Ets1 Enhances Context-Dependent Notch1 Activity in T-Cell Leukemia

Abstract The discovery of NOTCH1 as the most frequently mutated oncogene in T-ALL patients raised hopes for targeted therapy in this cancer. Unfortunately, in clinical trials, the pan-Notch inhibitor GSI caused excessive GI toxicity. Mice treated continuously with GSI die from intestinal stem cell loss and severe intestinal secretory cell metaplasia. Intermittent dosing of GSI is tolerable, but has weak anti-cancer effects. Thus, the challenge has been to find ways to selectively disable Notch in T-ALL. Our idea to meet this challenge stems from work by others showing that Notch cannot activate enhancers by itself. Notch requires a favorable "chromatin context" at its enhancers that is created by cooperating transcription factors. In theory, one could target cell-specific factors at these enhancers in order to avoid the intolerable effects of pan-Notch inhibition. In support of this, others showed that ubiquitous deletion of the T-cell specific Notch-dependent Myc enhancer in mice impairs T-ALL proliferation and thymopoiesis, but has no effect on other tissues. We previously showed that the transcriptional coactivator Zmiz1 is a direct cofactor of Notch1 that selectively promotes Notch activity at the T-cell Myc enhancer. However, it was unclear what other factors promote context-dependent Notch activity. Ets1 is an attractive candidate. It can bind nucleosome-occupied regions in T-cell precursors and most Notch response elements in T-ALL cells, including the T-cell MYC enhancer. To investigate its importance, we generated conditional Ets1 knockout mice. Deletion of Ets1 in hematopoietic cells using the VavCre transgene caused a 21-fold loss of thymocytes starting at the earliest stage. This was 4-fold more severe than the loss of thymocytes in Notch-deficient mice. Deletion of Ets1 using a ubiquitous tamoxifen-inducible Cre caused a Notch loss-of-function phenotype in the intestine with a 1.4 to 2.3-fold increase in goblet cells. This was milder than the effects of GSI (3.3 to 4.2-fold increase). ~64% of the Ets1-deleted mice died from unclear causes. In vivo deletion of Ets1 in Notch1-induced murine T-ALLs reduced blast counts by 30-fold and prolonged survival. In a panel of human T-ALL cell lines, on average, knockdown with two different shEts1 reduced proliferation by 2 and 9-fold respectively over ~1.5 weeks of culture. This was superior to the effects of GSI (up to 2-fold inhibition). A small molecule inhibitor of Usp9x, the deubiquitinase of Ets1, induced Ets1 protein degradation and impaired T-ALL cell proliferation with submicromolar GI50. In PDX models, shEts1 reduced circulating blasts by 44-fold and prolonged survival. To identify the mechanism by which Ets1 promotes T-ALL, we performed endogenous co-IP assays, which showed that Ets1 interacts with Notch1 and its cofactor Zmiz1. Further, Ets1 binding by ChIP correlated with Zmiz1 binding (R2=0.93). Knockdown of Ets1 reduced Zmiz1, Ets1, and Notch1 binding to enhancers of major T-ALL oncogenes, MYC and IL7R. RNA-Seq showed that Ets1 co-regulates the expression of ~30% of Notch1 target genes. Multiple MSigDB enrichment analyses of both Ets1 and Notch-regulated genes showed that the MYC and MTORC pathways were the #1 or #2 most enriched list. Enforced expression of Myc partially rescued the proliferation of human T-ALL cell lines deprived of Ets1. Based on these data, we predicted that Ets1 inhibition would sensitize enhancers to Notch inhibition. Accordingly, Ets1 withdrawal promoted the effects of GSI in repressing Myc expression and cell proliferation. Further, in our mouse model of Notch-induced T-ALL, Ets1 deletion in combination with intermittent doses of GSI reduced blast counts and prolonged survival more effectively than either treatment alone. Our data support an emerging model in which cofactors like Ets1 create a favorable chromatin context for Notch1 to activate a subset of response elements. The context dependence of Ets1 action, which promotes certain oncogenic signals of Notch1 in T cells, might be clinically relevant. Ets1 deprivation inhibited thymopoiesis and leukemic proliferation more effectively and with less intestinal toxicity than Notch deprivation. Our data suggest that inhibiting Ets1, possibly through targeted protein degradation, would combat important drivers of the Notch pathway with reduced adverse effects linked to pan-Notch inhibition. Disclosures No relevant conflicts of interest to declare.

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Myeloid Leukemias Directly Suppress T Cell Proliferation Through STAT3 and Arginase Pathways

Blood ◽

10.1182/blood.v122.21.3885.3885 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3885-3885 ◽

Cited By ~ 2

Author(s):

Samantha Miner ◽

Sawa Ito ◽

Kazushi Tanimoto ◽

Nancy F. Hensel ◽

Fariba Chinian ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Cell Lines ◽

Cd4 T Cells ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Suppressor Cells ◽

T Cell Proliferation ◽

Immune Editing

Abstract The immune-editing effect of myeloid leukemia has recently been reported in several studies. We previously demonstrated that the K562 leukemia-derived cell line suppresses T cell proliferation, which suggests that myeloid leukemia may function in a similar way to myeloid derived suppressor cells (MDSC). While the mechanism of suppression in leukemia is not fully understood, recent murine and human studies suggest that the STAT3 and arginase pathways play a key role in the immunosuppressive function of MDSC. We hypothesized that myeloid leukemia utilizes the MDSC STAT3 and arginase pathway to evade immune control, and block anti-leukemic immune responses. To evaluate the suppressive capacity of myeloid leukemia on T cell proliferation, we isolated CD34+ blasts and myeloid derived suppressor cells (MDSC: CD11b+CD14+) from blood of primary leukemia samples by FACS sorting (n=5). These cells were co-cultured with CFSE-labeled CD4+ T cells (n=9), previously isolated from healthy donor PBMCs using an automated cell separator (RoboSep). After stimulating with CD3/CD28 Dynabeads (Invitrogen, New York, USA) for 72 hours, proliferation was measured by CFSE dilution of the viable cell population. In three myeloid leukemias studied, CD4+ T cell proliferation was significantly suppressed in the presence of primary CD34 blasts and MDSC cells (p<0.001). Interestingly, CD34 blasts demonstrated a greater suppressive effect on T cells compared to MDSC cells for these samples (not statistically significant p=0.61). Next we repeated the proliferation assay using five leukemia cell lines: THP-1 and AML1 (derived from AML), K562 and CML1 (derived from CML), and the Daudi lymphoid-derived leukemia cell line. After staining with cell tracer dye and irradiating 100Gy, the cells were co-incubated with CFSE-labeled CD4+ T cells from healthy volunteers (n=6). We found that CD4+ T cell proliferation in the presence of the myeloid leukemia cell lines was significantly suppressed (mean proliferation 5.7±0.9% to 26.1±10.7%: p<0.0001 to 0.05) compared to lymphoid cell lines (mean proliferation 76.3±8.2%: p>0.05), consistent with the results obtained with the primary leukemia samples. To evaluate the impact of STAT3 and arginase on the immunosuppressive function of myeloid leukemia, the five cell lines were primed overnight with either arginase inhibitor (N(ω)-Hydroxy-nor-L-arginine; EMD Biosciences, Inc., California, USA) or two STAT3 inhibitors (STAT3 Inhibitor VI or Cucurbitacin I; EMD Millipore, Massachusetts, USA). Then, CD4+ T cells from healthy donors (n=3) were cultured with either (1) leukemia without any inhibitor (2) leukemia in the presence of inhibitor (3) leukemia primed with inhibitor. Priming leukemia with arginase inhibitor and STAT3 inhibitors almost completely abrogated their suppressive effect of T cell proliferation (p<0.001). We conclude that myeloid leukemia, like MDSC, directly immunosuppresses T cells, through STAT-3 and arginase. This finding may underlie the immune-editing of T cells by myeloid leukemia. Our results suggest that STAT3 inhibitors could be used to augment leukemia-targeted immunotherapy. Further investigation of T cell biology within the leukemia microenvironment is needed to further define immune editing mechanisms in myeloid leukemia. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures: No relevant conflicts of interest to declare.

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Hydroxyurea Is Most Suitable for Cytoreduction of AML Prior to CD33/CD3 Bispecific BiTE® Antibody (AMG 330) Therapy: Uncompromised T-Cell Proliferation Ex-Vivo and CD33 Upregulation on AML Cells

Blood ◽

10.1182/blood.v124.21.986.986 ◽

2014 ◽

Vol 124 (21) ◽

pp. 986-986 ◽

Cited By ~ 1

Author(s):

Christina Krupka ◽

Franziska Brauneck ◽

Felix S Lichtenegger ◽

Peter Kufer ◽

Roman Kischel ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Cell Lines ◽

Cell Function ◽

Expression Level ◽

T Cell Proliferation ◽

Hl60 Cells ◽

T Cell Function ◽

Equity Ownership

Abstract Bispecific T-cell engager (BiTE®) antibodies represent a promising tool for anti-leukemic immunotherapy. The CD19/CD3-bispecific antibody blinatumomab was shown to be active in refractory and relapse patients with B-precursor acute lymphoblastic leukemia (Topp et al, ASCO 2014). Transient, blinatumomab-mediated cytokine release syndrome has been linked to target cell numbers as this phenomenon is predominantly observed within the first treatment cycle. In our previous work, we demonstrated that the bispecific CD33/CD3 BiTE® antibody AMG 330 is able to induce activation and proliferation of residual autologous T-cells and effectively mediates lysis of primary acute myeloid leukemia (AML) cells (Krupka et al, Blood 2014; 123(3):356-65). We hypothesize that in AML patients with high initial leukocyte counts (WBC > 30.000/μl) a cytoreductive phase prior to AMG 330 therapy might be beneficial to reduce the incidence and severity of cytokine mediated toxicity. Ideally, the cytoreductive drug does not impair T-cell function or reduce target antigen expression level. In the current study, we evaluated the effect of cytarabine (20 µM), decitabine (5 µM), azacitidine (1 µM and 5 µM) and hydroxyurea (10 µM and 100 µM) on T-cell proliferation and function in close analogy to potential treatment algorithms for AML. Healthy donor (HD) T-cells were pre-incubated with the cytoreductive drugs for 72 hours. T-cells were CFSE-labeled and co-cultured with either HL60 or MV4-11 cells (effector cell:target (E:T) ratio 1:1) in the presence or absence of AMG 330 (5 ng/ml). After 3 days of co-culture, lysis of HL60 cells and T-cell proliferation was assessed by flow cytometry. Pretreatment of T-cells with cytarabine completely abrogated T-cell function (lysis of HL60 cells: untreated (UT): 96.9% vs 20 µM: 4.2%) and significantly impaired T-cell proliferation (UT: 31.2% vs 20 µM: 4.6%). These findings correlated to data using primary AML samples collected 3 and 6 days after discontinuation of cytarabine treatment. After a 3-day chemotherapy-free interval, we observed no relevant T-cell proliferation and lysis of AML cells upon the addition of AMG 330 to the ex-vivo long-term culture system (lysis of AML cells on day 12: 30%; fold change T-cell expansion 0.9). After a 6-day treatment-free interval, high T-cell proliferation and cytotoxicity against primary AML cells were observed (lysis of AML cells on day 12: 61%; fold change T-cell expansion: 3.1). In contrast to cytarabine, decitabine treatment only marginally impaired T-cell function. Similarly, pre-incubation with azacitidine did not convey a negative effect on T-cell function (lysis of HL60 cells: UT: 100% vs 1 µM: 94.9% vs 5µM: 86.8%; proliferation: UT: 90.9% vs 1 µM: 80% vs 5 µM: 66.8%). Pretreatment with hydroxyurea had the least impact on T-cell performance. It did not impair T-cell function (lysis of HL60 cells: UT: 100% vs 10 µM: 100% vs 100 µM: 100%) and proliferation compared to untreated controls (UT: 92.9% vs 100 µM 90.8% vs 10 µM 92.9%). As we have previously shown that the level of CD33 expression correlates to kinetics of AMG 330-mediated lysis (Krupka et.al, EHA 2014), we analyzed the effect of the cytoreductive agents on CD33 expression level in AML cell lines and primary AML cells. Five AML cell lines (HL60, MV4-11, PL21, OCI-AML3, KG1a) and a primary AML patient sample were cultured in the presence or absence of decitabine (5 µM and 50 µM), azacitidine (1 µM and 5 µM) or hydroxyurea (10 µM and 100 µM) for 72 hours. The change of CD33 expression level was evaluated by flow cytometry (median fluorescence intensity, MFI). No significant changes in CD33 expression level were observed after culture of AML cell lines and primary AML cells with decitabine or azacitidine. In contrast, hydroxyurea upregulated surface expression of CD33 on 2/5 cell lines (HL60 and PL21) in a dose dependent manner (HL 60 MFI Ratio: UT 134.9 vs 10 µM 171.3 vs 100 µM 210; PL21 MFI Ratio: UT 166.9 vs 10 µM 177.9 vs 100 µM 191.8). In summary, we could show that pretreatment with hydroxyurea did not impair T-cell function and proliferation. In addition, we observed an upregulation of CD33 expression on AML cell lines. As the BiTE® technology relies on T-cell function and target antigen expression level, sequential and combinatorial immuno-chemotherapeutic approaches need to address both issues. Our data support the use of hydroxyurea in AML patients that require cytoreduction prior to AMG 330 treatment. Disclosures Krupka: AMGEN Inc.: Research Funding. Kufer:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Kischel:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Zugmaier:AMGEN Inc.: Equity Ownership; AMGEN Research (Munich): Employment. Sinclair:AMGEN Inc.: Employment, Equity Ownership. Newhall:AMGEN Inc.: Employment, Equity Ownership. Frankel:AMGEN Inc.: Employment, Equity Ownership. Baeuerle:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership. Subklewe:AMGEN Inc.: Research Funding.

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Combination of Anti-PD-L1 Antibody with IMiD® Immunomodulatory Drugs, HDAC Inhibitor ACY-1215, Bortezomib, or Toll-like Receptor 9 Agonist Enhances Anti-Tumor Immunity and Cytotoxicity in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.3014.3014 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3014-3014 ◽

Cited By ~ 1

Author(s):

Arghya Ray ◽

Deepika Sharma DAS ◽

Yan Song ◽

Dharminder Chauhan ◽

Kenneth C Anderson

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Tumor Immunity ◽

Immune Suppression ◽

Nk Cell ◽

Fold Increase ◽

T Cell Proliferation ◽

Toll Like Receptor ◽

Ctl Activity

Abstract Introduction Dysfunctional T cells and Natural Killer (NK) cells in MM, together with functionally defective plasmacytoid dendritic cells (pDCs), contribute to the immune suppression in MM (Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia 2014, 28: 1716-1724). The mechanism and the role of immunoregulatory molecules mediating pDC-T cell and pDC-NK cell interactions in MM are now defined. Programmed cell death protein 1 (PD-1) is highly expressed on MM patient T cells and NK cells; and both pDCs and MM cells express PD-1 ligand PD-L1 (B7-H1). PD-L1 interaction with PD-1 results in bidirectional inhibitory responses in T cells. Our study showed that pDCs confer T cell and NK cell immune suppression in the MM BM milieu by engaging immune checkpoints via PD-L1/PD-1 signaling axis (Ray et al, Leukemia 2015, 29:1441-1444). Importantly, blockade of PD-L1-PD-1 using anti-PD-L1 Ab generates MM-specific CD8+ CTL activity, as well as enhances NK-cell-mediated MM cell cytolytic activity. Anti-MM therapies may modulate MM-host immune responses, which raises the possibility that efficacy of anti-PD-Ll Ab can be improved by combining these therapies with immune-stimulating agents. Here we examined the impact of combining immune checkpoint blockade with lenalidomide, pomalidomide, bortezomib, HDAC inhibitor ACY-1215, or Toll-Like Receptor 9 agonists on anti-tumor immunity and cytotoxicity in MM. Methods For combination studies, we utilized low concentrations of various drugs (pomalidomide, lenalidomide, ACY-1215, or bortezomib) that do not significantly decrease viability of MM cells. As in our prior studies, anti-PD-L1 Ab and TLR9 agonist are not cytotoxic against MM cells. T cell proliferation assay: MM patient pDCs were co-cultured with autologous T cells (pDC:T ratio; 1:10) in the presence of anti-PD-L1 Ab (5 μg/ml) alone, drug alone, or anti-PD-L1 Ab plus drug for 5-6 days, and proliferation was quantified with CellTrace Violet Cell proliferation Kit using FACS. CTL activity assays: MM patient CD8+ T cells were cultured with autologous pDCs (1:10 pDC/T ratio) with anti-PD-L1 Ab, drug alone, or anti-PD-L1 plus drug for 5 days; cells were washed to remove drug, and GFP+MM.1S cells (20:1 E/T ratio) were added for another 2-3 days, followed by quantification of viable GFP+MM.1S cells using FACS. NK-cell mediated cytotoxic activity was assessed using flow-based CFSE-stained K562 lysis assays, as well as degranulation assay quantifying cell surface CD107a. All statistical parameters were calculated using GraphPad Prism 6. Anti-PD-L1 Ab was purchased from eBiosciences, USA; and ACY-1215, bortezomib, lenalidomide, and pomalidomide were purchased from Selleck chemicals, USA. Results Combination of anti-PD-L1 Ab (5 μg/ml) with lenalidomide (50-100 nM) or pomalidomide (100 nM) triggered a more robust MM-specific CD8+ CTL activity than anti-PD-L1 Ab alone (1.5-2 and 2-3 fold increase in CTL activity for lenalidomide and pomalidomide combinations, repectively). Anti-PD-L1 Ab combination with lenalidomide or pomalidomide also significantly increased NK-cell-mediated MM cell cytotoxicity (p < 0.05). We next determined whether anti-PD-L1 Ab can be combined with histone deacetylase inhibitors ACY-1215 (250 nM) or Panobinostat (2 nM). Combination of anti-PD-L1 Ab with ACY-1215 or panobinostat enhanced MM-specific CD8+ CTL activity versus anti-PD-L1 Ab alone (1.5 and 2 fold increase in CTL activity for panobinostat and ACY-1215 combinations, respectively). Assessment of surface CD107a as a marker of NK cell functional activity showed that anti-PD-L1 Ab plus ACY-1215 markedly increased CD107a expression (>10 fold) versus anti-PD-L1 Ab alone. Our prior studies showed that TLR9 agonists can restore pDCs ability to trigger T cell proliferation. We found that a combination of anti-PD-L1 Ab and TLR9 agonists (1 μM) enhances MM-specific pDC-induced CTL activities (2-3 fold increase in CTL activity in combination regimen versus anti-PD-L1 Ab alone). Finally, a combination of bortezomib (2 nM) with anti-PD-L1 Ab increased the MM-specific CTL activity (1.5-2 fold increase). Conclusions Our study provides the basis for combining novel immunotherapies targeting PD-1/PD-L1 pathway with current anti-MM agents or pDC-activating TLR agonists, to both restore immune function and enhance cytotoxicity in MM. Corresponding Author: Dharminder Chauhan, PhD Disclosures Chauhan: Stemline Therapeutics: Consultancy.

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PRL2 Phosphatase Regulate Early Stage T-Cell Development through Fine-Tuning SCF/Kit Signaling

Blood ◽

10.1182/blood.v124.21.1569.1569 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1569-1569

Author(s):

Kobayashi Michihiro ◽

Yunpeng Bai ◽

Momoko Yoshimoto ◽

Rui Gao ◽

Chen Sisi ◽

...

Keyword(s):

Cell Proliferation ◽

T Cell ◽

Critical Role ◽

T Cell Development ◽

Fold Increase ◽

Cell Development ◽

Fine Tuning ◽

T Cell Proliferation ◽

Wild Type

Abstract The phosphatase of regenerating liver (PRL) family of phosphatases, consisting of PRL1, PRL2, and PRL3, represents an intriguing group of proteins being validated as biomarkers and therapeutic targets in human cancer. We have been investigating the role of PRL2 in normal / malignant hematopoiesis and found that PRL2 is important for HSC self-renewal (Kobayashi et al., Stem Cells, 2014). The receptor tyrosine kinase KIT can balance quiescence for HSC maintenance and proliferation for progeny supply. The defects seen in the PRL2-deficient hematopoietic and testis cells recapitulate the phenotype of c-Kit mutant mice, suggesting that the SCF/KIT signaling may be impaired in the absence of PRL2 (Kobayashi et al., Stem Cells, 2014; Dong et al., JBC, 2013). Given that KIT also plays critical role in maintaining postnatal T-lymphopoiesis in thymus, we hypothesized that PRL2 is important for T cell development. Here we report that loss of PRL2 impairs T-lymphopoiesis both in vitro and in vivo. PRL2 deficiency resulted in marked reduction of splenocyte and thymocyte counts compared to wild type (WT) mice. While we observed modest increase in the frequency of early T cell progenitor (ETP), DN2, and DN3 cells in PRL2 deficient thymus, T-cell reconstitution was dramatically decreased after HSC transplantation. T-cell number in the peripheral blood (PB) of recipient mice repopulated with PRL2-null HSCs was 30 times less than that of the WT HSCs (WT: 2288.6±579.8/µl vs PRL2 null: 69.5±22.1/µl, p<0.00001). Although the frequency of donor-derived thymocytes in recipient thymus was 91±6.1% in WT, PRL2 null HSCs contributed only 7.1±4.9% (p<0.00001) in the recipient thymus. By detailed fractionation, surprisingly, chimerism in ETP was comparable between WT and PRL2 null cells (WT: 91.8±10.1% vs PRL2 null: 59.6±13.5%, p<0.01). Importantly, the chimerism of PRL2-null thymocytes fell down to 10% in gated DN2, whereas WT HSCs consistently contributed around 90%, suggesting that the DN1-to-DN2 transition requires PRL2. Next, we evaluated the in vitro T-cell generation by utilizing the Delta-Like1 (DLL1) expressing OP9 (DL-OP9) stromal cells. While wild type KSLs produced massive amount of T-cells (fold increase: 33,000±3371) 22 days following plating onto the DL-OP9, PRL2 null KSLs only generated limited amount of T-cells (fold increase: 1765±665, p<0.0001), demonstrating that PRL2 is important for T-cell proliferation. We also monitored the generation of ETPs from KSLs in DL-OP9 cultures and observed significant expansion of ETPs derived from WT KSLs compared to that of the PRL2 null KSLs (fold increase: 183.8±14.4 vs 12.5±4.3, p<0.001). However, when sorted DN3 cells from WT and PRL2 KO thymus were plated onto DL-OP9, we saw similar increase in cell expansion, suggesting PRL2 regulate early T-cell development. WhilePRL2 is a dual specificity protein phosphatase, its substrates are unknown. To identifyPRL2 substrates in hematopoietic cells, we performed a protein phosphatase substrate trap assay. We utilized a GST-tagged PRL2/CS-DA mutant, in which the catalyticsite cysteine was mutated to serine, so that PRL2 binds to its substrates better, but is unable todephosphorylate them. We found that the mutant PRL2/CS-DA showed enhanced association with KIT than WT PRL2 in Kasumi-1 cells, suggesting that KIT is a potential PRL2 substrate. The PRL2 and KIT interaction was further confirmed by the Immunoprecipitation (IP) assay in 293T cells expressing KIT. We also detected the association of PRL2 with SHP2, CBL and PLC-g in Kasumi-1 cells, which are important regulators of KIT activation and stability. Moreover, PRL2 KO hematopoietic progenitor cells show decreased KIT phosphorylation at tyrosine 703 following SCF stimulation, suggesting that PRL2 may modulate KIT activation in these cells. To evaluate the impact of SCF signal strength on T-cell proliferation, we cultured sorted lympho-primed multipotent progenitor cells (LMPPs) from WT and KO mice onto DLL-Fc coated plates with graded doses of SCF (0.2, 1, 5, 25 ng/ml). The total number of cells generated from SCF treated WT LMPPs was significantly higher than that of the KO LMPPs in a dosage dependent manner. KO exhibited 6 times less sensitive to SCF than WT, indicating that PRL2 fine-tunes SCF signal intensity in early T-cell. Taken together, we have identified a critical role for PRL2 in T-cell proliferation and maintenance through fine-tuning SCF/KIT signaling. Disclosures No relevant conflicts of interest to declare.

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Zoledronic Acid Induces the Proliferation of Human Cord Blood Gamma Delta T Cells Ex Vivo

Blood ◽

10.1182/blood.v124.21.1427.1427 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1427-1427

Author(s):

Suzanne L Tomchuck ◽

Jin He ◽

Ross W. Perko ◽

Scarlett Evans ◽

Amy McKenna ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Ex Vivo ◽

Fold Increase ◽

T Cell Proliferation ◽

Gamma Delta T Cells ◽

Memory Cells ◽

Gamma Delta ◽

Gamma Delta T Cell

Abstract Cord blood (CB) T cells are known to be naïve cells, but can be activated to respond similar to adult peripheral blood (PB) T cells. Reports indicate that culture with aminobisphosphonate (NBP) stimulates CB gamma delta T cell proliferation ex vivo, specifically the TCRγ9δ2 subset, which has been extensively studied in PB gamma delta T cells. As CB gamma delta T cells are not well described, we compared CB gamma delta T cell proliferation, phenotype and genotype to PB gamma delta T cells when culturing cells with the NBP, Zometa (zoledronic acid), and IL-2. Fourteen days in culture resulted in significant fold increase in the proliferation of gamma delta T cells and in the percent of lymphocytes in both sample types. PB gamma delta T cells proliferated more robustly than CB with a 288.60 versus 21.32 fold increase, respectively. Additionally, in freshly isolated samples, CB gamma delta T cells comprised an average of 1.404% of the lymphocyte population, which was similar to PB gamma delta T cells, with an average of 2.319%. However, by day 14, PB gamma delta T cells increased to 70.15% of lymphocytes whereas CB gamma delta T cells increased to 12.49%. Phenotypically, both CB and PB had similar percent of CD45RA+ and CD45RO+ gamma delta T cell memory subsets in freshly isolated samples. Following culture, PB gamma delta T cells were mostly CD45RO+ memory cells, with significantly fewer CD45RA+ naïve cells, whereas more CB gamma delta T cells were of the intermediate CD45RA+CD45RO+ subset. Further phenotypic analysis of the memory subsets indicated that cultured PB gamma delta T cells were either effector memory cells (CD27-CD45RA-) or central memory cells (CD27+CD45RA-), while CB gamma delta T cells were mostly naïve (CD27+CD45RA+). The cytokines secreted by these cells were also assessed and the culture of PB and CB gamma delta T cells resulted in differing cytokine secretion profiles. After 14 days of culture, PB gamma delta T cells secreted more IFNγ and TNFα, while CB gamma delta T cells secreted more IL-10 and RANTES. We also examined TCRγ9 and TCRδ2 phenotypic expression and found that the TCRγ9δ2 was a common clone in freshly isolated PB gamma delta T cells, which predominated after 14 days in culture. However, while the TCRγ9δ2 variant was expressed in CB gamma delta T cells, it was low before and after culture, suggesting that Zometa may not stimulate gamma delta T cells in CB the same as PB. As limited TCRγδ phenotypic reagents are available, we developed a single cell PCR assay for genotypic analysis of the TCRγδ repertoire. PCR analysis suggests that the TCRγδ repertoire is diverse in both samples, yet TCRγ9δ2 is most prevalent. Further analysis of the variant subsets is warranted and may give insight into how each of these receptor pairings affects function. Disclosures No relevant conflicts of interest to declare.

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c-MYC Is a Major Downstream Target of NOTCH in T-Cell Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v106.11.3005.3005 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3005-3005

Author(s):

Arthur Lau ◽

Jon C. Aster ◽

Andrew P. Weng

Keyword(s):

T Cell ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Growth Arrest ◽

Notch Pathway ◽

Dominant Negative ◽

Cellular Growth ◽

Downstream Target ◽

Human T Cell ◽

Notch Inhibition

Abstract We recently reported that activating mutations in the NOTCH1 receptor occur in a high percentage of primary human T-cell acute lymphoblastic leukemias (T-ALL). Withdrawal of NOTCH signals by treatment with γ-secretase inhibitors (GSI) or by transduction with a dominant-negative Mastermind-like-1 polypeptide (a specific NOTCH pathway inhibitor) induces growth arrest of many T-ALL cell lines, suggesting that NOTCH supplies signals that are needed for maintenance of growth of T-ALL cells. In order to identify downstream targets of NOTCH that mediate these effects, we performed gene expression profiling on NOTCH signaling-dependent T-ALL cell lines before and after NOTCH inhibition. Among a number of identified candidate genes was c-MYC, which was of particular interest given its importance in promoting cellular growth and its known dysregulation in a number of hematolymphoid neoplasms. c-MYC was down-regulated following NOTCH inhibition, and rapidly up-regulated following release of NOTCH inhibition, even in the presence of protein synthesis inhibitors, suggesting that it is a direct NOTCH transcriptional target. Further, a subset of murine and human T-ALL cell lines were rescued from GSI-mediated growth arrest by c-MYC-expressing retroviruses. The failure of c-MYC to rescue some NOTCH-dependent cell lines likely stems from differences in cellular context, such as collaborating oncogenic lesions and/or the stage of T cell development the cell lines recapitulate. Nevertheless, these data implicate c-MYC as a major downstream target of NOTCH in T-ALL.

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Triggering of Toll-Like Receptor-4 In Human Multiple Myeloma Cells Promotes Proliferation and Alters Cell Responses to Immune and Chemotherapy Drug Attack

Blood ◽

10.1182/blood.v116.21.1905.1905 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1905-1905

Author(s):

Zhen Cai ◽

Hanying Bao ◽

Peilin Lu ◽

Lijuan Wang ◽

Donghua He ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

T Cell ◽

Tumor Cells ◽

Cell Lines ◽

Myeloma Cell ◽

Myeloma Cells ◽

Response To Chemotherapy ◽

Tlr4 Ligand ◽

Induced Apoptosis

Abstract Abstract 1905 Multiple myeloma (MM) is a fatal plasma cell malignancy mainly localized in the bone marrow. The clonal expansion of tumor cells is associated with the disappearance of normal plasma cells and with a marked depression in the production of normal immunoglobulin (Ig). This makes MM patients highly vulnerable to bacterial, fungal and viral infections and recurrent infections remain to be a major cause of death in MM patients. It has been shown that most primary myeloma cells and cell lines express multiple Toll-like receptors (TLRs). Among them, TLR4 is most frequently expressed. To investigate TLR-initiated responses in MM cells including proliferation, anti-apoptosis and immune escape, we first screened four commonly used human myeloma cell line (HMCL) for the expression of major TLRs by RT-PCR. Surprisingly, all the HMCL expressed multiple TLRs. We also examined primary myeloma cells from 4 patients with MM and our results showed that TLR4 was expressed by all the tumor cells. We incubated myeloma cells with LPS, the natural ligand for TLR4, and found that cell proliferation increased significantly. Targeting TLRs on malignant B cells can induce resistance to chemotherapeutic agents but can also be exploited for combined therapeutic approaches. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of HMCL cells. Myeloma cells were pretreated for four hours with LPS before being induced apoptosis by adriamycin. Results showed that LPS pretreatment partially protected the cells from adriamycin-induced apoptosis. The TLR signaling pathway activates several signaling elements, including NF-kB and ERK/JNK/p38 MAPKs, which regulate many immunologically relevant proteins. Time-dependent MAPK phosphorylation was measured to assess the activation of these kinases upon treatment with LPS in cell lines. ERK1/2, p38, and JNK phosphorylation and NF-kB were significantly up-regulated following LPS treatment. Moreover, our findings demonstrated that LPS-induced cell proliferation was dependent on JNK, ERK and p38 signaling. IL-18, a recently described member of the IL-1 cytokine superfamily, is now recognized as an important regulator of innate and acquired immune responses. In this study, we found that LPS induced IL-18 secretion and activated MAPK and NF-kB signaling simultaneously. Therefore, our results suggest that activation of the MAPK signaling and secretion of IL-18 are interconnected. Tumors evade immune surveillance by multiple mechanisms, including the production of factors such as TGF-β and VEGF, which inhibit and impair tumor-specific T cell immunity. Our study also showed that T cell proliferation induced by allostimulatory cells decreased when the HMCL were pre-treated with LPS. Moreover, immunoregulatory molecules on HMCL, such as B7-H1, B7-H2 and CD40, were upregulated after treatment with LPS, suggesting that TLR4 ligand LPS facilitates tumor cell evasion of the immune system. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks. Disclosures: No relevant conflicts of interest to declare.

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MAGE-A3 Specific T-Cell Receptor Adoptive Cell Transfer of Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.1843.1843 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1843-1843 ◽

Cited By ~ 1

Author(s):

Jeesun Park ◽

Shi Zhong ◽

Michelle Krogsgaard ◽

Amitabha Mazumder

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cell Lines ◽

Cd8 T Cells ◽

Fold Increase ◽

Cell Receptor ◽

Tumor Cell Lines ◽

Peptide Concentration

Abstract Abstract 1843 Background: Multiple myeloma (MM) is a cancer of plasma cells and the second most common blood cancer. Current treatment strategies such as high dose chemotherapy, autologous stem cell rescue, and allogeneic transplantation have improved response rates and increased survival. However, these treatments often include high procedure-related morbidity and mortality and can only be applied to a small minority of myeloma patients. Therefore, safe broadly applicable immunologic strategies for myeloma, such as Adoptive Cell Therapy (ACT) are urgently needed. Methods: In this study we focused on aHLA-A*0201-restricted cancer testis antigen MAGE-A3:112–120, which is widely expressed in many forms of cancers such as metastatic melanoma, non-small cell lung cancer and MM, but not expressed in most normal tissues. To develop a system of effective strategies for T-cell therapy of multiple myeloma, we employed T-cell engineering technology using a MAGE-A3specific T-cell receptor (TCR)obtained from Dr. Steven Rosenberg at the National Cancer Institute. MAGE-A3 specific TCR was sub-cloned into a lentiviral vector and tranduced into purified CD8+ T-cells from human peripheral blood mononucleocytes (hPBMCs). To test the effector functionality of the MAGE-A3 specific TCR, the MAGE-A3 TCR-transduced CD8+ T-cells were subjected to cytokine release and chromium release assays after being co-cultured with MAGE-A3 peptide-loaded T2 cells, and U266 (MAGE-A3+/HLA-A*0201+), MM1.r (MAGE-A3+/HLA-A*0201-), KAS6 (MAGE-A3-/HLA-A*0201+), and KMS11(MAGE-A3-/HLA-A*0201-) MM tumor cell lines. Results: We observedcytokine production of INF-g and IL-2 in the MAGE-A3 TCR-transduced CD8+ T-cells generally in a dose-dependent manner to the MAGE-A3 peptide-loaded T2 cells. For example, the difference of INF-g secretion bythe MAGE-A3 TCR-transduced CD8+ T-cells wasa 10-fold increase from 0.001 uM to 0.02 uM of the loaded MAGE-A3 peptide. IL-2 secretion was also increasedby 7-fold from 0.001 uM to 0.1 uM of the MAGE-A3 peptide concentration. At 10uM of the peptide concentration, there was a 29-fold increase of the IL-2 production as compared to the 0.001 uM peptide concentration. Between 10uM and 100 uMof the peptide concentration, there was a decrease in IL-2 secretion by 2-fold, which is commonly observed at high peptide concentrations presumably due to cytotoxicity. Specific lysis of tumor cells by the MAGE-A3 TCR-transduced CD8+ T-cellswas observed in all four MM tumor cell lines, and we detected higher percentage of cell lysisin U266 (38%) and MM1.r (51%) cell lines as compared to the KAS6 (11%) and KMS11(21%) cell lines. Conclusions: Our findings suggest that the MAGE-A3 TCR-engineered CD 8+ T-cells are able to specifically recognize MAGE-A3 antigen, produce IL-2 and IFN-g, and destroy MM tumor cells loaded with the MAGE-A3 antigen. This potentially could further translate into effective MAGE-A3 specific targeted tumor rejection in vivo. We also plan to transduce the MAGE-A3 TCR into hematopoietic stem cells to and test the effector function of those cells against MM tumor cells and eventually against MM patient samples. Disclosures: No relevant conflicts of interest to declare.

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Restoration of microRNA-212 causes a G0/G1 cell cycle arrest and apoptosis in adult T-cell leukemia/lymphoma cells by repressing CCND3 expression

Journal of Investigative Medicine ◽

10.1136/jim-2016-000233 ◽

2016 ◽

Vol 65 (1) ◽

pp. 82-87 ◽

Cited By ~ 3

Author(s):

Haihao Wang ◽

Qiannan Guo ◽

Peiwen Yang ◽

Guoxian Long

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

T Cell ◽

Cell Lines ◽

Cell Cycle Progression ◽

Cyclin D3 ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Cycle Progression ◽

Adult T Cell Leukemia

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive T-cell malignancy. This study was designed to explore the expression and functional significance of microRNA (miR)-212 in ATL. The expression of miR-212 in human ATL tissues and cell lines were investigated. Gain-of-function experiments were carried out to determine the roles of miR-212 in cell proliferation, tumorigenesis, cell cycle progression, and apoptosis. We also identified and functionally characterized the target genes of miR-212 in ATL cells. Compared with normal lymph node biopsies, lymphoma samples from ATL patients displayed underexpression of miR-212 (p=0.0032). Consistently, miR-212 was downregulated in human ATL cell lines, compared with normal T lymphocytes. Restoration of miR-212 significantly (p<0.05) inhibited ATL cell proliferation and tumorigenesis in mice. Overexpression of miR-212 led to an accumulation of G0/G1-phase cells and a concomitant reduction of S-phase cells. Moreover, enforced expression of miR-212-induced significant apoptosis in ATL cells. CCND3, which encodes a cell cycle regulator cyclin D3, was identified as a direct target of miR-212 in ATL cells. Rescue experiments with a miR-212-resistant variant of CCND3 demonstrated that overexpression of CCND3 restored cell-cycle progression and attenuated apoptotic response in miR-212-overexpressing ATL cells. Taken together, miR-212 exerts growth-suppressive effects in ATL cells largely by targeting CCND3 and may have therapeutic potential in ATL.

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Pentagalloyl glucose inhibits TNF‐α‐activated CXCL1/GRO-α expression and induces apoptosis‐related genes in triple-negative breast cancer cells

Scientific Reports ◽

10.1038/s41598-021-85090-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Patricia Mendonca ◽

Sumaih Alghamdi ◽

Samia Messeha ◽

Karam F. A. Soliman

Keyword(s):

Breast Cancer ◽

Cell Proliferation ◽

Cell Lines ◽

Cancer Progression ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Fold Increase ◽

Mapk Signaling ◽

Tnf Α ◽

Pentagalloyl Glucose

AbstractIn triple-negative breast cancer (TNBC), the tumor microenvironment is associated with increased proliferation, suppressing apoptotic mechanisms, an altered immune response, and drug resistance. The current investigation was designed to examine the natural compound pentagalloyl glucose (PGG) effects on TNF-α activated TNBC cell lines, MDA-MB-231 and MDA-MB-468. The results obtained showed that PGG reduced the expression of the cytokine GRO-α/CXCL1. PGG also inhibited IƙBKE and MAPK1 genes and the protein expression of IƙBKE and MAPK, indicating that GRO-α downregulation is possibly through NFƙB and MAPK signaling pathway. PGG also inhibited cell proliferation in both cell lines. Moreover, PGG induced apoptosis, modulating caspases, and TNF superfamily receptor genes. It also augmented mRNA of receptors DR4 and DR5 expression, which binds to TNF-related apoptosis-induced ligand, a potent and specific stimulator of apoptosis in tumors. Remarkably, PGG induced a 154-fold increase in TNF expression in MDA-MB-468 compared to a 14.6-fold increase in MDA-MB-231 cells. These findings indicate PGG anti-cancer ability in inhibiting tumor cell proliferation and GRO-α release and inducing apoptosis by increasing TNF and TNF family receptors' expression. Thus, PGG use may be recommended as an adjunct therapy for TNBC to increase chemotherapy effectiveness and prevent cancer progression.

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