Timmy O'Connell

The effects of DLEU1 gene expression in Burkitt lymphoma (BL): Potential mechanism of chemoimmunotherapy resistance in BL

Following a multivariant analysis we demonstrated that children and adolescents with Burkitt lymphoma (BL) and a 13q14.3 deletion have a significant decrease in event free survival (EFS) despite identical short intensive multi-agent chemotherapy. However, how this deletion in the 13q14.3 region is associated with a significant decrease in EFS in children and adolescents with BL is largely unknown. The gene Deleted in Lymphocytic Leukemia 1 (DLEU1) is located in the region of 13q14.3. Here, we report that DLEU1 expression is implicated in the regulation of BL programmed cell death, cell proliferation, and expression of apoptotic genes in transcription activator-like effector nuclease (TALEN)s-induced DLEU1 knockdown and DLEU1 overexpressing BL cell lines. Furthermore, NSG mice xenografted with DLEU1 knockdown BL cells had significantly shortened survival (p < 0.05 and p < 0.005), whereas those xenografted with DLEU1 overexpressing BL cells had significantly improved survival (p < 0.05 and p < 0.0001), following treatment with rituximab and/or cyclophosphamide. These data suggest that DLEU1 may in part function as a tumor suppressor gene and confer chemoimmunotherapy resistance in children and adolescents with BL.

Abstract 5575: Androgen receptor stimulation with 5α-dihydrotestosterone (DHT) decreases PD-L1 expression in androgen-responsive thyroid cancer cells

Thyroid cancer is the most rapidly increasing cancer in the US with 64,300 new cases expected in 2016. However, there is a disparity in the incidence of thyroid cancer between females and males, with women (49,350 expected cases) developing thyroid cancer three times more often than men (14,950 expected cases). Immune elimination of nascent tumor cells may explain the difference in disease incidence between men and women. To address this hypothesis, the effect of androgen on the expression of immune checkpoint molecules in an androgen responsive-thyroid cancer cell line was examined. The undifferentiated thyroid cancer cell line, 8505C, does not express a functional androgen receptor (AR). 84E7 is a clone of 8505C that was transfected with an AR containing plasmid resulting in constitutively expressed AR. Transcriptome analysis via RNASeq was performed on 8505C and 84E7, with and without 5α-dihydrotestosterone (DHT) treatment. Raw sequencing reads were aligned to the UCSC hg19 human reference genome with Tophat, and Cufflinks was used to measure transcript abundances in Reads Per Kilobase of exon model per Million mapped reads (RPKM) as well as to find genes with statistically significant changes in expression. DHT treatment of 84E7 resulted in >2 fold expression changes in 1,552 genes. We examined the immune checkpoint ligands expressed on 84E7 such as CD80, CD86, PD-L1, PD-L2, ICOSL, B7-H3, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GAL9. PD-L1 was the sole immune checkpoint molecule that exhibited a significant expression change in DHT-treated 84E7 cells with a 1.8 log2 fold decrease (p=0, q=0), or 72% reduction in mRNA content. Additional studies confirmed the RNASeq results and provide evidence of both qualitative (immunofluorescence) and quantitative (flow cytometry and western blotting) decreases in PD-L1 expression with DHT treatment. These results are significant in that PD-L1 is produced by tumor cells as a strategy for subverting and evading the immune response, specifically T cells, allowing for continued tumor growth and metastases. In the thyroid, the presence of activated androgen receptors could lead to an environment that is more favorable for immune system activation and may help eliminate nascent thyroid cancer cells. Thus, men may experience a decreased incidence of thyroid cancer due to an enhanced and less inhibited anti-tumor environment. Citation Format: Timmy J. O9Connell, Melanie Jones, Anvita Gupta, Tali Lando, Deya Jourdy, Edward Shin, Augustine Moscatello, Raj Tiwari, Jan Geliebter. Androgen receptor stimulation with 5α-dihydrotestosterone (DHT) decreases PD-L1 expression in androgen-responsive thyroid cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5575. doi:10.1158/1538-7445.AM2017-5575

Abstract 2608: Ibrutinib significantly improves survival in a human Burkitt lymphoma (BL) xenograft NSG mouse model: Ibrutinib may be a potential adjuvant agent in the treatment of BL

BACKGROUND: Burkitt lymphoma (BL) represents approximately 40% of all childhood and adolescent non-Hodgkin lymphoma (Miles/Cairo, BJH, 2012). Children with relapsed or progressive BL develop chemotherapy-resistant disease and can rarely be cured with salvage therapy (Cairo et al, JCO, 2012). Bruton9s tyrosine kinase (BTK) is a regulator of normal B-cell development and is activated upon B-cell receptor (BCR) stimulation. Chronic active BCR signaling through BTK activation can be inhibited by the selective and covalent BTK inhibitor, ibrutinib (Young et al, Nat Rev, 2013). Ibrutinib has been highly effective in the treatment of refractory patients with CLL and MCL and has been approved by the FDA for patients with CLL or MCL who have received at least one prior therapy (USPI) (Byrd et al, NEJM, 2013 and Wang et al, NEJM, 2013). BL, however, is associated with tonic or possibly chronic active BCR signaling; while, both CLL and MCL have chronic active BCR signaling. We have previously demonstrated ibrutinib significantly decrease BL proliferation and viability in vitro (Lee/Cairo et al, ASH, 2014) OBJECTIVE: We hypothesize that ibrutinib may be a potential adjuvant agent in the treatment of BL. Therefore, we investigated the in vivo anti-tumor activity of ibrutinib in BL xenografted NOD/SCID (NSG) mice. METHODS: The luciferase expression plasmid (ffluc-Zeo), kindly provided by L. Cooper, MD) was transfected into Raji cells. Cells were subcutaneously injected into NSG mice (6-8wks old, NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ, Jackson lab). Tumor cells engraftment and progression were examined by Bioluminescent Imaging using the Xenogen IVIS-200 (Caliper Life Sciences). Mice were orally gavaged with vehicle control or Ibrutinib (1.25, 12.5 and 25mg/kg/day, generously provided by Janssen RD 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2608. doi:10.1158/1538-7445.AM2015-2608

Abstract 3096: Obinutuzumab (GA101) significantly induces antiproliferative effects and programmed cell death, and significantly downregulates cell signaling pathways in primary mediastinal B-cell lymphoma (PMBL): Obinutuzumab may be a future potential targeted agent for treatment of PMBL

BACKGROUND: Primary Mediastinal large B-cell lymphoma (PMBL) is a rare form of non-Hodgkin Lymphoma (NHL) representing 2% of mature B-cell NHL in patients ≤ 18 years of age (Lones/Cairo et al., JCO, 2000). PMBL is classified as a distinct mature B-Cell lymphoma and is considered midway in the biologic spectrum between Diffuse Large B-Cell Lymphoma (DLBCL) and classical HL (Abramson et al., Blood, 2005). We have recently reported a significant decrease in EFS among children and adolescent PMBL patients compared with other stage III non-PMBL pediatric DLBCL patients following FAB/LMB 96 therapy, suggesting that children and adolescent PMBL may be an inherently different B-NHL (Gerrard/Cairo et al., Blood, 2013). Since 98% of PMBL express CD20, targeting the CD20 receptor with a CD20 antibody is of high clinical interest. Obinutuzumab (GA101) is a novel, anti-CD20 targeted monoclonal antibody recognizing a unique CD20 type II epitope and has been demonstrated to have efficacy in reducing tumor size and improving survival in other B-NHL xenograft models (Mossner et al., Blood, 2010). OBJECTIVES: We hypothesize that obinutuzumab may be a future potential targeted agent for the treatment of PMBL, and therefore, we investigated the effect of obinutuzumab on cell proliferation, programmed cell death, and cell signaling pathways in PMBL. METHODS: CD20+ PMBL, Karpas-1106P cells (Karpas) were obtained from the DSMZ, Germany and obinutuzumab was generously provided by C. Klein, Roche. Cell proliferation and apoptotic rate were assessed using MTS and FACS analysis, respectively. Statistical significance was determined by one-tailed Student9s t-test. RESULTS: There was a significant decrease of cell proliferation in obinutuzumab-treated Karpas vs PBS-treated Karpas (1.000 ± 0.000) at 48 hours with 1ug/ml (0.723 ± 0.005, p=0.0007), 10ug/ml (0.688 ± 0.025, p=0.0033), and 20ug/ml (0.627 ± 0.042, p=0.0092) obinutuzumab treatment. There was a significant increase in cell death in Karpas following 10ug/ml obinutuzumab treatment (37.790 ± 10.096, p=0.018) as compared to IgG isotope (1.040 ± 0.834, p=0.340) and PBS control (1.190 ± 0.762) at 48 hours by FACS analysis. We also observed significant decreases in the phosphorylation of Stat6 (0.730 ± 0.005, p=0.0001), Akt (0.691 ± 0.002, p CONCLUSIONS: Obinutuzumab significantly induced antiproliferative effects and cell death in PMBL and may be a future potential targeted agent for the treatment of PMBL. The effect(s) of obinutuzumab will be evaluated in vivo in a NOD/SCID PMBL xenograft mouse model. Citation Format: Changhong Yin, Timmy O9Connell, Janet Ayello, Carmella van de Ven, Sanghoon Lee, Mitchell Cairo. Obinutuzumab (GA101) significantly induces antiproliferative effects and programmed cell death, and significantly downregulates cell signaling pathways in primary mediastinal B-cell lymphoma (PMBL): Obinutuzumab may be a future potential targeted agent for treatment of PMBL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3096. doi:10.1158/1538-7445.AM2014-3096