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

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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Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development

PLoS ONE ◽

10.1371/journal.pone.0246197 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0246197

Author(s):

Jorge Marquez ◽

Jianping Dong ◽

Chun Dong ◽

Changsheng Tian ◽

Ginette Serrero

Keyword(s):

Flow Cytometry ◽

Western Blot ◽

Cell Lines ◽

Cancer Cell ◽

Cancer Cell Lines ◽

Negative Regulator ◽

Target Validation ◽

Antibody Drug

Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh’s T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.

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Expression of functional CD40 antigen on Reed-Sternberg cells and Hodgkin's disease cell lines

Blood ◽

10.1182/blood.v85.3.780.bloodjournal853780 ◽

1995 ◽

Vol 85 (3) ◽

pp. 780-789 ◽

Cited By ~ 151

Author(s):

A Carbone ◽

A Gloghini ◽

V Gattei ◽

D Aldinucci ◽

M Degan ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Lines ◽

Hodgkin's Disease ◽

Hodgkin’S Disease ◽

Growth Factor Receptor ◽

Large Cell ◽

Distinct Pattern ◽

Histologic Subtype ◽

Hodgkin's Lymphomas

CD40 is a member of the nerve growth factor receptor family, showing a significant homology to the Hodgkin's disease (HD)-associated antigen CD30 and is capable of transduce growth signals in a number of cell types. A series of 312 lymphoma samples, including 139 cases of HD, 32 cases of CD30+ anaplastic large cell (ALC) lymphomas, 141 cases of other non-Hodgkin's lymphomas (NHLs), and a panel of HD- or NHL-derived cell lines, were evaluated for CD40 expression by immunostaining of paraffin embedded sections, cell smears and flow cytometry. CD40 was strongly expressed with a highly distinct pattern of staining on Reed- Sternberg (RS) cells and variants in 100% (139/139) of HD cases, irrespective of their antigenic phenotype (T, B, non T-non B) and histologic subtype of HD. Conversely, CD40 was immunodetected on only one third (12/32; 37%) of ALC lymphoma cases and on 105 of 127 B-cell NHLs. The relative cell density of CD40 on HD cell lines (L-428, KM-H2, HDLM-2) as assessed by flow cytometry was significantly higher than on all other lymphoma cells analyzed. Engagement of CD40 by its soluble ligand (CD40L) enhanced both clonogenic capacity and colony cell survival of HD cell lines. Such effect was potentiated by interleukin-9 costimulation in KM-H2 cells. Finally, we have shown that in vitro rosetting of activated CD4+ T cells to HD cells (L-428) is mediated in part by the CD40/CD40L adhesion pathway. Our data indicate that CD40 is a useful antigen for immunodetection and identification of tumor cells in all subtypes of HD, and suggest that it may play a role in the regulation of RS cell expansion and the contact-dependent interactions of these cells with cytokine-producing T lymphocytes.

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P06.01 Delta24-ACT oncolytic adenovirus as a therapeutic approach for DIPG

Neuro-Oncology ◽

10.1093/neuonc/noz126.123 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii36-iii36

Author(s):

V Laspidea ◽

M Puigdelloses ◽

M García-Moure ◽

I Iñigo-Marco ◽

J Gallego ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Cell Lines ◽

In Vivo Studies ◽

Immunogenic Cell Death ◽

Murine Models ◽

Antitumoral Effect ◽

Infected Cells

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor, being the leading cause of pediatric death caused by cancer. We previously showed that administration of the oncolytic virus Delta-24-RGD to DIPG murine models was safe and led to an increase in the median survival of these animals. However, not all the animals responded, underscoring the need to improve this therapy. In order to increase the antitumoral effect of the virus, we have engineered Delta-24-RGD with the costimulatory ligand 4-1BBL (Delta24-ACT). 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. In this project, we evaluated the oncolytic effect of Delta24-ACT and the antitumor immune response in DIPG murine models. MATERIALS AND METHODS We use the NP53 and XFM murine DIPG cell lines. Flow cytometry was used to assess cell infectivity and ligand expression. We analyzed viral replication using a method based in hexon detection, and viral cytotoxic effect using the MTS assay. For immunogenic cell death analysis, we measured ATP secretion by a luminometric assay and calreticulin location by flow cytometry and immunofluorescence. For in vivo studies, cells and virus were injected in the pons of the mice, using the screw-guided system. RESULTS In vitro, Delta24-ACT was able to infect and induce cell death in a dose-dependent manner in murine DIPG cell lines. In addition, Delta24-ACT was able to replicate in these tumor cells and to express viral proteins. Moreover, infected cells expressed 41BBL in their membranes. Delta24-ACT could induce immunogenic cell death due to an increased secretion of ATP and calreticulin translocation to the membrane of infected cells (in no-infected cells it located in the ER), DAMPs that can trigger the immune response activation. In vivo, Delta24-ACT demonstrated to be safe in all the tested doses and was able to induce a significant increase in the median survival of the treated animals. Moreover, long-term survivors display immunological memory. CONCLUSIONS Delta24-ACT treatment led to antitumoral effect in DIPG murine cell lines in vitro. Of significance, we have demonstrated that in vivo administration of Delta24-ACT is safe and results in an enhanced antitumor effect. Future in vivo studies will explore the underlying immune mechanism of the virus.

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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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Combined Inhibition of Wee1 and Poly(ADP Ribose) Polymerase1/2 Synergistically Inhibits Acute Myeloid Leukemia Cells By Enhancing DNA Damage and the Induction of Apoptosis

Blood ◽

10.1182/blood.v124.21.3621.3621 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3621-3621 ◽

Cited By ~ 1

Author(s):

Jonathan C Snedeker ◽

Tamara M Burleson ◽

Raoul Tibes ◽

Christopher C. Porter

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

Dna Damage ◽

Cell Lines ◽

Conflicts Of Interest ◽

Combination Index ◽

Annexin V ◽

Concomitant Treatment ◽

Western Blots

Abstract Introduction: Successful treatment of AML remains dependent upon cytotoxic chemotherapy. However, traditional regimens are not well tolerated by older patients who are at highest risk of disease, and salvage rates after relapse are low, necessitating novel therapeutic strategies. Our groups identified Wee1 as a potential therapeutic target in AML, particularly in the context of concomitant treatment with cytarabine (Tibes et al, Blood, 2012; Porter et al, Leukemia, 2012). Wee1 inhibits CDK1&2 via phosphorylation thereby stalling cell cycle progression. One consequence of Wee1 inhibition/CDK1 activation is impairment of DNA repair via homologous recombination (Krajewska et al, Oncogene, 2013). Cells in which HR is impaired are dependent upon Parp1/2 function, and HR deficient cells are particularly sensitive to Parp1/2 inhibition. Therefore, we hypothesized that combined Wee1 and Parp1/2 inhibition may result in greater inhibition of AML cell proliferation and survival than either alone. Methods: Human AML cell lines, MV4-11 and Molm-13, and a mouse AML that expresses MLL-ENL/FLT3-ITD were cultured with various concentrations of a Wee1 inhibitor (AZ1775) and a Parp1/2 inhibitor (olaparib) and counted 72 hours later by propidium iodide exclusion and flow cytometry. In some experiments, cells were split into fresh media to recover for 72 more hours. Combination Index (CI) values were calculated by the method of Chou and Talalay. Apoptosis was measured using Annexin V/7AAD and flow cytometry. Western blots were used to confirm inhibition of CDK1/2 phosphorylation and to measure DNA damage induction (gamma-H2AX). Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 3 cell lines tested, with combination index values ranging from 0.3 to 0.9. When cells were allowed to recover after treatment, those treated by single agents were able to continue proliferating. However, those treated with the combination did not recover as well or at all, indicating greatly impaired proliferative capacity. Combined inhibition of Wee1 and Parp1/2 also resulted in a significant increase in apoptosis greater than either drug alone. Western blots for gamma-H2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone. Discussion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML cell proliferation, DNA damage and apoptosis than either drug alone. Future studies will include experiments with primary patient samples, as well as in vivo trials combining Wee1 inhibition with Parp1/2 inhibition. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in those for whom conventional chemotherapeutics may not be well tolerated. Disclosures No relevant conflicts of interest to declare.

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The BTK Inhibitor Ibrutinib Blocks SDF1/CXCR4 Mediated Migration of Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood.v124.21.915.915 ◽

2014 ◽

Vol 124 (21) ◽

pp. 915-915

Author(s):

Stuart A Rushworth ◽

Lyubov Zaitseva ◽

Megan Y Murray ◽

Matthew J Lawes ◽

David J MacEwan ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Cell Trafficking ◽

Acute Myeloid

Abstract Introduction Despite recent significant progress in the understanding of the biology of acute myeloid leukemia (AML) the clinical outcomes for the majority of patients diagnosed with AML presently remain poor. Consequently, there is an urgent need to identify pharmacological strategies in AML, which are not only effective but can be tolerated by the older, less well patient. Recently our group and others have shown that there is high Bruton’s Tyrosine Kinase (BTK) phosphorylation and RNA expression in AML. Moreover, our recent study described for the first time that ibrutinib and BTK-targeted RNA interference reduced factor-induced proliferation of both AML cell lines and primary AML blasts, as well as reducing AML blast adhesion to bone marrow stromal cells. Inhibition of BTK has been shown to regulate chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma cell migration by inhibiting SDF1 (stromal derived factor 1) induced CXCR4 regulated cell trafficking. Here we report that in human AML ibrutinib in addition functions in a similar way to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo. Methods To investigate the role of BTK in regulating AML migration we used both pharmacological inhibitor ibrutinib and genetic knockdown using a lentivirus mediated BTK targeted miRNA in primary AML blasts and AML cell lines. We examined migration of AML blasts and AML cells to SDF-1 using Transwell permeable plates with 8.0µM pores. Western blotting was used to examine the role of SDF-1 in regulating BTK, AKT and MAPK activation in primary AML blasts. Results We initially examined the expression of CXCR4 in human AML cell lines and found that 4/4 cell lines were positive for CXCR4 expression. Next we examined the effects of ibrutinib on the migration of the AML cell lines U937, MV4-11, HL60 and THP-1 in response to SDF1. We found that ibrutinib can inhibit the migration of all AML cell lines tested. We tested the in-vitro activity of ibrutinib on SDF-1 induced migration in a spectrum of primary AML blasts from a wide age spectrum of adult patients and across a range of WHO AML subclasses and found that ibrutinib significantly inhibits primary AML blast migration (n=12). Next we found that ibrutinib can inhibit SDF-1 induced BTK phosphorylation and downstream MAPK and AKT signalling in primary AML blast. Finally to eliminate the problems associated with off target ibrutinib activity we evaluated migration of AML cells lines using genetic inhibition of BTK. The introduction of BTK-specific miRNA dramatically inhibited the expression of BTK in THP-1 and HL60 and reduced SDF1 mediated migration confirming that BTK is involved in regulating AML migration in response to SDF1. Conclusions These results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL. Disclosures No relevant conflicts of interest to declare.

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siRNA for the Ig Light Chain Constant Region Reduces Light Chain Production and Secretion By Human Plasma Cells and in a Murine Xenograft Model

Blood ◽

10.1182/blood.v124.21.5722.5722 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5722-5722

Author(s):

Xun Ma ◽

Ping Zhou ◽

Monika Pilichowska ◽

Chakra P Chaulagain ◽

Sandy Wong ◽

...

Keyword(s):

Flow Cytometry ◽

Human Plasma ◽

Cell Lines ◽

Light Chain ◽

Plasma Cells ◽

Xenograft Model ◽

Myeloma Cell ◽

Constant Region

Abstract Background Ig light chain (LC) diseases such as AL amyloidosis and monoclonal light-chain deposition disease are caused by pathologic free LC. Treatment is aimed at eliminating LC production but success is limited. RNA interference (RNAi) can stop LC production but the diversity of LC variable region sequences poses a challenge that targeting consensus sequences in the constant region (CR) of LC mRNA may overcome (Blood 2014;123:3440). We have developed siRNA pools designed to target the κ or λ LC CR mRNA in human plasma cells and impair LC production and secretion, and have shown that the pool targeting the λ LC CR can do so, and can also trigger a terminal unfolded protein response in clones producing intact Ig due to intracellular accumulation of unpaired heavy chains (ibid). Here we report the results of continued in vitro and in vivo testing of these pools in patient specimens and in a murine xenograft model. Methods Pools of siRNA for the κ or λ LC CR (si[IGLCκCR], si[IGLCλCR]) were custom produced with a non-target control (si[-]). They were introduced in vitro into human plasma cells by an optimized streptolysin O-based method (SLO) and in a NOD.SCID xenograft flank plasmacytoma model by in vivo electroporation as per Gene Therapy 2011;18:1150. In vitro we evaluated LC gene expression, production and secretion at 24 hours in human myeloma cell lines and CD138-selected specimens from patients with plasma cell neoplasms, using real-time PCR (qPCR) for LC mRNA, flow cytometry for intracellular LC mean fluorescence intensity (MFI) and ELISA (Bethyl Laboratories) for LC secretion in 24-hour suspension cultures (106 cells/ml). In vivo we inoculated each of the flanks of NOD.SCID mice with 107 human myeloma cells (ALMC-1 or ALMC-2). When plasmacytomas were 0.5cm3 we injected si[IGLCλCR] or si[-] one time to each flank plasmacytoma respectively, allowing each mouse to serve as its own control. Two days later, the mice were sacrificed and the plasmacytomas excised for qPCR for λ LC mRNA and serum was obtained to measure human λ LC levels by ELISA. Results We have previously described results with siRNA targeting the λ LC CR in human cell lines that make λ LC (ALMC-1, ALMC-2, EJM, OPM2, MM.1S, and MM.1R) and in 16 AL λ patient specimens. We demonstrated significant decreases in LC mRNA, intracellular LC MFI, and λ LC secretion by cell lines (Blood 2014;123:3220); moreover, transcriptional profiling indicated minimal off-target effects (ibid; Supplement). We now report that in vitro secretion of λ LC by CD138-selected plasma cells from AL patients (n=3, newly diagnosed λ) treated with si[IGLCλCR] was reduced by 65% from a mean of 3.1 to 1.0µg/ml and that the residual λ LC mRNA was 49% of control. Similarly we treated κ LC secreting human myeloma cell lines with si[IGLCκCR] and si[-] (IM9, H929, JJN-3, and ARH77). By qPCR the residual κ LC mRNA was 13%, by flow cytometry the MFI was reduced by a median of 67.3% (22.5-90.8), and by ELISA mean κ LC secretion was reduced from 3.7 to 0.8µg/ml (P = 0.055, paired t test). We treated CD138-selected κ patient samples (AL 3, LCDD 1, MM 6) in the same way. By qPCR the residual κ LC mRNA was 57% control, by flow cytometry the MFI was reduced by a median of 37.5% (14-69.8), and by ELISA secretion was reduced from 9.4 to 6.5µg/ml (P = 0.02, paired t test). In the murine dual-flank xenograft model employing λ secreting cells, by qPCR there was a reduction in λ LC mRNA with si[IGLCλCR] treatment in 13 of 16 mice (ALMC-1 11/114, ALMC-2 2/2). In these 14, the median λ LC expression was 66% of control (range, 17-97). In 6/13 the average reduction in λ LC expression was 59%. Of note, measurable levels of human λ LC were found in the blood of all mice at sacrifice. Conclusion With one pool of siRNA targeting the constant region of the κ or λ LC we can significantly reduce production and secretion of LC by clonal human plasma cells, including patient cells, and also reduce the expression of LC in xenograft plasmacytomas in vivo. Two methods of siRNA delivery have been employed in this work thus far, SLO and in vivo electroporation, neither of which require endosomal escape. The specificity of the siRNA pools for plasma cell LC genes and the possible receptivity of plasma cells to RNAi are important positive aspects of this work. Further pre-clinical development of Ig LC CR RNAi employing lipid-based nanoparticle platforms is warranted in order to optimize cell-specific delivery, delivery efficiency and siRNA targeting. Disclosures No relevant conflicts of interest to declare.

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Heat Shock Protein 70 Inhibitor, Alone or in Combination with Bortezomib, Prevented Plasmacytoma Development in Immunodeficient Mice Transplanted with Myeloma Cell Lines

Blood ◽

10.1182/blood.v128.22.5658.5658 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5658-5658

Author(s):

Mariana Bleker de Oliveira ◽

Angela Isabel Eugenio ◽

Veruska Lia Fook Alves ◽

Daniela Zanatta ◽

Mihoko Yamamoto ◽

...

Keyword(s):

Cell Line ◽

Tumor Cells ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Conflicts Of Interest ◽

Control Group ◽

Immunodeficient Mice ◽

Late Apoptosis

Abstract Introduction: HSP70 has an integrative role in protein degradation due to the interaction with many pathways, such as ubiquitin proteasome (UPS), unfolded protein response (UPR) and autophagy. In multiple myeloma (MM) HSP70 is overexpressed and helps to prevent proteotoxic stress and cell death caused by overload of unfolded/misfolded proteins produced by tumor cells. Aims: To explore the role of HSP70 inhibition, isolated or in association with proteasome inhibitor, as therapeutic strategy for MM through in vitro and in vivo analyses. Methods: RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines were treated with HSP70 inhibitor (VER155008- 50 μM or 80μM) and proteasome inhibitor (bortezomib 100nM) for evaluation of apoptosis induction by flow cytometry using annexin V and propidium iodide. NOD.Cg-rkdcscid Il2rgtm1Wjl/SzJ immunodeficient mice were used for plasmacytoma xenograft model and treated with intravenous VER155008 (40mg/kg) and bortezomib (1mg/kg), immediately after transplant of RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines (N=3 for each group, including controls, bortezomib, VER155008, and combination of bortezomib and VER155008). Bioluminescence was measured in IVIS Kinetic (Capiler Life Science) once a day for seven days. Results: Bortezomib used as single treatment was able to induce apoptosis in RPMI8226-LUC-PURO cell line: the best result for in vitro studies RPMI8226-LUC-PURO was 65% of late apoptosis after treatment with bortezomib. On the other hand, U266-LUC-PURO cell line presented higher percentage of apoptosis when treated with bortezomib and VER155008 combination: U266-LUC-PURO cell line presented more than 60% of late apoptosis after VER155008 (80μM) combined with bortezomib, showing that inhibition of HSP70 could overcome U266-LUC-PURO resistance to bortezomib alone. Mice treated with VER155008, alone or in combination with bortezomib, showed complete inhibition of tumor growth (absence of bioluminescence) for both cell lines when compared with control group after one week of treatment (p<0.001, Two-way ANOVA). Therefore, in vivo studies using mice treated with VER155008, alone or in combination with bortezomib, prevented tumor development after one week of treatment, independent of the cell line used in the xenotransplant. Conclusion: Our study shows that HSP70 and proteasome inhibitors combination induced apoptosis in tumor cells in vivo for both MM cell lines. Since HSP70 is overexpressed in MM and connects several signaling pathways that maintain cell survival, such as UPS, UPR and autophagy, it can represent a key role to establish a new approach for the treatment of MM. Financial support: FAPESP 2010/17668-6 and CNPq (155272/2013-6). UNIFESP Ethics Committee (0219/12). Disclosures No relevant conflicts of interest to declare.

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Ionizing Irradiation Protection and Mitigation by Carbamazepine Is p53 and Autophagy Independent,

Blood ◽

10.1182/blood.v118.21.3400.3400 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3400-3400

Author(s):

Hyun Kim ◽

Mark E. Bernard ◽

Amy Farkas ◽

Frank Houghton ◽

Donna Shields ◽

...

Keyword(s):

Cell Lines ◽

Hind Limb ◽

Conflicts Of Interest ◽

Survival Curve ◽

Tumor Growth Inhibition ◽

Independent Manner ◽

Mitochondrial Membrane Depolarization ◽

Significant Difference

Abstract Abstract 3400 Introduction: Carbamazepine (CBZ), an established sodium channel blocker, used in treatment of epilepsy and trigeminal neuralgia, induces autophagy. CBZ has recently been demonstrated to be an ionizing radiation mitigator and protector (Kim,H.et. al IJRB-in press). CBZ protected C57BL/6NHsd female mice from irradiation if given 24 hr before total body irradiation (TBI) and mitigated if given 12 hours after irradiation. Materials and Methods: To elucidate the mechanism of CBZ action, autophagy incompetent (ATG5−/−) and competent (ATG5+/+) mouse embryo fibroblasts (MEF), p53−/− and p53+/+ bone marrow stromal cells and 32D cl 3 murine IL-3 dependent hematopoietic progenitor cells were tested for CBZ mediated radiation protection and mitigation in clonagenic irradiation survival curves. We also measured CBZ effect on irradiation-induced apoptosis, and depletion of antioxidant stores in vitro and after (TBI) in vivo in control mice and in hind limb irradiated mice with orthotopic tumors. Results: CBZ was a significant radiation protector and mitigator for both ATG5−/− and ATG5 +/+ cell lines by an increased ñ (a measurement of the shoulder on the clonogenic survival curve). CBZ treated ATG5 +/+ cells has an increased ñ of 11.1 +/− 0.2 or 8.8 +/− 0.2 for CBZ added before or after irradiation respectively compared to 5.4 +/− 0.9 for irradiation control cells (p = 0.0287 or 0.0119,respectively). ATG −/− cells were also protected and mitigated by CBZ (ñ of 16.1 +/− 2.6 as a radioprotector or 9.8 +/− 1.5 as a mitigator compared to 4.6 +/− 0.7 for irradiated control cells (p = 0.0002 or 0.0037, respectively). Thus, CBZ functions independent of autophagy. Incubation of p53 +/+ and p53−/− cell lines in 10 μM CBZ for one hour before irradiation protected (ñ of 5.6 +/− 0.9 compared to 1.9 +/− 0.5 for control irradiated p53 +/+ cells (p = 0.0126) and 3.7 +/− 1.9 compared to 1.8 +/− 0.4 for irradiated control p53 −/− cells (p = 0.0018). Thus, CBZ functions in a p53 independent manner. Other pro-autophagy drugs, Valproic Acid and Lithium Carbonate, were ineffective radiation protectors or mitigators. CBZ treatment of 32D cl 3 cells for one hour before or immediately following irradiation had no effect on mitochondrial membrane depolarization, apoptosis or viability but was protective and mitigative in clonagenic survival curve assays. Irradiation initially decreased antioxidant stores in both ATG5+/+ and ATG5−/− cells 10 to 30 min following irradiation. However, cells treated with CBZ had a faster recovery of antioxidant stores to pre-irradiation levels by 90 min compared to 120 min for control irradiated cells. To determine if CBZ protected both normal tissues and tumors in vivo mice with 3LL Lewis Lung carcinoma hind limb xenografts were treated with 10 mg/kg CBZ before or after 20 Gy limb irradiation. There was no significant difference in tumor growth inhibition by 20 Gy in mice treated with CBZ before or after irradiation (p = 0.2431 or 0.5439, respectively). Conclusion: Thus, CBZ is an effective radiation protector and mitigator of normal but not tumor tissues by a novel mechanism independent of autophagy, p53 and apoptosis. Acknowledgments: This project was supported by U191A168021–06. Disclosures: No relevant conflicts of interest to declare.

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Cytotoxic Activity of Anti-CD20-hIFN-α on Rituximab-Resistant B-NHL Clones and Synergy with Chemotherapy,

Blood ◽

10.1182/blood.v118.21.3499.3499 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3499-3499 ◽

Cited By ~ 1

Author(s):

Gabriel G Vega ◽

Mario I Vega ◽

Sara Huerta-Yepez ◽

Ali Jazirehi ◽

Hector Mayani ◽

...

Keyword(s):

Cell Lines ◽

Conflicts Of Interest ◽

Tumor Xenograft ◽

Antibody Concentration ◽

Wild Type ◽

Anti Cd20 ◽

Hodgkin's Lymphomas ◽

Apoptotic Effects

Abstract Abstract 3499 Rituximab (chimeric anti-CD20 mAb) (Rtx) has been successfully used in the treatment of patients with B-cell non-Hodgkin's lymphomas (B-NHLs). The combination treatment with chemotherapy results in achieving high response rates and prolongation of survival. However, a subset of patients does not initially respond to treatment and many responding patients relapse and no longer respond to further treatments. Currently, there are no alternative therapies for resistant patients. The mechanism of resistance in vivo is not clear. However, we have explored a potential mechanism by developing in vitro several clones of Rtx-resistant (RR) variants for several B-NHL cell lines and characterized their properties. Briefly, unlike the parental wild-type, the RR clones express CD20 but no longer respond to treatment with Rtx or combination with cytotoxic drugs. Further, these clones overexpress the activity of several survival/anti-apoptotic pathways [1]. It is not known whether chemical modification of Rtx might be necessary to exert its activity and signaling on the RR clones. Hence, a recent report demonstrated that a fusion protein consisting of Rtx and human IFN-α (anti-CD20-hIFN-α) exhibited superior activity over Rtx, IFN-α, or combination of Rtx and IFN-α, and exhibited significant anti-proliferative and apoptotic effects in vitro with several B-NHL cell lines and in vivo an anti-tumor xenograft response [2]. These findings prompted us to investigate the effect of anti-CD20-hIFN-α on the RR clones. We hypothesized that anti-CD20-hIFN-α may exert an anti-proliferative and apoptotic effects on the RR clones and may also synergize when used in combination with chemotherapy. In this study, we used the B-NHL line Ramos (Burkitt) and 2F7 (AIDS-related) and their respective Ramos RR1 and 2F7 RR1 clones as models. We examined the effects of anti-CD20-hIFN-α and Rtx on the wild-type and RR clones following treatment with IgG isotype control, Rtx, anti-CD20-hIFN-α, CDDP (10 mg/ml) and Treanda® (Bendamustine) (5 mg/ml), as well as combinations. Treatment of 2F7 with single agents alone had no cytotoxic effect; however, treatment with the combination of Rtx and CDDP or Treanda® or anti-CD20-hIFN-α plus CDDP or Treanda® resulted in significant cytotoxicity. Treatment of Ramos resulted in similar findings observed with 2F7, however, the anti-CD20-hIFN-α alone was significantly cytotoxic to Ramos cells. Importantly, whereas treatment of 2F7 RR1 or Ramos RR1 with Rtx or Rtx plus CDDP or Treanda® had no cytotoxic effects (as expected), the treatment with the anti-CD20-hIFN-α alone had significant cytotoxicity and synergy was observed when used in combination with CDDP or Treanda®. In all of the above experiments, the level of cytotoxicity was a function of the antibody concentration used (range 10–30 μg/ml). The mechanism by which anti-CD20-hIFN-α signals the RR clones for cytotoxicity and sensitization was examined. Preliminary findings show that treatment of the RR clones with anti-CD20-hIFN-α inhibits the activity of p38MAPK survival pathway and also inhibits the anti-apoptotic gene products, Bcl-2/BclXL and upregulates the pro-apoptotic expression of Bax. These findings established, for the first time, that modification of Rtx by fusion with IFN-α was cytotoxic on the RR clones and synergized with chemotherapy. The findings also show, unlike Rtx that, anti-CD20-hIFN-α signals the RR cells and inhibits survival/antiapoptotic pathways leading to direct cytotoxicity and chemo-sensitization. The molecular signaling mediated by anti-CD20-hIFN-α on the cell membrane of RR cells leading to inhibition of survival pathways will be presented. Disclosures: No relevant conflicts of interest to declare.

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