IKZF3 amplification predicts worse prognosis especially in intestinal-type gastric cancer.

PURPOSE: IKAROS family zinc finger 3 (IKZF3) is an oncogene involved in different malignancies, particularly in the development and malignant progression of lymphocytes. However, IKZF3 amplification and clinical significance in gastric cancers (GCs) remain unexplored. METHODS: We examined IKZF3 amplification status in 404 GCs with HER2 amplification status using tissue microarray (TMA) and fluorescence in situ hybridization (FISH) assays. RESULTS: IKZF3 amplification was detected in 6.9% (28/404) of all GC patients, with higher rates in intestinal-type gastric cancer (IGC) (11.22%, 22/196) compared to other types (2.88%, 6/208). HER2 amplification was identified in 16.09% (65/404) of all GC patients, with higher rates in IGC (20.92%, 41/196) compared to other types (11.54%, 24/208). Co-amplification of IKZF3 and HER2 was detected in 8.16% (16/196) of IGC patients and in 2.40% (5/208) of other types. IKZF3 amplification showed significant correlation with IGC (P = 0.001) and HER2 amplification (P = 0.0001). IKZF3 amplification exhibited significantly worse disease-free survival (DFS) (P = 0.014) and overall survival (OS) (P = 0.018) in GC patients, particularly in IGC (DFS: P < 0.001; OS: P < 0.001), rather than other types. Cox regression analysis demonstrate IKZF3 amplification as an independent poor prognostic factor in all GCs (P = 0.006, P = 0.004 respectively) and in IGC patients, regardless of stages I-II or III-IV (P = 0.007, P = 0.004 respectively). On the other hand, HER2 amplification was significantly associated with worse DFS (P = 0.008) and OS (P = 0.01) in IGC patients, but not in all GCs and in multivariate analysis. Within the subset of patients with HER2 amplification, those also exhibiting IKZF3 amplification displayed potential poorer prognosis (P = 0.08, P = 0.11 respectively). CONCLUSION: IKZF3 amplification was detected in minority of GC patients, especially in IGC, and was an independent indicator of poor prognosis. Our study, for the first time, found the prognostic value of IKZF3 was superior to HER2 for GC patients.

AIOLOS-Associated Inborn Errors of Immunity.

AIOLOS, encoded by the IKZF3 gene, belongs to the Ikaros zinc finger transcription factor family and plays a pivotal role in regulating lymphocyte development. Recently, heterozygous missense loss-of-function variants within the DNA-binding domain of the IKZF3 gene (G159R, N160S, and G191R) have been identified in patients with inborn errors of immunity (IEI). Additionally, a missense and a truncating variant (E82K and Q402X) leading to the AIOLOS haploinsufficiency have been documented. The majority of individuals with AIOLOS-associated IEI manifest recurrent sinopulmonary infections, as well as various bacterial and viral infections. The patients carrying the AIOLOSN160S variant exhibit severe immunodeficient phenotypes. In contrast, patients harboring AIOLOS haploinsufficient variants predominantly present with clinical phenotypes associated with immune dysregulation. A varying degree of B-lymphopenia and hypoimmunoglobulinemia was noted in approximately half of the patients. Mouse models of AIOLOSG159R and AIOLOSN160S variants (AiolosG158R and AiolosN159S in mice, respectively) recapitulated most of the immune abnormalities observed in the patients. Among these models, AiolosG158R mice prominently exhibited defects in early B cell differentiation resulting from mutant Aiolos interfering with Ikaros function through heterodimer formation. In contrast, AiolosN159S mice did not manifest early B cell differentiation defects. However, they displayed a distinct immune abnormality characterized by impaired induction of CD62L expression in lymphocytes, which is likely attributable to dysfunction of Ikaros, leading to defective lymphocyte homing to lymph nodes. Considering the diverse clinical phenotypes observed in the reported cases and the distinct molecular pathogenesis associated with each variant, further studies with more patients with AIOLOS-associated IEI would contribute to a better understanding of the clinical spectrum and underlying molecular mechanisms associated with this disorder.

Identification and Functional Analysis of a de novo IKZF3 Mutation in a Pediatric Patient with Combined Immunodeficiency.

AIOLOS, a vital member of the IKAROS protein family, plays a significant role in lymphocyte development and function through DNA binding and protein-protein interactions. Mutations in the IKZF3 gene, which encodes AIOLOS, lead to a rare combined immunodeficiency often linked with infections and malignancy. In this study, we evaluated a 1-year-4-month-old female patient presenting with recurrent infections, diarrhea, and failure to thrive. Laboratory investigations revealed decreased T lymphocyte and immunoglobulin levels. Through whole-exome and Sanger sequencing, we discovered a de novo mutation in IKZF3 (NM_012481; exon 5 c.571G > C, p.Gly191Arg), corresponding to the third DNA-binding zinc finger region of the encoded protein AIOLOS. Notably, the patient with the AIOLOS G191R mutation showed reduced recent thymic emigrants in naïve CD4+T cells compared to healthy counterparts of the same age, while maintaining normal levels of Th1, Th2, Th17, Treg, and Tfh cells. This mutation also resulted in decreased switched memory B cells and lower CD23 and IgM expression. In vitro studies revealed that AIOLOS G191R does not impact the expression of AIOLOS but compromises its stability, DNA binding and pericentromeric targeting. Furthermore, AIOLOS G191R demonstrated a dominant-negative effect over the wild-type protein. This case represents the first reported instance of a mutation in the third DNA-binding zinc finger region of AIOLOS highlighting its pivotal role in immune cell functionality.

Expression levels of GSDMB and ORMDL3 are associated with relapsing-remitting multiple sclerosis and IKZF3 rs12946510 variant.

Multiple sclerosis (MS), a noncurable autoimmune neurodegenerative disease, requires constant research that could improve understanding of both environmental and genetic factors that lead to its occurrence and/or progression. Recognition of the genetic basis of MS further leads to an investigation of the regulatory role of genetic variants on gene expression. Among risk variants for MS, Ikaros zinc finger 3 (IKZF3) gene variant rs12946510 was identified as one of the top-ranked and the expression quantitative trait loci (eQTL) for genes residing in chromosomal locus 17q12-21. The study aimed to investigate the association of gene expression of the immunologically relevant genes, which map to indicated locus, ORMDL3, GSDMB, and IKZF3, with MS and rs12946510 genotype, taking into account disease phase, clinical parameters of disease progression, and severity and immunomodulatory therapy. We used TaqMan® technology for both allelic discrimination and gene expression determination in 67 relapsing MS patients and 50 healthy controls. Decreased ORMDL3 and GSDMB mRNA levels had significant associations with MS and rs12946510 TT rare homozygote among patients. Significant positive correlations between ORMDL3 and GSDMB mRNA expression were observed in both patients and controls. We detected the significant between-effect of sex and rs12946510 on the expression of ORMDL3 in the patient group and interferon ß therapy and rs12946510 on GSDMB expression. Our results show the association of ORMDL3 and GSDMB mRNA expression with the clinical manifestation of MS and confirm that IKZF3 rs12946510 exerts the eQTL effect on both genes in multiple sclerosis. Besides providing novel insight related to MS phases and interferon ß therapy, the study results confirm previous studies on regulatory genetic variants, autoimmunity, and MS.

IKZF3 polymorphisms contribute to the increased risk of acute lymphoblastic leukemia in children.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common cancer in children. IKZF3 (IKAROS family zinc finger 3) is a hematopoietic-specific transcription factor, and it has been validated that it is involved in leukemia. However, the role of IKZF3 single-nucleotide polymorphisms (SNPs) remains unclear. In this case-control study, the authors investigated the association of IKZF3 SNPs with ALL in children. METHODS: Six IKZF3 reference SNPs (rs9635726, rs2060941, rs907092, rs12946510, rs1453559, and rs62066988) were genotyped in 692 patients who had ALL (cases) and in 926 controls. The associations between IKZF3 polymorphisms and ALL risk were determined using odds ratios (ORs) and 95% confidence intervals (CIs). The associations of rs9635726 and rs2060941 with the risk of ALL were further estimated by using false-positive report probability (FPRP) analysis. Functional analysis in silico was performed to evaluate the probability that rs9635726 and rs2060941 might influence the regulation of IKZF3. RESULTS: The authors observed that rs9635726C>T (adjusted OR, 1.49; 95% CI, 1.06-2.11; p = .023) and rs2060941G>T (adjusted OR, 1.51; 95% CI, 1.24-1.84; p = .001) were related to and increased risk of ALL in the recessive and dominant models, respectively. Furthermore, the associations of both rs9635726 (FPRP = .177) and rs2060941 (FPRP < .001) with ALL were noteworthy in the FPRP analysis. Functional analysis indicated that rs9635726 and rs2060941 might repress the transcription of IKZF3 by disrupting its binding to MLLT1, TAF1, POLR2A, and/or RAD21. CONCLUSIONS: This study revealed that IKZF3 polymorphisms were associated with increased ALL susceptibility in children and might influence the expression of IKZF3 by disrupting its binding to MLLT1, TAF1, POLR2A, and/or RAD21. IKZF3 polymorphisms were suggested as a biomarker for childhood ALL.

Single-cell epigenetic, transcriptional, and protein profiling of latent and active HIV-1 reservoir revealed that IKZF3 promotes HIV-1 persistence.

Understanding how HIV-1-infected cells proliferate and persist is key to HIV-1 eradication, but the heterogeneity and rarity of HIV-1-infected cells hamper mechanistic interrogations. Here, we used single-cell DOGMA-seq to simultaneously capture transcription factor accessibility, transcriptome, surface proteins, HIV-1 DNA, and HIV-1 RNA in memory CD4+ T cells from six people living with HIV-1 during viremia and after suppressive antiretroviral therapy. We identified increased transcription factor accessibility in latent HIV-1-infected cells (RORC) and transcriptionally active HIV-1-infected cells (interferon regulatory transcription factor [IRF] and activator protein 1 [AP-1]). A proliferation program (IKZF3, IL21, BIRC5, and MKI67 co-expression) promoted the survival of transcriptionally active HIV-1-infected cells. Both latent and transcriptionally active HIV-1-infected cells had increased IKZF3 (Aiolos) expression. Distinct epigenetic programs drove the heterogeneous cellular states of HIV-1-infected cells: IRF:activation, Eomes:cytotoxic effector differentiation, AP-1:migration, and cell death. Our study revealed the single-cell epigenetic, transcriptional, and protein states of latent and transcriptionally active HIV-1-infected cells and cellular programs promoting HIV-1 persistence.

IKZF3 modulates cerebral ischemia/reperfusion injury by inhibiting neuroinflammation.

Neuroinflammation is a key mediator to the pathogenic cascades induced by cerebral ischemia-reperfusion (I/R) injury. IKZF3, a key zinc finger transcription factor in the Ikaros family, has already been shown to modulate a wide range of cell functions and the production of inflammatory mediators. However, the effects of IKZF3 on inflammation and the potential mechanism after cerebral I/R injury remain unclear. In this study, we evaluated the effect of IKZF3 on HT-22 cells under oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro and in mice with MACO in vivo. We found that IKZF3 expression peaked at 12 h after MCAO and OGD/R, and there was high expression of IKZF3 in brain tissues and HT-22 cells. IKZF3 knockdown exacerbated the damage by OGD-induced HT-22 cells injury and MCAO-induced brain injury in mice by regulating the production of inflammatory factors, which promoted the phosphorylation and nuclear transfer of NF-ĸB and may bind with NF-ĸB-p65 in vivo and in vitro. Our results suggested that IKZF3 may provide a new target in improve neurological recovery and reducing neuroinflammation after cerebral I/R injury.

IKZF3 amplification frequently occurs in HER2-positive breast cancer and is a potential therapeutic target.

Breast cancer is one of the leading causes of cancer death in women, and although treatment outcome has substantially improved in the past decades, advanced or metastatic breast cancers still carry a poor prognosis. Gene amplification is one of the frequent genetic alterations in cancer, and oncogene amplification may be associated with cancer aggressiveness and oncogenicity. Targeting amplified genes such as HER2 has vastly improved disease outcome and survival, and anti-HER2 therapeutics have revolutionized the standard of care in HER2 breast cancer. Besides currently known druggable gene amplifications including ERBB2 and FGFR2, other frequently amplified genes are relatively less well known for function and clinical significance. By querying four large databases from TCGA and AACR-Genie, from a total of 11,890 patients with invasive ductal breast carcinoma, we discover IKZF3, CCND1, ERBB2 to be consistently amplified across different cohorts. We further identify IKZF3 as a frequently amplified gene in breast cancer with a prevalence of 12-15% amplification rate. Interestingly, IKZF3 amplification is frequently co-amplified with ERBB2/HER2, and is also associated with worse prognosis compared to IKZF3 non-amplified cancers. Analysis of HER2 breast cancer patients treated with trastuzumab revealed decrease in both ERBB2/HER2 and IKZF3 expression. Further investigation using the DepMap for gene dependency by genome-wide CRISPR screening revealed dependence on IKZF3 in HER2 breast cancer cell lines. Our study utilized an integrative analysis of large-scale patient genomics, transcriptomics and clinical data to reveal IKZF3 as a frequently amplified gene, and suggest a potential role of IKZF3 as a druggable target for HER2 breast cancer.

AIOLOS Variants Causing Immunodeficiency in Human and Mice.

AIOLOS is encoded by IKZF3 and is a member of the IKAROS zinc finger transcription factor family. Heterozygous missense variants in the second zinc finger of AIOLOS have recently been reported to be found in the families of patients with inborn errors of immunity. The AIOLOSG159R variant was identified in patients with B-lymphopenia and familial Epstein-Barr virus-associated lymphoma. Early B-cell progenitors were significantly reduced in the bone marrow of patients with AIOLOSG159R. Another variant, AIOLOSN160S was identified in the patients presented with hypogammaglobulinemia, susceptibility to Pneumocystis jirovecii pneumonia, and chronic lymphocytic leukemia. Patients with AIOLOSN160S had mostly normal B cell counts but showed increased levels of CD21lo B cells, decreased CD23 expression, and abrogated CD40 response. Both variants were determined to be loss-of-function. Mouse models harboring the corresponding patient's variants recapitulated the phenotypes of the patients. AIOLOS is therefore a novel disease-causing gene in human adaptive immune deficiency.

IKZF3 deficiency potentiates chimeric antigen receptor T cells targeting solid tumors.

Chimeric antigen receptor (CAR) T cell therapy has been successful in treating hematological malignancy, but solid tumors remain refractory. Here, we demonstrated that knocking out transcription factor IKZF3 in HER2-specific CAR T cells targeting breast cancer cells did not affect CAR expression or CAR T cell differentiation, but markedly enhanced killing of the cancer cells in vitro and in a xenograft model, which was associated with increased T cell activation and proliferation. Furthermore, IKZF3 KO had similar effects on the CD133-specific CAR T cells targeting glioblastoma cells. AlphaLISA and RNA-seq analyses indicate that IKZF3 KO increased the expression of genes involved in cytokine signaling, chemotaxis and cytotoxicity. Our results suggest a general strategy for enhancing CAR T efficacy on solid tumors.

Cereblon expression is a prognostic marker in newly diagnosed POEMS syndrome treated with lenalidomide plus dexamethasone.

POEMS syndrome is a rare plasma cell disorder. Lenalidomide has recently emerged as a therapeutic option for POEMS syndrome. Cereblon has been identified as the direct target of lenalidomide, and high cereblon expression is associated with better response and outcome to lenalidomide therapy in multiple myeloma patients. Here, we analyzed the predictive value of cereblon, IKZF1, and IKZF3 in CD138+ selected plasma cells from forty-one newly diagnosed POEMS syndrome patients treated with lenalidomide in combination with dexamethasone at both gene and protein levels. We found that patients with high cereblon expression tended to achieve better hematologic response compared to those with low expression (p = 0.024 for gene expression; p = 0.01 for protein expression). Multivariate Cox regression analysis revealed high cereblon mRNA expression as an independent prognostic marker for longer progression-free survival (hazard ratio 0.542; 95% CI 0.337-0.871; p = 0.011). In conclusion, our results emphasized the role of cereblon mRNA expression as a unique biomarker for predicting the clinical response and outcome of lenalidomide-based therapy in newly diagnosed POEMS syndrome patients.

A hotspot mutation in transcription factor IKZF3 drives B cell neoplasia via transcriptional dysregulation.

Hotspot mutation of IKZF3 (IKZF3-L162R) has been identified as a putative driver of chronic lymphocytic leukemia (CLL), but its function remains unknown. Here, we demonstrate its driving role in CLL through a B cell-restricted conditional knockin mouse model. Mutant Ikzf3 alters DNA binding specificity and target selection, leading to hyperactivation of B cell receptor (BCR) signaling, overexpression of nuclear factor κB (NF-κB) target genes, and development of CLL-like disease in elderly mice with a penetrance of ~40%. Human CLL carrying either IKZF3 mutation or high IKZF3 expression was associated with overexpression of BCR/NF-κB pathway members and reduced sensitivity to BCR signaling inhibition by ibrutinib. Our results thus highlight IKZF3 oncogenic function in CLL via transcriptional dysregulation and demonstrate that this pro-survival function can be achieved by either somatic mutation or overexpression of this CLL driver. This emphasizes the need for combinatorial approaches to overcome IKZF3-mediated BCR inhibitor resistance.

The association of genetic variants IL2RA rs2104286, IFI30 rs11554159 and IKZF3 rs12946510 with multiple sclerosis onset and severity in patients from Serbia.

An algorithm Probabilistic Identification of Causal SNPs, identified 434 causal variants for multiple sclerosis (MS) including IL2RA rs2104286, IFI30 rs11554159 and IKZF3 rs12946510. Analysis of individual and combined effects of these variants in the Serbian population identified that Il2RA rs2104286 G allele carriers had a lower risk for developing MS (gender adjusted OR = 0.63, p = .003). With regard to the IFI30 rs11554159 recessive genetic model, among HLA-DRB1*15:01 positive patients, the AA homozygote had a significantly higher MSSS compared to the G allele carriers (p = .003). This study confirms role of IL2RA rs2104286 in MS and suggest the role of IFI30 rs11554159 in disease severity, which needs validation.

The prognostic and predictive value of IKZF1 and IKZF3 expression in T-cells in patients with multiple myeloma.

Purpose: While recent studies described the role of IKZF1/3 proteins in multiple myeloma (MM) cells, few have highlighted the significance of IKZF1/3 expression in T-cells. In this study we examine the prognostic and predictive value of IKZF1/3 expression in T-cells in patients with MM stage III. Experimental design: We analysed the IKZF1/3 expression levels in T-cells from 45 MM stage I (MMI) and 50 newly diagnosed MM stage III (MMIII) patients, according to Durie-Salmon staging system, by flow cytometry to examine their prognostic and predictive value. We also combined in vivo observations with in vitro assays to determine the effect of IKZF1/3 expression on the T-cell immunophenotype and anti-tumour T-cell response in 162 MMIII patients. Results: We found that high IKZF3, but not IKZF1, expression in T-cells correlates with superior overall survival in MMIII patients treated with immunomodulatory drugs (thalidomide, lenalidomide and pomalidomide). Moreover, we show that higher IKZF3 expression in T-cells inhibits myeloma-specific T-cell response in vitro and that the immunophenotype of patients with high IKZF3 expression shows features that are contrary to the changes induced by immunomodulatory drugs. Although we observed higher IKZF3 expression levels in T-cells from patients with MMIII compared to MMI, IKZF3 expression was unaffected by the tumour microenvironment. Conclusion: In conclusion, IKZF3 expression in T-cells is a predictive value for clinical outcome in MMIII patients treated with immunomodulatory drugs due to its profound modulation of T-cell functionality.

Polymorphisms of IKZF3 Gene and Autoimmune Thyroid Diseases: Associated with Graves' Disease but Not with Hashimoto's Thyroiditis.

BACKGROUND/AIMS: The IKZF3 gene encodes a zinc-finger protein that plays an important role in the proliferation and differentiation of B lymphocytes. Autoimmune thyroid diseases (AITDs), mainly include Graves' disease (GD) and Hashimoto's thyroiditis (HT), are probably caused by the aberrant proliferation of B cells. The objective of this study was to explore the association between IKZF3 polymorphisms and AITDs. METHODS: We examined 915 AITD patients (604 GD and 311 HT) and 814 healthy controls. IKZF3 variants (rs2941522, rs907091, rs1453559, rs12150079 and rs2872507) were tested by PCR-ligase detection reaction. RESULTS: It was manifested that that the minor alleles of the five loci increased susceptibility to GD (p<0.05 for rs2941522, and p<0.01 for rs907091, rs1453559, rs12150079 and rs2872507) but in HT patients, these loci showed no significant difference compared with controls. Similarly, the genotype distributions of GD patients manifested obvious differences in all these loci compared with the control group, whereas no statistical differences were observed between HT patients and controls. Furthermore, bioinformatics tools were used to analyze rs1453559, rs12150079 and rs907091. These variants were believed to be the transcription regulator. CONCLUSION: It is the first time we reported the association between the IKZF3 polymorphisms and GD, indicating that IKZF3 gene tends to bean important risk factor for the development of GD.

Allelic imbalance of multiple sclerosis susceptibility genes IKZF3 and IQGAP1 in human peripheral blood.

BACKGROUND: Multiple sclerosis is a chronic inflammatory, demyelinating disease of the central nervous system. Recent genome-wide studies have revealed more than 110 single nucleotide polymorphisms as associated with susceptibility to multiple sclerosis, but their functional contribution to disease development is mostly unknown. RESULTS: Consistent allelic imbalance was observed for rs907091 in IKZF3 and rs11609 in IQGAP1, which are in strong linkage disequilibrium with the multiple sclerosis associated single nucleotide polymorphisms rs12946510 and rs8042861, respectively. Using multiple sclerosis patients and healthy controls heterozygous for rs907091 and rs11609, we showed that the multiple sclerosis risk alleles at IKZF3 and IQGAP1 are expressed at higher levels as compared to the protective allele. Furthermore, individuals homozygous for the multiple sclerosis risk allele at IQGAP1 had a significantly higher total expression of IQGAP1 compared to individuals homozygous for the protective allele. CONCLUSIONS: Our data indicate a possible regulatory role for the multiple sclerosis-associated IKZF3 and IQGAP1 variants. We suggest that such cis-acting mechanisms may contribute to the multiple sclerosis association of single nucleotide polymorphisms at IKZF3 and IQGAP1.

Differential effects of lenalidomide during plasma cell differentiation.

Thalidomide, lenalidomide and pomalidomide have greatly improved the outcome of patients with multiple myeloma. However, their effects on plasma cells, the healthy counterpart of myeloma cells, are unknown. Here, we investigated lenalidomide effects on normal human plasma cell generation using an in vitro model. Lenalidomide inhibited the generation of pre-plasmablasts and early plasma cells, while it moderately affected plasmablast production. It also reduced the expression level of Ikaros, Aiolos, and IRF4 transcription factors, in plasmablasts and early plasma cells. This suggests that their differential sensitivity to lenalidomide is not due to a difference in Ikaros or Aiolos degradation. Lenalidomide also inhibited long-lived plasma cell generation, but did not impair their long-term survival once generated. This last observation is in agreement with the finding that lenalidomide treatment for 3-18 months did not affect the bone marrow healthy plasma cell count in allografted patients with multiple myeloma. Our findings should prompt to investigate whether lenalidomide resistance in patients with multiple myeloma could be associated with the emergence of malignant plasmablasts or long-lived plasma cells that are less sensitive to lenalidomide.

Peripheral natural killer cell maturation depends on the transcription factor Aiolos.

Natural killer (NK) cells are an innate lymphoid cell lineage characterized by their capacity to provide rapid effector functions, including cytokine production and cytotoxicity. Here, we identify the Ikaros family member, Aiolos, as a regulator of NK-cell maturation. Aiolos expression is initiated at the point of lineage commitment and maintained throughout NK-cell ontogeny. Analysis of cell surface markers representative of distinct stages of peripheral NK-cell maturation revealed that Aiolos was required for the maturation in the spleen of CD11b(high)CD27(-) NK cells. The differentiation block was intrinsic to the NK-cell lineage and resembled that found in mice lacking either T-bet or Blimp1; however, genetic analysis revealed that Aiolos acted independently of all other known regulators of NK-cell differentiation. NK cells lacking Aiolos were strongly hyper-reactive to a variety of NK-cell-mediated tumor models, yet impaired in controlling viral infection, suggesting a regulatory function for CD27(-) NK cells in balancing these two arms of the immune response. These data place Aiolos in the emerging gene regulatory network controlling NK-cell maturation and function.