A multiply redundant genetic switch 'locks in' the transcriptional signature of regulatory T cells.

The transcription factor Foxp3 participates dominantly in the specification and function of Foxp3(+)CD4(+) regulatory T cells (T(reg) cells) but is neither strictly necessary nor sufficient to determine the characteristic T(reg) cell signature. Here we used computational network inference and experimental testing to assess the contribution of other transcription factors to this. Enforced expression of Helios or Xbp1 elicited distinct signatures, but Eos, IRF4, Satb1, Lef1 and GATA-1 elicited exactly the same outcome, acting in synergy with Foxp3 to activate expression of most of the T(reg) cell signature, including key transcription factors, and enhancing occupancy by Foxp3 at its genomic targets. Conversely, the T(reg) cell signature was robust after inactivation of any single cofactor. A redundant genetic switch thus 'locked in' the T(reg) cell phenotype, a model that would account for several aspects of T(reg) cell physiology, differentiation and stability.

Immunomodulatory drugs downregulate IKZF1 leading to expansion of hematopoietic progenitors with concomitant block of megakaryocytic maturation.

The immunomodulatory drugs, lenalidomide and pomalidomide yield high response rates in multiple myeloma patients, but are associated with a high rate of thrombocytopenia and increased risk of secondary hematologic malignancies. Here, we demonstrate that the immunomodulatory drugs induce self-renewal of hematopoietic progenitors and upregulate megakaryocytic colonies by inhibiting apoptosis and increasing proliferation of early megakaryocytic progenitors via down-regulation of IKZF1. In this process, the immunomodulatory drugs degrade IKZF1 and subsequently down-regulate its binding partner, GATA1. This results in the decrease of GATA1 targets such as ZFPM1 and NFE2, leading to expansion of megakaryocytic progenitors with concomitant inhibition of maturation of megakaryocytes. The down-regulation of GATA1 further decreases CCND1 and increases CDKN2A expression. Overexpression of GATA1 abrogated the effects of the immunomodulatory drugs and restored maturation of megakaryocytic progenitors. Our data not only provide the mechanism for the immunomodulatory drugs induced thrombocytopenia but also help to explain the higher risk of secondary malignancies and long-term cytopenia induced by enhanced cell cycling and subsequent exhaustion of the stem cell pool.

Analysis of Helios gene expression and Foxp3 TSDR methylation in the newly diagnosed Rheumatoid Arthritis patients.

BACKGROUND: The control of auto-reactive cells is defective in rheumatoid arthritis (RA). Regulatory T (Treg) cells which play a key role in the modulation of immune responses have an impaired function in RA. Foxp3 is a master regulator of Treg cells which its expression is under the tight control of epigenetic mechanisms. In the current study, we analyzed the epigenetic modulation of the Foxp3 Treg-specific demethylated region (TSDR) and Helios gene expression to determine Treg cells alteration in RA patients. METHODS: We have recruited 20 newly diagnosed patients with RA and 41 healthy controls in our study. The measurement of Foxp3 and Helios gene expression was performed by the real-time PCR technique and the methylation level of TSDR was analyzed by bisulfite treatment and quantitative methylation-specific PCR (Q-MSP). RESULTS: We found that RA patients had significantly lower level of Foxp3 gene expression and TSDR demethylation compared to healthy subjects (P < 0.001 and P = 0.006, respectively). Inversely, the Helios gene expression was elevated significantly in RA patients group (P = 0.048). We also observed a significant correlation between Foxp3 and Helios gene expression (P = 0.016) as well as a significant correlation between FoxP3 expression and demethylation rate of TSDR (P = 0.010). CONCLUSION: Our results suggested that both epigenetic modifications and Helios gene expression may have important roles in the pathogenesis of RA through their effects on Foxp3 gene expression.

Molecular characterization of acute lymphoblastic leukemia with high CRLF2 gene expression in childhood.

BACKGROUND: A high-level expression of the CRLF2 gene is frequent in precursor B-cell acute lymphoblastic leukemia (pB-ALL) and can be caused by different genetic aberrations. The presence of the most frequent alteration, the P2RY8/CRLF2 fusion, was shown to be associated with a high relapse incidence in children treated according to ALL-Berlin-Frankfurt-Münster (BFM) protocols, which is poorly understood. Moreover, the frequency of other alterations has not been systematically analyzed yet. PROCEDURE: CRLF2 mRNA expression and potential genetic aberrations causing a CRLF2 high expression were prospectively assessed in 1,105 patients treated according to the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP)-BFM ALL 2009 protocol. Additionally, we determined copy number alterations in selected B-cell differentiation genes for all CRLF2 high-expressing pB-ALL cases, as well as JAK2 and CRLF2 mutations. RESULTS: A CRLF2 high expression was detected in 26/178 (15%) T-cell acute lymphoblastic leukemia (T-ALL) cases, 21 of them (81%) had been stratified as high-risk patients by treatment response. In pB-ALL, a CRLF2 high expression was determined in 91/927 (10%) cases; the P2RY8/CRLF2 rearrangement in 44/91 (48%) of them, supernumerary copies of CRLF2 in 18/91 (20%), and, notably, the IGH/CRLF2 translocation was detected in 16/91 (18%). Remarkably, 7 of 16 (44%) patients with IGH/CRLF2 translocation had already relapsed. P2RY8/CRLF2- and IGH/CRLF2-positive samples (70 and 94%, respectively) were characterized by a high frequency of additional deletions in B-cell differentiation genes such as IKZF1 or PAX5. CONCLUSION: Our data suggest that this high frequency of genetic aberrations in the context of a high CRLF2 expression could contribute to the high risk of relapse in P2RY8/CRLF2- and IGH/CRLF2-positive ALL.

Th1/Th17 plasticity is a marker of advanced ß cell autoimmunity and impaired glucose tolerance in humans.

Upregulation of IL-17 immunity and detrimental effects of IL-17 on human islets have been implicated in human type 1 diabetes. In animal models, the plasticity of Th1/Th17 cells contributes to the development of autoimmune diabetes. In this study, we demonstrate that the upregulation of the IL-17 pathway and Th1/Th17 plasticity in peripheral blood are markers of advanced ß cell autoimmunity and impaired ß cell function in human type 1 diabetes. Activated Th17 immunity was observed in the late stage of preclinical diabetes in children with ß cell autoimmunity and impaired glucose tolerance, but not in children with early ß cell autoimmunity. We found an increased ratio of IFN-γ/IL-17 expression in Th17 cells in children with advanced ß cell autoimmunity, which correlated with HbA1c and plasma glucose concentrations in an oral glucose tolerance test, and thus impaired ß cell function. Low expression of Helios was seen in Th17 cells, suggesting that Th1/Th17 cells are not converted thymus-derived regulatory T cells. Our results suggest that the development of Th1/Th17 plasticity may serve as a biomarker of disease progression from ß cell autoantibody positivity to type 1 diabetes. These data in human type 1 diabetes emphasize the role of Th1/Th17 plasticity as a potential contributor to tissue destruction in autoimmune conditions.

Allogeneic Mature Human Dendritic Cells Generate Superior Alloreactive Regulatory T Cells in the Presence of IL-15.

Expansion of Ag-specific naturally occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloantigen-specific nTregs. A highly enriched nTreg fraction (CD4(+)CD25(bright)CD127(-) T cells) was alloantigen-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDCs), or PBMCs. The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the Treg-specific demethylation region of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4, and cytokines. In addition, the Ag-specific suppressive capacity of these expanded nTregs was tested. Allogeneic mature moDCs and skin-derived DCs were superior in inducing nTreg expansion compared with immature moDCs or PBMCs in an HLA-DR- and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2 could facilitate optimal mature moDC-induced nTreg expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloantigen-induced proliferation without significant suppression of completely HLA-mismatched, Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the Treg-specific demethylation region within the FOXP3 gene and highly expressed of FOXP3, HELIOS, and CTLA4. A minority of the expanded nTregs produced IL-10, IL-2, IFN-γ, and TNF-α, but few IL-17-producing nTregs were found. Next-generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs. Human allogeneic mature moDCs are highly efficient stimulator cells, in the presence of exogenous IL-15, for expansion of stable alloantigen-specific nTregs with superior suppressive function.

Insertional mutagenesis and deep profiling reveals gene hierarchies and a Myc/p53-dependent bottleneck in lymphomagenesis.

Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a 'progression network' that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes (Gfi1, Notch1). Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer genetics and the safety of vector-mediated gene therapy.

Regulatory T cells suppress CD4+ T cells through NFAT-dependent transcriptional mechanisms.

Regulatory T cells (Tregs) control autoreactive T cells by inhibiting activation-induced proliferation and cytokine expression. The molecular mechanisms responsible for the inactivation of effector T cells by Tregs remain yet to be fully characterized. We report that T-helper cells stimulated in the presence of Tregs quickly activate NFAT1 and have increased NFAT1-dependent expression of the transcription repressor Ikaros. NFAT1 deficiency or dominant-negative Ikaros compromises Treg-mediated inhibition of T-helper cells in vitro and in vivo. Thus, our results place NFAT-dependent mechanisms as general regulators of T-cell tolerance and show that Treg-mediated suppression of T-helper cells results from the activation of NFAT-regulated gene expression.

Forkhead box protein 3(+) regulatory T cells and Helios(+) subset in perinatally acquired HIV.

Forkhead box protein 3 (FoxP3)(+) regulatory T cells (Tregs ) are important not only in regulating the development of autoimmune conditions, but also in chronic infectious diseases. Given their cardinal function in suppressing immune activation, research has focused upon whether they play a detrimental role in chronic infections, particularly HIV. While the role of Tregs in HIV has been investigated intensively, it remains an unresolved topic. However, it is generally accepted that Tregs are susceptible to HIV infection and are preferentially preserved over conventional CD4(+) T cells. It is unknown whether the peripheral-induced or the thymic-derived Tregs are more susceptible to HIV cytotoxicity. It has been recognized that Tregs can be segregated into two subsets based on Helios expression, with the vast majority being Helios(+) . This study examines the impact of HIV infection on total Tregs and their Helios subsets in a perinatal-acquired HIV-infected paediatric population. The finding indicates a selective expansion or survival of Tregs in association with CD4 depletion and increased viraemia. The Helios(+) and Helios(-) subsets within Tregs appear to be equally affected. However, the Helios(+) Tregs seem to be more preserved in patients with low CD4(+) ≤ 25% and detectable plasma HIV RNA >20 copies/ml. In this group, the frequencies of Tregs are increased, but their numbers appear insufficient to restrain immune activation. In conclusion, our findings suggest that both Helios subsets of Tregs are susceptible to HIV infection and are preferentially preserved compared to conventional CD4(+) T cells.

Oligodeoxynucleotides stabilize Helios-expressing Foxp3+ human T regulatory cells during in vitro expansion.

Foxp3(+) regulatory T cells (Tregs) maintain self-tolerance and adoptive therapy, and using Foxp3(+) Tregs has been proposed as treatment for autoimmune diseases. The clinical use of Tregs will require large numbers of cells and methods for in vitro expansion of Tregs are being developed. Foxp3(+) Tregs can be divided into 2 subpopulations based on expression of the transcription factor, Helios. Foxp3(+)Helios(+) Tregs (70%) are thymic-derived, whereas Foxp3(+)Helios(-) Tregs (30%) are induced in the periphery. Foxp3(+)Helios(+) Tregs differ from Foxp3(+)Helios(-) Tregs in terms of epigenetic changes at the Foxp3 locus, their capacity to produce effector cytokines, and their stability of Foxp3 expression on days to weeks of expansion in vitro. Addition of a 25 mer DNA oligonucleotide of random composition for a short period during the expansion of Foxp3(+) Tregs in vitro results in prolonged stabilization of the Foxp3(+)Helios(+) subpopulation and yields an optimal population for use in cellular biotherapy.

Expression of Helios in peripherally induced Foxp3+ regulatory T cells.

The transcription factor Helios has been reported to be a marker of regulatory T cells (Treg) of thymic origin, distinguishing them from Treg induced in the periphery (iTreg). In this study, we demonstrate Helios expression in Foxp3(+) iTreg, both in vitro and in vivo. Following i.v. peptide injection, in vivo Helios expression in adoptively transferred TCR transgenic T cells was more rapid than Foxp3 induction but less stable at later time points without a second injection of peptide. Our in vitro data suggest that APC influence Helios expression in a manner distinct from stimuli required for Foxp3 induction. Thus, Helios expression in iTreg may reflect the context of stimulation during Foxp3 induction. In summary, the robust Helios expression we observe in iTreg precludes its use as a marker of thymic Treg.

Adult T-cell leukemia cells are characterized by abnormalities of Helios expression that promote T cell growth.

Molecular abnormalities involved in the multistep leukemogenesis of adult T-cell leukemia (ATL) remain to be clarified. Based on our integrated database, we focused on the expression patterns and levels of Ikaros family genes, Ikaros, Helios, and Aiolos, in ATL patients and HTLV-1 carriers. The results revealed profound deregulation of Helios expression, a pivotal regulator in the control of T-cell differentiation and activation. The majority of ATL samples (32/37 cases) showed abnormal splicing of Helios expression, and four cases did not express Helios. In addition, novel genomic loss in Helios locus was observed in 17/168 cases. We identified four ATL-specific short Helios isoforms and revealed their dominant-negative function. Ectopic expression of ATL-type Helios isoform as well as knockdown of normal Helios or Ikaros promoted T-cell growth. Global mRNA profiling and pathway analysis showed activation of several signaling pathways important for lymphocyte proliferation and survival. These data provide new insights into the molecular involvement of Helios function in the leukemogenesis and phenotype of ATL cells, indicating that Helios deregulation is one of the novel molecular hallmarks of ATL.

In vivo evidence for unbiased Ikaros retinal lineages using an Ikaros-Cre mouse line driving clonal recombination.

BACKGROUND: We showed previously that the transcription factor Ikaros is expressed in early but not late retinal progenitors cells (RPCs), and is necessary and sufficient for the production of early-born neurons. Preliminary evidence using retinal explant cultures qualitatively suggested that Ikaros-positive RPCs might share a common lineage with Ikaros-negative RPCs. RESULTS: To explore further this question in vivo in a quantitative manner, we generated BAC transgenic mouse lines expressing Cre recombinase under the regulatory elements of the Ikaros gene, and crossed them with Cre reporter lines. Different transgenic lines labeled a variable number of RPCs, resulting in either dense or sparse radial arrays of reporter-positive progenies. Analysis of over 800 isolated cell arrays, which are most likely clones, confirmed that Ikaros-expressing RPCs generate both early- and late-born cell types in the same lineage, and that the overall cell composition of the arrays closely resembles that of the population of the mature retina. Interestingly, another sparse line did not label arrays, but appeared to specifically reflect Ikaros postmitotic expression in amacrine and ganglion cells. CONCLUSIONS: These mouse lines confirm the unbiased potential of the Ikaros lineage in vivo and provide novel tools for clonal lineage tracing and single neuron tracking in the retina.

Cell cycle-specific function of Ikaros in human leukemia.

BACKGROUND: 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. PROCEDURES: Electromobility shift assay (EMSA), confocal microscopy, and phosphopeptide mapping were used to study Ikaros function during different stages of the cell cycle. RESULTS: 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. CONCLUSIONS: 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.

Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells.

Helios, a member of the Ikaros transcription factor family, is preferentially expressed at the mRNA level by regulatory T cells (Treg cells). We evaluated Helios protein expression using a newly generated mAb and demonstrated that it is expressed in all thymocytes at the double negative 2 stage of thymic development. Although Helios was expressed by 100% of CD4(+)CD8(-)Foxp3(+) thymocytes, its expression in peripheral lymphoid tissues was restricted to a subpopulation ( approximately 70%) of Foxp3(+) T cells in mice and humans. Neither mouse nor human naive T cells induced to express Foxp3 in vitro by TCR stimulation in the presence of TGF-beta expressed Helios. Ag-specific Foxp3(+) T cells induced in vivo by Ag feeding also failed to express Helios. Collectively, these results demonstrate that Helios is potentially a specific marker of thymic-derived Treg cells and raises the possibility that a significant percentage of Foxp3(+) Treg cells are generated extrathymically.

Expanded subpopulation of FoxP3+ T regulatory cells in renal cell carcinoma co-express Helios, indicating they could be derived from natural but not induced Tregs.

Conversion of conventional T cells into T regulatory cells (Tregs) has been proposed as a potential mechanism for Treg expansion in cancer. However, this evidence is supported by in vitro or mouse model studies with no data from in vivo or human studies to support its role in enriching peripheral and tumor-infiltrating Tregs. Recent work has shown that induced FoxP3+ Tregs (iTregs) do not express Helios; an Ikaros family transcription factor. We analyzed peripheral blood samples from untreated renal cell carcinoma (RCC) patients and following interleukin (IL)-2 treatment for the expression of FoxP3 and Helios. Our work shows that expanded peripheral FoxP3+ Tregs in untreated RCC patients co-express Helios. Interestingly, IL-2 administration results in expansion of FoxP3+ Helios+ natural Tregs (nTregs) significantly more than FoxP3+ Helios- iTregs. Our work shows that the increased FoxP3+ Treg subpopulation in RCC patients co-express Helios, indicating that they could be derived from natural but not induced Tregs.

Integration of high-throughput reporter assays identify a critical enhancer of the Ikzf1 gene.

The Ikzf1 locus encodes the lymphoid specific transcription factor Ikaros, which plays an essential role in both T and B cell differentiation, while deregulation or mutation of IKZF1/Ikzf1 is involved in leukemia. Tissue-specific and cell identity genes are usually associated with clusters of enhancers, also called super-enhancers, which are believed to ensure proper regulation of gene expression throughout cell development and differentiation. Several potential regulatory regions have been identified in close proximity of Ikzf1, however, the full extent of the regulatory landscape of the Ikzf1 locus is not yet established. In this study, we combined epigenomics and transcription factor binding along with high-throughput enhancer assay and 4C-seq to prioritize an enhancer element located 120 kb upstream of the Ikzf1 gene. We found that deletion of the E120 enhancer resulted in a significant reduction of Ikzf1 mRNA. However, the epigenetic landscape and 3D topology of the locus were only slightly affected, highlighting the complexity of the regulatory landscape regulating the Ikzf1 locus.

RNA-Binding Protein ZFP36L2 Downregulates Helios Expression and Suppresses the Function of Regulatory T Cells.

The zinc finger protein 36-like 2, ZFP36L2, is a member of a small family of RNA-binding proteins composed by ZFP36 (also known as tristetraprolin, TTP), ZFP36L1 and ZFP36L2 in humans, with corresponding murine orthologs. These proteins bind to adenine uridine-rich element (ARE) in the 3'untranslated region of target messenger RNA and stimulate target degradation. ZFP36 functions as an anti-inflammatory modulator in murine models of inflammatory diseases by down-regulating the production of inflammatory cytokines such as tumor necrosis factor-α. However, how ZFP36L1 and ZFP36L2 alter the function of CD4+ T cells is not completely understood. We addressed this issue by searching for the target genes of ZFP36L2 by comprehensive transcriptome analysis. We observed that ZFP36L2 is highly expressed in naïve CD4+ T cells; however, when CD4+ T cells are stimulated through their T cell receptors, ZFP36L2 expression is rapidly reduced in both humans and mice. Among CD4+ T cell populations, the expression levels of ZFP36L2 in regulatory T cells (Tregs) were significantly lower than those in naïve or effector CD4+ T cells. RNA-sequence analysis revealed that the forced expression of ZFP36L2 decreased Ikzf2 (encoding Helios) expression in Foxp3+ Tregs and inhibited the ability of induced Tregs (iTregs). ZFP36L2 directly bound to and destabilized the 3'untranslated region of Ikzf2 mRNA, which contains AU-rich elements. These results indicate that ZFP36L2 reduces the expression of Ikzf2 and suppresses iTreg function, raising the interesting possibility that the inhibition of ZFP36L2 in iTregs could be a therapeutic strategy for autoimmune diseases.

Cyclosporine enhances the sensitivity to lenalidomide in MDS/AML in vitro.

Our previous study revealed that expression of G protein-coupled receptor 68 (GPR68) was upregulated in MDSL cells, a cell line representing myelodysplastic syndromes (MDS), in response to lenalidomide (LEN), and mediated a calcium/calpain proapoptotic pathway. Isx, a GPR68 agonist, enhanced the sensitivity to LEN in MDSL cells. The fact that Isx is not a U.S. Food and Drug Administration-approved drug prompts us to look for alternative candidates that could enhance the sensitivity of LEN in MDS as well as other hematologic malignancies, such as acute myeloid leukemia (AML). In the study described here, we found that regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of calcineurin (CaN), was upregulated in MDSL cells in response to LEN, possibly through degradation of IKZF1. Consistently, cyclosporin (Cys), a pharmacological inhibitor of CaN, inhibited the activity of CaN and induced apoptosis in MDSL cells, indicating that CaN provided a prosurvival signal in MDSL cells. In addition, Cys enhanced the cytotoxic effect of LEN in MDS/AML cell lines as well as primary bone marrow cells from MDS patients and AML patient-derived xenograft models. Intriguingly, pretreatment with LEN reversed the suppressive effect of Cys on T-cell activation. Our study suggests a novel mechanism of action of LEN in mediating cytotoxicity in MDS/AML via upregulation of RCAN1, thus inhibiting the CaN prosurvival pathway. Our study also suggests that Cys enhances the sensitivity to LEN in MDS/AML cells without compromising T-cell activation.