion partners have been detected in ALL. These include E1A-binding protein P300 (EP300), CREB-binding protein (CREBBP), transcription factor 3 (TCF3), TATA-box binding protein associated factor 15 (TAF15), Ewing sarcoma breakpoint region 1 gene (EWSR1), AT-rich interactive domain-containing protein 1B (ARID1B), SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily A, member 4 (SMARCA4), SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily A, member 2 (SMARCA2), synergin gamma (SYNRG), clathrin heavy chain (CLTC), bone morphogenic protein 2-inducible kinase (BMP2K), Nipped-B-like protein (NIPBL), A Kinase Anchoring Protein 8 (AKAP8), Chromosome 11 Open Reading Frame 74 (C11orf74), DEAD-Box Helicase 42 (DDX42), ATP Synthase F1 Subunit Gamma (ATP2C1), Euchromatic Histone Lysine Methyltransferase 1 (EHMT1), Testic Expressed 41 (TEX41), etc. Patients diagnosed with ALL harboring ZNF384 rearrangements commonly had a good prognosis. The mechanisms, performance, and features of different ZNF384 rearrangements in acute lymphoblastic leukemia have been well evaluated.

Introduction

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and shows peak development during 1–4 years of age. ¹ B-cell acute lymphoblastic leukemia (B-ALL) has several subtypes classified according to genetic alterations, which include aneuploid and chromosome alteration. ² Distinct B-ALL subtypes are characterized by zinc finger protein 384 (ZNF384) rearrangement. ³ ZNF384 and its fusion partners are challenging to be detected by G-banding as both are located adjacent to the telomeres at the chromosome ends.^4,5 The clinical features of B-ALL with the ZNF384-related fusion gene hinges upon the fusion partner gene of ZNF384. ⁶ The distinct hematological/genetic types, accounting for approximately 5%–10% of all B-ALL cases, are characterized by ZNF384 rearrangement.^3,7 This fusion gene can always be detected in B cell/myeloid mixed phenotype acute leukemia (B/M MPAL), with an incidence of 48%. ⁸ The ZNF384 rearrangement subtype is common among adolescents and young adults. Patients with the ZNF384 fusion genes are more likely to experience monocytic switch. In this mechanism, the expression of lymphoid lineage markers (CD19 and CD34) decreases while the expression of myeloid lineage markers (CD33 and CD14) increases. During lineage conversion progression, gene fusions and transcripts did not change. Therefore, they can be a stable target for minimal residual disease monitoring. This phenomenon shows that the novel subgroup is the former subgroup presenting with new immunophenotype and molecule characteristics. Patients with B-ALL harboring the ZNF384-related fusion genes have a low CD10 expression and high CD13 and CD33 expression. ³ The ZNF384 rearrangements cause perturbed DNA binding and drives transcriptional deregulation.^9,10 Furthermore, ZNF384 fusion can prevent early B lymphocyte differentiation and induce leukemogenesis in vivo. ¹¹

ZNF384, also referred to Cas-interacting zinc finger protein and nuclear matrix protein 4, is located at 12p13. ¹² It has six well-conserved motifs, which are as follows: leucine zipper, serine/threonine-rich, and proline-rich domains at the N-terminus, and nuclear localization signal, zinc finger, and Gln-Ala repeat domains at the C-terminus. ¹³ The breakpoint of ZNF384 was found between exons 2 and 3. Further, exon 3 contains the start codon. Hence, the entire coding region of ZNF384 is well preserved. However, further research should be performed to evaluate whether the function domain was distorted. ¹⁴ ZNF384 encodes a C2H2-type zinc finger protein that plays a role in integrin binding and SH3 domain of p130Cas binding. ¹⁵ ZNF384, a nuclear matrix architectural transcription factor that can alter DNA structure, is shuttled between the nucleus and cytoplasm. ¹⁶ Furthermore, ZNF384 participates in bone metabolism and spermatogenesis.^13,17 The ZNF384 expression in hematopoietic stem cells decreases slightly and it did not change during B-cell differentiation. ¹⁸ ZNF384 rearrangements do not alter the ZNF384 gene expression and its fusion partner. ¹⁹ Although there was no significant difference between patients with high and low ZNF384 gene expression levels, the survival rate of high-level patients was significantly lower than that of other patients. ²⁰ In addition, it is characterized by hypermethylation.

Patients with ZNF384 rearrangement are associated with intermediate risk. ²¹ However, another study reported that pediatric and adult patients with ZNF384 fusions belong to the high- and intermediate-risk groups, respectively. ²² ZNF384 subtypes were found to be associated with better disease-free survival, favorable 5-year overall survival rate (74%), and a higher 3-year relapse-free survival rate.^23-25 However, the survival rate did not differ among patients in various age groups. ²⁶ Recently, Ma et al reported a 17-year-old ALL patient who had renal damage as the first manifestation, rearrangement of ZNF384 was confirmed by FISH detection. She had an unfavorable prognosis because she responded poorly to chemotherapy and developed a relapse shortly after reaching CR. ²⁷ ZNF384 rearrangement was significantly related to higher platelet counts and lower frequency of high-risk karyotype. ²⁸ For patients treated with chemotherapy alone, the RFS and OS of ZNF384 fusion patients were similar to those of non-ZNF384 fusion patients. For people who receive transplantation, those with ZNF384 fusion had significantly higher 3-year RFS than B-other ALL patients with no ZNF384 fusion. Adult patients with ZNF384 fusion have a better prognosis through receiving allo-HSCT24.

Patients with ZNF384 rearrangement harbored mutations in the RAS/FMS-like tyrosine kinase 3 (FLT3) pathway, which include NRAS, KRAS, neurofibromin 1 (NF1), protein tyrosine phosphatase, non-receptor type 11 (PTPN11), and FLT3 mutations. Moreover, epigenetic regulation (Lysine Methyltransferase 2D (KMT2D), CREB-binding protein (CREBBP), Lysine Demethylase 6A (KDM6A), and enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)) was common. ²⁹ Cell apoptotic response, mitogen-activated protein kinase (MAPK) signaling, and JAK-STAT signaling are significantly upregulated.^22,30 ZNF384 overexpression leads to the upregulation of matrix metalloproteinase (MMP) 1, MMP3, MMP7, MMP13, collagen type-1 (COLA1), and GATA-binding protein 3 (GATA3).^17,31,32 GATA3 overexpression activates the expression of CD13/CD33 in B-ALL. ²⁶

ZNF384 is also a poor prognostic predictor of hepatocellular carcinoma and a good prognosis predictor of breast cancer.^33,34 It promotes metastasis of hepatocellular carcinoma, colorectal cancer, and breast cancer.^33,35,36 Furthermore, ZNF384 is a potential therapeutic target for psoriasis and Alzheimer’s disease. ³⁷

In this review, we focus on the ZNF384 and its fusion partners and summarize the mechanisms, performance, and features of different ZNF384 rearrangements in acute lymphoblastic leukemia.

The ZNF384 Fusion parter

E1A-Binding Protein P300

E1A-binding protein P300 (EP300), a tumor suppressor gene, is located on 22q13. ³⁸ Patients with ALL harboring the EP300-ZNF384 fusion gene were first reported in 2015. ⁵ EP300 encodes the E1A binding protein P300, a histone acetyltransferase (HAT), which results in the acetylation of DNA-associated histone. ³⁹ Breakpoints are located between exon 6 of EP300 and exons 2 and 3 of ZNF384. ⁴⁰ EP300 and ZNF384 fusion leads to the loss of the HAT domain of EP300. However, the transcriptional adapter zinc finger 1 (TAZ1) domain was retained.^5,41-43 EP300 structure alteration could lead to inactivation of EP300 and ZNF384, companied by acetylation-mediated inactivation of the BCL6 and activation of the p53 tumor suppressor. ⁴⁴ Losing the HAT activity of EP300 leads to the proliferation of hematopoietic progenitors and stem cells, and this breaks the balance between proliferation and differentiation in hematopoietic processes.^44,45 It interacts with different transcription partners to regulate cell growth, differentiation, and cell cycle, and maintain genomic stability.^38,46 EP300 might increase the transcriptional activity of ZNF384 via its acetylation. ⁴⁷ Further, it fused with mixed lineage leukemia (MLL) in acute myeloid leukemia. ⁴⁸ EP300-ZNF384 induces leukemic transformation by altering the differentiation properties of hematopoietic stem and progenitor cells. ⁹ EP300-ZNF384 fusion genes prevent Pro-B cell differentiation and leukemia cells exhibit Pro-B features. ⁷ The incidence of EP300-ZNF384 is approximately 1% in pediatric B-others. ⁵ Adolescent and adult patients more commonly present with EP300-ZNF384 than children, with an incidence of 8.2% and 7.7%.^7,26 The median age at ALL diagnosis is 11 years in patients with EP300-ZNF384. ⁴⁹ However, elevated white blood cell (WBC) counts were not observed. Further, the patients showed a good responses to prednisolone. ⁵ Patients with EP300-ZNF384 have a lower risk of recurrence than other ALL patients with other types of ALL.^3,19

EP300-ZNF384 leads to the upregulation of cardiotrophin-like cytokine factor 1 (CLCF1), CAMP Responsive Element Binding Protein 5 (CREB5), STGALNAC2, CD33, and RUNX Family Transcription Factor 2 (RUNX2), and downregulation of ovochymase 2 (OVCH2), CAMP-regulated phosphoprotein 21 (ARPP21), neuropeptide Y (NPY), and bone morphogenetic protein 2 (BMP2). In addition, RAS mutants, FLT3 signaling axis, and cytokine receptor mutations were found. Liu et al showed that the expression of myeloid reprogramming genes GATA3, CEBPA, and CEBPB were up-regulated.^11,50 Gene set enrichment analysis (GSEA) revealed that the genes upregulated by EP300-ZNF384 are enriched in Janus-kinase signal transducers and activators of the transcription (JAK/STAT) signaling pathway, leukocyte adhesion and differentiation pathways, cell cycle, oxidative phosphorylation, and DNA repair. ⁵¹ In addition, the affinity of ZNF384 to the promoter of GATA3 can be enhanced, thereby leading to transcription activation. ¹¹ Yaguchi et al revealed that B-ALL cells have hematopoietic stem cell properties with EP300-ZNF384 expression. ¹¹

Several studies have demonstrated that patients with EP300-ZNF384 present with weak CD10 expression and aberrant expression of CD13 and CD33.^3,9,26 The expression of CD10 reduces the number of apoptotic cells, and it is correlated with inferior clinical outcome.^52,53 However, Jing et al have reported that half of the patients with EP300-ZNF384 present with a positive CD10 expression. Surprisingly, men with EP300-ZNF384 were CD10⁺, and women were CD10⁻. ⁴⁰ However, EP300-ZNF384 overexpression in REH cells did not alter immunophenotypic features, such as CD10 and CD13/CD33. Thus, downstream regulation of GATA3 might be required. ¹¹

CREB-Binding Protein

CREB-binding protein (CREBBP) is located at 16p13.3, and it belongs to the p300/CBP coactivator family. CREBBP is strongly correlated with EP300. Both CREBBP and EP300 have the same function as lysine acetyltransferase and can target histone acetyltransferase, which can alter chromatin conformation.^39,54,55 Copy-number aberration and CREBBP mutation in B-cell precursor (BCP) ALL lead to the loss of HAT function.^41,42 CREBBP-ZNF384 combines exons 4,5, or 6 of CREBBP with exons 2 or 3 of the ZNF384 gene. If ZNF384 and CREBBP juxtaposed, CREBBP contains only the KIX domain for interacting with CREB and MYB. However, CREBBP lost most of the domains associated with HAT.^56,57 CREBBP-ZNF384 decreases HAT activity compared to CREBBP protein alone. Unlike CREBBP alone, CREBBP-ZNF384 induces leukemic transformation by altering the differentiation properties of hematopoietic stem and progenitor cells. ⁹

Transcription Factor 3

Transcription factor 3 (TCF3), also known as E2A, is located at 19p13.3. A patient with B-ALL harboring TCF3-ZNF384 was first reported in 2005. ⁵⁸ The TCF3 gene is the second most common translocation partner for ZNF384 in B-ALL, resulting in TCF3-ZNF384 gene fusion. In addition, the TCF3-ZNF384 fusion gene is observed in 50% of B cell/myeloid mixed phenotype acute leukemia (B/M MPAL) cases.^10,49 In cases of TCF3-ZNF384, exons 11 and 13 of TCF3 were fused with exons 2 and 3 of ZNF384. The transactivation domain of TCF3 and the coding region of ZNF384 were well reserved. ⁵⁹ Gene set enrichment analysis (GSEA) showed that the genes regulated by TCF3-ZNF384 are involved in hematopoietic stem cell features. Most cases of TCF3-ZNF384 ALLs have additional alterations driving the RAS signaling pathway genes and CDKN2A/B deletion in patients with B-ALL harboring TCF3-ZNF384. The expression of GATA3 and NCOR1 was upregulated in TCF3-ZNF384-positive ALL.^3,19 This mutation did not emerge in other ZNF384 rearrangements. Further, TCF3-ZNF384 can induce 3T3 fibroblast transformation. ⁶⁰ The transcriptomic analysis of TCF3-ZNF384 is more similar to that of EP300-ZNF384 and CREBBP-ZNF384, but different from that of TCF3-PBX1 and ZNF384-fusion-negative. ³ However, patients with B-ALL harboring TCF3-ZNF384 present with different clinical features. That is, they have a higher white blood cell count and an earlier age of onset (5 years) than EP300-ZNF384-positive patients. ⁴⁹ In addition, TCF3-ZNF384-positive patients respond poorly to steroids, and they had a high frequency of relapse. ³ These patients are more likely to be classified in the high-risk group. A patient with TCF3-ZNF384 experienced a lineage switch from ALL to AML after chimeric antigen receptor T (CAR-T) therapy. ⁸ Kapadia et al reported a patient with BCP-ALL harboring TCF3-ZNF384 but with a lineage switch from ALL to MPAL after steroid therapy. This confirms that patients with TCF3-ZNF384 showed a poor response to steroid.^3,61 Further, Oberley et al reported that two patients with TCF3-ZNF384 relapsed after 10 years. ⁶ The inherent plasticity of leukemia stem cells causes divergent differentiation. ⁶²

TATA-Box Binding Protein Associated Factor 15

TATA-box binding protein associated factor 15 (TAF15) is located at chromosome 17q12. A case of B-ALL harboring TAF15-ZNF384 was first reported in 2002. ¹² It is a member of the TET (TLS/FUS, Ewing sarcoma breakpoint region 1 gene (EWSR1), TAF15) family, which is involved in fusion genes. ⁶³ TAF15 encodes transcription factors IID, which promotes the formation of RNA polymerase II, and participates in pre-mRNA splicing.^64,65 It has four well-conserved motifs: a transactivation domain at the N-terminal domain and RNA recognition motif (RRM), zinc finger (ZF), and RGG domain at the C-terminus domain. ⁶⁶ The breakpoint in the TAF15 gene is commonly located between exons 4 and 10, especially exons 6 and 7, ranging from q11 to q21. ⁶⁷ However, the most possible breaking point takes place on 17q12. TAF15-ZNF384 comprises the SYQG N-terminal domain from TAF15 and a complete sequence of ZNF384. ¹² TAF15-ZNF384 occurs in an early common progenitor which may differentiate into the myeloid or the lymphoid lineages. ⁶⁸ Several cases have a relatively poor outcome.^58,69 TAF15-ZNF384 can trigger an immune-mediated effect and accelerate disease progression by altering the COL1A1 gene expression but not the metalloproteinase levels. ⁷⁰ TAF15-ZNF384 induces 3T3 fibroblast transformation. ¹² The metalloproteinases expression could not be altered after TAF15-ZNF384 was transfected into HEK293 T cells. ¹² A patient with TAF15-ZNF384 was found to have FLT3 mutations. ⁶⁷

Ewing Sarcoma Breakpoint Region 1 Gene

Ewing sarcoma breakpoint region 1 gene (EWSR1) is a member of the TET family genes. ¹² It is located on 22q12.2. A B-ALL patients harboring EWSR1-ZNF384 was first reported in 2002. ¹² EWSR1-ZNF384 combines exon 7 of EWSR1 to exons 2, 3, or 7 of the ZNF384 gene. ⁷¹ This fusion gene disrupts transcriptional and epigenetic processes and drives self-renewal abnormalities at the early stage of leukemogenesis. Thereafter, IKAROS family zinc finger 1 (IKZF1), ETS Variant Transcription Factor 6 (ETV6), and BTG anti-proliferation factor 1 (BTG1) deletions were observed in the development differentiation of lymphoid.^12,71 EWSR1-ZNF384 induces 3T3 fibroblast transformation without affecting metalloproteinases expression as well. ¹²

AT-Rich Interactive Domain-Containing Protein 1B

AT-rich interactive domain-containing protein 1B (ARID1B) is located at 6q25 and is part of the human Brg1/Brm-associated factor (BAF) chromatin remodeling complex. Further, it binds to the DNA sequence via its AT-rich DNA-binding domain. ⁷² ARID1B mutation is found in different types of tumors. ⁷² Shago et al reported a patient presenting with ARID1B-ZNF384. ⁴ This fusion gene contains 666 N-terminal amino acids from ARID1B and 555 amino acids lacking the AT-rich interaction domain of the ARID1B protein. ⁷³

Transcription Factor 4

Transcription factor 4 (TCF4), a basic helix-loop-helix transcription factor, is located at 18q21.2. If TCF4 fuses with ZNF384, the functional domains of TCF4 are commonly lost. The genetic characteristics of TCF4-ZNF384 fusion were different from those of other ZNF384-rearrangements. ⁷⁴ Treatment was smooth and the prognosis was believed to be good. Wu et al reported a case of B-ALL with TCF4-ZNF384 fusion without abnormal CD13 and CD33 expression. ⁷⁴

Bone Morphogenic Protein 2-Inducible Kinase

Bone morphogenic protein 2 inducible kinase (BMP2K) plays an important role in skeletal system development. In the case of BMP2K-ZNF384, exons 15 and 14 of BMP2K were fused with exon 3 of ZNF384. Rearrangements retain the BMP2K kinase domain at the 5’ end, and the C-terminal inhibitory domain is lacking. ³ However, the BMP2K-ZNF384 mechanism remains unclear.

Others

The rest fusion partners are rare events in B-ALL. There are no detailed reports on the structure of these genes and the clinical outcomes of conditions associated with them. SMARCA2 and SMARCA4 are members of the SWI/SNF family, which has helicase and ATPase activities and can regulate gene expression by altering the chromatin landscape surrounding genes. ⁷² Synergin gamma (SYNRG) is involved in endocytosis and trafficking from the trans-Golgi network. ⁷⁵ Clathrin heavy chain (CLTC) is the major component of the coated vesicles. ⁷⁶ Nipped-B-like protein (NIPBL) plays a “porter” role in cohesin loading onto the DNA. ⁷⁷ A-kinase anchoring protein 8 (AKAP8) targets proteins that mediate the subcellular compartmentation of cAMP-dependent protein kinase (PKA) type II. ⁷⁸ Chromosome 11 open reading frame 74 (C11orf74) is a pseudogene. DDX42, a member of the Asp-Glu-Ala-Asp (DEAD) box protein family, encodes RNA helicases. ⁷⁹ ATP synthase F1 subunit gamma (ATP5C1) encodes a mitochondrial ATP synthase subunit. ⁸⁰ Euchromatic histone lysine methyltransferase 1 (EHMT1) encodes a histone methyltransferase that methylates the lysine 9 of histone H3, thereby enabling gene repression. ⁸¹ Testis expressed 41 (TEX41) is an RNA gene and is affiliated with the lncRNA class.

Conclusion

The emergence of high-throughput sequencing technology has significantly facilitated the detection of ZNF384 fusion genes. The B-ALL patients with ZNF384 rearrangement typically exhibit a favorable prognosis. However, patients harboring the TCF3-ZNF384 fusion have a less favorable prognosis. The number of B-ALL patients harbored ZNF384 is small, more relevant mechanism researches still need to be done to reveal the exact mechanism. Therefore, conducting further research to understand the biological function and molecular mechanisms of ZNF384 rearrangement is of great significance.