BCL1 Cells Research Articles

Evaluating the role of Ubiquitin D gene expression in types of leukemia.

Using animal models to develop new treatments is essential, especially in diseases like cancer. In this study, we induced leukemia by intravenous injection of cancer cells (BCL1 cell line) and the examination of cell markers in the animal's blood to study the changes in the expression of the UBD gene as a biomarker for diagnosing and examining the progress of the disease. For this purpose, five million BCL-1 cells were injected into the tail vein of BALBIe mice of the same breed. Fifty mice were killed after four weeks, and we examined peripheral blood cells and histological changes. Then RNA of the samples was extracted, and cDNA synthesis was done with the help of MMuLV enzyme, Oligo dT, and Random hexamer primers. Specific primers for UBD were designed using Primer Express software, and the expression level of the UBD gene was measured by the method. The results showed that in the CML group, the lowest expression level was 1.70 times, and in the ALL group, the highest expression level was 7.97 times compared to the control group. The average increase in UBD gene expression was 3.21 times in the CLL group and 4.94 times in the AML group. The UBD gene can be further investigated so that it may be used as a proposed biomarker for the diagnosis of leukemia. Therefore, the evaluation of the expression level of this gene can be used to diagnose leukemia. However, more studies than the currently applied methods are needed in cancer diagnosis, which has many errors compared to the technique used in this study, and to prove its accuracy and sensitivity.

The Pt(S-pr-thiosal)2 and BCL1 Leukemia Lymphoma: Antitumor Activity In Vitro and In Vivo.

B cell malignancies are, despite the development of targeted therapy in a certain percentage of the patients still a chronic disease with relapses, requiring multiple lines of therapy. Regimens that include platinum-based drugs provide high response rates in different B cell lymphomas, high-risk chronic lymphocytic leukemia (CLL), and devastating complication of CLL, Richter’s syndrome. The aim of this study was to explore the potential antitumor activity of previously synthetized platinum(IV) complex with alkyl derivatives of thyosalicilc acid, PtCl2(S-pr-thiosal)2, toward murine BCL1 cells and to delineate possible mechanisms of action. The PtCl2(S-pr-thiosal)2 reduced the viability of BCL1 cells in vitro but also reduced the growth of metastases in the leukemia lymphoma model in BALB/c mice. PtCl2(S-pr-thiosal)2 induced apoptosis, inhibited proliferation of BCL1 cells, and induced cell cycle disturbance. Treatment of BCL1 cells with PtCl2(S-pr-thiosal)2 inhibited expression of cyclin D3 and cyclin E and enhanced expression of cyclin-dependent kinase inhibitors p16, p21, and p27 resulting in cell cycle arrest in the G1 phase, reduced the percentage of BCL1 cells in the S phase, and decreased expression of Ki-67. PtCl2(S-pr-thiosal)2 treatment reduced expression of phosphorylated STAT3 and downstream-regulated molecules associated with cancer stemness and proliferation, NANOG, cyclin D3, and c-Myc, and expression of phosphorylated NFκB in vitro and in vivo. In conclusion, PtCl2(S-pr-thiosal)2 reduces STAT3 and NFκB phosphorylation resulting in inhibition of BCL1 cell proliferation and the triggering of apoptotic cell death.

Tween 80 improves the infectivity of BCL1 cell-adapted infectious bursal disease virus

Non-ionic surfactants have the ability to alter the cell membrane's permeability for enhancing virus replication. The impact of non-ionic surfactant Tween 80 (TW80) on the infectivity of infectious bursal disease virus (IBDV) was studied in BCL1 cells. The toxicity of different concentrations of TW80 for BCL1 cells was determined for five-time passages. The confluent monolayer of BCL1 was infected by IBDV and subsequently passaged. The adaptation was confirmed by virus titration and RT-PCR assay. Replication kinetics of the cell-adapted IBDV was evaluated in pre-treatment and simultaneous treatment with TW80 at 0.01% concentration. The IBDV infectivity patterns were determined by virus titration, FRAP assay, and transmission electron microscopy. Sequence analysis, RNA secondary structure, and potential N-glycosylation site were conducted for IBDV VP2. Despite the similar cytopathic effects found in both TW80-treated cells and similar ROS levels, the IBDV titer was higher in TW80 pre-treated cells compared to the simultaneous treatment one. Such an increase in IBDV titer did not associate with changes in the VP2 sequence and RNA secondary structure. The possible antioxidant capacity of TW80 can attenuate the ROS damage and improve the cell viability, thereby improving IBDV infectivity.

Comparing the effects of essential oils of Origanum Vulgare L. and Origanum Majorana L. on cancerous BCL-1 and immune cells (lymphocyte and macrophage)

Background: Plants of the Lamiaceae family are a rich source of medicinal compounds with anticancer properties. In many studies, only the anticancer properties of the compounds have been investigated and their toxic effects on healthy cells have not been addressed. The present study was designed to evaluate and compare the toxic effects of essential oil of two species from the genus Origanum (Lamiaceae family) include of Origanum Vulgare L. and Origanum Majorana L. on BCL-1 and normal lymphocyte and macrophage in vitro. Methods: The plant samples were collected and the essential oils were obtained using the hydro-distillation method (Clevenger). The essential oils were analyzed by GC.MS. BCL-1 cell line, lymphocyte, and macrophage from normal mice co-culture with various concentrations of essential oils for 24h. Cell viability and cytotoxic effect of the essential oils were determined by MTT and LDH assays. Results: GC.MS results show the diverse components of the two essential oils so that it has only 12 shared parts. Both essential oils at a concentration of 0.01-1% (O.Vulgare 47-79% and O.Majorana 30-82%) inhibited the growth of the BCL-1 cancer cell line. (O.Vulgare essential oil IC50:0.01% and O.Majorana essential oil IC50:0.03%). Whereas these two essential oils at the highest concentration (1%) do not decrease the cell viability of macrophages and lymphocyte. Also, according to LDH test results, none of the concentrations of both essential oil had a cytotoxic effect on normal lymphocyte and macrophage cells. Conclusion: The essential oils of O.Vulgare and O.Majorana are a proper candidate for cancer treatment because, in addition to having strong anticancer effects, they had no cytotoxic effect on normal cells of the immune system.

Role of Glucose Transporter 1 in Graft-Versus-Host Disease and Graft-Versus-Tumor Effect

Graft-versus-host disease (GVHD) is a major obstacle to allogeneic hematopoietic cell transplantation. GVHD is induced when donor mature T cells encounter host antigens and get activated. Activated T cells require high metabolic flux through growth-promoting pathways to support clonal expansion. It is generally thought that glycolysis is employed in this process. However, conflicting data have been reported by different groups regarding the role of glycolysis in GVHD. Since glucose transporter 1 (Glut1) is essential for T cell glycolysis, we attempted to define the role of glycolysis in GVHD using donor T cells that are deficient of Glut1.One million T cells from Glut1-knockout (CD4CreGlut1fl/fl) or wild-type (control) C57BL/6 donors were transplanted into lethally irradiated (9.5 Gy) C3H/HeJ mice along with 1×107 donor T-cell-depleted bone marrow (TCDBM) cells. While all recipients of control T cells developed lethal GVHD and died within 20 days after transplantation, eight out of ten CD4CreGlut1fl/fl T cell recipients survived more than 60 days after transplantation (Figure 1, P<0.01). Body weights were slightly lower and clinical scores were slightly higher (P=not significant) in the surviving CD4CreGlut1fl/fl T cell recipients at day +56 when compared to those in the TCDBM alone group, suggesting that CD4CreGlut1fl/fl T cell recipients developed very mild GVHD if any. We further studied the role of Glut1 in CD4 and CD8 T cell mediated GVHD separately using C57BL/6 into BALB/c model. Similar to the results from the total T cell experiment, all CD4CreGlut1fl/fl CD4 and CD8 T cell recipients survived more than 60 days after transplantation. By contrast, 100% control CD4 and 50% CD8 T cell recipients died of lethal GVHD within 60 days after transplantation. These data demonstrate that Glut1 is required for alloresponses and glycolysis is indispensasble in the development of lethal GVHD.To understand the mechanism by which CD4CreGlut1fl/fl T cells mediate less GVHD, we first studied their ability to respond to alloantigens in mixed lymphocyte reaction. The results indicated that CD4CreGlut1fl/fl T cells have dramatically decreased ability to respond to BALB/c antigens when compared with the control T cells. Consistent with the in vitro data, T cell expansion was 59.4% less in CD4CreGlut1fl/fl T cell recipients than that in control T cell recipients when measured at day +7 after GVHD induction. Apoptosis was increased in alloantigen-activated (CD69+) CD4 but not in CD8 T cells from CD4CreGlut1fl/fl mice when compared with that from control mice when measured 16 hours after the initiation of culture. However, more cell death was observed in both activated CD4 and CD8 T cells from CD4CreGlut1fl/fl mice than that from control mice. In addition, alloreactive CD4CreGlut1fl/fl T cells failed to sustain activated mTORC1 signaling, as assessed by phosphorylation of the downstream p70 S6 kinase target, small ribosomal subunit S6. These results indicate that, upon response to alloantigens, T cells are prone to apoptosis and cell death and lead to decreased clonal expansion in the absence of Glut1, suggesting a mechanism by which CD4CreGlut1fl/fl T cells have decreased ability to induce GVHD.To determine whether absence of Glut1 in T cells affects graft-versus-tumor (GVT) effects, we transplanted 1×106 T cells from Glut1-knockout (CD4CreGlutfl/fl) C57BL/6 donors into lethally irradiated (8.5 Gy) BALB/c mice along with 1×107 donor TCDBM cells. At the time of transplantation, the recipients were challenged with 1×105 BCL1 cells, a BALB/c-derived leukemia/lymphoma cell line. While all TCDBM alone recipients died of leukemia/lymphoma and all control T cell recipients died of lethal GVHD, all CD4CreGlut1fl/fl T cell recipients survived more than 100 days after transplantation without leukemia/lymphoma (Figure 2), indicating that T cells deficient of Glut1 preserve GVT effect.The results from this study definitely demonstrate that glycolysis is essential for the development of GVHD. Inhibition of glycolysis in T cells increases apoptosis and cell death and suppresses clonal expansion of allospecific T cells. Interestingly, GVT effect is preserved despite the inhibition of glycolysis. These data suggest that glycolysis could potentially be targeted for prevention and treatment of GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Shikonin Derivatives from Onsoma visianii Decrease Expression of Phosphorylated STAT3 in Leukemia Cells and Exert Antitumor Activity.

Antitumor effects of shikonins on chronic lymphocytic leukemia (CLL) and B-cell prolymphocytic leukemia (B-PLL) are mostly unexplored. The antitumor activity of shikonins, isolated from Onosma visianii Clem (Boraginaceae), in BCL1, mouse CLL cells and JVM-13, human B-PLL cells was explored in this study. The cytotoxicity of shikonin derivatives was measured by an MTT test. Cell death, proliferation, cell cycle, and expression of molecules that control these processes were analyzed by flow cytometry. Expression of STAT3-regulated genes was analyzed by real-time q-RT-PCR (Quantitative Real-Time Polymerase Chain Reaction). The antitumor effects of shikonin derivatives in vivo were analyzed, using flow cytometry, by detection of leukemia cells in the peripheral blood and spleens of mice intravenously injected with BCL1 cells. The two most potent derivatives, isobutyrylshikonin (IBS) and α-methylbutyrylshikonin (MBS), induced cell cycle disturbances and apoptosis, inhibited proliferation, and decreased expression of phospho-STAT3 and downstream-regulated molecules in BCL1 and JVM-13 cells. IBS and MBS decreased the percentage of leukemia cells in vivo. The link between the decrease in phosphorylated STAT3 by MBS and IBS and BCL1 cell death was confirmed by detection of enhanced cell death after addition of AG490, an inhibitor of Jak2 kinase. It seems that IBS and MBS, by decreasing STAT3 phosphorylation, trigger apoptosis, inhibit cell proliferation, and attenuate leukemia cell stemness.

Microrna-191 Regulates T-Cell Clonal Expansion during Graft-Versus-Host Disease

Allogeneic hematopoietic cell transplantation is a potentially curative treatment choice for a wide variety of hematological malignancies. However, graft-versus-host disease (GVHD), which is mediated by donor alloreactive T cells, limits the success of this procedure. Previous studies have demonstrated that several microRNAs (miRs) modulate graft-versus-host disease. miR-191 was previously reported to be able to support T cell survival after TCR stimulation. We hypothesize that miR191 regulates T cell response during GVHD. To test this hypothesis, we first studied miR-191 expression in alloreactive T cells. The result demonstrated that miR-191 was up-regulated in donor T cells isolated from murine GVHD recipients, suggesting that miR-191 may play a role in GVHD induction. We further studied the role of miR-191in GVHD using miR-191 deficient T cells (KO). Lethally irradiated (8.5 Gy) BALB/c mice were injected intravenously with 1×107 T cell-depleted bone marrow (TCDBM) cells along with 1×106 purified T cells from wild-type (WT) or KO mice, which are in C57BL/6 background. Interestingly, all recipients in the WT group died within 35 days after transplantation, while only one out of ten animals died in the KO group during an observation period of 56 days. Body weights and clinical scores were also improved in KO T cell recipients when compared with the WT controls. Similar results were also observed in a second GVHD model (C57BL/6→C3H/HeJ). To understand the mechanism by which miR-191 KO T cells have decreased ability to mediate GVHD, we first measured the ability of KO T cells to respond to alloantigens in vitro in a mixed lymphocytes reaction assay. Dramatically decreased alloresponse was observed with KO T cells as compared with WT T cells. Similarly, decreased clonal expansion was observed in KO T cells in vivo upon challenge with alloantigens as measured by bioluminescent imaging (Figure 1A). These results were further supported by data from a co-transfer experiment, in which equal numbers of WT and KO T cells were transplanted into the same GVHD recipient. At day7 after transplantation, KO T cells showed significantly reduced expansion in the spleen and liver compared with WT T cells. Reduced alloresponses mediated by KO T cells may not due to decreased proliferative capability directly as an in vivo carboxyfluorescein succinimidyl ester (CFSE) assay showed a comparable cell division between WT and KO T cells upon challenge with alloantigens. Rather, increased cell death is responsible for decreased alloresponse observed in KO T cells because dramatically increased number of dead cells was observed in KO group compared with WT group upon response to alloantigens in vitro and vivo. To determine the genes that are regulated by miR-191, we did a screening based on the prediction. Humans and mice share more than 100 predicted targets for miR-191. We chose top 20 of these targets for RT-qPCR screening. The result demonstrated that Taf5 was a target gene of miR-191. Expression of TAF5 protein was down-regulated in activated KO T cells when compared with the WT T cells. Finally, we investigated whether miR-191 KO T cells preserve graft-versus-leukemia effects. 1×106 T cells from WT or KO mice were transplanted into lethally irradiated BALB/c mice along with 1×107 TCDBM cells and 1×105 host-type BCL-1 cells. While all recipients that received only TCDBM and tumor cells developed lethal leukemia/lymphoma, none of WT and KO T cells recipients developed tumor. In conclusion, our findings reveal a critical role of miR-191 during GVHD process and demonstrate that miR-191 is a novel therapeutic target for GVHD. Figure 1 Disclosures No relevant conflicts of interest to declare.

The Apoptotic Pathway Induced by Vincristine in Mouse Proliferating and Resting Normal Lymphocytes and Lymphoma Cell Line

Background: Most anti-cancer drugs target mitosis and induce apoptosis in cancerous cells. In the immune system, proliferation and apoptosis of lymphocytes are indeed essential modulating elements. Objectives: In this study, we have investigated the effect of vincristine on normal resting and normal proliferating lymphocytes comparing with cancerous cells. Methods: Resting and proliferating splenocytes from mice and BCL1 (mouse lymphoma cell line) were cultured with different concentrations of vincristine for 48 hours, and cell lysates were prepared. The activity of caspases 3, 8, and 9 in cell lysates was measured using specific chromogenic substrates DEVD-pNA for caspase 3, IETD-pNA for caspase 8, and LEHD-pNA for caspase 9, the activity calculated as µmol/min/mg protein. Results: In the BCL1 cell line, the activity of both caspases 8 and 9 and caspase 3 increased at the presence of vincristine (5 µg/mL). In resting splenocytes, however, only a mild increase in caspase 9 activity was observed without any change in the activity of caspases 8 or 3. In the same situation, the activity of caspase 3 and 9 (but not caspase 8) elevated in proliferating cells exposed to vincristine. Nearly similar results were obtained with higher concentrations of vincristine (up 20 µg/mL). Conclusions: The results suggesting that vincristine may induce internal pathways of apoptosis in normal and cancerous cells while extrinsic pathway was induced in cancerous cells. On the other hand, the effects are highly dependent on the activation status of normal cells, and affirms that responding immune cells should be more seriously noticed when side effects of anticancer drugs are estimated.

Neutralizing IL-2 Early after HCT Specifically Prevents Expansion of Donor GM-CSF-Producing CD4+ T Cells That Initiate Acute Gut Gvhd

Acute graft versus host disease (GVHD) in gut has a critical impact on the outcome after allogenic hematopoietic cell transplantation (allo-HCT). It was recently reported that BATF-dependent GM-CSF-producing donor CD4+ T cells play a critical role in initiating acute gut GVHD (Ullrich et al: JCI 2018; Zeng: JCI; 2018). We recently reported that IL-2 from donor CD4+ T cells made infiltrating T cells resistant to host-tissue PD-L1-mediated tolerance (Ni & Song et al: JCI 2017). In the current studies, we tested whether in vivo neutralization of IL-2 early after allo-HCT regulates the expansion of GM-CSF-producing CD4+ T cells and the development of acute gut GVHD, and if so, whether host-tissue PD-L1 is required for this effect. We transplanted spleen (5x106) and BM cells (2.5x106) from C57BL/6 (H-2b) donors into irradiated wild-type (WT) or PD-L1-/- BALB/c (H-2d) recipients bearing BCL1 leukemia/lymphoma cells and into recipients without BCL1 cells. Recipients were treated with anti-IL-2 mAb IP (500 µg/mouse) on days 0, 2, 4 and 6, and controls were treated with isotype-matched IgG. We observed the following results. 1) Administration of anti-IL-2 fully prevented acute gut GVHD while preserving GVL activity in WT recipients. 2) Administration of anti-IL-2 did not prevent acute gut GVHD in PD-L1-/- recipients, indicating that GVHD prevention depends on expression of PD-L1 in recipient tissues. 3) Prevention of GVHD in WT recipients was associated with a marked reduction in the number of donor-derived GM-CSF-producing CD4+ T cells infiltrating gut tissue, together with decreased Paneth cell damage and bacterial translocation to the liver. These results indicate that neutralizing IL-2 early after allo-HCT specifically prevents expansion of acute gut GVHD-initiating GM-CSF-producing CD4+ T cells and prevents acute gut GVHD in a host-tissue PD-L1-dependent manner. These results also indicate that a short-term administration of anti-IL-2 following allo-HCT prevents induction of acute gut GVHD while preserving GVL effects. Current experiments are testing the molecular mechanisms whereby neutralizing IL-2 decreases the numbers of GM-CSF-producing CD4+ T cells in gut tissues via tissue expression of PD-L1. (Grant support: NIH 1R01CA228465-01 to Zeng). DisclosuresNo relevant conflicts of interest to declare.

Stat3 Regulates Alloreactive T Cell Susceptibility Towards PD-L1-Mediated Tolerance Signaling, Gvhd and GVL Activity

Prevention of graft versus host disease (GVHD) while preserving graft-versus-leukemia/lymphoma (GVL) activity of alloreactive donor T cells remains an elusive and long-sought goal. We and others reported that Stat3 deficiency in donor T cells results in expansion of Foxp3+ Treg cells and reduction of pathogenic Th17 cells, leading to prevention of chronic GVHD. We have now tested whether Stat3 deficiency in donor T cells can prevent acute GVHD while preserving GVL activity.We transplanted wild-type (WT) or Stat3-/- T cells from C57BL/6 (H-2b) donors into irradiated WT BALB/c recipients bearing B cell lymphoma line BCL1 or aggressive acute lymphocytic leukemia (ALL) cells. WT donor T cells caused acute GVHD, as expected. Stat3-/- donor T cells did not cause acute GVHD but still had strong GVL activity. This separation of GVL activity from GVHD was associated with augmented expansion and impaired tolerance by Stat3-/- T cells in lymphoid tissues such as the spleen. In contrast, tolerance by Stat3-/- T cells was enhanced in GVHD target tissues by different mechanisms. The yield of Stat3-/- T cells in gut tissues was lower than the yield of WT T cells due to the markedly higher apoptosis of Stat3-/- T cells compared to WT T cells. The yield of Stat3-/- T cells in the lung and liver was not lower than the yield of WT T cells, but expression of anergy/exhaustion markers such as PD-1 and KLRG1 was markedly higher in Stat3-/- T cells than in WT T cells.We recently reported that GVHD tissue expression of PD-L1 can tolerize donor CD8+ T cells in the absence of donor CD4+ T cells, but not when CD4+ and CD8+ T cells were given together. In contrast, PD-L1 expression in recipient GVHD target tissues tolerized Stat3-/- donor T cells when both CD4+ T cells and CD8+ T cells were given together. The induction of tolerance in Stat3-/- T cells by GVHD target tissue expression of PD-L1 depended on PD-L1/PD-1 interaction, which led to marked reduction of glycolysis in Stat3-/- T cells but not in WT T cells. These observations fit with previous findings that enhanced glycolysis was associated with enhanced GVHD activity of alloreactive T cells, as reported by Xue-zhong Yu's group. Our results indicate that Stat3 deficiency in donor T cells can augment their susceptibility to tissue PD-L1-mediated tolerance mechanisms such as down-regulation of glycolysis. Current experiments are testing whether Stat3 knockdown in mature T cells in the graft can prevent GVHD while preserving GVL activity. (Grant support: R01AI066008 to Zeng) DisclosuresNo relevant conflicts of interest to declare.

DNA binding and antitumor activities of platinum(IV) and zinc(II) complexes with some S-alkyl derivatives of thiosalicylic acid

A series of complexes of platinum(IV) (C1–C5) and zinc(II) (C6–C10) with S-alkyl derivatives of thiosalicylic acid were prepared and characterized. The interactions of the complexes with calf thymus DNA were analyzed by absorption (UV–Vis) and emission spectral studies (ethidium bromide displacement studies). The cytotoxic activities of complexes C1–C10 were determined against mouse B cell lymphocytic leukemia cells (BCL1), human B-prolymphocytic leukemia (JVM-13), mouse mammary carcinoma cells (4T1), and human mammary carcinoma cells (MDA-MB-468) and compared to the activities of the free ligand precursors and cisplatin. The cytotoxicities of the platinum(IV) and zinc(II) complexes toward mouse tumor cell lines were higher compared with their effects on human tumor cell lines. The zinc(II) complex C9 showed the highest antitumor activity toward the tested human cell lines, while the platinum(IV) complex C4 exhibited the highest antitumor activity toward mouse BCL1 and 4T1 cells. Both C4 and C9 have ligands derived from S-propyl thiosalicylic acid.

The Role of Regulatory B Cell-Like Malignant Cells and Treg Cells in the Mouse Model of BCL1 Tumor Dormancy

Cancer dormancy is a clinical state in which residual tumor cells persist for long periods of time but do not cause detectable disease. In the mouse B cell lymphoma model (BCL1), dormancy can be induced and maintained by immunizing mice with a soluble form of the IgM expressed on the surface of the tumor cells. Immunization induces an anti-idiotype antibody response that maintains dormancy. Mice with dormant tumor have low numbers of BCL1 cells in their spleens that divide and are killed at the same rate. When the anti-Id antibodies wane, the tumor cells grow rapidly and kill the host. Spleens from tumor-bearing mice contain both effector (CD4+ and CD8+) and regulatory T cells (Tregs). In other tumor models, it has been reported that Tregs promote tumor progression by preventing effector cells from killing the tumor. In this report, we demonstrate that the tumor site with rapidly dividing BCL1 cells has fewer Tregs than the tumor site harboring dormant BCL1 cells. In both cases, the Tregs were equally suppressive in vitro. In spleens from mice with actively growing tumor, CD8+ but not CD4+ T cells were virtually absent. In vitro analysis demonstrated a tumor-mediated elimination of CD8+ T cells that was contact dependent and involved the caspase-3 pathway. Most importantly, we found that the BCL1 cells expressed characteristics of B10 regulatory B cells, i.e., they were CD1dhiCD5+ and secreted high levels of IL-10. These BCL1 tumor cells can inhibit anti-tumor immune responses by depleting CD8+ effector T cells.

Influence of miR-30b regulating humoral immune response by genetic difference.

Investigation of genetic difference will be beneficial to researchers to understand the origins and nature of diseases. Previous studies have revealed that L-kynurenine (L-Kyn) level was changed significantly in patient with cancer and that miR-30b play different role in tumor cells and immune cells. Moreover, it has been also conformed that miR-30b involved in the process of L-Kyn-mediated suppression of humoral immune responses induced by lipopolysaccharide (LPS) in human normal B cells separated from volunteers' peripheral blood. Nevertheless, the miR-30b role regulating humoral immune response in B lymphoma cells has been still unclear due to the genetic difference between normal cells and tumor cells. The current study demonstrated that the selected concentration of L-Kyn (100, 1000 μM) significantly reduced the immunoglobulin M secretion induced by LPS when compared with the control group in B lymphoma, CH12.LX, and BCL-1 cells, which had, at least, incomplete dependence on Aryl hydrocarbon receptor, the receptor of L-Kyn. In addition, although L-Kyn (100 μM) significantly attenuated the expression of miR-30b in BCL-1 cells rather than in CH12.LX cells, no significant differences in the strength of L-Kyn-mediated suppression of humoral immune responses induced by LPS were detected by enzyme-linked immunosorbent assay between the LPS (10 μg/ml) + L-Kyn (100 μM) group and the LPS (10 μg/ml) + L-Kyn (100 μM) + miR-30b mimics/miR-30b inhibitor group in CH12.LX and BCL-1 cells, respectively. Further data also showed that mouse Bach2 mRNA was a novel target of miR-30b. These results suggest that genetic difference among cells has a great influence on the miR-30b role in the process of L-Kyn-mediated suppression of humoral immune responses induced by LPS.

Abstract 253: Synergistic antitumor activity of PD-1 signaling blockade and CD27 costimulation correlates with enhanced ratio of effector to regulatory T cells at the tumor site

Abstract Varlilumab (CDX-1127) is a fully human agonist antibody to CD27, a TNF receptor superfamily member expressed on the majority of T cells and subsets of NK cells and B cells. As such, varlilumab is an immunotherapy designed to harness the body's natural immune response by enhancing the activation of T cells that can specifically recognize and kill cancer cells. In an ongoing Phase 1 clinical trial, varlilumab has been very well tolerated and has demonstrated clear biologic activity and promising signs of clinical activity in advanced, treatment-refractory patient populations. This clinical data provides the rationale for combination studies with other immunomodulatory therapies, including inhibitors of PD-1 signaling. In this study we investigated the activity of varlilumab in combination with anti-PD-L1 using a disseminated murine B cell tumor, BCL1 in human CD27 transgenic mice. In this model of i.v. inoculated BCL1 cells, we have previously shown that varlilumab can delay tumor growth, but rarely results in cures. Despite expression of PD-L1, the BCL1 tumors were completely resistant to anti-PD-L1 monotherapy. However, the combination of varlilumab and anti-PD-L1 resulted in a significant improvement in survival and approximately 50% long-term survivors. To investigate the mechanism resulting in the synergistic activity, we collected spleens (the major site of tumor growth) from tumor bearing animals treated with individual or combined regimens and processed them for flow cytometry analysis. We found that animals treated with the combination of varlilumab and anti-PD-L1 had a higher ratio of infiltrating immune cells to tumor cells relative to monotherapy or saline controls. In particular, the ratio of effector (CD8+) T cells to Treg and the number of IFNγ+ CD4 and CD8 T cells were highly increased with the combination treatment. These data demonstrate that CD27 costimulation combined with checkpoint inhibition with anti-PD-L1 can result in improved immune profile in the tumor and synergistic survival benefit. This study, along with the good safety profile of varlilumab reported in the Phase 1 study, support the design of future combination studies in patients with cancer. Citation Format: Lawrence J. Thomas, Li-Zhen He, Anna Wasiuk, Lauren E. Gergel, James M. Boyer, Sarah M. Round, Henry C. Marsh, Tibor Keler. Synergistic antitumor activity of PD-1 signaling blockade and CD27 costimulation correlates with enhanced ratio of effector to regulatory T cells at the tumor site. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 253. doi:10.1158/1538-7445.AM2015-253

Bcl-xL Protein Protects from C/EBP Homologous Protein (CHOP)-dependent Apoptosis during Plasma Cell Differentiation

Although it is known that the unfolded protein response (UPR) plays a significant role in the process of plasma cell differentiation, the contribution of the individual sensors of the UPR to this process remains unclear. In this study we examine the death signals and compensatory survival signals activated during B cell activation and the first stages of plasma cell differentiation. During in vitro differentiation of both primary murine B cells and the Bcl1 cell line, we demonstrate that in addition to activation of the physiological UPR, changes in the expression of several Bcl-2 proteins occur, which are consistent with a lowering of the apoptotic threshold of the cell. Specifically, we observed decreased expression of Bcl-2 and Mcl-1 and increased expression of the proapoptotic protein Bim. However, these changes were countered by Bcl-xL induction, which is necessary to protect differentiating cells both from ER stress-induced death by tunicamycin and from the death signals inherent in differentiation. Consistent with differentiating cells becoming dependent on Bcl-xL for survival, the addition of ABT-737 resulted in apoptosis in differentiating cells through the inhibition of sequestration of Bim. Confirming this result, differentiation in the context of RNAi-mediated Bcl-xL knockdown also induced apoptosis. This cell death is C/EBP homologous protein (CHOP)-dependent, connecting these events to the UPR. Thus plasma cell differentiation proceeds through a Bcl-xL-dependent intermediate.

Central Memory T Cells That Express High Level Of CD44 Mediate Strong Anti-Tumor Response Without Gvhd

Several groups have demonstrated directly and indirectly that central memory T cells (TCM) have decreased ability to induce graft-versus-host disease (GVHD) compared with naïve T cells (TN). We have previously demonstrated that TCM expressing high level of CD44 (CD44high TCM) do not cause GVHD. In this study, we further defined the role of CD4+ and CD8+ subsets of this population in GVHD using the same GVHD model as we used previously (C57BL/6→BALB/c). While all CD4+ TN recipients developed lethal GVHD and died within 28 days after transplantation, none of the recipients of CD4+CD44high TCM developed GVHD and all survived more than 100 days after transplantation (P<0.01). Similarly, 36% of CD8+ TN recipients developed lethal GVHD but none of CD8+CD44high TCM recipients developed GVHD (P<0.05). One of the unique features of effector memory T cells (TEM) is that these cells are able to facilitate stem cell engraftment and mediate some degree of anti-tumor response without causing GVHD. To test the ability of TCM to deplete host radioresistant T cells, BALB/c recipients were lethally irradiated (8.5 Gy) and transplanted with 1x107 T-cell-depleted (TCD) bone marrow cells together with graded numbers of T cell subsets from C57BL/6. Host T cells persist in all mice receiving only TCD bone marrow cells. Addition of 1x105 of either TN or CD44high TCM to the graft led to 100% depletion of all host radioresistant T cells. In contrast, none of 1x105 TEM recipients and only 50% of 5x105 TEM recipients depleted all host radioresistant T cells. These data indicate that TN and CD44high TCM have equivalent abilities to deplete host radioresistant T cells and both of them are at least 5 times more potent than TEM. To test the ability of CD44high TCM to mediate anti-tumor effects, host-type leukemia/lymphoma cells (BCL1) cells were added to the stem cell graft. All recipients transplanted with TCD bone marrow together with BCL1 cells developed tumor and died within 28 days after transplantation. Addition of 1x104 TN and CD44high TCM protected 42% and 36% of the recipients from developing tumor respectively. In contrast, only 7% of TEM recipients were protected (P<0.05) at the same dose level. Similarly, addition of 1x105 TN and CD44high TCM protected 79% and 80% of the recipients from developing tumor respectively. However, only 25% of 1x105 TEM recipients were protected (P<0.01). These data indicate that TN and CD44high TCM have equal anti-tumor activity and both of them are at least 10 times more potent than TEM. Similar results were obtained in CD8 subset of CD44high TCM. A critical question is why CD44high TCM are able to kill host radioresistant T cells and tumor cells so efficiently without inducing GVHD. Even though CD44high TCM were able to respond to alloantigens, bioluminescence imaging analyses suggest that CD44high TCM also mediate an abortive alloresponses upon transfer into allogeneic recipients. Using an in vivo functional assay, we further demonstrated that alloreactive CD44high TCM lost the reactivity over time, suggesting a mechanism by which CD44high TCM were able to eliminate host resistance without causing GVHD. In conclusion, these data demonstrate that selective transplantation of CD44high TCM is able to efficiently separate anti-tumor effects from GVHD. Disclosures:No relevant conflicts of interest to declare.

Abstract A85: Peptides specific for chronic lymphocytic leukemic cells: Characterization and use in targeted drug delivery.

Abstract The ability to selectively target drugs to Chronic Lymphocytic Leukemia (CLL) cells may significantly improve the efficacy of current chemotherapy protocols for this disease. Our studies in model leukemic systems have already demonstrated that a strategy involving Peptide-Drug Conjugates (PDCs) may achieve this goal. In a previous preliminary study, we used a phage display peptide library to isolate a series of peptides that bind CLL cells. The current research has expanded on this work and began by absorbing a 7-mer linear and 7-mer cyclic phage display peptide libraries on a series of normal human or murine primary cells. Rigorous pre-absorption reduces the original vast phage library population (1011 clones) and enhances the ability to select peptides specific for the target CLL cells. The cells used included endothelial, heart, kidney, lung, liver, skin, fibroblasts and several subpopulations of blood cells. These absorptions resulted in a 7-log reduction in the size of the phage libraries. The stock libraries were also injected into mice. Recovery of blood circulating phage 24hrs later showed that in vivo absorption reduced the pool size of viable phage by approximately 8-logs. These "absorbed" libraries now form the basis for isolating CLL cell-bound and cell-internalizing phage. Target cells include murine cell lines derived from spontaneously-developed aggressive CLL (BCL-1 and A20) and human patient CLL cells. Analysis of peptide sequences of A-20 binding or internalizing phage clones revealed overlap in amino acid usage but not one consistent consensus sequence, suggesting that these peptides target different CLL-specific membrane components, some of which can induce phage internalization and should be applicable for PDCs. Using a similar approach, we are isolating peptides for BCL-1 cells as well as peptides to cells from an initial cohort of CLL patients. The bioinformatic and biochemical results for all these peptides will be presented. Peptides selected on the basis of uptake kinetics will be conjugated to cytotoxic drugs using our dendrimer technology. This technology has already allowed us to demonstrate improved efficacy of known CLL drugs. Progress on this stage of the project will also be presented. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A85. Citation Format: Michael A. Firer, Bat-Chen Lubin, Sharon Cohen, Galia Luboshits. Peptides specific for chronic lymphocytic leukemic cells: Characterization and use in targeted drug delivery. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A85.

Bcl-xL protects from UPR-associated apoptosis during plasma cell differentiation. (P1457)

Abstract Understanding factors that control plasma cell survival is important for the development of therapeutic approaches to diseases including multiple myeloma and autoimmune disorders. Plasma cell differentiation requires activation of the unfolded protein response, a set of signaling pathways in which prolonged activation leads to apoptosis in other cell types. Plasma cells activate this pathway during differentiation suggesting compensatory survival signals are activated. Consistent with this hypothesis, we found that LPS-driven differentiation protects Bcl1 cells against tunicamycin-activated ER stress-induced death. LPS plus cytokine-induced differentiation was associated with an increase in Bcl-xL expression accompanied by a decrease in Mcl-1 or Bcl-2 in Bcl1 cells and primary B cells respectively. Moreover, differentiation-induced protection against ER stress was shown to be Bcl-xL-dependent as tunicamycin-induced death was restored by the addition of the Bcl-xL/Bcl-2 inhibitor, ABT-737. Importantly, we also showed that during differentiation, Bcl1 cells and primary B cells are Bcl-xL-dependent as ABT-737 resulted in significant apoptosis in the absence of any additional death signal. Finally, we found that differentiation was accompanied by a change in Bim binding from primarily Mcl-1 and/or Bcl-2 to Bcl-xL in differentiating cells. These data demonstrate that Bcl-xL induction protects from death signals activated during plasma cell differentiation.

Polymer Therapeutics with a Coiled Coil Motif Targeted against Murine BCL1 Leukemia

The specificity of polymer conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) bearing cytostatic drugs for cancer cells could be significantly increased by the incorporation of a suitable targeting ligand, such as a monoclonal antibody (mAb). However, direct binding of the protein to the polymer carrier could cause considerable problems, such as decreasing the binding capacity of mAb to its target. Here, we introduce a novel strategy of joining a targeting moiety to a polymeric conjugate with cytostatic drug. The scFv of B1 mAb (specific for BCL1 leukemia cells) was tagged with peptide K ((VAALKEK)4). Peptide E ((VAALEKE)4), which forms a stable coiled coil structure heterodimer with peptide K, was assembled with the HPMA copolymers bearing doxorubicin. Such targeted polymeric conjugates possess very selective and high binding activity toward BCL1 cells. Similarly, targeted polymeric conjugates exert approximately 100 times higher cytostatic activity toward BCL1 cells in comparison to nontargeted conjugates in vitro. At the same time, the conjugates have comparable and rather low cytostatic activity for 38C13 cells, which are used as a negative control, in vitro.

Drug resistance to chlorambucil in murine B-cell leukemic cells is overcome by its conjugation to a targeting peptide

Targeting drugs through small-molecule carriers with a high affinity to receptors on cancer cells can overcome the lack of target cell specificity of most anticancer drugs. These targeted carrier-drug conjugates are also capable of reversing drug resistance in cancer cells. Although many targeted drug delivery approaches are being tested, the linkage of several and different drugs to a single carrier molecule might further enhance their therapeutic efficacy, particularly if the drugs are engineered for variable time release. This report shows that murine B-cell leukemic cells previously resistant to a chemotherapeutic drug can be made sensitive to that drug as long as it is conjugated to a targeting peptide and, in particular, when the conjugate contains multiple copies of the drug. Using a 13mer peptide (VHFFKNIVTPRTP) derived from the myelin basic protein (p-MBP), dendrimer-based peptide conjugates containing one, two, or four molecules of chlorambucil were synthesized. Although murine hybridomas expressing antibodies to either p-MBP (MBP cells) or a nonrelevant antigen (BCL-1 cells) were both resistant to free chlorambucil, exposure of the cells to the p-MBP-chlorambucil conjugate completely reversed the drug resistance in MBP, but not BCL-1 cells or normal spleen cells. Moreover, at equivalent drug doses, there was significant enhancement in the cytotoxic activity of multidrug versus single-drug copy conjugates. On the basis of these results, the use of multifunctional dendrone linkers bearing several covalently bound cytotoxic agents allows the development of more effective targeted drug systems and enhances the efficacy of currently approved drugs for B-cell leukemia.