IKZF1 Mutations Research Articles

Multifaceted roles of IKZF1 gene, perspectives from bench to bedside.

The IKZF1 gene encodes a transcription factor that belongs to the family of zinc-finger DNA-binding proteins associated with chromatin remodeling. The protein product, IKAROS, had been proved to regulate lymphopoiesis. Subsequent mouse model studies have further confirmed its regulating role in lymphopoiesis as well as in hematopoiesis; besides, it associates with immune function, certain immune disorders like common variable immunodeficiency and dysgammaglobulinemia have been proved to be associated with germline IKZF1 mutations. Dysfunction of IKAROS also bears paramount significance in leukemic transformation and alterations of IKZF1 gene predicts a poor prognosis in hematological malignancies. As an independent prognostic marker, IKZF1 has been incorporated in the risk stratification of BCP-ALL and stratification-guided therapy has also been generated. In this review, we provide a concise and comprehensive overview on the multifaceted roles of IKZF1 gene.

A novel IKZF1 variant in a family with autosomal dominant CVID: A case for expanding exon coverage in inborn errors of immunity

Common variable immune deficiency (CVID) is a heterogenous group of disorders characterized by varying degrees of hypogammaglobulinemia, recurrent infections, and autoimmunity. Currently, pathogenic variants are identified in approximately 20–30% of CVID cases. Here we report a 3-generation family with autosomal dominant Common Variable Immunodeficiency (CVID) diagnosed in 9 affected individuals. Although primary immune deficiency panels and exome sequencing were non-diagnostic, whole genome sequencing revealed a novel, pathogenic c.499C > T: p.His167Tyr variant in IKZF1, a critical regulator of B cell development. Functional testing done through pericentromeric heterochromatin localization and light shift chemiluminescent electrophoretic mobility shift assay confirmed the variant's deleterious effect via a haploinsufficiency mechanism. Our findings expand the spectrum of known IKZF1 mutations and contribute to a more comprehensive understanding of CVID's genetic heterogeneity. Furthermore, this case underscores the importance of considering whole genome sequencing for comprehensive genetic diagnosis when concern for a monogenic inborn errors of immunity is high.

Dependence of lymphopoiesis on efficient β-catenin degradation

Abstract Wnt/β-catenin pathway is tightly controlled by β-catenin destruction complex to regulate lineage specification and differentiation. Compared to epithelial lineage, lymphoid cells display higher rates of β-catenin degradation to achieve constitutively low levels of β-catenin. Emphasizing the reliance of lymphoid cells on β-catenin degradation, expression of degradation resistant form of β-catenin or deletion of APC or CK1 suppressed lymphoid development. Similarly, small molecules inhibiting GSK3β mediated phosphorylation or proteasomal degradation of β-catenin induced higher toxicity selectively in lymphoid cells compared to other lineages. Our interactome and CHIP-seq analysis highlighted that the unique dependency of lymphoid cells on efficient degradation of β-catenin is mediated by Ikaros zinc finger (IKZF) proteins and nucleosome remodeling and deacetylase (NuRD) complexes. Unlike activating TCF/β-catenin complexes in epithelial cells, recruitment of repressive β-catenin/IKZF/NuRD complexes to MYC superenhancer (SE) regions lead to suppression of Myc expression and induced cell death. Deletion of both IKZF1 and IKZF3 or mutation of Ikaros binding motifs within the Myc-SE subverted the β-catenin mediated toxicity. Upon loss of Ikaros factors, β-catenin interaction with TCF-factors resulted in de novo activation of epithelial specific genes. Our findings suggest that β-catenin degradation is an evolutionary requirement for Ikaros mediated protection of lymphoid identity.

Impact of Mutations in Blood Cancer-Related Genes on Clinical Outcomes in Chronic Myeloid Leukemia in Chronic Phase (CML-CP) after ≥2 Tyrosine Kinase Inhibitors (TKIs) in the Ascembl Trial

CONCLUSIONS Somatic mutations in blood cancer-related genes are commonly detected in CML-AP. The most frequently mutated genes are RUNX1, ASXL1IKZF1 (submicroscopic deletions), and BCR:: ABL1 (kinase domain mutations). ASXL1 is the most frequently detected mutation at CML diagnosis (≈9%). However, the incidence and impact of mutations in blood cancer-related genes in patients (pts) with CML-CP previously treated with ≥2 TKIs have not been described since genomic studies have focused on pts at diagnosis or at progression. With >2 years of follow-up in the phase 3 ASCEMBL study, asciminib (ASC) has continued to demonstrate superior efficacy vs bosutinib (BOS) in pts with CML-CP after ≥2 prior TKIs, and analysis of cancer gene somatic mutations in this population has the potential to reveal additional insights into pts' response to study treatments or characteristics of the disease in later lines of therapy. Here we present a descriptive analysis of variants in the ASCEMBL pt population, including frequencies, co-occurrences, and association of mutations with clinical outcomes, specifically major molecular response (MMR) and treatment failure (TF; defined as discontinuation due to lack of efficacy or intolerance). Adults with CML-CP previously treated with ≥2 TKIs with intolerance of their last TKI or lack of efficacy per 2013 European LeukemiaNet recommendations and without BCR:: ABL1 T315I or V299L mutations were randomized 2:1 to ASC 40 mg twice daily or BOS 500 mg once daily. DNA was isolated from blood collected at baseline (BL) and end of treatment (EOT; defined as treatment discontinuation for any reason or completion of the study). A next-generation sequencing panel of 89 commonly mutated genes in blood cancers, including ABL1 and IKZF1 deletion, was used to detect somatic mutations that met criteria for pathogenicity. Only samples with BCR::ABL1IS ≥2%, which was the level demonstrated to detect mutations in leukemic cells, were tested. A multivariable Cox proportional hazards regression model adjusted for age, sex, treatment, and reason for prior TKI discontinuation (intolerance vs lack of efficacy) was used to determine the prognostic value of BL mutations. A total of 233 pts were randomized to ASC (n=157) or BOS (n=76). Of 162 pts (ASC, n=110; BOS, n=52) with available sequencing data at BL, 102 had ≥1 mutation: 69 (63%) on ASC and 33 (63%) on BOS. Overall, 159 mutations were detected in 23 genes at BL, ranging from 0 to 5 per pt. The median variant allele frequency (VAF) was 19.6% (range, 0.9%-58%). ASXL1 (in 31% of pts) and ABL1 kinase domain (in 17% of pts) mutations were the most frequently detected. Of 27 pts with ≥1 ABL1 mutation at BL, 85% had ≥1 other mutated cancer gene. Co-occurrence of ASXL1 and ABL1 mutations at BL was reported in 12 of 162 pts (7.4%), which was higher than the expected 2.9% based on these genes' individual frequencies in this data set. RUNX1 and IKZF1 mutations were rare in CML-CP at BL. Of 162 pts tested at BL, 73 had TF and available samples for analysis at EOT with only 3 pts discontinuing due to disease progression. The presence of any mutation at BL was significantly prognostic of TF in the total cohort of 162 pts (Figure 1). In an unadjusted analysis, the presence of an ASXL1 mutation with VAF >5% at BL was a prognostic factor of TF. In 73 pts with TF, most BL mutations were also detected at EOT (110/116 mutations in 48/54 carriers) (Figure 2). Of 15 pts who acquired ≥1 mutation at EOT, 9 acquired ABL1 mutations. No single mutations at an individual or gene level were prognostic of MMR. Mutations were not predictive of MMR or TF when pts were examined by their assigned treatment of ASC or BOS. ASXL1 mutations are most frequently detected at CML diagnosis and were enriched at BL in this study of pts with CML-CP previously treated with ≥2 TKIs, which is consistent with a role in TKI resistance. RUNX1 and IKZF1 mutations, which are associated with progression to accelerated or blast phase in CML, were very rare in this pt population, supporting their role in CML disease progression. The high frequency of co-occurrence of BCR:: ABL1 mutations and blood cancer gene mutations in CML-CP is an important finding since cancer gene mutations could modify response to TKIs in individual pts. The presence of any somatic blood cancer mutation at BL was prognostic of TF. These mutations may serve as potential biomarkers that can identify pts who may be at higher risk of TF in later lines of therapy.

Real World Outcomes in Patients with Ph-like ALL at the University of New Mexico

Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a heterogenous subtype of ALL characterized by different translocations and molecular mutations, resulting in a gene expression profile similar to Philadelphia chromosome positive (Ph+) ALL without an identified BCR::ABL1 translocation. Unfortunately, Ph-like ALL is characterized by worse response rates to conventional therapies and poor overall survival compared to other ALL subtypes. It is reported that 20-30% of patients with newly diagnosed ALL in the United States have Ph-like ALL and the incidence appears to vary according to ethnicity. While many therapies are under investigation including the addition of a tyrosine kinase inhibitor or ruxolitinib to conventional chemotherapy, a standardized and molecularly-based approach to treating patients with Ph-like ALL is not yet defined. There also remain questions about the efficacy of integrating inotuzumab ozogamicin or blinatumomab into first-line therapy for these patients. In addition, recent retrospective data has demonstrated promising effects of using traditional salvage therapies for ALL such as Car T cells, inotuzumab, or blinatumomab in patients with Ph-like ALL. However, the optimal sequence of treatment as well the efficacy of combining salvage therapies with agents such as ruxolitinib remains unknown. Herein, we present our institutional experience with diagnosing and treating adult patients with Ph-like ALL at the University of New Mexico. Our goal was to characterize the demographics, molecular and cytogenetic landscape, and treatment outcomes at a medical center that serves a patient population enriched for this disease. Methods: After IRB approval, we performed a retrospective chart review of patients diagnosed and treated at the University of New Mexico between January 1, 2018 and April 1, 2023. We included patients who were 18 years or older at the time of diagnosis, received first-line therapy at the University of New Mexico, and had gene expression profiling or FISH testing that was consistent with Ph-like ALL. Statistical analysis was performed using SPSS Statistics v.27 (IBM, 2020). Results: We identified 9 patients who met inclusion criteria. All 9 patients self-identified as either ‘American Indian or Alaska Native’ or ‘Hispanic or Latino.‘ The median age at diagnosis was 34 and the mean BMI at diagnosis was 34.6. 5 patients were CD20 positive and 1 patient had a concurrent IKZF1 mutation. All patients received induction chemotherapy with either a BFM regimen or HyperCVAD. 5 (55%) patients were refractory to induction and 6 (66%) patients eventually underwent allogeneic stem cell transplant as part of their treatment. 1 patient received Car T cell therapy with Brexucabtagene autoleucel following relapse after allogeneic stem cell transplant. 1 patient died 124 days after diagnosis and 8 patients are alive currently. 4 patients had IGH:: CRLF2, 2 patients had P2RY8::CRLF2,1 patient had GTF2I::PDGFRB,1 patient had IGH::BCL2,and one patient had both a positive low-dose array and a positive gene expression profile without an identified molecular or cytogenetic abnormality consistent with Ph-like ALL. Discussion: We present our preliminary experience of patients with Ph-like ALL diagnosed and treated at the University of New Mexico. Our patients were young with a median age of 34. Consistent with published data describing patients with Ph-like ALL, our patients had poor responses to traditional ALL treatments and most commonly had an identified fusion with CLRF2. Our data supports the urgent need for the development of further treatment options for patients with Ph-like ALL as well as clinical trials aimed at standardizing management.

The clinical features and prognostic implications of PTPN11 mutation in adult patients with acute myeloid leukemia in China.

The clinical significance of protein tyrosine phosphatase nonreceptor type 11 mutation (PTPN11mut ) in acute myeloid leukemia (AML) is underestimated. We collected the data of AML patients with mutated PTPN11 and wild-type PTPN11 (PTPN11wt ) treated at our hospital and analyzed their clinical characteristics and prognosis. Fifty-nine PTPN11mut and 124 PTPN11wt AML patients were included. PTPN11mut was more common in myelomonocytic and monocytic leukemia, and was more likely to co-mutate with KRAS, KMT2C, NRAS, U2AF1, NOTCH1, IKZF1, and USH2A mutations than PTPN11wt . The overall survival for AML patients with PTPN11mut was significantly shorter than that for those with PTPN11wt (p = 0.03). The negative impact of PTPN11mut on overall survival was pronounced in the "favorable" and "intermediate" groups of ELN2017 risk stratification, as well as in the wild-type NPM1 group (p = 0.01, p = 0.01, and p = 0.04). PTPN11mut is associated with distinct clinical and molecular characteristics, and adverse prognosis in AML patients.

Ikaros is a principal regulator of Aire+ mTEC homeostasis, thymic mimetic cell diversity, and central tolerance.

Mutations in the gene encoding the zinc-finger transcription factor Ikaros (IKZF1) are found in patients with immunodeficiency, leukemia, and autoimmunity. Although Ikaros has a well-established function in modulating gene expression programs important for hematopoietic development, its role in other cell types is less well defined. Here, we uncover functions for Ikaros in thymic epithelial lineage development in mice and show that Ikzf1 expression in medullary thymic epithelial cells (mTECs) is required for both autoimmune regulator-positive (Aire+) mTEC development and tissue-specific antigen (TSA) gene expression. Accordingly, TEC-specific deletion of Ikzf1 in mice results in a profound decrease in Aire+ mTECs, a global loss of TSA gene expression, and the development of autoimmunity. Moreover, Ikaros shapes thymic mimetic cell diversity, and its deletion results in a marked expansion of thymic tuft cells and muscle-like mTECs and a loss of other Aire-dependent mimetic populations. Single-cell analysis reveals that Ikaros modulates core transcriptional programs in TECs that correlate with the observed cellular changes. Our findings highlight a previously undescribed role for Ikaros in regulating epithelial lineage development and function and suggest that failed thymic central tolerance could contribute to the autoimmunity seen in humans with IKZF1 mutations.

Mutated IKZF1 is an independent marker of adverse risk in acute myeloid leukemia

Genetic lesions of IKZF1 are frequent events and well-established markers of adverse risk in acute lymphoblastic leukemia. However, their function in the pathophysiology and impact on patient outcome in acute myeloid leukemia (AML) remains elusive. In a multicenter cohort of 1606 newly diagnosed and intensively treated adult AML patients, we found IKZF1 alterations in 45 cases with a mutational hotspot at N159S. AML with mutated IKZF1 was associated with alterations in RUNX1, GATA2, KRAS, KIT, SF3B1, and ETV6, while alterations of NPM1, TET2, FLT3-ITD, and normal karyotypes were less frequent. The clinical phenotype of IKZF1-mutated AML was dominated by anemia and thrombocytopenia. In both univariable and multivariable analyses adjusting for age, de novo and secondary AML, and ELN2022 risk categories, we found mutated IKZF1 to be an independent marker of adverse risk regarding complete remission rate, event-free, relapse-free, and overall survival. The deleterious effects of mutated IKZF1 also prevailed in patients who underwent allogeneic hematopoietic stem cell transplantation (n = 519) in both univariable and multivariable models. These dismal outcomes are only partially explained by the hotspot mutation N159S. Our findings suggest a role for IKZF1 mutation status in AML risk modeling.

Identification of IKZF1 genetic mutations as new molecular subtypes in acute myeloid leukaemia.

Genetic mutations of IKZF1 have been frequently delineated in B-lineage acute leukaemia (B-ALL) but rarely elucidated in acute myeloid leukaemia (AML). IKZF1 mutations confer a poor prognosis in AML, and hotspot mutations of IKZF1, N159Y and N159S tend to occur in B-ALL and AML respectively. However, the pathogenesis of IKZF1 N159S in AML and IKZF1 lineage susceptibility are largely unknown. The genetic and clinical characteristics of IKZF1-mutated AML patients were evaluated. Multi-omics analysis and functional assays were performed in vitro using IKZF1 mutations knock-in AML cell lines. 23 (4.84%) small sequence variants of IKZF1 were identified in 475 newly diagnosed AML (non-M3) patients. Based on RNA sequencing, three classes of IKZF1-related AML were defined, including 9 patients (39.13%) with IKZF1 N159S mutations, 10 (43.47%) with CEBPA mutations and 4 others (17.39%). IKZF1 N159S may define a unique subgroup with higher HOXA/B expression and native B-cell immune fractions. Gene expression data of multiple knock-in cell lines indicate that the lymphocyte differentiation-related MME and CD44 kept high expression in IKZF1 N159Y but were downregulated in N159S. CUT&TAG sequencing showed that IKZF1 N159S reshaped the binding profiles of IKZF1. Integration analysis suggested that the pathogenesis of IKZF1 N159S may depend on the deregulation of several cofactors, such as oncogenic MYC and CPNE7 targets. Collectively, we dissected the molecular spectrum and clinical features of IKZF1-related AML, which may promote an in-depth understanding of the pathogenesis, lineage susceptibility and clinical research of AML.

Identification of a novel NPM1 mutation in acute myeloid leukemia.

e19019 Background: Nucleophosmin (NPM1) is predominant nucleolar localization. NPM1-mutant acute myeloid leukemia (AML) account for 25-35% of adult AML patients, has been defined as a distinct AML entity in the 2022 WHO classification. The majority of NPM1 mutations reported affect exon 12 (classical type A mutation), account for 75-80% of adult NPM1-mutated AML cases, which generate an extra nuclear export signal (NES) in the C-terminal leading to aberrant cytoplasmic localization. Similar aberrant cytoplasmic translocation has also been reported in NPM1 mutants that identified in exons 9 and 11. Identification of novel non-type A mutants is paramount for diagnosis, risk stratification, and prognosis for targeted population. Methods: Somatic mutation analysis is performed on 566 AML patients using next-generation sequencing (NGS). Immunofluorescence and immunohistochemistry were used to identify the cytological localization of mutant NPM1. HEK-293T cells transiently transfected with plasmids for ectopic expression of the GFP-mutated NPM1 fusion proteins were treated with the specific Crm1/XPO1 inhibitor leptomycin B and to evaluate the NES dependence of their subcellular localization. Results: We identified one AML patient with NPM1 mutant located on exon 5. This patient was a 59-year-old female with de novo AML, with an 18-nucletide in-frame insertion at position 405 (c.405_406insGCCCTGGAACTGGGGAAC, named MutSong) in the middle of exon 5 [variant allele fraction (VAF), 11.9%]. It generated a new NPM1 mutant protein (p.135insALELGN), and containing a leucine-rich NES. Notably, different with all NPM1 mutant in other exons reported so far, our exon 5 mutant retained the functional c-terminal but insert one leucine-rich NES, similar finding was recently reported in NPM1 mutants that identified in exon 5 but on different position. This NPM1 mutant in exon 5 resulted in cytoplasmic localization both by confocal and immunohistochemistry in AML patient sample. HEK-293T overexpressing the new GFP-NPM1 exon 5 fusion protein indicated the aberrant localization in the cytoplasm and partially in nucleoli. Moreover, NES-dependent cytoplasmic localization was inhibited by exportin-1 inhibitor leptomycin B, indicated that the novel NES generated by exon 5 mutant is responsible for cytoplasmic localization. This patient carried WT1, IKZF1, JAK2, and NUP98 mutations, and was intravenously treated with daunorubicin plus cytarabine for induction followed by 3 cycles of intermediate-dose cytarabine as consolidation. Then the patient received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, the patient died of severe pulmonary infection and viral encephalitis 14 months after the initial remission, 7 months after allo-HSCT. Conclusions: Our finding of the novel NPM1 mutation in exon 5 supported that beside exon 12, exon 5 mutant is another NPM1 “born to be exported” mutant critical for leukemogenesis.

Acute leukemias with complex karyotype show a similarly poor outcome independent of mixed, myeloid or lymphoblastic immunophenotype: A study from the Bone Marrow Pathology Group

Mixed phenotype acute leukemia (MPAL) is a heterogenous group of acute leukemias characterized by leukemic blasts that express markers of multiple lineages. The revised 4th edition WHO classification of MPAL excludes AML with myelodysplasia related changes (AML-MRC), including those with complex karyotype (CK), from a diagnosis of MPAL. Abnormal karyotype is frequent in MPAL with the reported rate of CK in MPAL ranging from 19% to 32%. Due its rarity, the clinical and genetic features of MPAL with CK remain poorly characterized. This study aims to further characterize the genetic features of MPAL with CK in comparison to cases of AML and ALL with CK. Cases of de novo MPAL, AML, and B- and T-ALL patients with CK were collected from 8 member institutions of the Bone Marrow Pathology Group. We found no significant difference in overall survival between MPAL with CK compared to AML and ALL with CK. AML with CK was more strongly associated with TP53 mutations, however the presence of TP53 mutations conferred a worse prognosis regardless of lineage. ALL with CK seems to show increased IKZF1 mutation rates which is known to confer a worse prognosis in ALL. Additionally, MPAL with CK showed similarly poor outcomes regardless of whether a lymphoid or myeloid chemotherapy regimen is chosen. Our results suggest that acute leukemias with complex karyotype show a similarly poor outcome regardless of lineage differentiation and that mutation in TP53 confers a poor prognosis in all lineages. Our results support the exclusion of immunophenotypic MPAL with CK from MPAL and appear to confirm the approach proposed in the revised 4th edition WHO to include them as AML with myelodysplasia-related changes and similar myelodysplasia-related AML categories of newer classifications.

Transcriptome-based molecular subtypes and differentiation hierarchies improve the classification framework of acute myeloid leukemia

The current classification of acute myeloid leukemia (AML) relies largely on genomic alterations. Robust identification of clinically and biologically relevant molecular subtypes from nongenomic high-throughput sequencing data remains challenging. We established the largest multicenter AML cohort (n = 655) in China, with all patients subjected to RNA sequencing (RNA-Seq) and 619 (94.5%) to targeted or whole-exome sequencing (TES/WES). Based on an enhanced consensus clustering, eight stable gene expression subgroups (G1-G8) with unique clinical and biological significance were identified, including two unreported (G5 and G8) and three redefined ones (G4, G6, and G7). Apart from four well-known low-risk subgroups including PML::RARA (G1), CBFB::MYH11 (G2), RUNX1::RUNX1T1 (G3), biallelic CEBPA mutations or -like (G4), four meta-subgroups with poor outcomes were recognized. The G5 (myelodysplasia-related/-like) subgroup enriched clinical, cytogenetic and genetic features mimicking secondary AML, and hotspot mutations of IKZF1 (p.N159S) (n = 7). In contrast, most NPM1 mutations and KMT2A and NUP98 fusions clustered into G6-G8, showing high expression of HOXA/B genes and diverse differentiation stages, from hematopoietic stem/progenitor cell down to monocyte, namely HOX-primitive (G7), HOX-mixed (G8), and HOX-committed (G6). Through constructing prediction models, the eight gene expression subgroups could be reproduced in the Cancer Genome Atlas (TCGA) and Beat AML cohorts. Each subgroup was associated with distinct prognosis and drug sensitivities, supporting the clinical applicability of this transcriptome-based classification of AML. These molecular subgroups illuminate the complex molecular network of AML, which may promote systematic studies of disease pathogenesis and foster the screening of targeted agents based on omics.

High-Throughput Screening to Elucidate In Vivo Oncogenicity of Loss-of-Function Alterations in Lymphoid Malignancies

Large-scale genetic studies utilizing next-generation sequencing have identified many recurrently altered genes and revealed their overall picture in lymphoid malignancies. Although limited genes mutated at high frequency have been extensively investigated using genetically engineered mouse models, less attention has been directed to hundreds of rarely mutated genes (so-called long tail driver genes). Here we performed in vivo CRISPR loss-of-function screening to examine oncogenicity of somatic alterations, cooperability between them, and association between genotype and lineage in lymphoid malignancies. As many driver candidates mutated with various frequencies have been reported in diffuse large B-cell lymphomas (DLBCL), we constructed a custom lentiviral CRISPR library targeting 86 genes (12 sgRNAs each) in which loss-of-function alterations (mutations and deletions) are recurrently (> 5%) observed in DLBCL. These sgRNA-targeted genes also covered the majority of driver candidates of other precursor and mature lymphoid neoplasms, such as 71% and 87% of those observed in B-acute lymphoblastic leukemia (B-ALL/LBL) and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). Transplantation of the CRISPR library-transduced murine hematopoietic stem/progenitor (Lin-c-Kit+Sca-1+) cells caused a variety of hematologic malignancies in 180 (90%) out of 199 recipient mice, resulting in earlier death (with a median of 228 [range, 37‒476] days) of transplanted mice than control mice. Hematologic, immunophenotypic, and pathologic analyses of 132 tumors revealed that these malignancies consisted of B- and T-cell lymphomas (17% and 20%), B- and T-ALL/LBL (14% and 15%) and myeloid malignancy (14%) according to Bethesda criteria, although the diagnosis of 22 (17%) tumors could not be determined. sgRNA sequencing of 109 tumors showed that at least 1 sgRNA-targeted genes were detected in almost all (>99%) tumors, with a median of 3 (range, 1‒10) genes per tumor. Enrichment analysis of sgRNA-targeted genes demonstrated significant overrepresentation of 13 genes, such as Trp53 and Irf2bp2, pointing to stronger oncogenic capacity of these driver genes. These sgRNA-targeted genes showed lineage and tissue specificity, such as Pax5, Kmt2d, Atm, and S1pr2 for B-cell lineage and Zc3h12a and Socs1 for T-cell lineage, while only weak enrichment was observed in myeloid neoplasms. These tumors showed characteristic disease phenotype according to sgRNA-targeted genes: Pax5-targeted tumors were usually B-ALL/LBL, whereas Kmt2d-targeted tumors developed as B-cell lymphomas with earlier onset than those harboring other targeted genes. All of Zc3h12a-targeted tumors were T-cell lymphoma with CD4+CD8- immunophenotype and low clonality, while Socs1-targeted tumors primarily developed as T-ALL/LBL with CD4+CD8+ immunophenotype and frequent thymus involvement. Whole-exome sequencing (n = 108) not only confirmed targeted disruption by sgRNAs but also identified additional driver alterations in 76% and 40% of B-cell and T-cell neoplasms, respectively, suggesting the necessity of second-hit alterations in these tumors. These additional driver alterations were associated with a specific lineage. Frequent C-terminal truncating mutations of Notch1 and loss-of-function mutations of Ikzf1 were found in T-cell malignancies, while Jak1 and Jak3 hotspot missense mutations and Flt3 internal tandem duplications were common in B-cell malignancies, suggesting cooperative relationship between specific alterations and their association with lineage. In addition, hierarchical clustering of RNA-sequencing data (n = 105) identified several clusters associated with disease phenotypes and sgRNA-targeted genes. These clusters showed distinct expression signatures, including germinal center B-cell-like signatures in Kmt2d-targeted tumors, MYC target signatures in Pax5-targeted tumors, and inflammation-related signature in Zc3h12a-targeted tumors. In summary, our in vivo CRISPR loss-of-function screening enables high-throughput evaluation of the tumorigenic potential of many genetic abnormalities, their cooperability and association with lineage in lymphoid malignancies. Such approach accelerates understanding of genotype-phenotype association and discovery of potential therapeutic targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cancer-Related Gene Mutations Drive Resistance to the Third-Generation Tyrosine Kinase Inhibitor Therapy in Chronic Myeloid Leukaemia

Objectives Interrogate the dynamic evolution of cancer-related gene mutations in subjects with chronic myeloid leukaemia (CML) receiving the third generation- (3G-) tyrosine kinase inhibitor (TKI) therapy, and define those mediating resistance. Methods 176 consecutive subjects with chronic or accelerated phase CML failing imatinib and/or the second generation- (2G-) TKI and receiving a 3G-TKI (ponatinib or olverembatinib) were studied. Deep targeted sequencing was used to screen cancer-related gene mutations and Sanger sequencing for ABL mutations. Results 176 subjects in chronic (n = 136) or accelerated (n = 41) phases received ponatinib (n = 147) or olverembatinib (n = 29) therapy. 19 subjects were exposed to imatinib priorly; 16, the 2G-TKI; 141, imatinib and the 2G-TKI. 142 (81%) had ABL mutations including ABLT315I (n = 87, 61%), ABLT315I+additional (n = 30, 21%) or ABLothers (n = 25, 18%) at baseline. 157 (89%) subjects had other cancer-related gene mutations including ASXL1 (n = 121, 69%), KMT2D (n = 14, 8%), RUNX1 (n = 12, 7%), PHF6 (n = 9, 5%), KMT2C (n = 8, 5%), IKZF1 (n = 8, 5%), STAT5A (n = 8, 5%), TET2 (n = 8, 5%), DNMT3A (n = 7, 4%) and KDM6A (n = 7, 4%) and so on at baseline. In multivariable analyses, ASXL1G646Wfs*12 and PHF6 mutations were significantly-associated with the poor cytogenetic and molecular responses; RUNX1 and PHF6 mutations with poor progression-free survival; IKZF1 and STAT5A mutations with poor survival. ABLT315I mutation was significantly-associated with good responses. Follow-up analyses were done in 105 subjects. According to the therapy response of 3G-TKI, 51 subjects were classified into responsive cohort; 54 subjects, resistant cohort including primary (n = 44) and secondary (n = 10) resistance. In the responsive cohort, only 1 subject retained their ABL mutation and none developed a new ABL mutation (Figure 1B). In the resistant cohort, 6 subjects developed new ABL mutations including E255K/V (n = 2), F359V/I (n = 2), G250E and T315M (Figure 1C). There was a significantly-higher proportion of subjects with ABL mutations in the resistant cohort compared with the responsive cohort (52% vs. 2%, p < 0.001) despite similar ABL mutation status at baseline. As for other cancer-related gene mutations, 92 (88%) had other cancer-related gene mutations with a median number of 2 (range, 0 - 7) at baseline. Compared with the responsive cohort, the detected frequency and the median number of mutations in the resistant cohort were significantly-higher both at the baseline (p < 0.001 - 0.027) and at the end of observation period (p < 0.001 - 0.002) (Figure 2&3). More subjects developed new mutations in the resistant cohort than those in the responsive cohort (74% vs. 39%, p < 0.001, Figure 2&3). As the most frequently detected of ASXL1 mutation, despite similar frequency of ASXL1 mutation between the responsive and resistant cohorts at baseline (p = 0.68), more subjects in the resistant cohort had ASXL1G646Wfs*12 mutation than those in the responsive cohort (p = 0.03), while similar frequency of ASXL1non-G646Wfs*12 mutations. At the end of observation, 52 (50%) subjects (including 40 in the resistant cohort and 12 in the responsive cohort) had the ASXL1 mutation. In subjects with ASXL1 mutation, 21 subjects were detected to the ASXL1G646Wfs*12 mutation, with only 1 (2%) subject in the responsive cohort, 20 (37%) in the resistant cohort (p < 0.001). Frequencies of ASXL1non-G646Wfs*12 were similar. In addition, the variant allele frequencies (VAFs) of ASXL1G646Wfs*12 and ASXL1non-G646Wfs*12 mutations were significantly-decreased during 3G-TKI therapy in the responsive cohort (p < 0.001 - 0.002, Figure 4) but not in the resistant cohort. There were no significant differences in the primary resistance cohort during the 3G-TKI therapy. However, the VAFs of ASXL1G646Wfs*12 mutation significantly-decreased when in the temporarily response (p = 0.05), while significantly-increased when the secondary resistance occurred (p = 0.015, Figure 4A) in the secondary resistant cohort. In contrast, the VAFs of ASXL1non-G646Wfs*12 mutation presented the decreased trend when in both temporarily response and secondary resistance (Figure 4B). The patterns of the dynamic evolution in RUNX1, IKZF1, SETBP1 and PHF6 mutations were similar as those in ASXL1 G646Wfs*12 mutation. ConclusionsASXL1G646Wfs*12, RUNX1, IKZF1, SETBP1 and PHF6 mutations are the key drivers of resistance to 3G-TKI therapy in CML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MAP4K2 Inhibition Reinforces the Iberdomide Sensitivity in MM Cells By Inducing IKZF1 Degradation through a CRBN Independent Mechanism

MAP4K2 is predominantly and highly expressed in the germinal center of B cells, also called Germinal Center Kinase (GCK). Recently we have shown that MAP4K2 knockdown in K- or N-RAS mutated MM cells induces MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins. IKZF1 is a critical transcriptional factor regulating IRF4 and c-MYC transcription in MM, therefore we expect that IKZF1 degradation is one of the major mechanisms that contribute to the inhibition of MAP4K2 induced MM cell death. To address the role of IKZF1 degradation in MAP4K2 inhibition induced cytotoxicity in MM, we generated a mutant IKZF1 by substituting Glutamine Q146 to Histidine, which abrogates IKZF1 ubiquitination induced by CRBN. MM.1S cells were transduced with lentiviral constructs to overexpress flag-IKZF1WT or flag-IKZF1Q146H. Lenalidomide failed to induce a complete IKZF1-degradation in IKZF1Q146H MM.1S cells, indicating IMiDs induced IKZF1-degradation is mediated by CRBN binding and subsequent ubiquitination. In contrast, IKZF1 mutation did not rescue MM.1S from MAP4K2 inhibitor (TL4-12) induced IKZF1 downregulation. Proliferation assays further confirmed that IKZF1Q146H MM.1S cells were resistant to LEN induced cell growth inhibition, but not MAP4K2 inhibitor, demonstrating that MAP4K2 inhibition causes the degradation of IKZF1 and cell growth inhibition through a mechanism different from that of lenalidomide in myeloma cells. We further evaluated the effects of MAP4K2 silencing in IKZF1Q146H MM.1S cells. MAP4K2 knockdown resulted in significant inhibition of cell proliferation and increase of apoptotic cells in both cell lines (flag-IKZF1WT or flag-IKZF1Q146H MM.1S). Flag-IKZF1WT or flag-IKZF1Q146H were also overexpressed in IMiDs resistant (RPMI-8266) MM cell line by lentiviral infection, and no differences of MAP4K2 knockdown induced change of IKZF1 levels, cell proliferation, or apoptosis were observed between two cell lines. These data suggest that MAP4K2 inhibition induces anti-MM effects by targeting IKZF1 via an CRBN independent mechanism and subsequently overcomes IMiDs resistance. Our recent work also showed that the combination of iberdomide and MAP4K2 silencing led to synergetic anti-MM effects in vitro. In previous biochemical and structural studies, it was demonstrated that iberdomide binds to cereblon with a higher affinity than lenalidomide or pomalidomide. To investigate the combined effects of Iberdomide with MAP4K2 silencing on MM tumor growth, we generated subcutaneous MM xenografts in non-obese diabetic severe combined immunodeficient (NOD/SCID) mice using the inducible MAP4K2 shRNA MM.1S cells. Combination of Iberdomide with MAP4K2 silencing further enhanced tumor inhibition compared to the single treatment. Furthermore, we found that combination treatment significantly prolonged mice survival, compared to the single treatment. Taken together, our findings demonstrate that MAP4K2 is a novel therapeutic target to bypass IMiDs resistance in RAS mutated MM. Combination of MAP4K2 inhibition with Iberdomide results in synergetic anti-cancer effects in MM, therefore will provide a novel strategy to manage the relapsed or refractory patients with multi-drug resistance.

Mutations of TP53 Gene in Acute Lymphoblastic Leukemia Do Not Affect the Survival Outcomes after Haploidentical Stem Cell Transplantation

Introduction: Mutations and deletions in TP53 are found in all hematological malignancies, Whereas TP53 mutations (TP53mut) were described to occur quite frequently in acute lymphocytic leukemia (ALL). Previous studies have demonstrated that TP53 mutation was associated with poor prognosis in ALL. Allogeneic hematopoietic stem cell transplantation (HSCT) represents an effective therapy for reducing the chances of relapse of ALL. Recent medical progress has led to haploidentical-SCT (haplo-SCT) achieving transplant outcomes similar to those with matched-sibling donor SCT and matched-unrelated donor SCT in patients with hematological malignancies, haplo-SCT provided an alternative treatment for ALL patients for whom identical donors are unavailable. However, the impact of TP53mut in ALL on survival outcomes in haplo-HSCT setting remains unknown. This study aims to investigate the effect of TP53 mutations on survival outcomes of ALL treated with haplo-SCT at a single institution. Methods: 78 subjects with available TP53 status at diagnosis were retrospectively considered. We excluded 11 subjects who received HLA-matched related transplantation and 2 subjects with HLA-matched unrelated donor transplantation. The remaining 65 subjects with haplo-HSCT were enrolled. TP53 mutation analysis was assessed on bone marrow (BM) samples. LFS and OS were estimated using Kaplan and Meier method and tested between groups using a log-rank test. Cumulative incidence of relapse (CIR) was treated as a competing risk for non-relapse mortality (NRM). Results: A cohort of 65 patients was included with a median(range) age of 30 years (14-51) and was followed up for a median duration of 602 days (range;42-2017 days). 60% of patients were male. 24 of 65 patients showed TP53 mutations. The baseline characteristics including sex, age, white blood cell, diagnosis, Ph/Ph-like chromosomes, IKZF1 mutation, donor-recipient gender, donor-recipient relation, ABO mismatched, CR status before SCT, pre-MRD status were similar between the two groups. 7 (10.8%) patients suffered platelet engraftment failure, the cumulative incidence of grade II-IV aGVHD or cGVHD was comparable between the two groups (TP53mutvs TP53wt:38.8% vs 30.2%, P=0.80; 69.0% vs 63.4%, P=0.98). The 2-year cumulative of CIR rates were 13.1% and 12.5% (P=0.96) among TP53mut and TP53wt patients. The 2-year LFS rates of TP53mut patients were similar to patients with TP53wt (74.2% vs 77.4%, P=0.80). No significant differences in 2-year OS rates (82.9% vs 87.3%, P=0.61) or 2-year NRM rates (12.7% VS 10.2%, P=0.69) were observed in TP53mut and TP53wt patients. TP53 played no prognostic role in multivariate analysis. WBC count at diagnosis (>50x109/L: HR=3.860, P=0.016) and age (>40 years old: HR=4.120, P=0.012) were independent risk factors associated with 2-year LFS, additionally, cGVHD showed protective effect for 2-year LFS in univariate analysis (P= 0.06), but significant was not confirmed in a multivariate analysis. WBC count at diagnosis (>50 x109/L: HR=5.483, P=0.042) was the only independent risk factor of 2-year CIR. To assess the prognostic impact of TP53mut in a homogeneous population, we reanalyzed patients with B-cell immunophenotype. There was no significant difference in survival outcomes between TP53mut and TP53wt groups when only B-ALL were assessed (TP53mutvs TP53wt: 2-year OS: 82.2% vs 93.0%, P=0.23; 2-year LFS: 73.0% VS 82.7%, P=0.44; 2-year NRM:13.3% vs 7.1%, P=0.40; 2-year CIR:13.7% vs 10.2%, P=0.81). Conclusion: Our study showed that TP53 mutations may not be related to the unfavorable impact on survival in ALL patients after treatment with haplo-HSCT. The results imply that haplo-HSCT may be a viable treatment for this subgroup of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cup-like blasts in an adult patient with BCR-ABL1 positive B-cell acute lymphoblastic leukemia.

A 68-year-old female was presented to the hospital with complainment of asthenicus for 2 months. Initial hematological results showed leukocyte of 80.26 × 109/L, hemoglobin of 134 g/L, and platelet of 401 × 109/L. Peripheral blood smear revealed 80% blast cells with small or medium size, and 27% blast cells with frequent cup-like nuclei (Figure 1A). Further bone marrow smear revealed hypercellular with 83% small- to medium-sized blast cells, and 10% blast cells with cup-like nuclei (Figure 1B). Myeloperoxidase (MPO) and nonspecific esterase were all negative. Further bone marrow immunophenotyping by flow cytometry were positive for CD34, CD19, HLA-DR, CD22, CD10, CD58, and cyCD79a on blast cells, but other myeloid and T-cell phenotype were negative. Then this patient was diagnosed with acute B-cell lymphoblastic leukemia (B-ALL). Furthermore, BCR-ABL1 p190 fusion gene was found in this patient, and 32 common lymphoid leukemia genes were sequenced and result showed the IKZF1 mutation (exon5, c.4.88A>G, p.His163Arg). Blasts with cup-like morphology are well recognized in acute myeloid leukemia (AML), being frequently associated with a normal karyotype and mutated NPM1 and/or FLT3-ITD. Recently, a novel association between cup-like nuclei and IKZF1 deletion in pediatric B-ALL had reported [1]. And these pediatric cup-like nuclei cases are more likely to have an early B-precursor phenotype, normal karyotype, or nonrecurrent cytogenetic abnormalities and are less likely to have favorable cytogenetic abnormalities [1]. Herein, we describe an unusual adult case of BCR-ABL1- p190 positive B-ALL with cup-like morphology. Whether this morphology of adult B-ALL patient is related to the BCR-ABL1 p190 and/or IKZF1 mutations needs further investigation. The authors would like to thank the encouragement and support of the members who take advanced study in the unit of cytology of the Department of Clinical Laboratory, Zhejiang Provincial People's Hospital. All authors reviewed this manuscript and agreed to submit this manuscript. The authors declare that they have no conflict of interest. Zhenni Wang and Xing Jin provide the picture and clinical data. Jinlin Liu analyzed the data and wrote the manuscript. This study has approved by the ethical committee of Zhejiang Provincial People's Hospital. This study did not involve the personal information, only report the laboratory data. Then the patient consent was waived, and this waive was approved by the ethical committee of Zhejiang Provincial People's Hospital. The authors received no funding for this article. Data are available on request from the authors.

P1408: MONOALLELIC AND BIALLELIC GERMLINE MUTATIONS AFFECTING THE TRANSCRIPTION FACTOR HELIOS CAUSE PLEIOTROPIC DEFECTS OF IMMUNITY

Background: Helios, encoded by IKZF2, is a member of the Ikaros family of transcription factors with pivotal roles in T-follicular helper, NK- and T-regulatory cell physiology. Somatic IKZF2 mutations are frequently found in lymphoid malignancies. Aims: Although germline mutations in IKZF1 and IKZF3, encoding Ikaros and Aiolos, have recently been identified in patients with phenotypically similar immunodeficiency syndromes, the effect of germline mutations in IKZF2 on human hematopoiesis and immunity remains to be elucidated. Methods: We performed whole exome sequencing in six patients with immune dysregulation and their family members. We then applied Co-immunoprecipitation, electrophoretic mobility shift assay and immunofluorescence microscopy techniques to assess dimerization and DNA binding capabilities of the mutations. Proteomic analysis by proximity-dependent Biotin Identification (BioID) was used to assess affinity of mutant Helios to its interaction partners, while single-cell RNA sequencing was applied to peripheral blood mononuclear cells (PBMCs) of two patients to study the transcriptional effect of the truncating and biallelic IKZF2 mutations. Results: We identified six patients with monoallelic germline IKZF2 mutations (one nonsense (p.R291X)- and 4 distinct missense variants) and one patient with a biallelic germline IKZF2 mutation (p.I325V). While patients with heterozygous variants were affected by immune dysregulation (systemic lupus erythematosus, immune thrombocytopenia or EBV-associated hemophagocytic lymphohistiocytosis), the patient with the homozygous variant suffered from recurrent respiratory tract infections complicated by bronchiectasis. Immunophenotype analysis revealed reduced propensities of NK cells, CD4+ T-cells with an inverted CD4/CD8 ratio, T-follicular helper cells and hypogammaglobulinemia across patients, while the p.I325V patient also had a reduction in relative and absolute B-cells and low relative proportions of Treg, MAIT and iNKT populations. Single-cell RNA sequencing of PBMCs from the patients carrying the monoallelic R291X truncating variant or the biallelic I325V variant revealed upregulation of pro-inflammatory genes associated with T-cell receptor activation and T-cell exhaustion. Our biochemical and proteomic analysis has revealed that only the truncating R291X mutation results in loss of Helios DNA binding and dimerization properties. However, BioID analysis revealed aberrant interaction of 4/6 Helios mutants with core components of the Nucleosome Remodeling histone Deacetylase (NuRD) complex conveying Helios-mediated epigenetic and transcriptional dysregulation. Summary/Conclusion: Thus, germline mutations in IKZF2 cause a novel syndrome associating immunodeficiency and profound immune dysregulation and disrupt interaction with the NuRD complex.

Clinical and Genetic Characteristics of IKZF1 Mutation in Chinese Children With B-Cell Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a malignancy associated with altered lymphoid precursor hyperplasia and accompanied with different genetic mutations. Few studies have been reported on the association between gene mutations and clinical features of IKZF1 mutation in children with B-cell ALL (B-ALL). We investigated clinical and genetic characteristics in 200 newly diagnosed pediatric B-ALL through multiplex ligation-dependent probe amplification (MLPA) and targeted next-generation sequencing (NGS) method. We found that IKZF1 mutations, including large segment deletions, small insertions or deletions (InDels) and single nucleotide variations (SNVs), were detected in 22 patients with a positive mutation rate of 11.0%. IKZF1 mutation was significantly associated with higher WBC count (19.38 × 109/L vs. 5.80 × 109/L, p = 0.002). Compared with IKZF1 wild-type cases, a higher frequency of IL7R gene mutation was discovered in IKZF1 mutant cases (9.1% vs. 0.0%, p = 0.012). Patients with IKZF1 mutation were less sensitive to glucocorticoid induction than patients without IKZF1 mutation (63.6% vs. 9.0%, p < 0.001). On the 15th day of induction, minimal residual disease (MRD) > 10−3 level were higher in IKZF1 mutant patients than wild-type patients (45.5% vs. 22.3%, p = 0.018). In conclusion, our study reveals the association between genetic mutations and clinical features in Chinese children with B-ALL, which might contribute to molecular classification, risk stratification and prognosis evaluation, and provide new ideas for targeted therapy in ALL.

Genomic heterogeneity contributed to different prognosis between adult and pediatric acute lymphoblastic.

The prognosis of acute lymphoblastic leukemia (ALL) in adults is inferior to that in children. Hence, ALL remains challenging to cure in the adult population. Aberrant genetic alterations have been observed in ALL, although the patterns of differential gene alterations in adult and pediatric ALL have not been comprehensively determined on a genome-wide scale. We investigated the biologic differences in genomic profiles between adults (n=64) and children (n=54) with ALL and relationship between genomic heterogeneity and prognosis. The 2 populations showed similar common mutation types but an increased prevalence of genetic alterations in adult ALL. The median numbers of gene mutations were 17 (range: 1-53) and 4.5 (range: 1-19) per sample in adult and pediatric ALL, respectively (p<0.001). An increased number of gene mutations and age were significantly correlated (R2 =0.5853, p<0.001). We identified 122 and 53 driver genes in adult and pediatric ALL samples, respectively. IKZF1, IDH1, and TTN mutations were significantly enriched in adult patients with ALL. KRAS, ARID1A, and CREBBP mutations were significantly enriched in pediatric patients with ALL (p<0.05). The incidence of relapse was 40.0% and 9.6% in adult and pediatric patients with ALL, respectively (p=0.003). The overall survival and relapse-free survival of adult patients with ALL were poorer than those of pediatric patients with ALL (p=0.002 and p<0.001, respectively). This genomic landscape enhances the understanding of the biologic differences in ALL between the 2 populations and provides insight for developing therapeutic approaches.