Helios serves as a suppression marker to reduce regulatory T cell function in pancreatic cancer patients.

Destabilizing and reprogramming regulatory T (Treg) cells have become a potential strategy to treat tumor. Mounting evidence indicates that the transcription factor Helios is required for the stable differentiation of Treg lineage. Hence, we investigated whether Helios suppression could be a potential treatment option for pancreatic cancer patients. We found that Helios+ cells were predominantly in Foxp3+ Treg cells. By contrast, Foxp3+ Treg cells can be Helios+ or Helios-, but the level of Foxp3 expression was significantly higher in Helios+Foxp3+ Treg cells than in Helios-Foxp3+ Treg cells. Resected pancreatic tumors were highly enriched with both Helios+Foxp3+ Treg cells and Helios-Foxp3+ Treg cells. Also, the proportion of Helios+ cells in total Foxp3+ Treg cells was significantly higher in peripheral blood mononuclear cells (PBMCs) of patients than in PBMCs of healthy controls and further increased in patient tumors. Using shRNA, we knocked down Helios expression without significant downregulation of Foxp3. After Helios knockdown, CD4+CD25+CD127- Treg cells presented significantly lower levels of TGF-ß secretion, lower levels of IL-10 secretion, and higher levels of IFN-γ secretion. In addition, Helios shRNA-transfected CD4+CD25+CD127- Treg cells presented lower capacity to inhibit CD4+CD25-CD127+ T conventional cell proliferation than control shRNA-transfected CD4+CD25+CD127- Treg cells. Of note, CD4+CD25+CD127- Treg cells from pancreatic cancer patients demonstrated higher TGF-ß expression and higher suppression capacity than the cells from healthy controls. Overall, these results suggest that in pancreatic cancer patients, Helios may serve as a candidate to suppress Treg function, which could be used as a target to treat pancreatic cancer.

IL-6 and TNFα Drive Extensive Proliferation of Human Tregs Without Compromising Their Lineage Stability or Function.

Treg therapies are being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs remains controversial. We challenged human Tregs ex-vivo with pro-inflammatory cytokines IL-6 and TNFα and observed greatly enhanced proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads or CD28 superagonist (CD28SA). The cytokine-exposed Tregs maintained high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low IFNγ, IL-4, and IL-17 secretion. Blocking TNF receptor using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNFα without aCD3/28 beads (beadless) as an alternative protocol for therapeutic Treg manufacturing. Metabolomics profiling revealed more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential during beadless Treg expansion. Finally, beadless expanded Tregs maintained suppressive functions in vitro and in vivo. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. This property can be harnessed for therapeutic Treg manufacturing.

Monitoring tumour burden and therapeutic response through analysis of circulating tumour DNA and extracellular RNA in multiple myeloma patients.

Monitoring tumour burden and therapeutic response through analyses of circulating cell-free tumour DNA (ctDNA) and extracellular RNA (exRNA) in multiple myeloma (MM) patients were performed in a Phase Ib trial of 24 relapsed/refractory patients receiving oral azacitidine in combination with lenalidomide and dexamethasone. Mutational characterisation of paired BM and PL samples at study entry identified that patients with a higher number of mutations or a higher mutational fractional abundance in PL had significantly shorter overall survival (OS) (p = 0.005 and p = 0.018, respectively). A decrease in ctDNA levels at day 5 of cycle 1 of treatment (C1D5) correlated with superior progression-free survival (PFS) (p = 0.017). Evaluation of exRNA transcripts of candidate biomarkers indicated that high CRBN levels coupled with low levels of SPARC at baseline were associated with shorter OS (p = 0.000003). IKZF1 fold-change <0.05 at C1D5 was associated with shorter PFS (p = 0.0051) and OS (p = 0.0001). Furthermore, patients with high baseline CRBN coupled with low fold-change at C1D5 were at the highest risk of progression (p = 0.0001). In conclusion, this exploratory analysis has provided the first demonstration in MM of ctDNA for predicting disease outcome and of the utility of exRNA as a biomarker of therapeutic response.

Mild hypothermia provides Treg stability.

Regulatory T cells (Tregs) play crucial role in maintenance of peripheral tolerance. Recent clinical trials confirmed safety and efficacy of Treg treatment of deleterious immune responses. However, Tregs lose their characteristic phenotype and suppressive potential during expansion ex vivo. Therefore, multiple research teams have been studding Treg biology in aim to improve their stability in vitro. In the current paper, we demonstrate that mild hypothermia of 33 °C induces robust proliferation of Tregs, preserves expression of FoxP3, CD25 and Helios, and prevents TSDR methylation during culture in vitro. Tregs expanded at 33 °C have stronger immunosuppressive potential and remarkably anti-inflammatory phenotype demonstrated by the whole transcriptome sequencing. These observations shed new light on impact of temperature on regulation of immune response. We show that just a simple change in temperature can preserve Treg stability, function and accelerate their proliferation, responding to unanswered question- how to preserve Treg stability in vitro.

IKAROS expression in distinct bone marrow cell populations as a candidate biomarker for outcome with lenalidomide-dexamethasone therapy in multiple myeloma.

Immunomodulatory drugs (IMiDs) are a cornerstone in the treatment of multiple myeloma (MM), but specific markers to predict outcome are still missing. Recent work pointed to a prognostic role for IMiD target genes (e.g. CRBN). Moreover, indirect activity of IMiDs on immune cells correlated with outcome, raising the possibility that cell populations in the bone marrow (BM) microenvironment could serve as biomarkers. We therefore analysed gene expression levels of six IMiD target genes in whole BM samples of 44 myeloma patients treated with lenalidomide-dexamethasone. Expression of CRBN (R = 0.30, P = .05), IKZF1 (R = 0.31, P = .04), IRF4 (R = 0.38, P = .01), MCT-1 (R = 0.30, P = .05), and CD147 (R = 0.38, P = .01), but not IKZF3 (R = -0.15, P = .34), was significantly associated with response. Interestingly, IKZF1 expression was elevated in BM environmental cells and thus selected for further investigation by multicolor flow cytometry. High IKAROS protein levels in total BM mononuclear cells (median OS 83.4 vs. 32.2 months, P = .02), CD19+ B cells (median OS 71.1 vs. 32.2 months, P = .05), CD3+ CD8+ T cells (median OS 83.4 vs 19.0 months, P = .008) as well as monocytes (median OS 53.9 vs 18.0 months, P = .009) were associated with superior overall survival (OS). In contrast, IKAROS protein expression in MM cells was not predictive for OS. Our data therefore corroborate the central role of immune cells for the clinical activity of IMiDs and built the groundwork for prospective analysis of IKAROS protein levels in distinct cell populations as a potential biomarker for IMiD based therapies.

Preclinical activity of CPI-0610, a novel small-molecule bromodomain and extra-terminal protein inhibitor in the therapy of multiple myeloma.

Inhibition of the bromodomain and extra-terminal (BET) proteins is a promising therapeutic strategy for various hematologic cancers. Previous studies suggest that BET inhibitors constrain tumor cell proliferation and survival mainly through the suppression of MYC transcription and activity. However, suppression of the transcription of additional genes also contributes to the antitumor activity of BET inhibitors but is less well understood. Here we examined the therapeutic potential of CPI-0610, a potent BET inhibitor currently undergoing phase I clinical testing, in multiple myeloma (MM). CPI-0610 displays potent cytotoxicity against MM cell lines and patient-derived MM cells through G1 cell cycle arrest and caspase-dependent apoptosis. CPI-0610-mediated BET inhibition overcomes the protective effects conferred by cytokines and bone marrow stromal cells. We also confirmed the in vivo efficacy of CPI-0610 in a MM xenograft mouse model. Our study found IKZF1 and IRF4 to be among the primary targets of CPI-0610, along with MYC. Given that immunomodulatory drugs (IMiDs) stabilize cereblon and facilitate Ikaros degradation in MM cells, we combined it with CPI-0610. Combination studies of CPI-0610 with IMiDs show in vitro synergism, in part due to concomitant suppression of IKZF1, IRF4 and MYC, providing a rationale for clinical testing of this drug combination in MM patients.

High IKZF1/3 protein expression is a favorable prognostic factor for survival of relapsed/refractory multiple myeloma patients treated with lenalidomide.

The aim of this study is to assess nucleoprotein expression of IKZF1/3 in patients with relapsed/refractory multiple myeloma (MM) who received lenalidomide-based therapy and correlated them with their clinical outcomes. A total of 50 patients diagnosed with MM were entered in the study with the median follow-up of 86.4 months. By immunohistochemistry (IHC), IKZF1 and IKZF3 were expressed in 72 and 58% of the cases, respectively. IKZF1 and IKZF3 expressions were associated with longer median progression free survival (P = 0.0029 and P < 0.0001) and overall survival (P = 0.0014 and P < 0.0001). IKZF3 expression also appears predicted a favorable response to the lenalidomide-based therapy.

Dissecting the regulatory microenvironment of a large animal model of non-Hodgkin lymphoma: evidence of a negative prognostic impact of FOXP3+ T cells in canine B cell lymphoma.

The cancer microenvironment plays a pivotal role in oncogenesis, containing a number of regulatory cells that attenuate the anti-neoplastic immune response. While the negative prognostic impact of regulatory T cells (Tregs) in the context of most solid tissue tumors is well established, their role in lymphoid malignancies remains unclear. T cells expressing FOXP3 and Helios were documented in the fine needle aspirates of affected lymph nodes of dogs with spontaneous multicentric B cell lymphoma (BCL), proposed to be a model for human non-Hodgkin lymphoma. Multivariable analysis revealed that the frequency of lymph node FOXP3(+) T cells was an independent negative prognostic factor, impacting both progression-free survival (hazard ratio 1.10; p = 0.01) and overall survival (hazard ratio 1.61; p = 0.01) when comparing dogs showing higher than the median FOXP3 expression with those showing the median value of FOXP3 expression or less. Taken together, these data suggest the existence of a population of Tregs operational in canine multicentric BCL that resembles thymic Tregs, which we speculate are co-opted by the tumor from the periphery. We suggest that canine multicentric BCL represents a robust large animal model of human diffuse large BCL, showing clinical, cytological and immunophenotypic similarities with the disease in man, allowing comparative studies of immunoregulatory mechanisms.

Identification of different Ikaros cDNA transcripts in Philadelphia-positive adult acute lymphoblastic leukemia by a high-throughput capillary electrophoresis sizing method.

BACKGROUND: Ikaros is the prototypic member of a Kruppel-like zinc finger transcription factor subfamily that is required for normal hematopoietic cell differentiation and proliferation, particularly in the lymphoid lineages. Alternative splicing can generate multiple Ikaros isoforms that lack different numbers of exons and have different functions. Shorter isoforms, which lack the amino-terminal domain that mediates sequence-specific DNA binding, exert a dominant negative effect and inhibit the ability of longer heterodimer partners to bind DNA. DESIGN AND METHODS: In this study, we developed a high-throughput capillary electrophoresis sizing method to detect and quantify different Ikaros cDNA transcripts. RESULTS: We demonstrated that Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressed high levels of the non-DNA-binding isoform Ik6 that was generated following IKZF1 genomic deletions (19/46 patients, 41%). Furthermore, a recurring 60 bp insertion immediately upstream of exon 5, at the exon 3/exon 5 junction, was frequently detected in the Ik2 and Ik4 isoforms. This insertion occurred either alone or together with an in-frame ten amino acid deletion that was due to a 30 bp loss at the end of exon 7. Both the alterations are due to the selection of alternative cryptic splice sites and have been suggested to cause impaired DNA-binding activity. Non-DNA-binding isoforms were localized in the cytoplasm whereas the DNA-binding isoforms were localized in the nucleus. CONCLUSIONS: Our findings demonstrate that both aberrant splicing and genomic deletion leading to different non-DNA-binding Ikaros cDNA transcripts are common features of Philadelphia chromosome-positive acute lymphoblastic leukemia.

Ikaros DNA-binding proteins as integral components of B cell developmental-stage-specific regulatory circuits.

Ikaros DNA-binding proteins are critical for the development of lymphocytes and other hematopoietic lineages, but it remains unclear how they cooperate with other regulators of signaling and transcription to achieve ordered gene expression during development. Here, we show that Ikaros proteins regulate the pre-BCR component lambda5 in a stage-specific manner. In pre-BI cells, Ikaros modulated lambda5 expression in competition with the transcriptional activator EBF. This required Ikaros binding to the Igll1 (lambda5) promoter and was abolished either by mutation of the Ikaros DNA-binding domain or by deletion of a single Ikaros site from the Igll1 promoter. At the transition from the pre-BI to pre-BII stage, the expression of the Ikaros family member Aiolos was upregulated and required for the efficient silencing of Igll1. Aiolos expression was controlled by pre-BCR signals via the adaptor protein SLP-65. Thus, pre-BCR signaling regulates Aiolos and the silencing of Igll1 via a developmental-stage-specific feedback loop.

An essential role for the hematopoietic transcription factor Ikaros in hypothalamic-pituitary-mediated somatic growth.

Ikaros transcription factors play critical functions in the control of lymphohematopoiesis and immune regulation. Family members contain multiple zinc fingers that mediate DNA binding and homooligomerization or heterooligomerization. Ikaros is abundantly expressed in pituitary mammosomatotrophs, where it deacetylates histone 3 sites on the proximal growth hormone (GH) promoter to silence gene expression. Ikaros-null mice display stunted growth with reduced circulating levels of the GH target factor insulin-like growth factor I (IGF-I). Ikaros-deficient mice have small anterior pituitary glands with a disproportionately reduced somatotroph population. Systemic administration of GH results in increased IGF-I levels and enhanced somatic growth. In contrast, reconstitution with WT lymphocytes was not sufficient to rescue the stunted growth phenotype of Ikaros-deficient mice. Ikaros was identified in mouse hypothalamic arcuate nuclei, where it colocalized with GH-releasing hormone (GHRH); in contrast, Ikaros-null mice lack GHRH immunoreactivity in the hypothalamus. Overexpression of Ikaros enhanced GHRH promoter activity and induced endogenous GHRH gene expression. These findings unmask a wider role for Ikaros in the neuroendocrine system, highlighting a critical contribution to the development of the hypothalamic-pituitary somatotrophic axis.

BCR-ABL1 induces aberrant splicing of IKAROS and lineage infidelity in pre-B lymphoblastic leukemia cells.

Pre-B lymphoblastic leukemia cells carrying a BCR-ABL1 gene rearrangement exhibit an undifferentiated phenotype. Comparing the genome-wide gene expression profiles of normal B-cell subsets and BCR-ABL1+ pre-B lymphoblastic leukemia cells by SAGE, the leukemia cells show loss of B lymphoid identity and aberrant expression of myeloid lineage-specific molecules. Consistent with this, BCR-ABL1+ pre-B lymphoblastic leukemia cells exhibit defective expression of IKAROS, a transcription factor needed for early lymphoid lineage commitment. As shown by inducible expression of BCR-ABL1 in human and murine B-cell precursor cell lines, BCR-ABL1 induces the expression of a dominant-negative IKAROS splice variant, termed IK6. Comparing matched leukemia sample pairs from patients before and during therapy with the BCR-ABL1 kinase inhibitor STI571 (Imatinib), inhibition of BCR-ABL1 partially corrected aberrant expression of IK6 and lineage infidelity of the leukemia cells. To elucidate the contribution of IK6 to lineage infidelity in BCR-ABL1+ cell lines, IK6 expression was silenced by RNA interference. Upon inhibition of IK6, BCR-ABL1+ leukemia cells partially restored B lymphoid lineage commitment. Therefore, we propose that BCR-ABL1 induces aberrant splicing of IKAROS, which interferes with lineage identity and differentiation of pre-B lymphoblastic leukemia cells.