SCID-hu Mice Research Articles

RING box protein-1(RBX1), a key component of SCF E3 ligase, induced multiple myeloma cell drug-resistance though suppressing p27

ABSTRACT Multiple myeloma (MM) is a clonal disease of plasma cells that remains, for the most part, incurable despite the advent of several novel therapeutics. The elevated expression of p27 and its association with cell-cycle arrest is speculated to be one of the major mechanisms by which MM cells escape the cytotoxic effects of therapeutic agents. In this study, we demonstrated that RBX1 silencing could inhibit MM cell growth and promote cell drug resistance. RBX1 directly interacted with and triggered the ubiquitination and degradation of p27, ultimately causing p27 reduction. Additionally, cell growth and apoptosis analysis indicated that the role of RBX1 in regulating myeloma cell proliferation and drug resistance resulted from p27 accumulation, which occurred in a Thr187 phosphorylation-dependent manner. Furthermore, the cell-cycle analysis demonstrated that RBX1 overexpression induced cells to enter the cell cycle (S-phase) and partially inhibited chemotherapeutic drugs-mediated cell cycle arrest. Notably, the forced expression of RBX1 also inhibited the cell adhesion-mediated elevation of p27 and induced the accumulation of adherent cells in apoptosis, especially the proteolytic cleavage of caspase-3. Additionally, RBX1 knockdown significantly inhibited myeloma development in SCID-Hu mice and in a human MM xenotransplant model. Overall, these in vitro and in vivo experiments indicated that the RBX1-p27 axis could be a central molecular mechanism by which RBX1 functions as a tumor promoter and stimulates cell growth in chemotherapeutic drugs treated MM cells.

Comparative Pharmacokinetics of a Dual Inhibitor of HIV-1, NBD-14189, in Rats and Dogs with a Proof-of-Concept Evaluation of Antiviral Potency in SCID-hu Mouse Model.

We earlier reported substantial progress in designing gp120 antagonists. Notably, we discovered that NBD-14189 is not only the most active gp120 antagonist but also shows antiviral activity against HIV-1 Reverse Transcriptase (RT). We also confirmed its binding to HIV-1 RT by X-ray crystallography. The dual inhibition is highly significant because, intriguingly, this compound bridges the dNTP and NNRTI-binding sites and inhibits the polymerase activity of isolated RT in the enzymatic assay. This novel finding is expected to lead to new avenues in designing a novel class of HIV-1 dual inhibitors. Therefore, we needed to advance this inhibitor to preclinical assessment. To this end, we report the pharmacokinetics (PK) study of NBD-14189 in rats and dogs. Subsequently, we assessed the toxicity and therapeutic efficacy in vivo in the SCID-hu Thy/Liv mouse model. The PK data indicated a favorable half-life (t1/2) and excellent oral bioavailability (%F = 61%). NBD-14189 did not show any measurable toxicity in the mice, and treatment reduced HIV replication at 300 mg/kg per day in the absence of clear evidence of protection from HIV-mediated human thymocyte depletion. The data indicated the potential of this inhibitor as an anti-HIV-1 agent and needs to be assessed in a non-human primate (NHP) model.

A First in Class APRIL Fully Blocking Antibody Targets Novel Immune Regulation of APRIL in Multiple Myeloma: Further Therapeutic Implication

A proliferation inducing ligand (APRIL), a critical paracine factor for human multiple myeloma (MM), is mainly secreted by myeloid cells, including macrophages and osteoclasts in the bone marrow (BM) microenvironment. Recently, an antagonistic APRIL monoclonal antibody (mAb) 01A significantly reduced myeloma burden in SCID-hu mice (Blood. 2016;127:3225-3236). This data suggests that the humanized 01A named BION-1301, which also potently (IC50 <1 nM) blocks APRIL binding to its receptors B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator (TACI) is likely to exert anti-MM activity in vivo. Here, we further characterize the novel inhibition by 01A and a hIgG4 chimeric version C4 on multiple immune cells affected by APRIL in the immunosuppressive MM BM milieu. First, using gene-modified RPMI8226 MM cells, 01A potently and selectively blocks growth and viability of APRIL-overexpressing but not BCMA-overexpressing RPMI8226 transfectants. Significantly, 01A inhibits MM cell growth and survival induced by osteoclasts, as a physiologically relevant endogenous source of APRIL. Next, we demonstrate that pretreatment of APRIL decreases MM cell lysis induced by Daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). In addition, APRIL prevents anti-BCMA (J6M0)-induced ADCC against BCMA-expressing MM cell lines in a dose-dependent manner. Conversely, 01A or C4 (a surrogate of BION-1301) reverts APRIL-reduced MM cell lysis induced by J6M0 in the presence of PBMC or NK effector cells. These data indicate that targeting CD38 and BCMA may not be sufficient in MM. To further define the role of APRIL mediating anti-MM immune responses, we show that T cells do not express BCMA while natural T regulatory (Treg) cells significantly express higher TACI when compared with corresponding conventional T cells (Tcon) from the same individual (Abstract #102467). In parallel, detailed gene expression profiling in multiple solid tumors show that the tumor-resident Treg markers are significantly correlated with TACI, along with a strong association of APRIL expression levels with T cell inflamed signature. In addition to natural Treg, MM cell-induced Tregs (iTregs) also showed significantly increased TACI levels when compared with paired conventional T cells (Tcon) from the same individual in ex vivo cocultures. Importantly, APRIL further enhances MM cell-induced iTregs with IL-10-producing and CD15s+[JD2] FOXP3high fractions; conversely, 01A blocks APRIL-induced upregulation of iTreg. Osteoclasts also upregulated iTreg generation induced by MM cells. On the contrary, 01A or C4 partially blocks such induction. Importantly, APRIL directly promotes proliferation and viability of Treg via the induction of CCND1/2, BCL2, and BCL2L1/BCL-xL, which is blocked by 01A or C4. Finally, APRIL enhances the suppressive function of Treg on the proliferation of autologous conventional T cells, which could also be overcome by 01A or C4. Collectively, our data therefore demonstrate that APRIL, via TACI but not BCMA, critically modulates regulatory T cell function; and, importantly, 01A or C4 targets these immune regulatory subsets that have been linked to MM progression via APRIL-dependent manner. Moreover, since patients with active diseases have severe bone lesions induced by hyperactive osteoclasts, inhibiting the APRIL-TACI interaction may also overcome osteoclast-inhibited T cell killing on MM cells. Anti-APRIL Ab (BION-1301) will be entering clinical trial in late 2017 to target the immunosuppressive BM microenvironment and improve patient outcome in MM. DisclosuresDulos:Aduro Biotech Europe: Employment, Equity Ownership. Van Elsas:Aduro Biotech Europe: Employment, Equity Ownership. Richardson:Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees. Anderson:Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Transplantation of Purified Human Pericytes for Myocardial Repair in SCID-hu Mice.

The physiological, pathological, and regenerative roles of pericytes as microvascular mural cells and multipotent precursors have gained significant attention. The capacity to prospectively purify pericytes from multiple organs enables the investigation of their tissue-specific regenerative capabilities. Here, we describe the application of purified human pericytes for cardiac regeneration post-infarct in an immunodeficient mouse model. This protocol includes experimental details of pericyte isolation from both human skeletal and cardiac muscle, an immunodeficient mouse model of acute myocardial infarction, and xenogeneic pericyte transplantation.

Lentivirus-mediated RNA interference targeting programmed death receptor ligand 1 increases the immunologic anti-tumor effect of dendritic cell vaccination against pancreatic cancer in SCID-hu mice.

Programmed death receptor ligand 1 (PD-L1), which belongs to the B7 family, is overexpressed in a variety of human cancer types and serves a crucial role in immune escape by malignant cells. Programmed death receptor 1 (PD-1) is a specific PD-L1 receptor. PD-1/PD-L1 signaling inhibits the antitumor effects of dendritic cell (DC) immunization for tumor treatment. The aim of the present study was to determine whether inhibiting PD-L1 may increase the immunologic anti-tumor effect of dendritic cells against pancreatic cancer. In the present study, PD-L1 levels in non-cancerous and malignant tissue samples were compared, and the impact of PD-L1 downregulation on human pancreatic cancer PaTu8988 cells was determined by lentivirus-based RNA interference and DC immunotherapy. PD-L1 expression in pancreatic specimens was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. PaTu8988 cells expressing reduced levels of PD-L1 were generated by lentivirus-based knockdown to assess the mechanism by which the inhibition of PD-L1 signaling in DC immunization affects therapeutic outcomes in pancreatic cancer-bearing SCID-hu mice. PD-L1 levels were markedly elevated in pancreatic adenocarcinoma samples compared with in non-cancerous tissue. PD-L1 silencing in pancreatic adenocarcinoma cells resulted in improved treatment outcomes of DC immunization in vitro and in vivo compared with traditional DC immunization. PD-L1 silencing enhances the antitumor response of cytotoxic T cells by increasing interferon γ production in vitro. In vivo, this method prevented tumor growth and lung metastasis, and prolonged survival in the SCID-hu model. In conclusion, the results of the present study suggested that suppressing PD-L1 in malignant cells during DC immunization may be a useful tool for immunotherapy in pancreatic adenocarcinoma.

APRIL Is Significantly Elevated at All Stages of Multiple Myeloma (MM) and Interferes with Anti-Bcma Monoclonal Antibody-Mediated Cytolysis, Supporting the Clinical Evaluation of Bion-1301 As a Novel Therapeutic Approach in MM

A proliferation inducing ligand (APRIL) is a natural ligand with higher affinity than BAFF for both B cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), which are overexpressed on multiple myeloma (MM) cells. APRIL, which is abundantly secreted by myeloma-supporting osteoclasts and macrophages, promotes MM cell progression in vivo and further induces regulatory T cells (Treg) via TACI, but not BCMA, to promote an immunosuppressive MM bone marrow (BM) microenvironment (Blood 2017;130:3066). In preclinical studies, an antagonistic APRIL monoclonal antibody (mAb) significantly inhibited human MM cell growth in SCID-hu mice and abrogated APRIL-induced immunosuppression mediated by Tregs. Here we characterized the ability of BION-1301, a neutralizing APRIL mAb, currently under clinical development in MM (NCT03340883) to overcome protection conferred by APRIL against MM cell lysis induced by the anti-BCMA J6M0 mAb (Blood 2017 130:499).Our studies show that APRIL protects against MM cell lysis induced by J6M0 in the presence of FcR-expressing effector cells (PBMC, monocytes or NK), an effect which is blocked by neutralizing anti-APRIL mAb. APRIL also downregulates J6M0-induced cell membrane CD107a expression on NK cells co-cultured with BCMA-expressing MM cells, which is similarly abrogated by anti-APRIL mAb. Importantly, anti-APRIL mAb also significantly decreases protection conferred by osteoclasts against MM cell lysis induced by J6M0. These data indicate that blocking APRIL with anti-APRIL mAb may enhance BCMA mAb targeted (J6M0)-induced MM cell lysis. Next, using anti-human IgG1 to detect J6M0 binding to MM cell surface BCMA, we found that APRIL in a dose-dependent manner directly competes with J6M0 for binding to BCMA, which was confirmed by ELISA. In addition, APRIL effectively inhibits J6M0 binding to BCMA at 4°C, which argues against APRIL-induced BCMA receptor shedding and/or internalization. In contrast, BAFF affects J6M0 binding to BCMA only at higher concentrations (>1 µg/mL), consistent with > 2-log higher affinity of APRIL vs. BAFF for BCMA.We further assessed APRIL, BAFF, BCMA, and TACI levels in the serum of patients with MM at various stages of disease. Specifically, we used ELISA to measure free APRIL and the ELLA automated immunoassay platform to determine the levels of soluble BAFF, soluble BCMA, and soluble TACI in serum samples from patients with MM (n=193) as well as serum samples from healthy volunteers (HV, n=100). Patient samples included monoclonal gammopathy of undetermined significance (MGUS, n=12), smoldering MM (SMM, n=20), newly diagnosed MM (ND, n=39), post induction pre-autologous stem cell transplant (ASCT, n=55), post-ASCT (n=6), and relapsed refractory MM (RR, n=61). We found that free APRIL levels are significantly increased in serum samples from patients with MM at all stages of disease, when change from baseline levels were compared to those from HVs (post-ASCT: p=0.0003; other groups (MGUS, SMM, ND, pre-ASCT, and RR): p<0.0001). In contrast, average soluble BAFF levels gradually increase with progression of disease from 818.47 pg/mL at MGUS to 1685.50 pg/mL at RR MM. Both soluble BCMA and soluble TACI concentrations are highest in serum from a subset of patients with ND (593550 and 5999.63 pg/mL, respectively) and RR (1088897 and 5050.97 pg/mL, respectively) MM. A detailed analysis of clinical characteristics, treatment and response, as well as measurement of additional cytokines and chemokines is ongoing and will be reported when applicable.Our studies therefore indicate that therapies directed at the APRIL/BCMA and APRIL/TACI axes may simultaneously target MM cells and counteract APRIL-induced immunosuppression, and that combination strategies targeting APRIL with BCMA directed therapy may augment anti-MM activity. Moreover, elevated free APRIL serum levels in MGUS and all stages of MM suggest a role for APRIL in mediating immunosuppression during the development and in the pathogenesis of MM. DisclosuresDulos:Aduro Biotech: Employment. Guelen:Aduro Biotech Europe: Employment. van Zandvoort:Aduro Biotech Europe: Employment. van de Wiel:Aduro Biotech Europe: Employment. van de Crommert:Aduro Biotech Europe: Employment. Lejeune:Aduro Biotech Europe: Employment. Namini:Aduro Biotech: Employment. Eenennaam:Aduro Biotech Europe: Employment, Equity Ownership. van Elsas:Aduro Biotech Europe: Employment. Richardson:Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Munshi:OncoPep: Other: Board of director. Anderson:Takeda Millennium: Consultancy; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Oncopep: Equity Ownership.

Activity of nucleic acid polymers in rodent models of HBV infection

Nucleic acid polymers (NAPs) block the release of HBsAg from infected hepatocytes. These compounds have been previously shown to have the unique ability to eliminate serum surface antigen in DHBV-infected Pekin ducks and achieve multilog reduction of HBsAg or HBsAg loss in patients with chronic HBV infection and HBV/HDV coinfection. In ducks and humans, the blockage of HBsAg release by NAPs occurs by the selective targeting of the assembly and/or secretion of subviral particles (SVPs).The clinically active NAP species REP 2055 and REP 2139 were investigated in other relevant animal models of HBV infection including woodchucks chronically infected with WHV, HBV transgenic mice and HBV infected SCID-Hu mice. The liver accumulation of REP 2139 in woodchucks following subcutaneous administration was examined and was found to be similar to that observed in mice and ducks. However, in woodchucks, NAP treatment was associated with only mild (36–79% relative to baseline) reductions in WHsAg (4/10 animals) after 3–5 weeks of treatment without changes in serum WHV DNA. In HBV infected SCID-Hu mice, REP 2055 treatment was not associated with any reduction of HBsAg, HBeAg or HBV DNA in the serum after 28 days of treatment. In HBV transgenic mice, no reductions in serum HBsAg were observed with REP 2139 with up to 12 weeks of treatment.In conclusion, the antiviral effects of NAPs in DHBV infected ducks and patients with chronic HBV infection were weak or absent in woodchuck and mouse models despite similar liver accumulation of NAPs in all these species, suggesting that the mechanisms of SVP assembly and or secretion present in rodent models differs from that in DHBV and chronic HBV infections.

Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia

Syncytia formation has been considered important for cell-to-cell spread and pathogenesis of many viruses. As a syncytium forms, individual nuclei often congregate together, allowing close contact of nuclear membranes and possibly fusion to occur. However, there is currently no reported evidence of nuclear membrane fusion between adjacent nuclei in wild-type virus-induced syncytia. Varicella-zoster virus (VZV) is one typical syncytia-inducing virus that causes chickenpox and shingles in humans. Here, we report, for the first time, an interesting observation of apparent fusion of the outer nuclear membranes from juxtaposed nuclei that comprise VZV syncytia both in ARPE-19 human epithelial cells in vitro and in human skin xenografts in the SCID-hu mouse model in vivo. This work reveals a novel aspect of VZV-related cytopathic effect in the context of multinucleated syncytia. Additionally, the information provided by this study could be helpful for future studies on interactions of viruses with host cell nuclei.

Osteocyte Apoptosis Attracts Myeloma Cells to Bone and Supports Progression through Regulation of the Bone Marrow Microenvironment

Multiple Myeloma (MM) pathogenesis and progression is intricately tied to the bone marrow (BM) microenvironment, and understanding this microenvironment is of upmost importance. Osteocytes are the most abundant cell type in bone, comprising over 90% of the cellular population. These cells were long thought to be primarily inert mechanical sensors, however osteocyte regulation of the BM is increasingly appreciated. Still, very little is known about the role of osteocytes in the context of cancer. We initially evaluated MM patient BM biopsies and determined that osteocyte apoptosis is increased compared to MGUS and healthy BM. We further validated this finding in the SCID-hu mouse model - increased osteocyte death was seen in the primary human bone grafts that were injected with aggressive MM cells compared to those injected with less aggressive MM cells. Surprisingly, and more interestingly, increased osteocyte apoptosis/death was also found in the contralateral, un-injected human bone grafts from mice with aggressive MM. Importantly, these changes in the secondary bone grafts occurred prior to detection of MM cells. In the present study, we investigated if and how osteocyte apoptosis at distant bone sites, before MM metastasis, feeds back to MM cells to support dissemination and progression.We developed a unique, syngeneic mouse model of MM in which osteocytes can be specifically ablated in vivo. Mice expressing the Diphtheria toxin receptor (DTR) in mature osteocytes (RIKEN BioResource Center, Japan) were crossed with C57Bl/KaLwRij mice, a strain that when injected with syngeneic 5TGM1 MM cells develops many characteristics of human MM. The resulting 8-week old progeny expressing the DTR gene and on the C57Bl/KaLwRij background were intraperitoneally (i.p.) injected with Diphtheria toxin (DT,12.5 μg/kg) or PBS as control (n=7 per group). After 7 days, mice were injected intravenously (i.v.) with 2x106 5TGM1 MM cells expressing firefly Luciferase. Weekly bioluminescent imaging and ELISA for IgG2bκ (secreted by 5TGM1 cells) tracked tumor location and growth over time. Remarkably, compared to control mice (DTR+PBS), osteocyte ablated (DTR+DT) mice showed significantly enhanced tumor homing to bone by bioluminescent imaging and significantly increased total tumor burden by both bioluminescent imaging and sera IgG2bκ ELISA.To understand mechanistically how osteocyte apoptosis enhances MM-bone homing and tumor progression, we evaluated BM microenvironmental changes after osteocyte apoptosis was induced. Syngeneic DTR mice were injected at 8 weeks with DT or PBS (n=3-4 per group). Mouse legs were collected after 7 days: one leg for sectioning and one leg for isolating BM cells and BM extracellular fluid. Flow cytometry was performed on BM cells and BM extracellular fluid was evaluated using a cytokine/chemokine array. Leg sections were stained via immunohistochemistry (IHC). Cytokine arrays of BM extracellular fluid revealed substantial increases of multiple cytokines/chemokines important for MM dissemination and progression (i.e. IL-6, HGF) in DTR+DT mice. Additionally, bone destruction-associated molecules DKK1, RANKL, and M-CSF were also increased in DTR + DT mice compared to DTR+PBS control mice. IHC showed increased numbers of osteocytes positive for sclerostin, HIF-1α and RANKL. Finally, flow cytometric analysis suggested heightened immune suppression in the BM as multiple immune-suppressive cell populations were enhanced in DTR+DT mice compared to DTR+PBS mice. These include myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and a regulatory B cell subset (Breg).In conclusion, we have successfully generated a syngeneic model of MM in which osteocytes are selectively ablated in vivo after a single injection of DT. Using this model, our data demonstrate that osteocyte apoptosis supports MM homing and growth in bone, likely as a result of an altered BM microenvironment. Interestingly, these changes include enhanced MM supportive/attractive soluble molecules, support of bone destruction, and enhanced immune suppression. Combined, our data suggests that osteocyte apoptosis is important for developing a pre-metastatic niche and contributes to MM progression in bone. Therefore, preventing osteocyte apoptosis may help stall and prevent dissemination of MM, and possibly other cancers, to bone. DisclosuresNo relevant conflicts of interest to declare.

Thymidine Phosphorylase Exerts Complex Effects on Bone Resorption and Formation in Myeloma

Thymidine phosphorylase (TP), an enzyme that can reversibly catalyze the conversion of thymidine to thymine and 2-deoxy-D-ribose (2DDR), has been shown to participate in tumor angiogenesis and proliferation. Yet little is known regarding its function in bone. The goal of this study is to elucidate the role and mechanism of myeloma-expressed TP in the activation of osteoclast-mediated bone resorption and the suppression of osteoblast-mediated bone formation. We hypothesized that myeloma-expressed TP plays an important role in the pathogenesis of myeloma bone disease. We observed that TP is highly expressed in myeloma cells but not in normal plasma cells. To examine the role of myeloma-expressed TP in lytic bone lesions, we categorized all tested patient-derived myeloma cells and human myeloma cell lines into two groups: TP-high and TP-low expressing cells. These myeloma cells, as well as human myeloma cells with overexpressed or knocked downed levels of TP, were injected into the implanted human bone chips of SCID-hu mice or the femurs of SCID mice. Analysis of radiography and histomorphometry were used for assessing lytic lesions. Our results showed that injection of TP-high expressing myeloma cells into mice caused more lytic lesions than injection of TP-low cells. To examine its role in osteoclast and osteoblast differentiation, the progenitors were co-cultured with the myeloma cells, and analyzed with staining of TRAP and Alizarin red S. We observed that co-culture with TP-high expressing myeloma cells induced more osteoclast differentiation and less osteoblast formation than those co-cultured with TP-low cells. Mechanistic studies further showed that TP-high expressing myeloma cells secreted more 2DDR than TP-low cells. The secreted 2DDR bound to the integrin aVb3 in osteoclast progenitors, activated the PI3K/Akt signaling, and enhanced DNMT3A expression and methylation of IRF8, leading to increased NFATc1 expression and osteoclast differentiation. The secreted 2DDR could also bind to the integrins aVb3 and a5b1 in osteoblast progenitors, activated the PI3K/Akt signaling, and enhanced DNMT3A expression and methylation of RUNX2 and osterix, leading to decreased osteoblast differentiation. We further examined the patient bone marrow samples, and demonstrated a positive correlation between TP expression in myeloma cells and osteolytic bone lesions in myeloma patients. Thus, our study not only elucidates a novel mechanism of myeloma-induced increased osteoclast-mediated bone resorption and suppressed osteoblast-mediated bone formation, but also implicates a potential therapeutic approach for myeloma bone disease. DisclosuresNo relevant conflicts of interest to declare.

Modulation of host CD59 expression by varicella-zoster virus in human xenografts in vivo

Varicella-zoster virus (VZV) is the causative agent of both chickenpox (varicella) and shingles (zoster). VZV survives host defenses, even with an intact immune system, and disseminates in the host before causing disease. To date, several diverse immunomodulatory strategies used by VZV to undermine host immunity have been identified; however, few studies have addressed the complement evasion strategies used by this virus. Here, we show that expression of CD59, which is a key member of host regulators of complement activation (RCA), is significantly upregulated in response to VZV infection in human T cells and dorsal root ganglia (DRG) but not in human skin xenografts in SCID-hu mice in vivo. This is the first report demonstrating that VZV infection upregulates host CD59 expression in a tissue-specific manner in vivo, which may aid VZV in complement evasion and pathogenesis.

Functional analysis of human cytomegalovirus UL/b' region using SCID-hu mouse model.

Human cytomegalovirus (HCMV) attenuated strains, Towne, and AD169, differ from prototypic pathogenic strains, such as Toledo, in that they are missing a ∼15-kb segment in the UL/b' region. In contrast to the attenuated strains, Toledo can replicate in human tissue implants in SCID (SCID-hu) mice. Thus, this model provides a unique in vivo system to study the mechanism of viral pathogenesis. Twenty-two ORFs have been annotated in the UL/b' region, including tissue-tropic genes encoded in a pentameric gH/gl complex. To differentiate the role of the pentameric gH/gl complex versus the functions of other ORFs in the 15-kb region in supporting viral growth in vivo, a series of recombinant viral strains were constructed and their ability to replicate in SCID-hu mice was tested. The mutations in the Towne and AD169 strains were repaired to restore their pentameric gH/gl complex and it was found that these changes did not rescue their inability to replicate in the SCID-hu mice. Subsequently four deletion viruses (D1, D2, D3, and D4) in the 15-kb region from the Toledo strain were created. It was demonstrated that D2 and D3 were able to grow in SCID-hu mice, while D1 and D4 were not viable. Interestingly, co-infection of the implant with the D1 and D4 viruses could compensate their respective growth defect in vivo. The results demonstrated that rescuing viral epithelial tropism is not sufficient to revert the attenuation phenotype of AD169 or Towne, and pathogenic genes are located in the segments missing in D1 and D4 viruses. J. Med. Virol. 88:1417-1426, 2016. © 2016 Wiley Periodicals, Inc.

Insights into the function of tegument proteins from the varicella zoster virus.

Chickenpox (varicella) is caused by primary infection with varicella zoster virus (VZV), which can establish long-term latency in the host ganglion. Once reactivated, the virus can cause shingles (zoster) in the host. VZV has a typical herpesvirus virion structure consisting of an inner DNA core, a capsid, a tegument, and an outer envelope. The tegument is an amorphous layer enclosed between the nucleocapsid and the envelope, which contains a variety of proteins. However, the types and functions of VZV tegument proteins have not yet been completely determined. In this review, we describe the current knowledge on the multiple roles played by VZV tegument proteins during viral infection. Moreover, we discuss the VZV tegument protein-protein interactions and their impact on viral tissue tropism in SCID-hu mice. This will help us develop a better understanding of how the tegument proteins aid viral DNA replication, evasion of host immune response, and pathogenesis.

Patient-Derived 3D Tissue-Engineered Bone Marrow Cultures Support Primary MM Growth

INTRODUCTIONIn vitro culturing of primary myeloma cells has been a major challenge because the lack of an in vitro technology capable of recreating the complicated bone marrow (BM) microenvironment which multiple myeloma (MM) cells depend on for its survival. While primary myeloma cells cannot grow ex vivo in SCID mice, they are able to growth in SCIDhu mice. However, animal models are expensive, time-consuming, and often have limited reproducibility. In addition, classic laboratory models cannot take into consideration the variability of disease in every patient, and the MM patient population is highly variable, both genetically and epigenetically, and the biological characteristics of patients are widely different, which demonstrates sensitivity of individual patients to different therapies. Typical two-dimensional (2D) models rely on a limited number of MM cell lines which cannot reflect the enormous heterogeneity and variations present in individual patients. The goal of this study is to create a patient derived three-dimensional tissue-engineered bone marrow (3DTEBM) culture system based on cross-linked MM-derived BM supernatant, including endogenous soluble growth factors and cytokines, and by incorporating mononuclear cells, including MM (CD138+ population) and accessory cells (CD138- population), from the same patient. METHODSThe 3DTEBMwas formed through calcium cross-linking of BM supernatants combined with the CD138+ and CD138- population from the same MM patient. We tested the growth of fresh primary CD138+ MM cells with CD138- cells in 2D vs patient-derived 3DTEBM cultures based on the original BM ratio (CD138-/CD138+) of the patient at 3 days by flow cytometry. We further selected a patient with not very aggressive tumor and evaluated the effect of CD138- increasing densities on CD138+ MM growth in 3DTEBM cultures at day 3 by flow cytometry. In addition, the effect of patient-derived 3DTEBM and 2D cultures with and without CD138- cells on CD markers expression of the CD138+ population was tested at day 3 by flow cytometry. Finally, the growth of frozen primary CD138+ MM cells with and without CD138- cells in 2D vs patient-derived 3DTEBM cultures was analyzed for 14 days by flow cytometry. RESULTSWe found that patient-derived 3DTEBM cultures support primary MM cell growth. CD138+ MM cells from patients with less aggressive tumors (high BM ratio CD138-/CD138+) showed better growth in 3DTEBM than in 2D systems at day 3. However, more aggressive tumors (low BM ratio CD138-/CD138+), in which MM cells are less dependent of accessory cells, the 3DTEBM cultures showed similar CD138+ MM growth as in 2D cultures. After that, we detected in the patient-derived 3DTEBM cultures a direct correlation between positive and negative fractions, with increased CD138- densities the CD138+ MM growth increased.Next, we analyzed the effect of 3D cultures in CD markers expression and we showed that CD138+ MM cells expressed loss of the plasma cells markers (CD38, CD56, and CD138), reduction of B cells markers (CD19, CD20 and CD22), and increased of the stem cell marker CD34 in 3DTEBM compared to 2D cultures.Finally, patient-derived 3DTEBM supported the growth of frozen primary CD138+ MM cells better than 2D cultures, and the addition of CD138- cells enhanced even more CD138+ MM growth after 14 days in culture. CONCLUSIONSPatient-derived 3DTEBM cultures support primary fresh and frozen CD138+ MM growth better than classic systems, and induced de-differentiation of MM cells while increased a stem-cell like phenotype. These results highlight the importance of the BM microenvironment (BM supernatant, including endogenous soluble growth factors and cytokines, and the CD138- population from the same patient) to facilitate primary MM cell growth in vitro. Therefore, patient-derived 3DTEBM cultures allowed long-term culture of primary fresh and frozen myeloma cells ex vivo, and these findings indicate that patient-derived 3DTEBM can be utilized for studying multiple myeloma biology and for testing patient-targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

Potent Inhibition of the Growth and Induction of Apoptosis of Mantle Cell Lymphoma By the Novel Anthracycline Berubicin in Vitro and in Vivo

Rituximab in combination with hyperfractionated cyclophosphamide, vincristine, doxorubicin (DOX), and dexamethasone has been a successful frontline therapy for aggressive mantle cell lymphoma (MCL), a distinct and heterogeneous subtype of non-Hodgkin lymphoma, with complete remission rates as high as 87% in patients at or below 65 years of age. However, the efficacy of rituximab in combination with hyperfractionated cyclophosphamide, vincristine, DOX, and dexamethasone comes at the expense of severe grade III-IV toxic effects, and its use is limited by de novoor acquired multidrug resistance. Berubicin (WP744; BRN) is a clinically evaluated analog of DOX that, like DOX, stabilizes cleavable complexes between DNA and topoisomerase II, leading to irreversible double-strand DNA breaks and abrogation of DNA religation. In contrast to DOX, BRN overcomes multidrug resistance by circumventing efflux mediated by ATP-binding cassette proteins like P-glycoprotein and MRP-1. BRN is also the first anthracycline derivative to effectively cross the blood-brain barrier, with demonstrated phase 1 responses in brain cancer patients. Our MCL studies demonstrated that treatment with BRN decreased cell growth threefold to sixfold more potently than DOX in all tested MCL cell lines (Mino, Jeko-1, SP53, and Granta 519) and four MCL patient samples by arresting MCL cell-cycle progression at the G2/M phase and inducing extensive apoptosis. Moreover, in a Western blot assay, Jeko-1 and Mino cells exhibited activation of caspase-3 and caspase-9 and cleavage of poly(ADP-ribose) polymerase over time, pointing to the presence of a mitochondria-mediated apoptosis pathway in BRN-treated MCL cell lines. We then assessed the in vivo activity of BRN, using DOX as a control, in MCL-bearing severe combined immunodeficient (SCID) and SCID-hu mice. Our results indicated that SCID mice given 10 mg/kg DOX survived no more than 15 days after treatment, whereas all mice given 10 mg/kg BRN survived up to 63 days after treatment. On the other hand, when BRN and DOX were administered at lower doses of 5 mg/kg in a SCID-hu MCL mouse model, similar survival durations for BRN and DOX were observed. In summary, our studies demonstrate that BRN is highly potent against MCL in vitro and displays a wider therapeutic window than DOX, delivering very promising in vivo activity in both SCID and SCID-hu MCL models. These results indicate that BRN should be evaluated in the treatment of other lymphomas and seriously considered as an effective replacement for DOX in MCL treatment. BRN’s success in Phase 1 studies in glioma patients and observed safety and effectiveness in MCL models warrant further investigation of its clinical use and combination with existing therapies in MCL patients. DisclosuresPriebe:MD Anderson: Waldemar Priebe holds patents and has a financial interest in the development of berubicin. Patents & Royalties.

Anti-CD20 and B-Cell Receptor-Mediated Growth Inhibition and Apoptosis: A Preclinical Study of Ibrutinib and Rituximab Combination Therapy in Mantle Cell Lymphoma in Vitro and in Vivo

Abstract Intruduction Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor outcome and therapeutic challenge. Oral single-agent ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, elicited a response rate of 68% in phase II clinical trial and has been approved by FDA for the treatment of MCL patients who received at least one prior therapy. To increase the response rate, the combination of ibrutinib with rituximab would be an ideal regimen because: (1) Both BTK and CD20 are effective therapeutic targets for MCL; (2) CD20-dependent apoptosis occurs associated with B-cell receptor (BCR) activity. Regardless rituximab-mediated immune response (ADCC and CDC) and ibrutinib-mediated ITK inhibition (Th1 polarization), an important potential effect of ibrutinib and rituximab may enhance the direct anti-MCL cytotoxicity of the combination. Methods and Results We investigated the effects of ibrutinib and/or rituximab in 6 established MCL cell lines and 3 freshly isolated primary MCL cells in vitro. First, ibrutinib, as well as BTK-siRNA, inhibited the growth of MCL cells. Then we found that ibrutinib induced the growth inhibition and apoptosis of most MCL cell lines and primary MCL cells in a dose-dependent manner during 5-7 days of culture (IC50 range from 1.5 to 15 µM). On the other hand, rituximab alone induced 10-30% growth inhibition and apoptosis of both established and primary MCL cells in vitro. Importantly, our results showed that ibrutinib and rituximab combination synergistically induced apoptosis of MCL cells during 5-7 days of culture in vitro. Next, our animal study confirmed the compartmental shift phenomenon mediated by ibrutinib which was similar with the data from clinical study. Briefly, SCID mice were subcutaneously implanted with human fetal bone chips (SCID-hu). After 6 weeks of bone implantation, the freshly isolated MCL cells from patients were directly engrafted into the human fetal bone chips. The engrafted MCL cells produced measurable levels of human β2-microglobulin (β2M) in mouse serum. Once human β2M had been detected in mouse serum, the primary MCL-bearing SCID-hu mice were treatment with 25 mg/kg ibrutinib oral gavage daily. A transient increase of human CD5+CD20+ cells in mouse peripheral blood was detected by flow cytometry from day 8 of treatment, representing a shift of human MCL cells from human fetal bone chip to mouse peripheral blood. At this time, 10 mg/kg rituximab was intravenously administrated every 3 days for total 7 doses. Our results demonstrated that rituximab and ibrutinib combination eradicated MCL cells in vivo and kept mice survival longer than either ibrutinib or rituximab alone. Conclusions Based on our preclinical data of rituximab and ibrutinib combination in vitro and in primary MCL-bearing SCID-hu mice, ibrutinib plus rituximab could be an effective regimen in a clinical trial of relapsed or refractory MCL. Disclosures Off Label Use: Ibrutinib plus rituximab may enhance the direct anti-MCL cytotoxicity. Wang:Pharmacyclics: Honoraria, Research Funding.

β-l-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (l-BHDU) prevents varicella-zoster virus replication in a SCID-Hu mouse model and does not interfere with 5-fluorouracil catabolism

The alphaherpesvirus varicella-zoster virus (VZV) causes chickenpox and shingles. Current treatments are acyclovir (ACV) and its derivatives, foscarnet and brivudine (BVdU). Additional antiviral compounds with increased potency and specificity are needed to treat VZV, especially to treat post-herpetic neuralgia. We evaluated β-l-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (l-BHDU, 1) and 5′-O-valyl-l-BHDU (2) in three models of VZV replication: primary human foreskin fibroblasts (HFFs), skin organ culture (SOC) and in SCID-Hu mice with skin xenografts. The efficacy of l-BHDU in vivo and its drug–drug interactions were previously not known. In HFFs, 200μM l-BHDU was noncytotoxic over 3days, and l-BHDU treatment reduced VZV genome copy number and cell to cell spread. The EC50 in HFFs for l-BHDU and valyl-l-BHDU were 0.22 and 0.03μM, respectively. However, l-BHDU antagonized the activity of ACV, BVdU and foscarnet in cultured cells. Given its similar structure to BVdU, we asked if l-BHDU, like BVdU, inhibits 5-fluorouracil catabolism. BALB/c mice were treated with 5-FU alone or in combination with l-BHDU or BVdU. l-BHDU did not interfere with 5-FU catabolism. In SCID-Hu mice implanted with human skin xenografts, l-BHDU and valyl-l-BHDU were superior to ACV and valacyclovir. The maximum concentration (Cmax) levels of l-BHDU were determined in mouse and human tissues at 2h after dosing, and comparison of concentration ratios of tissue to plasma indicated saturation of uptake at the highest dose. For the first time, an l-nucleoside analog, l-BHDU, was found to be effective and well tolerated in mice.

Varicella Zoster Virus and the Neuro-Attenuated Vaccine Necessity

Varicella Zoster Virus and the Neuro-Attenuated Vaccine Necessity

Efficacy of broadly neutralizing monoclonal antibody PG16 in HIV-infected humanized mice

Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clades A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70–80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV inoculation. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier.

Macrophages Activation By ICAM1 Antibody Combined With Lenalidomide Has Enhanced Anti-Myeloma Activity In a Supportive Microenvironment In Vivo and In Vitro

Tumor associated macrophages are reportedly accumulated in myelomatous bones and support myeloma cell survival and growth. Myeloma cells from the majority of patients express ICAM1, a cell surface receptor associated with the interaction of myeloma cells with stromal cells. BI-505, a humanized IgG1 lambda antibody against ICAM1, has been recently reported to inhibit growth of ICAM1-expressing myeloma cells by specifically activating macrophages (Veitonmäki et al, Cancer Cell, 2013) while lenalidomide is a known clinical immunomodulatory agent. The aim of the study was to test the effect of combination treatment with BI-505 and lenalidomide on growth of myeloma cells in vivo and in vitro. ICAM1-expressing myeloma cells from 4 patients with progressive disease were engrafted in SCID-hu mice and treated with control antibody, BI-505 (10 mg/kg, weekly, i.p.), lenalidomide (2 mg/kg, daily, p.o.) or BI-505 and lenalidomide for 5-8 weeks (5-6 mice/group in each set of patient's cells). Myeloma growth was assessed by monitoring circulating human kappa light chain immunoglobulin or soluble syndecan-1 in the mice sera, or using live-animal imaging of myeloma cells infected with lentiviral particles containing EGFP/luciferase construct. Myeloma growth was inhibited by the two drugs in all experiments. At the end of each experiment, tumor burden in the BI-505, lenalidomide and drug combination groups was 13±4% (p<0.0001), 21±6% (p<0.0001) and 3±1% (p<0.0001) relative to control groups, respectively. Myeloma burden was also significantly lower in the drug combination group compared to BI-505 (p<0.03) or lenalidomide (p<0.008) groups. These data are well in concordance with results from experiments with myeloma cell lines RPMI8226 and U266 in immunodeficient mice where the combination of BI-505 and lenalidomide showed significantly more anti-tumor effect compared to either treatment alone. In vitro, we established a coculture system in which whole donor bone marrow (BM) cells were cultured for 7 days with serum pooled from myeloma patients, followed by coculture with CD138-selected myeloma cells for an additional 7 days (BM:myeloma cell ratio 4:1). Phenotypically, the cultured BM contained cells of various hematopoietic lineages including macrophages. Growth of myeloma cells was assessed by CD45/CD38 flow cytometry or bioluminescence analysis of luciferase-expressing myeloma cells in coculture. In this system, primary myeloma cells survived and propagated regardless of their molecular risk signature or subsets while BI-505 (1-5 μg/ml) inhibited growth of ICAM1-expressing myeloma cells by 54±6% (n=5, p<0.003) but had no effect on myeloma cells that do not express ICAM1. In the coculture setting, combination of BI-505 (2.5 μg/ml) and lenalidomide (0.5 μM) had higher anti-tumor efficacy than each of the agents alone. These data indicate that macrophages can be activated by BI-505 to induce anti-myeloma response and that the anybody activity is enhanced in combination with lenalidomide. Disclosures:Teige:BioInvent International: Employment. Frendéus:BioInvent International: Employment.