Ikaros Family Members Research Articles

Transcriptional function of E2A, Ebf1, Pax5, Ikaros and Aiolos analyzed by in vivo acute protein degradation in early B cell development.

Early B cell lymphopoiesis depends on E2A, Ebf1, Pax5 and Ikaros family members. In the present study, we used acute protein degradation in mice to identify direct target genes of these transcription factors in pro-B, small pre-B and immature B cells. E2A, Ebf1 and Pax5 predominantly function as transcriptional activators by inducing open chromatin at their target genes, have largely unique functions and are essential for early B cell maintenance. Ikaros and Aiolos act as dedicated repressors to cooperatively control early B cell development. The surrogate light-chain genes Igll1 and Vpreb1 are directly activated by Ebf1 and Pax5 in pro-B cells and directly repressed by Ikaros and Aiolos in small pre-B cells. Pax5 and E2A contribute to V(D)J recombination by activating Rag1, Rag2, Dntt, Irf4 and Irf8. Similar to Pax5, Ebf1 also represses the cohesin-release factor gene Wapl to mediate prolonged loop extrusion across the Igh locus. In summary, in vivo protein degradation has provided unprecedented insight into the control of early B cell lymphopoiesis by five transcription factors.

Cytotoxic Programming of CD4+ T Cells Is Regulated by Opposing Actions of the Related Transcription Factors Eos and Aiolos.

In contrast to the "helper" activities of most CD4+ T effector subsets, CD4+ cytotoxic T lymphocytes (CD4-CTLs) perform functions normally associated with CD8+ T and NK cells. Specifically, CD4-CTLs secrete cytotoxic molecules and directly target and kill compromised cells in an MHC class II-restricted fashion. The functions of these cells have been described in diverse immunological contexts, including their ability to provide protection during antiviral and antitumor responses, as well as being implicated in autoimmunity. Despite their significance to human health, the complete mechanisms that govern their programming remain unclear. In this article, we identify the Ikaros zinc finger transcription factor Eos (Ikzf4) as a positive regulator of CD4-CTL differentiation during murine immune responses against influenza virus infection. We find that the frequency of Eos+ cells is elevated in lung CD4-CTL populations and that the cytotoxic gene program is compromised in Eos-deficient CD4+ T cells. Consequently, we observe a reduced frequency and number of lung-residing, influenza virus-responsive CD4-CTLs in the absence of Eos. Mechanistically, we determine that this is due, at least in part, to reduced expression of IL-2 and IL-15 cytokine receptor subunits on the surface of Eos-deficient CD4+ T cells, both of which support the CD4-CTL program. Finally, we find that Aiolos, a related Ikaros family member and known CD4-CTL antagonist, represses Eos expression by antagonizing STAT5-dependent activation of the Ikzf4 promoter. Collectively, our findings reveal a mechanism wherein Eos and Aiolos act in opposition to regulate cytotoxic programming of CD4+ T cells.

IKAROS and AIOLOS directly regulate AP-1 transcriptional complexes and are essential for NK cell development.

Ikaros transcription factors are essential for adaptive lymphocyte function, yet their role in innate lymphopoiesis is unknown. Using conditional genetic inactivation, we show that Ikzf1/Ikaros is essential for normal natural killer (NK) cell lymphopoiesis and IKZF1 directly represses Cish, a negative regulator of interleukin-15 receptor resulting in impaired interleukin-15 receptor signaling. Both Bcl2l11 and BIM levels, and intrinsic apoptosis were increased in Ikzf1-null NK cells, which in part accounts for NK lymphopenia as both were restored to normal levels when Ikzf1 and Bcl2l11 were co-deleted. Ikzf1-null NK cells presented extensive transcriptional alterations with reduced AP-1 transcriptional complex expression and increased expression of Ikzf2/Helios and Ikzf3/Aiolos. IKZF1 and IKZF3 directly bound AP-1 family members and deletion of both Ikzf1 and Ikzf3 in NK cells resulted in further reductions in Jun/Fos expression and complete loss of peripheral NK cells. Collectively, we show that Ikaros family members are important regulators of apoptosis, cytokine responsiveness and AP-1 transcriptional activity.

Distinct Role of STAT3 and STAT5 Factors in MDS and AML

Introduction: Myelodysplastic Syndrome (MDS) refers to a heterogeneous group of clonal hematologic disorders characterized by hematopoietic dysfunctions and impaired differentiation in the hematopoietic stem and progenitor cells compartment. MDS is commonly referred to as a pre-leukemic stage due to the increased risk of progression to Acute Myeloid Leukemia (AML). AML is an aggressive hematologic malignancy characterized by the accumulation of immature myeloblasts in the bone marrow and the peripheral blood. Signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) are important regulators of several cellular processes including cell proliferation and survival. Abnormal STAT3 and STAT5 signaling has been implicated in various solid and hematologic malignancies, including MDS and AML, which renders them appealing targets for the development of novel therapeutic strategies. Materials: STAT3, STAT5A or STAT5B knock-downs have been generated in two AML (MOLM-13 and Kasumi-1) and two MDS (MDS92 and MDS-L, gift of Prof. Kaoru Tohyama) cell lines, using shRNAs through lenti-viral delivery. The 4 biological replicates with the highest levels of knock-downs for STAT5A, STAT5B or STAT3 have been selected for each cell line and mRNA sequencing has been performed. To define the genome-wide binding of STAT5A, STAT5B and STAT3 in the two conditions, CUT&Tag has been performed in wild type MOLM-13, Kasumi-1 and MDS-L cells. Additionally, ATAC sequencing has been carried out in the same cell lines to investigate the chromatin conformation changes between the two conditions. Furthermore, STAT3, STAT5A and STAT5B knock-downs have been conducted using shRNAs through lenti-viral delivery in CD34+ enriched cells, isolated from bone marrow mononuclear cells of 14 high-risk MDS patients and 14 secondary (MDS-related) AML patients. The samples have been processed for mRNA-Sequencing to model the leukemic transformation in primary human cells. Results: To elucidate the role of STAT3 and STAT5 in MDS to AML transformation, changes in STAT3 and STAT5 target gene networks have been identified. mRNA sequencing data from STAT3, STAT5A or STAT5B knock-downs in MDS and AML cell lines revealed a distinct role of the three factors in each state. The differential binding of each factor, explored through CUT&Tag, and the gene expression profiles in MDS and AML reciprocally reveal a different function of each factor, including pathways involved in cell cycle and apoptosis. Also, the direct and indirect target genes of STAT3, STAT5A and STAT5B in each condition, as well as a possible interplay between STAT3 and STAT5 activation in AML has been uncovered. The exploration of the chromatin accessibility landscapes between the MDS and AML conditions through ATAC sequencing has provided an insight into the chromatin conformation states, unveiling potential leukemia promoting networks, as well as the involvement of STAT3 and STAT5 pathways in the malignant transformation. Furthermore, a potential cross talk of STAT3 and STAT5 factors with the IKAROS family members, has been linked to AML transformation, as significant differential regulation has been detected between MDS and AML conditions after knock-down of STAT5A and STAT5B. The results are currently validated in primary hematopoietic cells of MDS and AML patients after STAT3, STAT5A or STAT5B knock-downs. Conclusions: Our results show a different role between the two STAT5 factors (STAT5A and STAT5B) in MDS and AML conditions and delineate the role of STAT5A, STAT5B and STAT3 factors and their target gene networks in leukemic transformation, thus providing novel targets for therapeutic management of MSD and AML. Funding: This project is funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813091 (ARCH)

Eos is a critical transcription factor for T regulatory cell (Treg) function

Abstract Eos is a member of the Ikaros transcription factor family and is highly expressed in Tregs. Mice with a selective deficiency of Eos in Treg develop systemic or organ-specific autoimmune disease at at a young age. We have generated a specific anti-Eos monoclonal antibody. The percentage of Tregs expressing Eos rapidly increases with aging from almost 0% at birth to 30~50% of thymic Tregs and 35~60% of splenic Tregs at 3 weeks of life and then remains stable. Eos is expressed at very low levels on induced Tregs, conventional CD4 +T cells and CD8 +T cells in vivo or in vitro. In the thymus, Eos is only expressed on CD73 +CCR6 +CXCR4 +Tregs, which suggests that Eos +thymic Tregs represent peripheral Tregs that have recirculated to the thymus. In thymus, Eos expression on Tregs is also highly correlated with CXCR4 and CCR6. Using these markers, we sorted Eos +and Eos −Tregs and in vitro expanded them using anti-CD3/CD28 beads and IL-2; Eos expression remained stable. Using immunofluorescence microscopy, we have found that Eos is highly co-localized with Helios, another Ikaros family member and HDAC1, but not other Ikaros family members including Ikaros or Aiolos, suggesting that Eos and Helios may form heterodimers and together with HDAC1 collaborate in Treg function. Nevertheless, expression of Eos is independent of Helios and vice versa, as Treg from Helios deficient mice express normal or elevated levels of Eos, and Treg from Eos deficient mice express normal levels of Helios. Taken together, these strongly suggest that Eos plays a related, but distinct role, from Helios in Treg function. This work was supported by Division of Intramural Research (DIR), NIAID, NIH.

Ikaros family proteins redundantly regulate temporal patterning in the developing mouse retina.

Temporal identity factors regulate competence of neural progenitors to generate specific cell types in a time-dependent manner, but how they operate remains poorly defined. In the developing mouse retina, the Ikaros zinc-finger transcription factor Ikzf1 regulates production of early-born cell types, except cone photoreceptors. In this study we show that, during early stages of retinal development, another Ikaros family protein, Ikzf4, functions redundantly with Ikzf1 to regulate cone photoreceptor production. Using CUT&RUN and functional assays, we show that Ikzf4 binds and represses genes involved in late-born rod photoreceptor specification, hence favoring cone production. At late stages, when Ikzf1 is no longer expressed in progenitors, we show that Ikzf4 re-localizes to target genes involved in gliogenesis and is required for Müller glia production. We report that Ikzf4 regulates Notch signaling genes and is sufficient to activate the Hes1 promoter through two Ikzf GGAA-binding motifs, suggesting a mechanism by which Ikzf4 may influence gliogenesis. These results uncover a combinatorial role for Ikaros family members during nervous system development and provide mechanistic insights on how they temporally regulate cell fate output.

Dual IKZF2 and CK1α Degrader Targets Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive hematologic disease for which several epigenetic regulators have been identified as therapeutic targets. We previously found that a transcription factor and Ikaros family member, IKZF2 (HELIOS) is required for myeloid leukemic stem cell activity (Park et al. Cell Stem Cell 2019). Additionally, IKZF2 enhances self-renewal by increasing HOXA9 gene expression and in parallel blocks myeloid differentiation by reducing C/EBP expression. Thus, our studies suggested that targeting IKZF2 could be a new therapeutic strategy in AML. Immunomodulatory drugs (IMiDs; lenalidomide and others) have been used clinically to specifically target and degrade other Ikaros family members. However, these have been unable to target IKZF2. Utilizing a structure-guided approach, we developed a small molecule with low nanomolar degrader activity against IKZF2 (DEG-35). DEG-35 treatment in different AML cell lines induce apoptosis and differentiation with IC50 values of 5 ±0.4 nM in MOLM-13 cells to 27± 2.5 uM in NB4 cells. To understand if DEG35 has activity against other substrates, we performed global quantitative proteomics and found casein kinase 1 alpha (CK1α), a serine/threonine kinase previously identified to be critical for myeloid leukemogenesis and a known IMiD substrate, to be the most depleted protein (Jaras et al. JEM). Also, we performed a PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) screen assay which utilizes 770 barcoded cell lines from 20 lineages and identified multiple correlations with the p53 pathway. Previous studies found that CK1α suppresses the activity of p53 phosphorylation or binding MDM proteins. To further probe the mechanism behind DEG35 activity, we performed RNA sequencing at 24hrs in MOLM-13 cells. Gene set enrichment analysis (GSEA) using the rank list of differentially expressed genes from the MOLM-13 cells treated with DMSO and DEG-35 revealed enrichment for genes upregulated in CK1α KO keratinocytes and p53 targets. Additionally, we found enrichment for the MLL-AF9 IKZF2 KO LSC gene sets, myeloid differentiation, loss of MEIS1-HOXA9 and MYC targets. Immunoblotting validated the reduction of MYC and HOXA9 protein in DEG-35 treated MOLM-13 cells. To determine the contribution of CK1α and IKZF2 to the cellular phenotypes in MOLM13 cells, we developed CK1α and IKZF2 nondegradable mutants. CK1α, G40N was able to significantly reduce the cell killing activity of DEG-35 by 94% compared to vector cells (**p<0.01). Moreover, p53 deletion was also able to block DEG-35 induced apoptosis by 98% and differentiation by 88% compared to control. Additionally, expression of IKZF2 H141Q rescued DEG-35 mediated differentiation by 55% (***p<0.001), but not apoptosis. Thus, we demonstrate that the degradation of IKZF2 and CK1α by DEG-35 blocks cell growth and induces myeloid differentiation in human AML cell lines through CK1α-p53- and IKZF2-dependent pathways. To further probe the efficacy and therapeutic index of targeting both IKZF2 and CK1α. We found that AML cells were more sensitive to DEG-35 compared to normal bone marrow cells (murine MLL-AF9 Crbn I1391V cells has 10 fold and AML PDX cells have 2-5 fold higher sensitivity to normal mouse or human bone marrow cells).Treatment of DEG-35 at 50 mg/kg in AML PDX-transplanted mice led to significant prolonged survival compared to vehicle treated mice (median survival of DMSO mice: 45 days vs DEG-35 mice: 61 days; ***p< 0.001) and reduced disease burden. To improve pharmacological properties, we developed analog DEG-77, which has 10-fold increased solubility than DEG-35 in DMSO. DEG77 exhibited better in vivo efficacy in the MLL-AF9 Crbn I1391V model (Increase in median survival by DEG-35: 2 days *p<0.05 vs DEG-77: 31 days ****p<0.0001). Importantly, the increase of median survival of 30 days (****p<0.0001) was also observed in the secondarily transplanted DEG-77 mice compared to DMSO mice, suggesting that DEG-35 can reduce LSC activity. In summary, we report the development of nanomolar monofunctional cereblon-dependent degraders of IKZF2 and casein kinase 1 alpha (CK1α). DEG-35 or the analog DEG-77 delays leukemia progression in murine and human AML mice models. Thus, we provide a novel strategy for multi-targeted degradation of IKZF2/CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

Aiolos restrains the acquisition of cytotoxic features by CD4+ T cells

Abstract Classically, CD4+ T cells have been defined as “helper” type cells, providing aid to other immune cell populations via the secretion of cytokines and direct cell-cell interactions. More recently, a subset of CD4+ T cells with cytotoxic capabilities, termed CD4+ cytotoxic T lymphocytes (CD4-CTLs), have been observed in both mice and humans, performing protective functions in the settings of infection and cancer while conversely contributing to the pathogenesis of autoimmunity. Despite their well-documented importance in several disease contexts, the complete mechanisms that underlie their differentiation and function remain unknown. Here, we identify the Ikaros family member, Aiolos, as a novel regulator of CD4+ CTL differentiation and function. We find that Aiolos deficiency results in increased expression of key CD4+ CTL transcription factors and effector molecules both in vitro and in an in vivo murine model of influenza infection. Mechanistically, we find that Aiolos deficiency results in increased IL-2/STAT5 signaling, supporting a repressive role for Aiolos in CD4+ CTL differentiation via negative regulation of the IL-2/STAT5 pathway. Collectively, this work identifies Aiolos as a novel negative regulator of CD4+ cytotoxic gene programming, and thus may represent a therapeutic target for the treatment of autoimmune diseases and enhanced anti-tumor immunity. Sponsored by a grant from NIAID (NIH-RO1 AI134972) and funds through The Ohio State University College of Medicine

Established and emergent roles for Ikaros transcription factors in lymphoid cell development and function

Ikaros zinc finger transcription factors are important regulators of the gene programs underlying the development of hematopoietic cell lineages. The family consists of five members: Ikaros, Helios, Aiolos, Eos, and Pegasus, which engage in both homo- and heterotypic intrafamilial interactions to exert diverse functional effects. Pioneering studies focused on the role of these factors in early lymphoid development, as their absence resulted in severe defects in lymphocyte populations. More recent work has now begun to define nuanced, stage-specific roles for Ikaros family members in the differentiation and function of mature T, B, and innate lymphoid cell populations including natural killer (NK) cells. The precise transcriptional mechanisms by which these factors function, both independently and collaboratively, is an area of active investigation. However, several key themes appear to be emerging regarding the pathways influenced by Ikaros family members, including the end-to-end regulation of cytokine signaling. Here, we review roles for Ikaros factors in lymphoid cell development, differentiation, and function, including a discussion of the current understanding of the transcriptional mechanisms they employ and considerations for the future study of this important transcription factor family.

Effect of Notch and PARP Pathways' Inhibition in Leukemic Cells.

Differentiation of blood cells is one of the most complex processes in the body. It is regulated by the action of transcription factors in time and space which creates a specific signaling network. In the hematopoietic signaling system, Notch is one of the main regulators of lymphocyte development. The aim of this study was to get insight into the regulation of Notch signalization and the influence of poly(ADP-ribose)polymerase (PARP) activity on this process in three leukemia cell lines obtained from B and T cells. PARP1 is an enzyme involved in posttranslational protein modification and chromatin structure changes. B and T leukemia cells were treated with Notch and PARP inhibitors, alone or in combination, for a prolonged period. The cells did not show cell proliferation arrest or apoptosis. Analysis of gene and protein expression set involved in Notch and PARP pathways revealed increase in JAGGED1 expression after PARP1 inhibition in B cell lines and changes in Ikaros family members in both B and T cell lines after γ-secretase inhibition. These data indicate that Notch and PARP inhibition, although not inducing differentiation in leukemia cells, induce changes in signaling circuits and chromatin modelling factors.

Normal Plasma Cell Biology: Natural Achilles Heels as Targets in Myeloma

Normal Plasma Cell Biology: Natural Achilles Heels as Targets in MyelomaÊ Primary exposure to antigen leads to affinity maturation, selection and terminal differentiation of mature B cells to antibody-secreting plasma cells. Long-lived high-affinity plasma cells and their immediate precursors reside in the bone marrow and are responsible for rapid recall reactions and a life-time production of high affinity antibodies. Members of the IKAROS gene family have been specifically implicated in the generation of long-lived plasma cells. Mice deficient for Aiolos, the B cell specific member of the IKAROS gene family fail to produce high affinity plasma cells in the bone marrow and to sustain serum antibody titers after both primary or secondary immunization with a range of hapten concentrations. Chimera reconstitutions have demonstrated that the bone marrow plasma cell defect is B cell intrinsic. However, lack of AIOLOS does not alter expression of any of the previously described factors required for B cell differentiation into the plasma cell stage. No defect in somatic hyper-mutation, the generation of memory B cells, or short-lived high affinity plasma cells in the spleen is observed upon challenge or re-challenge. Thus this IKAROS family member is critically required for high-affinity bone plasma cells possibly by regulating their interaction with the bone marrow microenvironment, a process that is likely critical for long-term survival. High-affinity bone marrow plasma cells are the normal counterpart of the devastating B cell malignancy multiple myeloma. Multiple myeloma is treatable with immune modulatory drugs (IMiD) such as lenalidomide. Recent studies have shown that IMiD alter the specificity of the CRL4-CEREBLON (CRL4CRBN) E3 ubiquitin ligase complex. Among the new targets for the CRL4CRBN complex are two of the IKAROS family members, AIOLOS and IKAROS, whose degradation adversely affects the cellular fitness of multiple myeloma cells. Recent studies in B cell precursors have implicated the IKAROS gene family in the regulation of bone marrow stromal interactions. IKAROS and AIOLOS activities balance self-renewal, survival, and pre-B cell differentiation by engaging epigenetic and transcriptional machineries through discrete lineage-specific superenhancers. Such AIOLOS and IKAROS-dependent regulatory pathways engaged in early B cell precursors may also be involved in controlling bone marrow stromal interactions with high affinity plasma cells and their malignant counterparts thereby contributing to long-term growth and survival. Further exploitation of IKAROS family-targeted pathways in bone marrow plasma cells and multiple myeloma is warranted. DisclosuresNo relevant conflicts of interest to declare.

Expression and splicing of Ikaros family members in murine and human thymocytes

Abstract The Ikaros family of transcription factors consists of five highly homologous zinc-finger proteins: Ikaros, Helios, Eos, Aiolos and Pegasus. These family members can homodimerize or heterodimerize in any combination which can alter transcriptional activity. Ikaros family member function can further vary with alternative splicing that affects DNA-binding. Previous studies have demonstrated that abolishing the function of the entire Ikaros family blocks lymphocyte development. Whereas eliminating the function of one Ikaros family member results in relatively minor defects. These data indicate that multiple Ikaros family members play a role in lymphocyte development. The goal of this study was to fully characterize expression and splicing of Ikaros family members in murine and human T cell development from the CD4−CD8− double negative (DN) developmental stage to the CD4+CD8+ double positive (DP) stage. In both mice and humans, Aiolos and Ikaros mRNA levels increase during the progression of T cell development from the DN to DP stages. However, the corresponding increase in Aiolos and Ikaros protein levels was only observed in mice. Additionally, extensive alternative splicing of Ikaros and Aiolos was observed in mice, but only Ikaros was extensively spliced in humans. Helios mRNA and protein levels decreased during murine T cell development, but increased in human T cell development. These data indicate that differences in human and murine T cell development may be regulated by differences in Ikaros family member expression and splicing.

Expression and splicing of Ikaros family members in murine and human thymocytes

The Ikaros family of transcription factors includes five highly homologous members that can homodimerize or heterodimerize in any combination. Dimerization is essential for their ability to bind DNA and function as transcription factors. Previous studies showed that eliminating the function of the entire family blocks lymphocyte development while deletion of individual family members has relatively minor defects. These data indicate that multiple family members function during T cell development, so we examined the changes in expression of each family member as thymocytes progressed from the CD4−CD8− double negative (DN) to the CD4+CD8+ double positive (DP) developmental stage. Further, we compared the expression of each family member in murine and human thymocytes. In both species, Ikaros and Aiolos mRNA levels increased as thymocytes progressed through the DN to DP transition, but the corresponding increases in protein levels were only observed in mice. Further, Ikaros and Aiolos underwent extensive alternative splicing in mice, whereas only Ikaros was extensively spliced in humans. Helios mRNA and protein levels decreased during murine T cell development, but increased during human T cell development. These differences in the expression and splicing of Ikaros family members between human and murine thymocytes strongly suggest that the Ikaros family of transcription factors regulates murine and human T cell development differently, although the similarities across Ikaros family members may allow different proteins to fulfill similar functions.

The Ikaros Family of Transcription Factors Regulates the Expression of the Transcriptional Repressor Bcl-6 in CD4+ T Cells

Background:CD4+ T helper cells play critical roles in the regulation of pathogen-specific immune responses and immune tolerance. The formation of each individual T helper cell subset is dictated by the expression of a unique gene program. These gene programs are regulated by both the cytokine environment and cell-intrinsic, cytokine-responsive "lineage-defining" transcription factors, which imprint the conserved gene expression programs characteristic of a given T helper cell lineage. The transcriptional repressor Bcl-6 is one such factor, and has been identified as the lineage-defining transcription factor for the T follicular helper (TFH) cell subset. TFH cells participate in the generation of humoral immunity by providing help to B cells, which are responsible for the production of pathogen-neutralizing antibodies. Interestingly, Bcl-6 expression has also been implicated in the formation of CD4+ central memory T (TCM) cells, which play a critical role in long-term cell-mediated immunity. Recently, our laboratory has demonstrated that Bcl-6 expression can be induced in effector T helper 1 (TH1) cells in response to decreased interleukin 2 (IL-2) signaling. Consequently, TH1 cells are capable of upregulating Bcl-6-dependent TFH- and TCM-like gene programs, suggesting that these cells may be able to contribute to aspects of long-term humoral and cell-mediated immunity. Despite these insights, the upstream factor(s) that directly control the expression of Bcl-6 remain largely unknown. Preliminary RNAseq analysis indicated that the expression of members of the Ikaros family of zinc-finger transcription factors, which have been shown to play important roles in regulating gene expression during hematopoiesis, correlated with that of Bcl-6 in TH1 and TFH/TCM-like cells. As such, we hypothesized that Ikaros-family proteins may contribute to the regulation of Bcl-6 expression.Methods:Naïve CD4+ T cells isolated from the spleens and lymph nodes of 5-8 week old C57BL/6 mice were stimulated with α-CD3 and α-CD28 in TH1 polarizing conditions. Following the generation of TH1 cells, these cells were split into either high IL-2 conditions to maintain the TH1 phenotype or into low IL-2 conditions to induce the TFH/TCM-like phenotype. Total cellular RNA, total cellular protein, and chromatin samples were isolated for analysis.Results:In this study, we demonstrate that the Ikaros family members, Ikaros and Aiolos, are preferentially expressed in TFH/TCM-like cells when compared to TH1 cells. siRNA knockdown demonstrates that the expression of Bcl-6 correlates with that of Ikaros and/or Aiolos. To define the molecular mechanisms that lead to the aforementioned findings, we used chromatin immunoprecipitation (ChIP) assays to show that Ikaros and Aiolos directly bind to the Bcl-6 promoter region. Interestingly, co-immunoprecipitation experiments reveal that Ikaros and Aiolos physically interact, suggesting that they may act cooperatively to promote Bcl-6 expression. Finally, Ikaros and Aiolos siRNA experiments show that reduced expression of these transcription factors correlates with a reduction in the expression of a number of canonical TFH and TCM genes.Conclusion:Collectively, these results demonstrate that the Ikaros family members Ikaros and Aiolos are IL-2-sensitive transcription factors that positively regulate Bcl-6 expression and that of key TFH and TCM genes. These data support the possibility that Ikaros and Aiolos may be critical factors in the induction of the TFH and TCM cell types and thus, potentially, in the regulation of long-term humoral and cell-mediated immunity. DisclosuresNo relevant conflicts of interest to declare.

Proteomic/Transcriptomic Signatures of Infant MLL-r Rearranged B-ALL at Diagnosis and Relapse Reveal Lineage Plasticity and Diagnostic Heterogeneity

Infant leukemia, defined as leukemia before 1 year of age, is an aggressive set of diseases with dramatically poorer outcome when compared to childhood leukemia. A particularly intractable subset of infant leukemia patients, who are prone to relapse, are those with B-cell acute lymphoblastic leukemia (B-ALL) harboring a chromosomal translocations involving the epigenetic modifier gene mixed lineage leukemia (MLL), referred hereafter as MLL rearranged (MLL-r) B-ALL. Rearrangements in MLL occur in 70-80% of all infant B-ALL patients (compared to 5% in pediatric leukemia) and this cohort is characterized by a record setting dearth of cooperating lesions beyond MLL-r.To better understand the mechanism of relapse in this patient cohort we employed RNA sequencing and deep mass spectrometry based proteomics to characterize infant MLL-R B-ALL patient derived xenografts. Five infant MLL-r B-ALL patients with matched diagnosis and relapse primary sample were individually injected into NSG immunocompromised mice. For all patients >95% human engraftment purity was achieved in mouse bone marrow and spleen. Fresh cells were submitted for RNA sequencing and nuclear fractionated as well as whole cell lysates were generated for mass spec protein analysis. Protein and RNA quantitation at relapse and diagnosis across all patients was achieved on molecules that correspond to about 8,000 genes for protein and 17,000 genes for RNA.Hierarchal clustering as well as multi-dimensional scaling analysis of RNA sequencing data revealed a closer intra-patient relationship at the two treatment time points (diagnosis and relapse) than an inter-patient relationship at diagnosis or at relapse. A common relapse signature was simply non-existent according to the data, however this could be attributed to the small sample size. The integration of proteomic and transcriptomic data revealed a strong correlation of relapse/diagnosis fold changes at the level of RNA and protein. Intriguingly, a plasticity at relapse was observed in key developmental and differentiation marker genes associated with the myeloid, T, and B cell lineages. For example, in Patient 1 up-regulation at the level of protein and RNA of myeloid/acute myeloid leukemia (AML) associated genes; RUNX2, MPO, and CD33, and down-regulation of T and B cell associated genes; CD1A, CD2, and BCL6 where observed at relapse. Patient 2 showed an RNA/protein up-regulation of myeloid and AML markers CEBPB and MPO while a concurrent down-regulation of the Ikaros family member Helios, B/T cell attenuator BTLA, and the oncogene ETV6 at relapse. Patient 3 showed an RNA/protein up-regulation of Helios, the oft mutated in B-ALL; ARID5B, and the pre-B-cell leukemia associated transcription factor PBX3 at relapse. Patient 1 and 2 appear to show signs that myeloid programs are being greater utilized at relapse while in Patient 3 seems to intensify its commitment to the B-cell lineage. All patients showed some degree of lineage marker infidelity at relapse and interestingly, this plasticity was not reflected in the traditional flow cytometric markers used to differentiate AML from B-ALL suggesting that this lineage infidelity might be more common than previously thought.Despite the mutational uniformity of infant MLL-R B-ALL genomes it is surprising that such a diversity of RNA expression and subsequent protein levels is observed. This data suggests that MLL fusion proteins have great flexibility to control of the transcriptional regulation/protein expression and could in part help explain why these patients do not rely upon newly acquired mutations to evade treatment protocols. Furthermore, lineage switching in leukemia is rarely observed but as this data suggests that this is more common in MLL-r B-ALL than previously thought. DisclosuresNo relevant conflicts of interest to declare.

Variations in mRNA and protein levels of Ikaros family members in pediatric T cell acute lymphoblastic leukemia.

Pediatric T cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous disease in which the cells share phenotypic characteristics with normal human thymocytes. The Ikaros family of transcription factors includes five members that are required for normal T cell development and are implicated in leukemogenesis. The goal of this work was to correlate the pattern of expression of Ikaros family members with the phenotype of the T-ALL cells. We obtained twenty-four samples from pediatric T-ALL patients and used multi-parameter flow cytometry to characterize each sample, comparing the phenotype of the leukemic cells with normal human thymocytes. Then, we defined the expression levels of each Ikaros family member to determine whether the mRNA levels or splicing or protein levels were similar to the normal patterns seen during human T cell development. Multi-parameter analysis of the phenotype of T-ALL cells revealed that each patient's cells were unique and could not be readily correlated with stages of T cell development. Similarly, the pattern of Ikaros expression varied among patients. In most patients, Ikaros mRNA was the dominant family member expressed, but some patients' cells contained mostly Helios, Aiolos, or Eos mRNA. Despite that most patients had elevated mRNA levels of Ikaros family members and unique patterns of mRNA splicing, most patients had significantly reduced protein levels of Ikaros and Aiolos. Our analysis of the cell phenotype and Ikaros expression levels in T-ALL cells revealed the extent of heterogeneity among patients. While it is rarely possible to trace leukemic cells to their developmental origin, we found distinct patterns of Ikaros family mRNA levels in groups of patients. Further, mRNA and protein levels of Ikaros and Aiolos did not correlate, indicating that mRNA and protein levels are regulated via distinct mechanisms.

Expression patterns of Ikaros family members during positive selection and lineage commitment of human thymocytes.

The Ikaros family of transcription factors is essential for normal T-cell development, but their expression pattern in human thymocytes remains poorly defined. Our goal is to determine how protein levels of Ikaros, Helios and Aiolos change as human thymocytes progress through the positive selection and lineage commitment stages. To accomplish this goal, we used multi-parameter flow cytometry to define the populations in which positive selection and lineage commitment are most likely to occur. After human thymocytes express CD3 and receive positive selection signals, the cells down-regulate expression of CD4 to become transitional single-positive (TSP) CD8+ thymocytes. At this stage, there was a transient increase in the Ikaros, Helios and Aiolos protein levels. After the TSP CD8+ developmental stage, some thymocytes re-express CD4 and become CD3hi double-positive thymocytes before down-regulating CD8 to become mature single-positive CD4+ thymocytes. Except for regulatory T cells, Helios protein levels declined and Aiolos protein levels transiently increased during CD4+ T-cell maturation. For thymocytes progressing toward the CD8+ T-cell lineage, TSP CD8+ thymocytes increase their expression of CD3 and maintain high levels of Aiolos protein as the cells complete their maturation. In summary, we defined the TSP CD8+ developmental stage in human T-cell development and propose that this stage is where CD4/CD8 lineage commitment occurs. Ikaros, Helios and Aiolos each undergo a transient increase in protein levels at the TSP stage before diverging in their expression patterns at later stages.

Role of the transcription factor Ikaros in development of autoimmune disease

Abstract The etiology of autoimmune disease is not fully understood, but disease development is believed to be due to a combination of genetic predispositions and environmental factors. Ikaros is a transcription factor that is an important regulator of hematopoiesis and critical for development of B cells. Recent genome wide association studies have found that several autoimmune diseases are associated with SNPs annotated to the Ikaros gene (IKZF1), as well as the closely related Ikaros family member Aiolos (IKZF3). Mouse models of autoimmunity are important research tools, and we recently reported the generation of two new Ikaros-mutant mice, with targeted deletions of the exons encoding the DNA-binding zinc finger 1 (ZnF1) or ZnF4 (Schjerven et al., 2013). These deletions did not abolish B-cell development, but rather illuminated differential roles of Ikaros at different stages of early B-cell development. Interestingly, we found that one of these mouse strains displayed very high levels of serum Anti-Nuclear Antibodies (ANA), indicative of autoimmune disease. Furthermore, we found by ex vivo studies that while wt spleen B cells require a “second signal” such as CD40L in addition to BCR stimulation (by anti-IgM), Ikaros-mutant B cells became activated and proliferated upon BCR stimulation alone without any externally added “second signal”. We hypothesize that Ikaros is important to limit autoimmunity and prevent auto reactive B cells by setting a requirement for the “second signal” in adaptive immune-recognition. Results will be presented from ongoing investigation using in vivo analysis of the autoimmune-related phenotype, and ex vivo analysis of Ikaros-dependent molecular mechanisms underlying the response to stimuli in B cells.

Ikaros, Helios, and Aiolos protein levels increase in human thymocytes after β selection.

In human T cell development, the mechanisms that regulate cell fate decisions after TCRβ expression remain unclear. We defined the stages of T cell development that flank TCRβ expression and found distinct patterns of human T cell development. In half the subjects, T cell development progressed from the CD4(-)CD8(-) double-negative stage to the CD4(+)CD8(+) double-positive (DP) stage through an immature single-positive (ISP) CD4(+) intermediate. However, in some patients, CD4 and CD8 were expressed simultaneously and the ISP population was small. In each group of patients, CD3(-) ISP and DP thymocytes were subdivided into ISP1, ISP2, DP1, DP2, DP3, DP4, and DP5 developmental stages according to their expression of CD28, CD44, CD1a, CD7, CD45RO, and CD38. The ISP2, DP2, and DP3 thymocyte populations proliferated more robustly than ISP1 and DP1 and expressed markers consistent with TCRβ expression. After the DP3 stage, proliferation returned to baseline levels. We then analyzed protein levels of Ikaros, Helios, and Aiolos, the three Ikaros family members most abundantly expressed in human thymocytes. Ikaros and Helios expression increased transiently at the ISP2, DP2, and DP3 populations. Aiolos expression also increased at the ISP2, DP2, and DP3 stages, but its expression remained elevated throughout the DP4 and DP5 stages. In summary, we propose a model of human T cell development that reflects the asynchronous nature of TCRβ expression and we define the subpopulations of thymocytes that are highly proliferative and express Ikaros family members.

Non-Functional ("haploinsufficient"), but Not Dominant Negative Clonal IKZF1 Deletions Confer an Adverse Prognosis in Adult BCR-ABL-Negative Acute Lymphoblastic Leukemia

Background: IKAROS (IKZF1) is a key transcription factor for B-cell development. The IKZF1 protein exerts its functions as a dimer (homodimer or heterodimer with other IKAROS family members). Various IKZF1 intragenic deletions have been identified as recurrent aberrations in acute lymphoblastic leukemia: those resulting in the loss of transcription of one allele or at least loss of the dimerization domain ("haploinsufficient", "non-functional") and those involving only the DNA-binding domain but retaining the dimerization domain of the transcription factor, leading to so-called "dominant negative" isoforms.Several studies in pediatric ALL have confirmed the negative prognostic impact of IKZF1 alterations although there is still no consensus if they are independent risk factors in a multivariate analysis. The situation in adult ALL is less clear and few larger studies have addressed this issue, in particular the prognostic impact of different types of IKZF1 alterations.Objectives: We investigated archived patient samples obtained in the context of the German Multicenter ALL Study Group (GMALL) therapy studies for different IKZF1 alterations to assess their prognostic implications and their molecular pattern.Methods: We used RT-PCR to determine the presence of aberrant IKZF1 transcript variants and additionally investigated all patients by DNA-based PCRs for the IKZF1 aberrations D4-7, D2-7, D4-8, D2-8. Rare transcript variants, such as D2-3, D2, D5-7 were molecularly characterized on the DNA level by specific PCRs. IKZF1 aberrations were quantified using gel densitometry and/or quantitative real time PCR such that clonal and subclonal aberrations could be distinguished. All chromosomal breakpoints were molecularly characterized.Results: In total, 482 B cell precursor ALL patients aged between 15 and 65 years were included in the analysis. Samples were obtained at primary diagnosis and tested BCR-ABL -negative. Other molecular aberrations were tested according to the GMALL standards: 39 MLL-AF4-positive, 4 MLL-ENL, 1 MLL-AF9, 30 TCF3-PBX1, 3 ETV6-RUNX1. The immunophenotypes were the following: 424 pre B/common, 58 pro B.One hundred and twenty-eight out of 482 (27%) patients carried at least one IKZF1 deletion, with 37 patients carrying more than one deletion. Altogether there were 175 IKZF1 deletions, 71 cases of D4-7, 47 with D2-7, 26 with D4-8, 19 with D2-3, 10 with D2-8, and 1 with D5-7 and D2 each. Taken together 56 patients (12%) carried only non-functional mutations while 50 (10%) had dominant-negative mutations only. There was a group of 22 patients with both types of mutations (5%).Evaluating the prognostic impact, we considered the effect of non-functional and dominant-negative mutations separately. Patients with a non-functional IKZF1 mutation had a reduced overall survival (OS at 5 ys 37% vs 59% in IKZF1-unmutated patients, N=78/404, p=0.001) while dominant-negative mutations had no effect on OS (OS at 5 ys 54% compared to 56%, N=72/410, p=0.95). However, solely present subclonal non-functional mutations did not affect survival (OS at 5 ys 59% compared to 59%, N=23/404). In the dominant-negative group, clonality had no influence on OS (p=0.56, N=45/73/351). Taken together, patients with clonal non-functional IKZF1 had a significantly worse OS compared to those without (0.28 vs 0.59, N=53/427, p<.0001). This was true for patients with standard risk as defined by GMALL risk stratification (0.37 vs 0.68, N=24/243, p.0002). High risk patients also tended to do worse, which however did not reach significance (0.26 vs 0.46, N=30/184, p=.06).With regard to molecular analysis 193 chromosomal fusion sites were characterized. In the great majority (>95%) putative cryptic recombination sites were identified in the near vicinity thus underlining the causal role of the VDJ recombination machinery in the generation of these aberrations.Conclusions: We conducted a detailed and large-scale molecular analysis of intragenic IKZF1 deletions and identified clonal non-functional IKZF1 deletions as adverse prognostic factor in adult and adolescent B cell precursor ALL patients treated according to the GMALL protocols. Subclonal, or dominant negative IKZF1 mutations had no prognostic effect. It remains open for further analysis whether clonal non-functional IKZF1 deletions represent an independent prognostic factor in MRD-based risk stratification. DisclosuresWäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.