Apoptosis Induction in Cutaneous T-Cell Lymphoma Cells after Treatment with Lenalidomide

Abstract Introduction: Cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas, derived from skin-homing mature T-cells. The two most common types of CTCL are Mycosis fungoides (MF) (50%-72%), which is generally indolent in behavior, and Sézary syndrome (SS) (1%-3%), an aggressive leukemic form of the disease. Currently, there is no effective treatment for CTCL patients. Research on new therapies for CTCL is largely centered on defining novel therapeutic targets, but in most cases the response is short and the survival rate remains the same. Therefore, the CTCL's resistance to chemotherapy and the lack of full understanding of their pathogenesis request further investigation. Aims: With the view of a more targeted therapy, we evaluated in vitro the effectiveness of lenalidomide, an immunomodulatory agent (IMID) with clinical efficacy, among others, in plasma cell dysplasias and lymphoproliferative disorders. Methods: Four CTCL cell lines were used: MJ and MyLa (derived from MF), SeAx and Hut-78 (both derived from peripheral blood of patients with Sézary syndrome). SeAx and Hut-78 cells were cultured in RPMI 1640, supplemented with 10% FBS and 2 mM L-glutamine. MJ and MyLa cells were cultured in RPMI 1640 and IMDM, respectively, supplemented with 20% FBS. All cell lines were maintained at 37°C in a humid atmosphere of 5% CO2. Cells were treated with various concentrations of lenalidomide (1μΜ, 10μΜ and 100μΜ) for 24, 48 and 72h. Apoptosis was determined by flow cytometry using the Annexin V/PI method. The proliferative capacity of untreated and lenalidomide- treated cells was measured using the BrdU assay. One-way Anova and LSD/ Bonferroni methods were applied for the statistical analysis of the results. Results: Our data indicate that all cell lines responded with enhanced apoptosis at various lenalidomide treatment conditions. Between the two MF lines tested, MyLa cells were affected the most. Specifically, MyLa cells exhibited a statistically significant augmentation on their apoptosis compared to untreated cells after treatment with 10μΜ and 100μΜ lenalidomide for 24h (9.7 and 8.66 vs 4.83, p=0.000 and p<0,001, respectively) and 48h (6.1 and 4.46 vs 3,36, p=0.000 and p<0,007, respectively), as well as after treatment with 10μΜ Lenalidomide for 72h (5.3 vs 4.7, p=0.000). Similarly, MJ cells also responded with enhanced apoptosis after 24h and 48h of treatment with 100μΜ lenalidomide (8.8 vs 7.43 and 11.13 vs 8.66, p=0.000, respectively) and 72h of treatment with 1 μM lenalidomide (10.63 vs 7.96, p=0.000). Regarding the response of the two SS cell lines to lenalidomide treatment, SeAx cells were significantly affected, exhibiting high apoptotic rates compared to untreated cells after treatment with 1μΜ lenalidomide for 48h (13.76 vs 5.1, p=0.000) and 1μΜ, 10μΜ and 100 μΜ lenalidomide for 72h (1.5, 2.4 and 7.03 vs 0.13, p=0.000, respectively). Lenalidomide had a rather moderate effect on the apoptosis of Hut-78 cells, which presented with statistically significant enhanced apoptosis only after treatment with 1μΜ lenalidomide for 72h (1.33 vs 0.8, p<0,009). The proliferation capacity of the CTCL cell lines tested presented no statistically significant changes under any of the treatment conditions. Conclusions: Our observations demonstrate that lenalidomide doesn't appear to have any significant effect on the proliferation of the CTCL cell lines. On the contrary, it seems to lead both MF and SS cell lines to significantly enhanced apoptosis. Interestingly, the MF cell lines seem to be more sensitive to lenalidomide treatment in terms of apoptosis induction compared to SS cell lines. Moreover, in all CTCL cell lines tested, high concentrations of lenalidomide contributed to enhanced apoptosis after a shorter period of treatment, compared to lower concentrations, which seemed to be more effective after a longer period, with the exception of SeAx cells. Although these initial results need to be further confirmed both in vitro and in vivo, they appear very encouraging for the integration of lenalidomide treatment, alone or in combination, in CTCL therapy. Disclosures No relevant conflicts of interest to declare.

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Targeting the PI3K/mTOR Pathway in Lymphoma with PQR309 and PQR620: Single Agent Activity and Synergism with the BCL2 Inhibitor Venetoclax

Blood ◽

10.1182/blood.v128.22.3017.3017 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3017-3017

Author(s):

Chiara Tarantelli ◽

Eugenio Gaudio ◽

Petra Hillmann ◽

Filippo Spriano ◽

Ivo Kwee ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Mtor Inhibitor ◽

Cell Lymphoma ◽

Single Agent ◽

Apoptosis Induction ◽

In Vivo Experiments

Abstract Background. The PI3K/AKT/mTOR pathway is an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor that has shown in vitroanti-lymphoma activity (Tarantelli et al, ASH2015) and is in phase 2 trial (NCT02249429, , NCT02723877, NCT02669511). PQR620 is a novel mTORC1/2 inhibitor that has shown preclinical activity in solid tumor models (Beaufils et al, AACR 2016). Here, we present the in vitro and in vivo anti-lymphoma activity of PQR620 as single agent and also the in vivo results of PQR620 or PQR309 containing combinations with the BCL2 inhibitor venetoclax. Materials and Methods. The drug concentration causing 50% inhibition of cell proliferation (IC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well plates. For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/J) mice were subcutaneously inoculated with 10 x106 (RIVA) or with 5 x106(SU-DHL-6) cells. Results. PQR620 had a median IC50 of 250 nM (95%CI, 200-269 nM) when tested on 44 lymphoma cell lines. Activity was higher in B cell (no.=36) than in T cell tumors (no.=8) (median IC50s: 250 nM vs 450 nM; P=0.002). At 72h, anti-tumor activityof PQR620 was mostly cytostatic and apoptosis induction was seen only in 6/44 cell lines (13%), Sensitivity to PQR620 or apoptosis induction did not differ between DLBCL and MCL, and they were not affected by the DLBCL cell of origin, by TP53 status or by the presence of MYC or BCL2 translocations. The activity of PQR620 as single agent underwent in vivo evaluation in two DLBCL models, the germinal center B cell type DLBCL (GCB-DLBCL) SU-DHL-6 and the acivated B cell-like DLBCL (ABC-DLBCL) RIVA. Treatments with PQR620 (100mg/kg dose per day, Qdx7/w) started with 100-150 mm3 tumors and were carried for 14 (SU-DHL-6) or 21 days (RIVA). In both models, PQR620 determined a 2-fold decrease of the tumor volumes in comparison with control, with significant differences in both SU-DHL-6 (D7, D9, D11, D14; P < 0.005) and RIVA (D14, D16, D19, D21; P < 0.005). Based on the previously reported synergy between the dual PI3K/mTOR inhibitor PQR309 and venetoclax (Tarantelli et al, ASH 2015), we evaluated the combination of the PQR620 or PQR309 with the BCL2 inhibitor venetoclax (100 mg/kg, Qdx7/w) in the SU-DHL-6 model. Both the venetoclax combination with the dual PI3K/mTOR inhibitor and the venetoclax combination with mTORC1/2 inhibitor were superior to the compounds given as single agents, leading to the eradication of the xenografts. The combination of PQR620 with venetoclax showed highly significant differences either versus control or single agents during all days of the experiment (D4, D7, D9, D11, D14; P < 0.001). Similarly, the combination of PQR309 with venetoclax showed highly significant differences versus venetoclax (D7, D9, D11, D14; P < 0.001) and PQR309 (D7, D9, D11; P < 0.005) alone. Conclusions. The novel mTORC1/2 inhibitor PQR620 had in vitro and in vivo anti-lymphoma activity as single agent. In vivo experiments showed that both PQR620 and the dual PI3K/mTOR inhibitor PQR309 can strongly benefit from the combination with the BCL2 inhibitor venetoclax. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Fabbro:PIQUR Therapeutics AG: Employment. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees.

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Roles of OX40 and OX40 Ligand in Mycosis Fungoides and Sézary Syndrome

International Journal of Molecular Sciences ◽

10.3390/ijms222212576 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12576

Author(s):

Yuki Kawana ◽

Hiraku Suga ◽

Hiroaki Kamijo ◽

Tomomitsu Miyagaki ◽

Makoto Sugaya ◽

...

Keyword(s):

Tumor Cells ◽

Mycosis Fungoides ◽

Cell Lines ◽

Survival Rates ◽

Clinical Samples ◽

Sézary Syndrome ◽

Sezary Syndrome ◽

And Function

Mycosis fungoides (MF) and Sézary syndrome (SS), the most common types of cutaneous T-cell lymphoma (CTCL), are characterized by proliferation of mature CD4+ T-helper cells. Patients with advanced-stage MF and SS have poor prognosis, with 5-year survival rates of 52%. Although a variety of systemic therapies are currently available, there are no curative options for such patients except for stem cell transplantation, and thus the treatment of advanced MF and SS still remains challenging. Therefore, elucidation of the pathophysiology of MF/SS and development of medical treatments are desired. In this study, we focused on a molecule called OX40. We examined OX40 and OX40L expression and function using clinical samples of MF and SS and CTCL cell lines. OX40 and OX40L were co-expressed on tumor cells of MF and SS. OX40 and OX40L expression was increased and correlated with disease severity markers in MF/SS patients. Anti-OX40 antibody and anti-OX40L antibody suppressed the proliferation of CTCL cell lines both in vitro and in vivo. These results suggest that OX40–OX40L interactions could contribute to the proliferation of MF/SS tumor cells and that the disruption of OX40–OX40L interactions could become a new therapeutic strategy for the treatment of MF/SS.

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Sézary syndrome cells overexpress syndecan-4 bearing distinct heparan sulfate moieties that suppress T-cell activation by binding DC-HIL and trapping TGF-β on the cell surface

Blood ◽

10.1182/blood-2010-08-302034 ◽

2011 ◽

Vol 117 (12) ◽

pp. 3382-3390 ◽

Cited By ~ 24

Author(s):

Jin-Sung Chung ◽

Lisa H. Shiue ◽

Madeleine Duvic ◽

Amit Pandya ◽

Ponciano D. Cruz ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

Heparan Sulfate ◽

Cell Lines ◽

T Cell Activation ◽

Cell Activation ◽

Sézary Syndrome ◽

Sezary Syndrome

Abstract Because syndecan-4 (SD-4) on effector and memory T cells inhibits T-cell activation by binding dendritic cell–associated heparan sulfate proteoglycan-integrin ligand (DC-HIL) on antigen presenting cells and because malignant cells of the cutaneous T-cell lymphoma (CTCL) subset, Sézary syndrome (SS), exhibit memory T-cell phenotype, we posited SS cells to express SD-4. Indeed, malignant T cells from patients with SS and from CTCL cell lines constitutively expressed SD-4 at high levels, in contrast to T cells from healthy volunteers and patients with other inflammatory skin diseases and to non-CTCL cell lines that did not. SS cells also bound to DC-HIL at a level higher than normal T cells activated in vitro, resulting in their inhibited proliferation to anti–CD3 antibody. SD-4 on SS cells also trapped transforming growth factor-β1 to their cell surface, enhancing their ability to inhibit activation of syngeneic and allogeneic normal T cells. All of these inhibitory properties were dependent on overexpression of distinct heparan sulfate (HS) moieties by SD-4 on SS cells. Finally, we showed toxin-conjugated DC-HIL to abrogate the ability of SS cells to proliferate in vitro. These findings indicate that SD-4 bearing distinct HS moieties plays a pathogenic role in SS and may be targeted for treatment.

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Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.810.810 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 810-810 ◽

Cited By ~ 3

Author(s):

Haiying Qin ◽

Sang M Nguyen ◽

Sneha Ramakrishna ◽

Samiksha Tarun ◽

Lila Yang ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Chain ◽

Dual Targeting ◽

Single Antigen ◽

Therapeutic Function

Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

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Enhanced In Vivo activation of Adoptively Transferred Genetically Targeted T Cells Following Cyclophosphamide Chemotherapy: Initial Results From a Phase I Clinical Trial Treating CLL Patients with Autologous CD19-Targeted T Cells.

Blood ◽

10.1182/blood.v114.22.3436.3436 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3436-3436

Author(s):

Renier J. Brentjens ◽

Daniel Hollyman ◽

Jae Park ◽

Elmer Santos ◽

Raymond Yeh ◽

...

Keyword(s):

Clinical Trial ◽

T Cells ◽

T Cell ◽

High Efficiency ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Low Grade ◽

Autologous Tumor

Abstract Abstract 3436 Poster Board III-324 Patient T cells may be genetically modified to express chimeric antigen receptors (CARs) targeted to antigens expressed on tumor cells. We have initiated a clinical trial treating chemotherapy-refractory chronic lymphocytic leukemia (CLL) patients with autologous T cells modified to express the 19-28z CAR targeted to the CD19 antigen expressed on most B cell malignancies. In the first cohort of this trial, patients were infused with the lowest planned dose of modified T cells alone. All patients treated in this cohort experienced low-grade fevers following modified T cell infusion, and 2 of 3 treated patients exhibited subjective and laboratory evidence of transient reductions in tumor burden. The first patient treated on the second cohort of this study received prior cyclophophamide chemotherapy followed by the same dose of modified T cells administered to the first cohort of patients. This patient experienced persistent fevers, dyspnea, hypotension, renal failure, and died 44 hours following modified T cell infusion, likely secondary to sepsis. Modified T cells were not detectable in the peripheral blood of treated patients at 1 hour following completion of T cell infusion. However, post mortem analyses revealed a rapid infiltration of targeted T cells into anatomical sites of tumor involvement. Serum levels of the inflammatory cytokines IL-5, IL-8, and GM-CSF, but not TNFα, markedly and rapidly increased following infusion of genetically targeted T cells in this patient, mirroring the in vitro cytokine secretion profile of this patient's T cells, and consistent with marked in vivo activation of the modified T cells. Similar cytokine signatures were not found in patients from the first cohort. Significantly, serum cytokine analyses from the second cohort patient revealed a marked increase in the pro-proliferative cytokines IL-2, IL-7, IL-12, and IL-15 following cyclophosphamide therapy, in contrast to the baseline levels found in the first cohort. This report demonstrates the high efficiency trafficking of CD19-targeted T cells and in vivo activation of T cells encoding a second generation CD28/zeta chain-based chimeric antigen receptor. Furthermore, these data highlight mechanisms whereby cyclophosphamide may generate an in vivo milieu that enhances the anti-tumor efficacy of autologous tumor targeted T cells. Disclosures No relevant conflicts of interest to declare.

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Differential Effects of Milatuzumab On Human Antigen-Presenting Cells in Comparison to Malignant B Cells.

Blood ◽

10.1182/blood.v114.22.2744.2744 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2744-2744

Author(s):

Xiaochuan Chen ◽

Rhona Stein ◽

Chien-Hsing Chang ◽

David M. Goldenberg

Keyword(s):

T Cell ◽

B Cells ◽

Cell Lines ◽

Hairy Cell ◽

Employment Equity ◽

Cross Presentation ◽

Equity Ownership ◽

Antigen Presenting

Abstract Abstract 2744 Poster Board II-720 Introduction: The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, is in clinical evaluation as a therapeutic mAb for non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and multiple myeloma after preclinical evidence of activity in these tumor types. In addition to its expression in malignant cells, CD74 is also expressed in normal B cells, monocytes, macrophages, Langerhans cells, follicular and blood dendritic cells. A question therefore arises whether milatuzumab is toxic to or affects the function of these immune cells. This has important implications, not only for safe therapeutic use of this mAb, but also for its potential application as a novel delivery modality for in-vivo targeted vaccination. Methods: We assessed the binding profiles and functional effects of milatuzumab on human antigen-presenting cell (APC) subsets. Studies on the effect of milatuzumab on antigen presentation and cross-presentation are included. In addition, binding and cytotoxicity on a panel of leukemia/lymphoma cell lines and CLL patient cells were tested to demonstrate the range of malignancies that can be treated with this mAb. Results: Milatuzumab bound efficiently to different subsets of blood dendritic cells, including BDCA-1+ myeloid DCs (MDC1), BDCA-2+ plasmacytoid DCs (PDC), BDCA-3+ myeloid DCs (MDC2), B lymphocytes, monocytes, and immature DCs derived from human monocytes in vitro, but not LPS-matured DCs, which correlated well with their CD74 expression levels. In the malignant B-cells tested, milatuzumab bound to the surface of 2/3 AML, 2/2 mantle cell (MCL), 4/4 ALL, 1/1 hairy cell leukemia, 2/2 CLL, 7/7 NHL, and 5/6 multiple myeloma cell lines, and cells of 4/6 CLL patient specimens. Significant cytotoxicity (P<0.05) was observed in 2/2 MCL, 2/2 CLL, 3/4 ALL, 1/1 hairy cell, 2/2 NHL, and 2/2 MM cell lines, and 3/4 CD74-positive CLL patient cells, but not in the AML cell lines following incubation with milatuzumab. In contrast, milatuzumab had minimal effects on the viability of DCs or B cells that normally express CD74. The DC maturation and DC-mediated T-cell functions were not altered by milatuzumab treatment, which include DC-induced T-cell proliferation, CD4+CD25+FoxP3+ Treg expansion, and CD4+ naïve T-cell polarization. Moreover, milatuzumab had little effect on CMV-specific CD8- and CD8+ T cell interferon-g responses of peripheral blood mononuclear cells stimulated in vitro with CMV pp65 peptides or protein, suggesting that milatuzumab does not influence antigen presentation or cross-presentation. Conclusion: These results demonstrate that milatuzumab is a highly specific therapeutic mAb against B-cell malignancies with potentially minimal side effects. It also suggests that milatuzumab may be a promising novel delivery mAb for in vivo targeted vaccinations, given its efficient binding, but lack of cytotoxicity and functional disruption on CD74-expressing normal APCs. (Supported in part by NIH grant PO1-CA103985.) Disclosures: Chang: Immunomedics Inc.: Employment, Equity Ownership, Patents & Royalties. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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The Combination of Histone Deacetylase Inhibitors and Hypomethylating Agents Exhibits Marked Synergy In Preclinical Models of T-Cell Lymphoma

Blood ◽

10.1182/blood.v116.21.3937.3937 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3937-3937 ◽

Cited By ~ 2

Author(s):

Enrica Marchi ◽

Danielle C Bongero ◽

Matko Kalac ◽

Luigi Scotto ◽

Owen A. O'Connor

Keyword(s):

T Cell ◽

Cell Lines ◽

Histone Deacetylase Inhibitors ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Hypomethylating Agents ◽

Cytotoxicity Assays ◽

Nanomolar Range

Abstract Abstract 3937 CHOP and CHOP-like chemotherapy programs remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite often sub-optimal results. Histone deacetylase inhibitors (HDACIs) are epigenetic agents known to be active in T-cell lymphoma. Recently romidepsin (R) was approved for patients with relapsed or refractory CTCL. Both R and belinostat (B) are being investigated in patients with relapsed or refractory PTCL. We have previously shown that hypomethylating agents as decitabine (D) produce synergistic interactions with HDACIs in B-cell lymphomas. We investigated the in vitro and in vivo activity of D, R and B alone or in combination in different T-cell lymphoma and leukemia cell lines including CTCL (H9, HH), and T- acute lymphoblastic leukemia (T-ALL) lines resistant to gamma-secretase inhibitors (GSI) (P12, PF-382). For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo™ followed by acquisition on a Biotek Synergy HT. The IC50s for D, B and R were calculated using the Calcusyn software (Biosoft). Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR) based on the GraphPad software (RRR<1 are defining synergism). Apoptosis was assessed by staining with Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Whole cell lysate proteins were extracted and quantified according to Bradford assay. After electrophoresis on a gradient 4–20% SDS-PAGE gels the proteins were transferred to nitrocellulose membrane. After blocking and incubation with the primary and the secondary antibodies, the chemiluminescent agent was added and the x-ray films were exposed to the membranes. The IC50s for belinostat alone at 24, 48 and 72 hours were generally in the nanomolar range: H9: 108.1nM – 35.7nM – 29.1nM; HH: 240.1nM - 67.6nM – 39.01nM; P12: 386.9nM – 99.9nM – 99.8nM; PF 382: 267.1nM – 135nM – 118.3nM. The IC50s for romidepsin alone at 24, 48 and 72 hours were generally in the low nanomolar range: H9: 5nM – 2.1nM – 2.2nM; HH: 14nM – 2.6nM - 2.5nM; P12: 6.2nM – 2.4nM – 2.1nM; PF382: 6.1nM – 1.7nM – 1.5nM. The IC50s for D alone at 72 and 96 hours were in the micromolar range: H9: 7.4uM – 3.7uM; HH: > 20 uM. In the cytotoxicity assays, the combination of D and B or R at 72 hours showed synergism in all the cell lines studied. The most representative RRRs are showed in table 1. Table 1 D 0.5 uM 1uM B (nM) RRR H9 50 0.7 0.7 70 0.6 0.6 100 0.4 0.5 PF 382 150 0.8 0.7 0.5 uM 1 uM R (nM) RRR H9 0.5 0.9 0.9 1 0.8 0.8 2 0.3 0.3 PF 382 1 0.8 0.7 1.5 0.4 0.4 2 0.1 0.1 When H9, HH, P12 and PF382 cell lines were treated with D and B or R for 72 hours, all the combination groups showed significantly more apoptosis than the single drug exposures and controls. Table 2 displays the range of apoptosis induction for B, R and D or for them used in combination and the RRR value after the analysis for the most significant data. Table 2 B D B + D RRR (% Apoptotic + Dead Cells) H9 100nM (22.9%) 500nM (17.9%) 51.5% 0.7 HH 100nM (42.9%) 1uM (46.9%) 61.3% 0.8 P 12 150nM (16%) 1uM (42.7%) 80.1% 0.4 PF 382 100nM (8.3%) 1uM (27.9%) 40.1% 0.8 R D R + D H9 2nM (22.2%) 500nM (17.9%) 63.6% 0.5 HH 2nM (80%) 1uM (46.9%) 89.7% 0.6 P 12 2nM (9.9%) 10uM (58.7%) 98% 0.03 PF 382 2nM (54.5%) 500nM (17.9%) 88.7% 0.2 Increased acetylation of H3 was observed when H9 cells were treated with R alone and synergistically increased after exposing cells to the combination of D + B and D + R. The expression of phosphorylated Stat3 was decreased after exposure of H9 cells to the combination of D and R. Additional interrogation of the effects of this epigenetic therapy on the JAK-STAT signaling pathway are now underway. An in vivo xenograft study in six to eight weeks old female SCID beige mice injected subcutaneously with 2 × 107 HH cells has also begun and will be reported. Mice were separated into different cohorts and treated with intraperitoneal injections of D or B or their combination according to the following schedules: D alone at 1.5 mg/kg on days 1, 5; B alone at 35 mg/Kg/day for 7 days. Collectively, the data suggest that the combination of a hypomethylating agent like D and a HDACI (B and R) are synergistic in in vitro models of human T-cell lymphoma, and may lead to a new platform for the treatment of these diseases. Disclosures: O'Connor: Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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MHC Class II and T Cell Receptor Signals Are Dispensable for IL-2-Induced Regulatory T Cell Proliferation In Vivo

Blood ◽

10.1182/blood.v118.21.2181.2181 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2181-2181

Author(s):

Tao Zou ◽

Atsushi Satake ◽

Jonathan Maltzman ◽

Taku Kambayashi

Keyword(s):

T Cell ◽

Conflicts Of Interest ◽

Interleukin 2 ◽

Adaptor Protein ◽

Class Ii ◽

Sh2 Domain ◽

Tcr Signaling ◽

Mhc Ii

Abstract Abstract 2181 Regulatory T cells (Tregs) protect the host from autoimmunity and inappropriate immune activation. Thus, to ensure immune tolerance in the steady state, an adequate number of peripheral Tregs must be constantly maintained. Prior work has suggested that major histocompatibility class II (MHC II) and interleukin-2 (IL-2) are both necessary to maintain peripheral Treg homeostasis and proliferation in vivo. However, we have recently reported that Treg proliferation may not strictly depend on MHC II, as the provision of IL-2 was sufficient to drive proliferation of Tregs in an MHC II-independent manner in vitro, as long as the Tregs interacted with dendritic cells (DC)s. Here, extending our previous in vitro observations, we tested the dependence of Treg proliferation on IL-2, DCs, and TCR signaling in vivo. Proliferation of adoptively transferred Tregs was detected in wildtype (WT) mice. This proliferation was markedly enhanced when the mice were injected with IL-2 immune complexes (IC)s but not when the IL-2 IC-injected mice lacked DCs, suggesting that IL-2-induced Treg proliferation was dependent on DCs in vivo. As previously reported, adoptively transferred Tregs did not proliferate in MHC II-deficient hosts. However, the injection of IL-2 ICs into these mice induced Treg proliferation comparable to those transferred into IL-2 IC-injected WT mice, suggesting that IL-2 signaling by Tregs obviated the need of MHC II for their proliferation. Furthermore, while the ablation of TCR signaling by conditional deletion of the adaptor protein SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) rendered Tregs unable to proliferate by themselves, IL-2 IC treatment partially rescued this deficiency. We next examined the signaling pathways involved in Treg proliferation downstream of the IL-2 receptor. Despite the importance of the Stat5 pathway in IL-2 receptor signaling during Treg development in the thymus, activation of Stat5b alone was insufficient to rescue proliferation of SLP-76-deficient Tregs, indicating that alternative pathways must also be activated for Treg proliferation. Additional studies investigating the role of other signaling molecules downstream of the IL-2 receptor are currently underway. In summary, we have demonstrated for the first time that Tregs do not require TCR signaling through interaction with MHC II for their proliferation in vivo. We propose that this MHC II-independent mode of Treg proliferation allows Tregs with multiple antigen specificities to proliferate, which ensures that a diverse TCR repertoire is continuously maintained in the Treg pool. Furthermore, we believe that exploitation of these pathways may be therapeutically beneficial in autoimmunity and in transplantation. Disclosures: No relevant conflicts of interest to declare.

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Preclinical Activity of Brentuximab Vedotin (SGN-35) in Primary Effusion Lymphoma (PEL),

Blood ◽

10.1182/blood.v118.21.3728.3728 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3728-3728 ◽

Cited By ~ 1

Author(s):

Shruti Bhatt ◽

Brittany Ashlock ◽

Yaso Natkunam ◽

Juan Carlos Ramos ◽

Enrique Mesri ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Lines ◽

Conflicts Of Interest ◽

Primary Effusion Lymphoma ◽

Large Cell ◽

Brentuximab Vedotin ◽

Antibody Drug Conjugate ◽

Human Herpes Virus 8

Abstract Abstract 3728 Primary effusion lymphoma (PEL) is a distinct and aggressive subtype of non-Hodgkin lymphoma (NHL) commonly presenting with pleural, peritoneal, or pericardial malignant effusions usually without a contiguous tumor mass. PEL is most commonly diagnosed in HIV-positive patients, accounting for 4% of all NHLs in this population, yet may also develop in immunosuppressed HIV-negative individuals. While Human Herpes Virus 8 (HHV8 or Kaposi's sarcoma-associated herpesvirus) is directly implicated in the oncogenesis of this lymphoma, most PEL cases are also associated with Epstein-Barr virus and the combination of the two may facilitate transformation. The tumor cells exhibit plasmablastic features and express CD45, CD38, CD138, HHV8 and CD30. PEL is an aggressive tumor characterized by a short median survival of only 6 months with current therapeutic approaches underscoring the urgent need for development of new therapeutics. Brentuximab vedotin (SGN-35) is an antibody-drug conjugate (ADC) comprised of an anti-CD30 monoclonal antibody cAC10 conjugated by a protease-cleavable dipeptide linker to a potent cell killing agent monomethyl auristatin E (MMAE). Following binding to CD30, brentuximab vedotin is rapidly internalized and is transported to lysosomes, where the peptide linker is selectively cleaved allowing binding of the released MMAE to tubulin and leading to cell cycle arrest and apoptosis. Brentuximab vedotin was recently reported to have promising antitumor activity in CD30 expressing tumors, such as Hodgkin and Anaplastic large cell lymphomas. Since PEL tumors are reported to express CD30, we have hypothesized that brentuximab vedotin might be effective in the treatment of this NHL subtype. Initially, we have confirmed by flow cytometry the expression of CD30 on PEL cell lines (UM-PEL 1, UM-PEL 3, BC-1 and BC-3), and by review of immunohistochemistry and flow cytometry results in patients with previous diagnosis of PEL at our institution. To examine in vitro potency of brentuximab vedotin, UM-PEL 1, UM-PEL 3, BC-1 and BC-3 PEL cell lines were treated with brentuximab vedotin at concentration ranging from 0–100 micrograms/ml. Staining with YO-PRO and Propidium Iodide (PI) demonstrated dose dependent cell apoptosis and death in all the cell lines at 72 hours post treatment. In contrast, control IgG conjugated with MMAE failed to induce apoptosis and cell death of PEL cell lines confirming specific brentuximab vedotin cytotoxicity. Furthermore, brentuximab vedotin decreased proliferation of PEL cells at 48 hours leading to a complete proliferation arrest at 72 hours, as measured by MTS assay. These effects were absent after equivalent doses of control IgG conjugated drug treatment. Supportive to this, labeling of cells with PI to detect active DNA content by flow cytometry showed that bretuximab vedotin induced growth arrest in G2/M phase. To further establish the anti-tumor potential of brentuximab vedotin in vivo, we used the direct xenograft UM-PEL 1 model, established in our laboratory (Sarosiek, PNAS 2010), which mimics human PEL tumors. UM-PEL 1 bearing mice were injected intraperitoneally 3 times a week with brentuximab vedotin or control IgG conjugated MMAE for 4 weeks. Brentuximab vedotin treatment markedly prolonged overall survival of UM-PEL-1 bearing mice compared to controls (p Disclosures: No relevant conflicts of interest to declare.

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Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

Blood ◽

10.1182/blood.v120.21.2417.2417 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2417-2417

Author(s):

Olga Ritz ◽

Jochen K Lennerz ◽

Karolin Rommel ◽

Karola Dorsch ◽

Elena Kelsch ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Ectopic Expression ◽

B Cell Lymphoma ◽

Proximal Promoter ◽

Constitutively Active

Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.

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