ALBERT

Description: A broad-spectrum antibiotic, DCAP, reduces uropathogenic Escherichia coli infection and enhances vorinostat anticancer activity by modulating autophagy A broad-spectrum antibiotic, DCAP, reduces uropathogenic 〈i〉Escherichia coli〈/i〉 infection and enhances vorinostat anticancer activity by modulating autophagy, Published online: 13 July 2018; doi:10.1038/s41419-018-0786-4 A broad-spectrum antibiotic, DCAP, reduces uropathogenic Escherichia coli infection and enhances vorinostat anticancer activity by modulating autophagy

Description: We have developed a murine model for mantle cell lymphoma (muMCL) that occurs after pristane injection of Eμ-cyclin D1 transgenic mice that are 〉 1 year of age (Leukemia 20:891, 2006). Eμ-cyclin D1 transgenic mice are healthy, implying that cyclin D1 may be necessary, but not sufficient, for MCL decvelopment. We are using this model to identify genes that coooperate with cyclin D1 in lymphomagenensis. Comparing gene expression in B cells from spleens of these muMCL with B cells from non-pristane injected age-matched Eμ-cyclin D1 transgenic mice by differential gene expression, we identified up-regulation of RNA for the Huntingtin interacting protein HIP-1 in the muMCL. We confirmed that this translated into up-regulation at the protein level by Western blot. HIP-1 is an inositol lipid, clathrin and actin binding protein that can directly transform fibroblasts. HIP-1, located on 7q, was previously found in a fusion protein with PDGFRβ in a case of CMML with t(5;7), suggsting a role in hematopoietic malignancies. To test the generalizability of the finding of HIP-1 overexpression in lymphoma, we performed immunohistochemistry using the monoclonal anti-HIP-1 antibody 4B10 on a lymphoma tissue microarray (Cybridi). Positive antibody staining was found in 79 of 116 (68.1%) non-Hodgkin’s lymphomas (NHL). Based on: our identification of HIP-1 as a potential cooperating gene in MCL development; overexpression of HIP-1 in many NHL; and that the presence of antibodies to HIP-1 in serum correlates with overexpression of the protein, we tested serum samples from lymphoma patients. We found circulating HIP-1 antibodies in sera from 23 of 39 (59%) lymphoma patients. The highest antibody titer was in serum from a patient with T-ALL in remission during maintenance chemotherapy who had a cytogenetic abnormality of 7q near the site of the HIP-1 gene. Studies are ongoing to expand this data set to see if HIP-1 antibodies correlate with disease type or outcome. In summary, HIP-1 appears to be involved in lymphoma development in a murine model of MCL and is frequently overexpressed in other types of NHL. Its role in lymphomagenesis, and potentially in treatment response and prognosis needs to be further explored.

Description: TRAIL-R1 and -R2 signaling induces apoptosis via a pathway that activates caspase 8. The proteosome inhibitor bortezomib may act via several pathways. Agonistic antibodies to TRAIL-R1 and -R2 and bortezomib are in clinical trials in solid tumors and hematologic malignancies. To develop rational combinations for future clinical studies, we investigated the actions of these agents on non-Hodgkin s lymphoma (NHL) cell lines. The t(14;18)+, EBV- NHL cell lines DoHH2 and WSU-FSCCL were treated with agonistic monoclonal antibodies to TRAIL-R1 (HGS-ETR1) and -R2 (HGS-ETR2) (Human Genome Sciences, Rockville, MD) and/or bortezomib. While HGS-ETR 1 and HGS-ETR 2 are effective inducers of apoptosis in FSCCL, DoHH2, which expresses dim TRAIL-R1 (DR4, HGS-ETR1 target) and TRAIL-R2 (DR5, HGS-ETR2 target), shows minimal growth inhibition or apoptosis induction by HGS-ETR1 or HGS-ETR2. Bortezomib has modest effects on DoHH2 cells in growth inhibition and apoptosis assays. HGS-ETR1 and HGS-ETR2 induction of apoptosis in WSU-FSCCL is efficiently blocked by the caspase inhibitor ZVAD. In contrast, bortezomib effects are not blocked by ZVAD, indicating an independent mechanism of action. To determine if these separate pathways would provide enhanced combination activity, DoHH2 cells were pre-treated with bortezomib for 30 min, followed by incubation with HGS-ETR1 or HGS-ETR2. This led to supra-additive induction of apoptosis (annexin V staining). We conclude that bortezomib sensitizes DoHH2 cells to the action of HGS-ETR1 and HGS-ETR2. Further, bortezomib induces apoptosis in DoHH2 cells by an independent mechanism, and the combination of TRAIL-receptor signaling and bortezomib may be a useful combination to explore.

Description: Birinapant (TL32711), a Smac mimetic in clinical testing, potently targets Inhibitor of Apoptosis Proteins (IAPs, including cIAPs and XIAP) to unblock intrinsic and extrinsic pathways, enabling caspase-dependent apoptosis via multiple signals. Birinapant also inactivates canonical NF-kB signaling through cIAPs. We investigated the pro-apoptotic effects of birinapant, alone and in combination with bendamustine (BDM), an active lymphoma therapeutic agent, in a panel of B cell lymphoma cell lines representing germinal center/follicular (GC) vs. activated B cell (ABC) subtypes. We hypothesized that the efficacy of this potential combination therapeutic strategy might differ between GC and ABC lymphoma types, as ABC are reported to be NF-kB-dependent. We used the following EBV negative cell lines: WSU-FSCCL t(14:18)+ follicular lymphoma (FL), FC-TxFL2 t(14:18)+ transformed FL, and SU-DHL4 GC-type diffuse large B cell lymphoma (DLBCL) as examples of GC origin lymphomas. U2932 and TMD8 cell lines represent ABC-type DLBCL.  Apoptosis was determined by annexin V staining and confirmed by caspase-3 activation, each assessed by flow cytometric methods following 48 h incubation. Birinapant had little effect (50% annexin V+) of apoptosis induced by 10 uM BDM in WSU-FSCCL and FC-TxFL2,  and only slightly enhanced the low level of BDM-induced apoptosis in the GC DLBCL cell line DHL-4 (to 10-15%). In the ABC DLBCL cell lines, however, whereas 10uM BDM induced

Description: Background: Mantle cell lymphoma (MCL) is characterized by t(11;14) which dysregulates cyclin D1 expression. Eμ-cyclinD1 transgenic mice, however, are healthy. Additional genetic events must be necessary for lymphomagenesis, and knowledge of these would enhance understanding and therapy of MCL. In addition, a mouse model of MCL would be helpful in drug development. Alterations in p53 have been described in MCL, often associated with the blastic variant. Objectives and Methods: To determine whether p53 and cyclin D1 can cooperate in lymphomagenesis, we cross bred Eμ-cyclinD1 transgenic mice (Bodrug et al EMBO J, 1996, courtesy of Alan Harris) with mice transgenic for mutant p53 (Jackson Labs, Jacks et al Curr Biol, 1994). Progeny mice were monitored for presence of the transgenes by PCR of tail vein DNA and observed for development of disease. Results: Of mice carrying both aberrant genes, 24 of 38 developed B cell lymphoma. Mice did not become visibly ill until at least 12 months of age, with median age at sacrifice 15.5 (range 12–23) months. The lymphoma was generally disseminated, involving spleen, liver, diffuse adenopathy and marrow with occasional extranodal sites. Histology varied between small and large cell, with some having a vaguely follicular growth pattern. T cell lymphomas occurred in 2 other mice, while 5 developed osteosarcoma (1 of these in a mouse that also had B cell lymphoma). The B cell lymphomas were clonal by Cμ-VH PCR. Cyclin D1 expression was documented by Western analysis. A cell line has also been developed from one of the B cell lymphomas and this line rapidly grows into disseminated lymphoma in syngeneic mice. These B cell lymphomas differ from the thymic T cell lymphomas seen in heterozygous p53 mutant mice that do not co-express cyclin D1. The latency period differs from cyclin D1 x myc double transgenic mice. Conclusions: This model demonstrates cooperation between p53 and cyclin D1 pathways in B cell lymphomagenesis and should prove useful in delineating how these signals interact. The cell line may prove useful in pre-clinical testing of new agents for MCL.

Description: Monoclonal antibodies and chemotherapy can be effective, but not curative, therapy for non-Hodgkin s lymphoma (NHL). Rituximab acts by several mechanisms, including directly signaling apoptosis of CD20+ cells via the mitochondrial pathway involving caspase 9. Chemotherapy also activates this apoptotic pathway. TRAIL-R1 and -R2 signaling induces apoptosis via a pathway that activates caspase 8. Thus, we investigated the effects of agonistic monoclonal antibodies to TRAIL-R1 and -R2 on NHL cell lines (DoHH2, WSU-FSCCL and FC-TxFL2, each t(14;18)+, EBV-). TRAIL-R1 (HGS-ETR1) and -R2 (HGS-ETR2) antibodies were from Human Genome Sciences (Rockville, MD). FC-TxFL2 expressed the highest cell surface levels of TRAIL-R1 (DR4, target of HGS-ETR1) and TRAIL-R2 (DR5, target of HGS-ETR2). WSU-FSCCL expressed lower, but significant, levels of each. DoHH2 expressed dim TRAIL-R1 and the lowest levels of TRAIL-R2 of the 3 cell lines. IC50 (MTT assay after 72 hr incubation with antibody) was 0.25mcg/ml for WSU-FSCCL and FC-TxFL2 with either HGS-ETR1 or HGS-ETR2, while DoHH2 was minimally inhibited by either antibody. Both HGS-ETR1 and HGS-ETR2 antibodies induced dose dependent increases in apoptosis (annexin V assay) of WSU-FSCCL and FC-TxFL-2, but not DoHH2. Caspase activation (flow cytometry using fluorescent substrates; Western analysis) was seen 4–6 hr after antibody addition. As expected, caspases 3 and 8 were activated by both antibodies in the two sensitive cell lines. Caspase 9, however, was also activated. Thus, TRAIL-R1 and TRAIL-R2 antibody binding inhibits growth, induces apoptosis and activates caspases 3, 8 and 9 in NHL cells expressing the targets. The induction of caspase 9 suggests cross-talk between the extrinsic and intrinsic pathways, in which activation of caspase 8 leads to cleavage and translocation of bid, which in turn leads to activation of caspase 9, and ultimately caspase 3. We have confirmed that bid cleavage does occur in these NHL cells after HGS-ETR1 and HGS-ETR2 binding. We have further shown that both the pan-caspase inhibitor ZVAD and a specific caspase 8 inhibitor block HGS-ETR1 and HGS-ETR2 induced apoptosis. As predicted, a specific inhibitor of caspase 9 only partially blocks apoptosis. This suggests that combination of the agonist HGS-ETR1 and HGS-ETR2 antibodies with agents that act via the caspase 9 pathway would be rational combinations to test for therapeutic potential in NHL.

Description: Abstract 3735 Agonist monoclonal antibodies (mAbs) to CD137, a co-stimulatory TNF receptor family member expressed on activated T and NK cells, can induce immune-mediated rejection of multiple murine tumor types, and a fully human anti-CD137 mAb, BMS-663513, is in early-phase clinical trials in solid tumors. Significant activity has been seen in murine lymphoma models, both alone and in combination with anti-CD20 mAbs, providing rationale for clinical studies in lymphoma patients. Recently, however, CD137 up-regulation on activated human B cells has been reported, with CD137 ligation causing enhanced B cell proliferation and survival. This raises the concern that mAb binding to CD137, if present, on B cell neoplasms may promote tumor cell proliferation and/or resistance to apoptosis that may counteract the beneficial effects on T and NK cells. We therefore sought to assess the expression of CD137 on a series of human cell lines and primary tumor samples from patients with B-cell neoplasms, and if expressed, to explore the consequences of ligation with the anti-CD137 agonist BMS-66513. First, archived paraffin-embedded lymph node specimens from patients with low-grade B-cell lymphoma (n=11: 5 follicular, 4 marginal zone, 2 small lymphocytic) and diffuse large B-cell lymphoma (n=15) were stained for CD137 by immunohistochemistry. Reactive tonsillar tissue served as a positive control. No CD137 expression was observed within any tumor cells. Next, fresh samples from 14 additional patients with known tumor involvement of peripheral blood or bone marrow (8 chronic lymphocytic leukemia, 1 mantle cell lymphoma, 3 myeloma, 2 marginal zone lymphoma) were analyzed by multi-color flow cytometry. Again, no CD137 expression was observed on the gated neoplastic cells. Baseline surface expression of CD137 was similarly absent in all B cell-derived lines tested (Raji, FCTxFL2, FSCCL, DoHH2, Jeko-1, RPMI8226). However, activation with PMA/Ionomycin could reproducibly induce CD137 expression (% positive: 0.17% → 91%) after 24 hours in 1 of the lines: the follicular lymphoma FSCCL. Interestingly, this was the only line tested that lacked constitutive expression of CD137 ligand (CD137L), suggesting some reciprocal regulation of ligand and receptor expression. Despite this up-regulation of CD137, in vitro ligation of PMA/Ionomycin-activated FSCCL cells with BMS-66513 did not further increase tumor cell proliferation, nor protect the cells from activation-induced cell death, in contrast to effects of CD137 ligation reported in normal B cells (Zhang et al, J Immunol 2010; 184:787). Similarly, BMS-663513 treatment of activated, CD137+ FSCCL cells did not diminish the apoptosis induced by doxorubicin or bortezomib treatment. In addition, FSCCL cells recovered from ascites 7 and 14 days following intraperitoneal injection in SCID mice did not express CD137, implying that CD137 up-regulation is not occurring in vivo during tumor growth. Finally, treatment of FSCCL cells with rituximab, either in vitro or in vivo, did not induce CD137 expression. In conclusion, we demonstrate a lack of steady-state CD137 expression on malignant B cells, confirming the prior study by Houot et al (Blood 2009; 114:3431) and extending these findings to include CLL/SLL for the first time. While CD137 could be induced in a single cell line upon non-specific activation, CD137 expression on FSCCL cells was not seen under physiologic conditions likely to be encountered in the clinical setting, consistent with the primary patient data. Furthermore, even when CD137 was expressed, ligation with the agonist anti-CD137 mAb BMS-663513 did not provide a pro-proliferative or anti-apoptotic signal. These studies provide reassurance and further rationale for exploring agonist anti-CD137 antibodies as therapies for B cell neoplasms. Disclosures: Borghaei: Lilly, Genentech, Amgen, Pfizer: Honoraria, Research Funding. Jure-Kunkel:Bristol Meyers Squibb: Employment.