Loss Of Ikaros Research Articles

IKZF1 Deletions Are Markers of Treatment Resistance in Adult Ph-Negative B-Cell Acute Lymphoblastic Leukemia Patients Treated within the Ongoing Risk-Adapted Pethema LAL19 Trial

Introduction IKZF1 (Ikaros) deletion has been related with poor outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL). However, its impact in adult patients (pts) treated with MRD-oriented trials has not been fully established. This study aimed to assess if IKZF1 deletions are markers of poor prognosis in adults with Ph-negative BCP ALL treated within the ongoing risk-adapted PETHEMA LAL19 trial (NCT04179929). The impact of IKZF1 loss in these pts and those from the EWALL Group (European Working group on ALL) is being evaluated in a HARMONY (Healthcare Alliance for Resourceful Medicine Offensive against Neoplasms in Hematology) project (Project Ref ALL2). Methods In the PETHEMA LAL19 trial, pts with poor end-induction MRD clearance (MRD>0.01%) or high-risk genetics (low-hypodiploid, KMT2Ar, TP53 biallelic alteration or IKZF1+CDKN2A/B deletion) are assigned to allogeneic stem cell transplantation, the remaining pts receive maintenance chemotherapy. All pts are analyzed by G-banding, FISH, NGS (DNA gene panel) and SNP array. Next generation flow cytometry is used for MRD assessment. Main outcome measures: i) Complete Remission (CR): absence of extramedullary disease, neutrophil level >1x109/L, platelet level >100x109/L, and bone marrow blast level < 5%; ii) Cumulative Incidence of Relapse (CIR) and non-relapse mortality (NRM): calculated using cumulative incidence rates to accommodate competing risks and compared by Gray's test; iii) Overall survival (OS): measured from the time of protocol entry to the time of death or last contact. Results 147pts were analyzed. Mean age 40y [range 18-60], 56% males, 10% CNS involvement, 78% common B, 15% pro-B and 7% pre-B ALL. Ph-like (12%) was the most common genetic subtype followed by KMT2Ar (11%), PAX5 P80R (8%), low-hypodiploid (7%), TCF3::PBX1 and high-hyperdiploid (6% each), iAMP21 and ETV6::RUNX1 (2% each). More uncommon biological entities such as ZNF384r, MEF2Dr, MYCr, IDH1 R132C and IDH2 R140Q were seen in single pts (<1% each).The remaining patients (41%) were B-other,no growth or normal karyotype. IKZF1 deletion (IK DEL) was observed in 66/147(45%) pts. Deletions involved the whole gene (37/66, 56%) or a subset of exons (partial gene deletions). The most recurrent partial gene deletions (n=29) were: loss of exons 4-7 (n=6, 21%), 4-8 (n=5, 17%), 2-8 and 1-7 (n=4, 14% each), 1-3 (n=3, 10%), 2&3 and 2-7 (n=2, 7% each). The IKZF1plus profile was observed in 37/147 (25%) pts. IK DEL were more frequent in low-hypodiploid (100%) and Ph-like ALL (88%), and less prevalent in KMT2Ar (19%), PAX5 P80R (18%) and TCF3::PBX1 ALL (11%), p<0.001. Median follow up was 11.4 months. IKDEL pts showed lower CR rate than pts without Ikaros loss (75% vs. 91%, p=0.027). The IKZF1plus signature showed similar CR rate to IK DEL without IKZF1plus profile (IKZF1not plus) (73% vs. 78%, p=ns). IKDEL pts showed poorer MRD clearance than WT individuals. Interestingly, this fact was mainly attributable to partial gene deletions (MRD≥0.01% partial 79% vs. whole 42% vs. WT 29%, p=0.003). IKZF1plus was also correlated with positive MRD, however IKZF1not plus pts showed higher frequency of MRD positivity than IKZF1plus pts (MRD≥0.01% plus 47% vs. not plus 71% vs. WT 29%, p=0.012). Pts with whole gene loss showed lower OS than pts with partial gene losses or IKZF1 WT (1y-OS 62% vs. 88% vs. 89%, p=0.012)(Figure 1A). Of note, relapse was not the main cause of death of IK DEL whole gene pts (n=26) as only 1 patient died due to disease progression. Instead, NRM was the main cause of death in whole gene-deleted pts (4 died in Ind-1, 3 in Ind-2, 1 due to TRM and 1 due to Covid19 infection). Accordingly, the 1y-CIR of IK DEL whole pts was lower than that of IKZF1 WT individuals, who showed similar CIR to IK DEL partial ones (1y-CIR whole 7% vs. WT 30% vs. partial 32%, p=0.059) (Figure 1B). Pts with IKZF1plus signature did not show differences in OS and CIR compared to IKZF1 WT or IKZF1not plus individuals (data not shown). Conclusions This preliminary data suggest that IK DEL may be marker of poor treatment response in adult Ph-negative BCP ALL. Further analyses including all patients of the HARMONY project may identify which isoforms or in which genetic ALL subtypes IKZF1 DEL are particularly deleterious. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting signaling pathways vulnerabilities for the treatment of IKZF1-deleted ph-negative B lymphoblastic leukemia.

7033 Background: Approximately 20% of children diagnosed with B lymphoblastic leukemia (B-ALL) will suffer a relapse, and most adults with B-ALL have a poor prognosis. Genome-wide association studies of B-ALL patients have identified frequent deletions of the gene IKZF1, encoding the master regulator of lymphoid development, IKAROS. These deletions are associated with therapy resistance, increased risk of relapse, and inferior survival. Currently, how loss of IKAROS function contributes to therapy resistance and increased risk of relapse is not fully understood. We used CRISPR-Cas9 genome editing to develop human B-ALL cell lines with various IKZF1 deletions that genetically and phenotypically recapitulate those occurring in patients. Using these isogenic cell lines, we have previously shown IKZF1 deletion results in cell-intrinsic chemoresistance and increased activation of the JAK/STAT signaling pathway (Rogers, Gupta et al. 2021). Methods: Given JAK/STAT is often dysregulated in poor-prognosis leukemia, we investigated the potential mechanisms of aberrant JAK/STAT activation and the therapeutic potential of targeting JAK/STAT in our engineered cell lines. We treated our cells with SH-4-54 (STAT3/5 inhibitor) or tofacitinib (JAK1 inhibitor) alone and in combination with dexamethasone. To elucidate how loss of IKAROS mediates an increase in JAK-STAT activity, we also performed RNAseq of known pathway regulators, comparing IKZF1 wild-type with IKZF1 knockout. Results: The JAK/STAT negative regulator Suppressor of Cytokine Signaling 2 (SOCS2) was significantly downregulated with IKZF1 deletion, validated by RTqPCR and immunoblotting. We further analyzed publicly-available RNAseq data from &gt; 650 pediatric B-ALL samples, finding that SOCS2 expression is significantly lower in patients with low IKZF1expression (likely corresponding to IKZF1 deletion) compared to those with high IKZF1 expression. When we treated our engineered cell lines with tofacitinib or SH-4-54, IKZF1 wild-type cells were sensitive to each compound, suggesting JAK/STAT signaling plays a vital role in cell survival. In contrast, the IKZF1-deleted cells were relatively resistant to JAK/STAT inhibitors alone. However, in combination with dexamethasone, treatment of cells with sub-IC50 levels of SH-4-54 or tofacitinib resulted in re-sensitization to glucocorticoid-induced apoptosis. Conclusions: Our findings support that IKZF1 deletion leads to a targetable upregulation of the JAK/STAT pathway that, when inhibited, results in relative re-sensitization to dexamethasone. JAK/STAT pathway upregulation in IKZF1 deleted cells may be mediated by decreased expression of SOCS2. These results provide initial promise for targeting these vulnerabilities for the treatment of this poor-prognosis disease.

Modeling IKZF1 lesions in B-ALL reveals distinct chemosensitivity patterns and potential therapeutic vulnerabilities

AbstractIKAROS family zinc finger 1 (IKZF1) alterations represent a diverse group of genetic lesions that are associated with an increased risk of relapse in B-cell acute lymphoblastic leukemia. Due to the heterogeneity of concomitant lesions, it remains unclear how IKZF1 abnormalities directly affect cell function and therapy resistance, and whether their consideration as a prognostic indicator is valuable in improving outcome. CRISPR/Cas9 strategies were used to engineer multiple panels of isogeneic lymphoid leukemia cell lines with a spectrum of IKZF1 lesions to measure changes in chemosensitivity, gene expression, cell cycle, and in vivo engraftment that can be linked to loss of IKAROS protein. IKZF1 knockout and heterozygous null cells displayed relative resistance to a number of common therapies for B-cell acute lymphoblastic leukemia, including dexamethasone, asparaginase, and daunorubicin. Transcription profiling revealed a stem/myeloid cell–like phenotype and JAK/STAT upregulation after IKAROS loss. A CRISPR homology-directed repair strategy was also used to knock-in the dominant-negative IK6 isoform into the endogenous locus, and a similar drug resistance profile, with the exception of retained dexamethasone sensitivity, was observed. Interestingly, IKZF1 knockout and IK6 knock-in cells both have significantly increased sensitivity to cytarabine, likely owing to marked downregulation of SAMHD1 after IKZF1 knockout. Both types of IKZF1 lesions decreased the survival time of xenograft mice, with higher numbers of circulating blasts and increased organ infiltration. Given these findings, exact specification of IKZF1 status in patients may be a beneficial addition to risk stratification and could inform therapy.

The Immunomodulatory Drugs, through Loss of Ikaros and Aiolos, Derepress CD38 and Functionally Synergise with Daratumumab

The transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) have recently been found to be targeted for destruction by the immunomodulatory drugs (IMiDs). However, the role of Ikaros and Aiolos and why their loss leads to multiple myeloma (MM) cell death remains unclear. We have used CRISPR-Cas9 genome editing to delete IKZF1 and IKZF3 in human MM cell lines to gain further insight into their downstream gene regulatory networks.Consistent with the action of the IMiDs, loss of either of these genes resulted in both a G1/S cell cycle arrest and induction of apoptosis. This was not dependent on the subsequent reduction of the IRF4-MYC “axis”, as neither IRF4 or MYC were consistently downregulated across all cell lines examined (despite cell death occurring) and overexpression of either factor failed to rescue for Ikaros loss. This led us to further investigate the transcriptional changes resulting from deletion of Ikaros, Aiolos or treatment with the IMiD lenalidomide using RNA-sequencing.Supporting their central role in the action of the IMiDs, significant overlap was seen on loss of Ikaros, Aiolos, or lenalidomide treatment; with loss of Ikaros/Aiolos accounting for approximately 75% of differential gene expression following lenalidomide.Importantly both Ikaros and Aiolos were found to repress the expression of interferon stimulated genes (ISGs) and their loss led to the activation of an interferon-like response, possibly contributing to cell death. In keeping with this, treatment with lenalidomide and low dose IFNb resulted in synergistic cell death. Furthermore, CD38 appeared to be both an ISG and repressed target of Ikaros/Aiolos through their interaction with the nucleosome remodelling and deacetylase complex, and its expression is increased at both mRNA and protein level on their loss. Given recent clinical studies have shown improved outcomes with combination treatment with lenalidomide and the anti-CD38 monoclonal antibody daratumumab, we wondered whether this represented a MM cell intrinsic mechanism explaining this synergy. Consistent with this hypothesis, loss of Ikaros, or treatment with lenalidomide or IFNb led to increased daratumumab induced NK cell mediated antibody-dependent cellular cytotoxicity.These results give further insight into the mechanism of action of the IMiDs, and provide mechanistic rationale for combination with anti-CD38 monoclonal antibodies. DisclosuresFedele:Celgene: Other: Travel grant to attend ASH. Low:Merck: Honoraria.

T-ALL can evolve to oncogene independence.

The majority of cases of T-cell acute lymphoblastic leukemia (T-ALL) contain chromosomal abnormalities that drive overexpression of oncogenic transcription factors. However, whether these initiating oncogenes are required for leukemia maintenance is poorly understood. To address this, we developed a tetracycline-regulated mouse model of T-ALL driven by the oncogenic transcription factor Lmo2. This revealed that whilst thymus-resident pre-Leukemic Stem Cells (pre-LSCs) required continuous Lmo2 expression, the majority of leukemias relapsed despite Lmo2 withdrawal. Relapse was associated with a mature phenotype and frequent mutation or loss of tumor suppressor genes including Ikzf1 (Ikaros), with targeted deletion Ikzf1 being sufficient to transform Lmo2-dependent leukemias to Lmo2-independence. Moreover, we found that the related transcription factor TAL1 was dispensable in several human T-ALL cell lines that contain SIL-TAL1 chromosomal deletions driving its overexpression, indicating that evolution to oncogene independence can also occur in human T-ALL. Together these results indicate an evolution of oncogene addiction in murine and human T-ALL and show that loss of Ikaros is a mechanism that can promote self-renewal of T-ALL lymphoblasts in the absence of an initiating oncogenic transcription factor.

Transcriptional circuits in B cell transformation.

Loss of IKAROS in committed B cell precursors causes a block in differentiation while at the same time augments aberrant cellular properties, such as bone marrow stromal adhesion, self-renewal and resistance to glucocorticoid-mediated cell death. B cell acute lymphoblastic leukaemias originating from these early stages of B cell differentiation and associated with IKAROS mutations share a high-risk cellular phenotype suggesting that deregulation of IKAROS-based mechanisms cause a highly malignant disease process. Recent studies show that IKAROS is critical for the activity of super-enhancers at genes required for pre-B cell receptor (BCR) signalling and differentiation, working either downstream of or in parallel with B cell master regulators such as EBF1 and PAX5. IKAROS also directly represses a cryptic regulatory network of transcription factors prevalent in mesenchymal and epithelial precursors that includes YAP1, TEAD1/2, LHX2 and LMO2, and their targets, which are not normally expressed in lymphocytes. IKAROS prevents not only expression of these 'extra-lineage' transcription factors but also their cooperation with endogenous B cell master regulators, such as EBF1 and PAX5, leading to the formation of a de novo for lymphocytes super-enhancer network. IKAROS coordinates with the Polycomb repression complex (PRC2) to provide stable repression of associated genes during B cell development. However, induction of regulatory factors normally repressed by IKAROS starts a feed-forward loop that activates de-novo enhancers and elevates them to super-enhancer status, thereby diminishing PRC2 repression and awakening aberrant epithelial-like cell properties in B cell precursors. Insight into IKAROS-based transcriptional circuits not only sets new paradigms for cell differentiation but also provides new approaches for classifying and treating high-risk human B-ALL that originates from these early stages of B cell differentiation.

Superenhancer reprogramming drives a B-cell-epithelial transition and high-risk leukemia.

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem-epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD-YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem-epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem-epithelial-B-cell phenotype that underlies high-risk B-ALL.

Pomalidomide Transcriptionally Reprograms Adult Erythroid Progenitors Independently of Ikaros Proteasomal Degradation

Pomalidomide, a second-generation immunomodulatory drug, is a fetal hemoglobin (HbF) inducing agent with potential implications for the treatment of β-hemoglobinopathies such as sickle cell disease (SCD). However, its mechanism of action remains unknown. Through an in-depth characterization of human erythropoiesis and globin gene regulatory networks, we now provide evidence that pomalidomide alters transcription networks involved in erythropoiesis and globin switching, thereby leading to a partial reprogramming of adult hematopoietic progenitors toward fetal-like erythropoiesis. Adult peripheral blood CD34+ cells from normal individuals were differentiated toward the red cell lineage using an adapted 3-phase culture system. At day 14 of culture, we observed a reciprocal globin gene switch at the mRNA and protein levels. These results were confirmed by high performance liquid chromatography of hemolysates (HbF/(HbF+HbA): 31.7 ± 1.4% vs. 6.5 ± 0.7% pomalidomide and vehicle, respectively). Next, we studied erythroid differentiation using flow cytometric analyses of the cell surface markers interleukin-3R (IL-3R), glycophorin A (GPA), CD34 and CD36 for early erythroid precursors (BFU-E and CFU-E) as well as GPA, α4-integrin and band3 for terminal erythroid differentiation. While there were no changes in terminal erythroblast maturation, an accumulation of BFU-E in pomalidomide-treated cultures at days 2 and 4 of differentiation was seen, indicating a delay at the BFU-E to CFU-E transition, and also, that pomalidomide exerts its effect in the early-stages of erythropoiesis. Indeed, treatment with pomalidomide during the phase of the culture system that generates erythroid progenitors led to significantly more γ-globin expression than treatment during the phase which proerythroblasts undergo terminal erythroid differentiation. At the molecular level, pomalidomide was found to rapidly and robustly decrease Ikaros (IKZF1) expression exclusively by post-translational targeting to the proteasome. Moreover, pomalidomide selectively reduced the expression of components of key globin regulatory pathways including BCL11A, SOX6, KLF1, GATA1 and LSD1 while not affecting others (e.g. CoREST, GATA2, GFI1B, and HDAC1). Pomalidomide had a transient effect on GATA1 and KLF1 expression. While shRNA knockdown of Ikaros using two different lentiviral constructs delayed erythroid differentiation, it failed to appreciably stimulate HbF production or alter BCL11A expression. These results suggest that the loss of Ikaros alone is insufficient to recapitulate the phenotype observed in pomalidomide-treated conditions. We next compared the expression levels of proteins involved in globin gene regulation among untreated peripheral blood, pomalidomide-treated peripheral blood and untreated cord blood-derived erythroid cells. We found striking similarities between cord blood and pomalidomide-treated adult cells at day 4 of differentiation. Indeed, BCL11A, KLF1, SOX6, LSD1 and GATA1 showed decreased expression levels both in cord blood and pomalidomide-treated adult peripheral blood, while the levels of CoREST, HDAC1 and GATA2 remained unchanged indicating that pomalidomide partially reprograms adult erythroid cells to a fetal-like state. Taken together, our results show that the mechanism underlying reactivation of HbF by pomalidomide involves Ikaros-independent reprogramming of adult erythroid progenitors. Finally, we found that this mechanism is conserved in SCD-derived CD34+ cells. Our work has broad implications for globin switching, as we provide direct evidence that Ikaros does not play a major role in the repression of γ-globin during adult erythropoiesis, and further supports the previously held notion that globin chain production is determined prior to or at the level of CFU-E. DisclosuresAllen:Celgene: Research Funding; Bristol Myers Squibb: Equity Ownership; Onconova: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

Ikaros mediates gene silencing in T cells through Polycomb repressive complex 2.

T-cell development is accompanied by epigenetic changes that ensure the silencing of stem cell-related genes and the activation of lymphocyte-specific programmes. How transcription factors influence these changes remains unclear. We show that the Ikaros transcription factor forms a complex with Polycomb repressive complex 2 (PRC2) in CD4−CD8− thymocytes and allows its binding to more than 500 developmentally regulated loci, including those normally activated in haematopoietic stem cells and others induced by the Notch pathway. Loss of Ikaros in CD4−CD8− cells leads to reduced histone H3 lysine 27 trimethylation and ectopic gene expression. Furthermore, Ikaros binding triggers PRC2 recruitment and Ikaros interacts with PRC2 independently of the nucleosome remodelling and deacetylation complex. Our results identify Ikaros as a fundamental regulator of PRC2 function in developing T cells.

Flying too close to the sun: loss of IKAROS accelerates CML

Flying too close to the sun: loss of IKAROS accelerates CML

Ikaros fingers on lymphocyte differentiation.

The Ikaros family of DNA-binding proteins are critical regulators of lymphocyte differentiation. In multipotent, hematopoietic progenitors, Ikaros supports transcriptional priming of genes promoting lymphocyte differentiation. Ikaros targets the Nucleosome Remodeling Deacetylase (NuRD)complex to lymphoid lineage genes, thereby increasing chromatin accessibility and transcriptional priming. After lymphoid lineage specification, Ikaros expression is raised to levels characteristic of intermediate B cell and T cell precursors, which is necessary to support maturation and prevent leukemogenesis. Loss of Ikaros in T cell precursors allows the NuRD complex to repress lymphocyte genes and extends its targeting to genes that support growth and proliferation, causing their activation and triggering a cascade of events that leads to leukemogenesis. Loss of Ikaros in B cell precursors blocks differentiation and perpetuates stromal adhesion by enhancing integrin signaling. The combination of integrin and cytokine signaling in Ikaros-deficient pre-B cells promotes their survival and self-renewal. The stages of lymphocyte differentiation that are highly dependent on Ikaros are underscored by changes in Ikaros transcription, supported by a complex network of stage-specific regulatory networks that converge upon the Ikzf1 locus. It is increasingly apparent that understanding the regulatory networks that operate upstream and downstream of Ikaros is critical not only for our understanding of normal lymphopoiesis, but also in placing the right finger on the mechanisms that support hematopoietic malignancies in mouse and human.

Ikaros inhibits megakaryopoiesis through functional interaction with GATA-1 and NOTCH signaling

AbstractThe transcription factor Ikaros regulates the development of hematopoietic cells. Ikaros-deficient animals fail to develop B cells and display a T-cell malignancy, which is correlated with altered Notch signaling. Recently, loss of Ikaros was associated with progression of myeloproliferative neoplasms to acute myeloid leukemia and increasing evidence shows that Ikaros is also critical for the regulation of myeloid development. Previous studies showed that Ikaros-deficient mice have increased megakaryopoiesis, but the molecular mechanism of this phenomenon remains unknown. Here, we show that Ikaros overexpression decreases NOTCH-induced megakaryocytic specification, and represses expression of several megakaryocytic genes including GATA-1 to block differentiation and terminal maturation. We also demonstrate that Ikaros expression is differentially regulated by GATA-2 and GATA-1 during megakaryocytic differentiation and reveal that the combined loss of Ikzf1 and Gata1 leads to synthetic lethality in vivo associated with prominent defects in erythroid cells and an expansion of megakaryocyte progenitors. Taken together, our observations demonstrate an important functional interplay between Ikaros, GATA factors, and the NOTCH signaling pathway in specification and homeostasis of the megakaryocyte lineage.

Cell cycle‐specific function of Ikaros in human leukemia

The loss of Ikaros is associated with the development of B and T cell leukemia. Data on Ikaros function, including its role as a tumor suppressor and a regulator of cell cycle progression, come almost exclusively from murine studies; little is known of the mechanisms that regulate human Ikaros function. Our studies are the first to examine the function and regulation of human Ikaros isoforms during the cell cycle in human ALL. Electromobility shift assay (EMSA), confocal microscopy, and phosphopeptide mapping were used to study Ikaros function during different stages of the cell cycle. The DNA-binding activity of human Ikaros complexes undergoes dynamic changes as the cell cycle progresses. In S phase, Ikaros DNA-binding affinity for regulatory regions of its target genes decreases, while its binding to pericentromeric heterochromatin is preserved and correlates with Ikaros pericentromeric localization. These S phase-specific changes in Ikaros function are controlled by phosphorylation via the CK2 kinase pathway. During cell cycle progression, the subcellular pericentromeric localization of the largest human Ikaros isoforms is different from that in mouse cells, suggesting unique functions for human Ikaros. Our results demonstrate that the function of Ikaros is cell cycle-specific and controlled by CK2-mediated phosphorylation during S phase of the cell cycle in human T-cell and B-cell ALL. The differences we observe in murine and human Ikaros function highlight the importance of using human cells in studies of ALL. These data identify the CK2 pathway as a target for therapies in ALL.

Role of Ikaros in T-cell acute lymphoblastic leukemia

Ikaros is a zinc finger transcriptional regulator encoded by the Ikzf1 gene. Ikaros displays crucial functions in the hematopoietic system and its loss of function has been linked to the development of lymphoid leukemia. In particular, Ikaros has been found in recent years to be a major tumor suppressor involved in human B-cell acute lymphoblastic leukemia. Its role in T-cell leukemia, however, has been more controversial. While Ikaros deficiency appears to be very frequent in murine T-cell leukemias, loss of Ikaros appears to be rare in human T-cell acute lymphoblastic leukemia (T-ALL). We review here the evidence linking Ikaros to T-ALL in mouse and human systems.

Ikaros Is a GATA Switch Target Gene That Restrains Megakaryopoiesis In Part by Blocking Notch Signaling.

AbstractAbstract 1602The hematopoietic transcription factor Ikaros regulates the development of lymphoid cells. In particular, Ikaros has been shown to suppress expression of Notch target genes in developing thymocytes. In addition, Ikaros-deficient animals fail to develop B cells and instead display a T cell lymphoproliferative disorder. Furthermore, alterations in Ikaros gene expression are associated with human lymphoid neoplasms and loss of Ikaros is associated with progression of myeloproliferative neoplasms to acute myeloid leukemia. However, the role of Ikaros in normal and malignant myelopoiesis is not well characterized. Therefore, we first analyzed myeloid development in Ikaros deficient mice and observed extramedullary hematopoiesis in the spleen and a striking thrombocytosis in the peripheral blood of 8–9 week-old Ikaros-null mice (2×106 platelets/μl, compared to 0.5×106 platelets/μl in wild-type littermates). Flow cytometry, histology and colony forming assays revealed that the bone marrow and spleen of young Ikaros-null mice harbored a substantial increase in the numbers of megakaryocytes and myeloid cells. We next investigated how Ikaros activity is reduced during terminal differentiation. Previous reports have shown that GATA1-deficient and GATA1s mutant megakaryocytes, which are associated with Down syndrome acute megakaryoblastic leukemia (DS-AMKL), express aberrantly high levels of Ikaros, suggesting that Ikaros is a target of GATA-1 repression during terminal megakaryocyte differentiation. By chromatin immunoprecipitation assays, we found multiple sites in the Ikaros locus that are bound by GATA-2 in proliferating progenitors and by GATA-1 in maturing megakaryocytes. Furthermore, we discovered that GATA-1s fails to occupy these conserved Ikaros loci. Together these results strongly suggest that GATA-1, but not GATA-1s, displaces GATA-2 from the Ikaros gene during differentiation and that this GATA switch leads to repression of Ikaros, in turn allowing for terminal maturation of megakaryocytes. Since Notch participates in specification of the megakaryocyte lineage, we then asked whether Ikaros inhibited megakaryopoiesis by interfering with Notch signaling. Retroviral transduction of full-length Ikaros in murine common myeloid progenitors, but not in LSK cells, reduced megakaryocyte development upon Notch stimulation in OP9-DL1 stromal co-cultures. Ectopic expression of Ikaros in the 6133 cell line, which models the t(1;22) subtype of AMKL and shows aberrant Notch target genes activation by the OTT-MAL fusion oncogene, inhibited proliferation and induced apoptosis. Genome wide expression profiling in transduced 6133 cells confirmed that Ikaros alters expression of several genes involved in the Notch and growth factor signaling pathways. These results indicate that Ikaros restricts megakaryocyte development and inhibits proliferation of OTT-MAL-transformed AMKL cells at least in part by suppressing Notch signaling. Together, our results suggest that a functional antagonism between the Notch pathway and Ikaros controls normal megakaryocyte development and that this axis is deregulated in AMKL, contributing to aberrant expansion of immature megakaryoblasts. Disclosures:No relevant conflicts of interest to declare.

Abstract 1961: Ikzf1 haploinsufficiency contributes to the pathogenesis of BCR-ABL1 positive acute lymphoblastic leukemia

Abstract Expression of the constitutively active tyrosine kinase BCR-ABL1 is the hallmark of two diseases with distinct pathologic and clinical features: chronic myeloid leukemia (CML), an expansion of relatively mature granulocytes that typically responds well to kinase inhibition, and pre-B cell acute lymphoblastic leukemia (ALL), an aggressive malignancy of lymphoid progenitors that has a dismal prognosis. The basis for this dichotomy has been poorly understood. We recently identified a near-obligate deletion of IKZF1 (encoding the lymphoid transcription factor IKAROS) in de novo BCR-ABL1 positive ALL, and at the progression of CML to lymphoid blast crisis, suggesting that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 lymphoid leukemia. To test this hypothesis, we have examined the effect of Ikaros haploinsufficiency in a retroviral bone marrow transplant model of murine BCR-ABL1 B-progenitor ALL. Unmanipulated marrow from either Ikzf1 wild type mice or mice heterozygous for an Ikzf1 null allele was transduced with MSCV bicistronic retrovirus expressing GFP and p185 BCR-ABL1 and transplanted into lethally irradiated C57BL/6 recipients. Recipients of MSCV-GFP-p185 BCR-ABL1-transduced Ikzf1+/− marrow developed leukemia with markedly increased penetrance and reduced latency in comparison to recipients of wild-type marrow (46 vs. 122 d, P&amp;lt;0.0001). The resulting leukemias resulted in bone marrow replacement by lymphoblasts, pancytopenia, extramedullary infiltration, and frequent clinical and histologic central nervous system involvement. The leukemias were of pre-B immunophenotype and uniformly transplantable into sublethally irradiated secondary recipients. Importantly, detailed enumeration of lymphoid subsets in Ikzf+/+ and +/− marrow did not demonstrate an expansion of lymphoid progenitors in Ikzf1+/− marrow, suggesting that the observed increased penetrance of B-ALL in Ikzf1+/− animals was not simply due to the expansion of targets of BCR-ABL1 transformation. Thus, Ikaros haploinsufficiency cooperates with BCR-ABL1 to induce an aggressive pre-B leukemia with clinical and pathologic features recapitulating human BCR-ABL1 ALL. These results support genomic analyses of human leukemia suggesting that IKAROS loss is important in the pathogenesis of this disease. Experiments evaluating the interaction of Ikzf1 loss with other targets of genomic alteration in BCR-ABL1 ALL (e.g. CDKN2A/B, PAX5), studies examining the role of Ikzf1 in responsiveness to kinase inhibitors, and genomic profiling studies are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1961.

Ikzf1 Haploinsufficiency Contributes to the Pathogenesis of BCR-ABL1 Positive Acute Lymphoblastic Leukemia.

Abstract 678Expression of the constitutively active tyrosine kinase BCR-ABL1 is the hallmark of two diseases with distinct pathologic and clinical features: chronic myeloid leukemia (CML), an expansion of relatively mature granulocytes that typically responds well to kinase inhibition, and pre-B cell acute lymphoblastic leukemia (ALL), an aggressive malignancy of lymphoid progenitors that has a dismal prognosis. The basis for this dichotomy has been poorly understood. Recent studies profiling genome-wide DNA copy number alterations in CML and ALL have identified a near-obligate deletion of IKZF1 (encoding the lymphoid transcription factor IKAROS) in de novo BCR-ABL1 positive ALL, and at the progression of CML to lymphoid blast crisis, suggesting that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 lymphoid leukemia. To test this hypothesis, we have examined the effect of Ikaros haploinsufficiency in a retroviral bone marrow transplant model of murine BCR-ABL1 B-progenitor ALL. Unmanipulated marrow from either Ikzf1 wild type mice or mice heterozygous for an Ikzf1 null allele was transduced with MSCV bicistronic retrovirus expressing GFP and p185 BCR-ABL1 and transplanted into lethally irradiated C57BL/6 recipients. Recipients of MSCV-GFP-p185 BCR-ABL1-transduced Ikzf1+/− marrow developed leukemia with markedly increased penetrance and reduced latency in comparison to recipients of wild-type marrow (46 vs. 122 d, P<0.0001). The resulting leukemias resulted in bone marrow replacement by lymphoblasts, pancytopenia, extramedullary infiltration, and frequent clinical and histologic central nervous system involvement. The leukemias were of pre-B immunophenotype and uniformly transplantable into sublethally irradiated secondary recipients. Importantly, detailed enumeration of lymphoid subsets in Ikzf+/+ and +/− marrow did not demonstrate an expansion of lymphoid progenitors in Ikzf1+/− marrow, suggesting that the observed increased penetrance of B-ALL in Ikzf1+/− animals was not simply due to the expansion of targets of BCR-ABL1 transformation. Thus, Ikaros haploinsufficiency cooperates with BCR-ABL1 to induce an aggressive pre-B leukemia with clinical and pathologic features recapitulating human BCR-ABL1 ALL. These results support genomic analyses of human leukemia suggesting that IKAROS loss is important in the pathogenesis of this disease. Experiments evaluating the interaction of Ikzf1 loss with other targets of genomic alteration in BCR-ABL1 ALL (e.g. CDKN2A/B, PAX5), and the role of dominant negative isoforms of Ikaros in leukemogenesis will be of interest. Disclosures:No relevant conflicts of interest to declare.

Genetic inactivation of Ikaros is a rare event in human T-ALL

The Ikaros (Ikzf1) gene, encoding a transcription regulator, is a major tumor suppressor in B-cell acute lymphoblastic leukemia (B-ALL). In the mouse, however, loss of Ikaros is primarily associated with T-ALL development. Whether Ikaros is also implicated in human T-ALL remains unclear. We studied Ikaros in 25 human T-ALL samples from diverse molecular subtypes at the mRNA, protein, sequence and genomic copy number level. We found that Ikaros was abnormal in only one sample: one allele was lost by genomic deletion, while proteins generated from the remaining allele were delocalized and concentrated at a single cytoplasmic structure. Thus, inactivation of Ikaros by deletion or mutation is rare in human T-ALL.

It's T-ALL about Notch

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subset of ALL with poor clinical outcome compared to B-ALL. Therefore, to improve treatment, it is imperative to delineate the molecular blueprint of this disease. This review describes the central role that the Notch pathway plays in T-ALL development. We also discuss the interactions between Notch and the tumor suppressors Ikaros and p53. Loss of Ikaros, a direct repressor of Notch target genes, and suppression of p53-mediated apoptosis are essential for development of this neoplasm. In addition to the activating mutations of Notch previously described, this review will outline combinations of mutations in pathways that contribute to Notch signaling and appear to drive T-ALL development by 'mimicking' Notch effects on cell cycle and apoptosis.

Antagonistic Interactions between Ikaros and the Chromatin Remodeler Mi-2β Determine Silencer Activity and Cd4 Gene Expression

Lineage commitment is induced by changes in gene expression dictated by the intimate interaction between transcription factors and chromatin regulators. Here, we revealed the antagonistic interplay between Ikaros and its associate the chromatin remodeler Mi-2beta during T cell development, as exemplified by the regulation of Cd4 expression. Loss of Ikaros or Mi-2beta led to activation or repression, respectively, of the Cd4 locus at inappropriate stages of development. Their combined mutation reverted to normal CD4 expression. In double-negative thymocytes, Ikaros binding to the Cd4 silencer contributed to its repressive activity. In double-positive thymocytes, concomitant binding of Mi-2beta with Ikaros to the Cd4 silencer caused silencer inactivation, thereby allowing for CD4 expression. Mi-2beta facilitated recruitment of histone acetyl transferases to the silencer. This recruitment possibly antagonized Ikaros and associated repressive activities. Thus, concomitant interactions between functionally opposing chromatin-regulating machineries are an important mode of gene regulation during lineage determination.