Influence of Cyclophosphamide on L-Asparaginase-Induced Allergy in Animal Model

BACKGROUND: L-Asparaginase (L-ASP) is one of the essential drugs for acute lymphoblastic leukemia and lymphoma. Since L-ASP proteins used clinically are derived from bacteria, they often cause allergies, including anaphylaxis. However, the characteristics of L-ASP-induced hypersensitivity are not well documented so far. Also, there is neither suitable treatment for hypersensitivity to L-ASP nor adequate experimental model of L-ASP allergy. In addition, some anti-cancer drugs, used concomitantly with L-ASP, are known to affect lymphocyte activities, although the role of such drugs on L-ASP hypersensitivity is not known. Previously, we have established mouse model of L-ASP allergy and found that a neutralizing antibody (Ab) of IgE is effective to inhibit L-ASP-induced allergic reaction. Aims: In order to establish in vitro model of L-ASP allergy, we used RBL-2H3 mast cells and evoked degranulation of the cells sensitized with L-ASP-allergic mouse serum using L-ASP as an antigen. Furthermore, we examined the effect of cyclophosphamide (CY), which reportedly impairs the functions of regulatory T (Treg) cells, on L-ASP allergy both in vivo and in vitro. Methods: Male BALB/c mice (7 week old) were sensitized with L-ASP (100 units) with Al(OH)3 gel (1 mg) on days 1 and 15. The right ears of the mice were locally sensitized on days 18, 21, 24 by i.d. injection of L-ASP (10 units). Antigen challenge was carried out on day 27 by i.d. injection (10 units). CY (75, 150, 300 mg/kg) was i.p. administrated at day -1 and day 13. Anti-IgE treatment (BD Bioscience, clone R35-92; 100 μg, i.p.) was carried out on the day before L-ASP challenge. The serum was collected on day 27. Total IgE level in the serum was measured by ELISA kit (Yamasa). RBL-2H3 cells were sensitized by the serum and stimulated by L-ASP to determine β-hexosaminidase (β-Hex) release in vitro. Results : L-ASP-sensitization induced allergic ear edema and an increase in serum IgE level in mice, both of which are augmented by an administration of CY at 150 mg/kg. L-ASP-induced allergic ear edema was inhibited by a pretreatment with anti-IgE Ab. L-ASP-induced CY-enhanced ear edema was also inhibited by anti-IgE Ab. On the other hand, at 300 mg/kg of CY, L-ASP-induced ear edema was much lower than 150 mg/kg group, though IgE concentration in the serum was as high as CY 150 mg/kg group. When RBL-2H3 cells were sensitized by anti-L-ASP serum, L-ASP challenge induced β-Hex release. Anti-L-ASP serum of CY 150 mg/kg-treated mice induced higher β-Hex release than normal anti-L-ASP sera in vitro, though that of CY 300 mg/kg-treated mice did not induce β-Hex release. After removing IgG from the serum of CY 300 mg/kg-treated mice using protein G Mag Sepharose (GE Healthcare), β-Hex release became higher than normally sensitized mice. Anti-IgE Ab treatment both ex vivo and in vitro inhibited L-ASP-induced β-Hex release from the RBL-2H3 cells sensitized by anti-L-ASP serum of CY-treated mice. Conclusions: In this study, we showed that L-ASP sensitization induced IgE production in vivo and this serum could bring about β-Hex release from RBL-2H3 cells in vitro; both allergic reactions were inhibited by an anti-IgE antibody which binds to Fc region of IgE molecule. From these results, we propose that non-anaphylactogenic neutralizing antibody of IgE, such as omalizumab, can be a candidate drug for the treatment of L-ASP hypersensitivity. Also in this study, CY induced biphasic effects on L-ASP allergy in mice, enhancing it at low dosage and attenuating it at high dosage. Since CY is reportedly impair the functions of Treg cells, CY-induced suppression of Treg may enhance Th2 responses so as to augment L-ASP hypersensitivity. Further investigations for the detection and avoidance of L-ASP hypersensitivity are under the way. Disclosures No relevant conflicts of interest to declare.

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Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.810.810 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 810-810 ◽

Cited By ~ 3

Author(s):

Haiying Qin ◽

Sang M Nguyen ◽

Sneha Ramakrishna ◽

Samiksha Tarun ◽

Lila Yang ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Chain ◽

Dual Targeting ◽

Single Antigen ◽

Therapeutic Function

Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

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IL-12 Enhances Tumor Immunity Via Differential Effects On Regulatory T Cells and Cytotoxic Lymphocytes.

Blood ◽

10.1182/blood.v114.22.1656.1656 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1656-1656

Author(s):

Xuefang Cao ◽

Karen Leonard

Keyword(s):

Tumor Growth ◽

Tumor Immunity ◽

Conflicts Of Interest ◽

Treg Cells ◽

Cell Contact ◽

Cytotoxic Lymphocytes ◽

Differential Effects ◽

Killing Activity

Abstract Abstract 1656 Poster Board I-682 To study the roles of IL-12 and Interferon-gamma (IFNg) in tumor immunity, we used RMAS lymphoma cells to challenge IL-12 receptor beta 2-deficient (IL-12Rb2-/-) and IFNg receptor 1-deficient (IFNgR1-/-) mice that are in the syngeneic C57BL/6J background. We intravenously injected mice with a dose of 1 × 104 RMAS cells that caused death in about 50% of wild-type (WT) mice. As shown in the Figure below, all of the WT mice treated with exogenous IL-12 were rescued from death caused by tumor growth; endogenous IL-12 was not sufficient to impact tumor growth since IL-12Rb2-/- mice showed a survival rate similar to that of WT mice. However, all of the IFNgR1-/- mice succumbed to tumor growth, indicating that endogenous IFNg is required for tumor immunity in this system. Furthermore, IL-12 treatment did not improve the survival of the IFNgR1-/- mice, suggesting that IFNg signaling is required for IL-12's anti-tumor effect. We previously showed that an IL-12/IFNg axis can inhibit tumor-induced regulatory T cell (Treg) proliferation in vitro (Cao et al, 2008 ASH Annual Meeting). We have subsequently examined their effects on Treg cells in vivo. Compared to naive mice, significant Treg expansion (4.9 ± 2.1 fold, n=5, p=0.025) was observed in the peritoneal cavity of WT mice within 2 weeks after an intraperitoneal injection of 1 × 104 RMAS cells. This expansion was completely blocked by treatment with exogenous IL-12. Treg cells in the IL-12Rb2-/- mice expanded to levels comparable to that in WT animals, suggesting that endogenous IL-12 was not sufficient to control Treg expansion. In contrast, significantly higher Treg expansion was observed in IFNgR1-/- mice (36.8 ± 11.8 fold, n=5, p=0.002), which was partially inhibited by IL-12 treatment (13.2 ± 3.5 fold, n=5, p=0.002), suggesting that an IFNg-independent mechanism may also account for IL-12's anti-Treg effect. To further study the effects of IL-12 and IFNg on cytotoxic T lymphocyte (CTL) function, we performed mixed lymphocyte reactions (MLR) and used flow-based killing assays (FloKA) to measure cell contact-dependent killing of allogeneic P815 tumor cells. MLR-activated CTLs were found to kill tumor targets via perforin/granzyme-mediated cytotoxicity. At a 10:1 (effector:target) ratio, granzyme AxB-deficient CTLs and perforin-deficient CTLs displayed significantly reduced killing (8.6 ± 1.2% and 4.5 ± 0.9%, respectively) compared to WT CTLs (36.1 ± 3.5%). IL-12 supplement (2ng/ml) to the MLR significantly increased the killing activity of WT CTLs (65.3 ± 4.2%), but had no significant effect on granzyme AxB-deficient CTLs or perforin-deficient CTLs. In contrast, IFNg supplement (10ng/ml) to the MLR had no significant effect on the killing activity of CTLs. Conversely, MLR-activated IFNgR1-/- CTLs killed P815 cells as efficiently as WT CTLs and responded to IL-12 treatment as efficiently as WT CTLs. Taken together, these data suggest that IL-12 treatment inhibits tumor-induced Treg expansion and stimulates IFNg-dependent anti-tumor immune responses. In addition, IL-12 also activates perforin/granzyme-dependent function of cytotoxic T lymphocytes. These differential effects on diverse immune components may collectively result in enhanced tumor immunity. Disclosures No relevant conflicts of interest to declare.

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The Small Molecule Inhibitor of VLA4 TBC3486 Sensitizes Resistant ALL to Chemotherapy

Blood ◽

10.1182/blood.v120.21.1500.1500 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1500-1500 ◽

Cited By ~ 2

Author(s):

Yao-Te Hsieh ◽

Eun Ji Gang ◽

Halvard Bonig ◽

Ronald J Biediger ◽

Peter Vanderslice ◽

...

Keyword(s):

Cell Viability ◽

Small Molecule ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Chemotherapy Resistance ◽

Leukemia Cell ◽

The Novel ◽

Small Molecule Inhibition

Abstract Abstract 1500 Significant progress notwithstanding, drug resistant acute lymphoblastic leukemia (ALL) remains a therapeutic challenge, as well as acute and long-term off-target toxicity of anti-ALL therapies can be dose-limiting or debilitating. Therefore, the development of more targeted therapies is desirable. We recently provided evidence that chemotherapy resistance of ALL cells can be partly overcome by interfering with the function of VLA4, the alpha4beta1 integrin, in vivo. In those studies, we used the anti-functional antibody Natalizumab. We extended our studies to an alternative VLA4 inhibitor, the novel non-peptidic small molecule TBC3486. Previous in vitro assays and molecular modeling studies indicate that TBC3486 behaves as a ligand mimetic, competing with VCAM-1 for the MIDAS site of VLA-4. As such, the compound has been shown to be efficacious in VLA-4 dependent models of inflammatory and autoimmune disease. The potential usefulness of this novel inhibitor in leukemia treatment was tested in our established in vitro and in vivo assays. LAX7R cells, primary pre-B-ALL with a normal karyotype from a patient with an early relapse, were used throughout for the studies reported here. LAX7R cells were treated with 25μM TBC3486 or THI0012 control, the inactive enantiomer of TBC3486, and seeded onto plates coated with human VCAM-1. Adhesion, scored after 2 days, was significantly inhibited by TBC3486 compared to control treated cells (7.9%±4.0 vs 95.4%±8.0; p=0.003). Proliferation rate and cell viability were unaffected by the treatments. In a co-culture system of LAX7R cells with OP9 stroma cells, which we use as an in vitro model of stroma-mediated chemotherapy resistance, we assessed differential effects of VDL (Vincristine, Dexamethasone, L-Asparaginase) on leukemia cell survival in the presence or absence of TBC3486. Stromal adhesion significantly protected LAX7R cells against VDL chemotherapy; this effect was significantly attenuated by TBC3486 compared to the control as determined by Trypan blue exclusion of dead cells (Cell viability of 39.9%±5.1 vs. 57.2±1.8; p=0.02). After these encouraging observations, we next evaluated the benefit of TBC3486 on leukemia progression in a xenotransplant assay. LAX7R cells were lentivirally labelled with luciferase for in vivo tracking and injected into NOD/SCID hosts. Three days after leukemia cell transfer, mice received either TBC3486 or THI0012 (control) (10mg/kg/d) daily for 2 weeks (intraperitoneally), with or without VDL chemotherapy. This experiment is in progress, but already survival of leukemia-bearing mice was significantly prolonged, from a median survival time (MST) for control mice of 33 days post-leukemia injection to a MST of 47 days post-leukemia injection for TBC3486 treated mice (p=0.02). Similarly, bioluminescence imaging revealed a marked delay of leukemia cell dissemination (p<0.0001). Taken together, our data demonstrate that small molecule inhibition of VLA4 using the novel TBC3486 is a suitable approach for targeting of chemotherapy-resistant leukemia. Further studies are warranted to understand and evaluate preclinically adjuvant small molecule inhibition of integrins to overcome relapse of ALL. Disclosures: No relevant conflicts of interest to declare.

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Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽

10.1182/blood.v120.21.1506.1506 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1506-1506

Author(s):

Marika Masselli ◽

Serena Pillozzi ◽

Massimo D'Amico ◽

Luca Gasparoli ◽

Olivia Crociani ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Map Kinases ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Significant Proportion ◽

Leukemic Cells ◽

K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

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CD138 Regulates Myeloma Cell Survival, Proliferation, BM Engraftment and Tumor Organization

Blood ◽

10.1182/blood.v126.23.2974.2974 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2974-2974

Author(s):

David R Fooksman ◽

Amitabha Mazumder ◽

Mark McCarron

Keyword(s):

Plasma Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Early Stage ◽

Serum Starvation ◽

High Dose ◽

Rapid Reduction ◽

Myeloma Cells

Abstract Multiple myeloma is the 2nd most common blood cancer in adults with a median survival time of 5 years despite high-dose chemotherapy and bone marrow transplantation interventions. Syndecan-1 or CD138, is a heparan-sulfate coated glycoprotein, which is highly expressed on the surface of plasma cells and myeloma cells, important for adhesion and accumulating survival signals. Expression of CD138 is heterogeneous in myeloma tumors, in vivo and in vitro leading some to speculate it may distinguish stem-like subpopulations. While this role is highly disputed, we investigated the effect of CD138 expression on tumor pathology in vivo. To characterize CD138neg and CD138high subpopulations, we used GFP+ Vk*myc myeloma model from Leif Bergsagel, which develops myeloma tumors in BM and spleen of C57Bl/6 mice. We found CD138high populations were more proliferative in vivo based on EdU incorporation experiments. We transferred equal numbers of sorted subpopulations into hosts and found that CD138high cells generated larger tumors in the BM than CD138neg cells after 12 weeks. Analysis of these tumor-bearing mice revealed that all tumors contained both subpopulations, indicating that these two subsets are hierarchically equivalent. We find that in mice with small tumors, the majority of cells (80% or more) are CD138high cells, while in large tumors, the level drops (to 30-50% of tumor) with higher composition of CD138neg cells. We also find lower CD138 levels on myeloma cells found in the blood compared to BM. Using intravital two-photon time-lapse imaging in the tibial BM, we find that tumor cells from smaller, early stage tumors are physically arrested within the BM parenchyma, while in larger, more advanced tumors, myeloma cells are more motile and active. CD138neg cells were more apoptotic based on ex vivo Annexin V staining following serum starvation. Interestingly, serum starvation led to rapid reduction in CD138 surface expression. Taken together, we propose a model where CD138 expression regulates localization and survival in the BM niche, but is downregulated from the plasma membrane when tumor size outgrows the necessary resources, allowing myeloma cells to migrate and metastasize to distant new locations. Disclosures No relevant conflicts of interest to declare.

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In vivo monitoring of JAK/STAT and PI3K/mTOR signal transduction inhibition in pediatric CRLF2-rearranged acute lymphoblastic leukemia (ALL).

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.9506 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 9506-9506

Author(s):

Sarah Kathleen Tasian ◽

Shannon L. Maude ◽

Junior Hall ◽

Tiffaney Vincent ◽

Charles Grenfell Mullighan ◽

...

Keyword(s):

Signal Transduction ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Nsg Mice ◽

Signal Transduction Inhibitors ◽

Background Therapy ◽

Pediatric All ◽

Therapeutic Responses

9506 Background: Therapy intensification for children with B-precursor ALL with high-risk genetic lesions has improved relapse-free survival. CRLF2 rearrangements and JAK2 and IL7RA mutations occur in 10-15% of adult and pediatric ALL patients, most of whom relapse. We and others identified aberrant kinase signatures and perturbed JAK/STAT and PI3K/mTOR signal transduction via in vitro studies of CRLF2-rearranged (CRLF2r) ALLs, suggesting the therapeutic relevance of signal transduction inhibitors (STIs). Our creation of CRLF2r ALL xenograft models has enabled rapid preclinical testing of STIs and measurement of in vivo target inhibition. We hypothesized that inhibition of JAK/STAT and PI3K/mTOR phosphosignaling correlates with therapeutic responses in these models. Methods: NOD/SCID/γc-null (NSG) mice well-engrafted with pediatric ALL samples were treated with the JAK inhibitor ruxolitinib, the mTOR inhibitor sirolimus, or vehicle for 72 hours (for signaling response) or 4 weeks (for therapeutic response). Splenocytes were briefly stimulated ex vivo with thymic stromal lymphopoietin (ligand for CRLF2) and stained with human-specific surface and intracellular phosphoantibodies for multi-parameter phosphoflow cytometry analysis. Results: Ruxolitinib-induced inhibition of phospho (p)-JAK2 and pSTAT5 was most pronounced in non-CRLF2r ALLs with novel JAK2-activating BCR-JAK2 and IL7RA/LNK mutations. Sirolimus potently inhibited pS6 and other PI3K/mTOR pathway phosphoproteins in the CRLF2r r ALLs. PSTAT5 and pS6 inhibition correlated with longer-term ruxolitinib- and sirolimus-induced decreases in ALL cell burden, demonstrating therapeutic responses to STIs. Conclusions: Ruxolitinib inhibited JAK/STAT phosphosignaling and markedly decreased leukemic burden in the JAK2-activating BCR-JAK2 and IL7RA/LNK mutant ALL xenografts. Sirolimus potently inhibited PI3K/mTOR (as well as some JAK/STAT) phosphosignaling and had greater therapeutic efficacy than ruxolitinib in the CRLF2r ALLs. The safety of ruxolitinib and of temsirolimus with cytotoxic chemotherapy are currently being established in Children’s Oncology Group Phase I trials.

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Factors Important to the Prioritization and Development of Successful Topical Microbicides for HIV-1

Molecular Biology International ◽

10.1155/2012/781305 ◽

2012 ◽

Vol 2012 ◽

pp. 1-12 ◽

Cited By ~ 4

Author(s):

Karen W. Buckheit ◽

Robert W. Buckheit

Keyword(s):

Clinical Trials ◽

Ex Vivo ◽

Sexual Transmission ◽

Virus Transmission ◽

Preclinical Development ◽

Sexual Transmission Of Hiv ◽

The Right ◽

Hiv 1

Significant advancements in topical microbicide development have occurred since the prevention strategy was first described as a means to inhibit the sexual transmission of HIV-1. The lack of clinical efficacy of the first generation microbicide products has focused development attention on specific antiretroviral agents, and these agents have proven partially successful in human clinical trials. With greater understanding of vaginal and rectal virus infection, replication, and dissemination, better microbicide products and delivery strategies should result in products with enhanced potency. However, a variety of development gaps exist which relate to product dosing, formulation and delivery, and pharmacokinetics and pharmacodynamics which must be better understood in order to prioritize microbicide products for clinical development. In vitro, ex vivo, and in vivo models must be optimized with regard to these development gaps in order to put the right product at the right place, at the right time, and at the right concentration for effective inhibition of virus transmission. As the microbicide field continues to evolve, we must harness the knowledge gained from unsuccessful and successful clinical trials and development programs to continuously enhance our preclinical development algorithms.

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Activation of Simultaneous Apoptosis and Necroptosis to Eradicate Drug Resistant Leukemia

Blood ◽

10.1182/blood.v126.23.1283.1283 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1283-1283

Author(s):

Scott McComb ◽

Julia Aguadé-Gorgorió ◽

Blerim Marovca ◽

Lena Harder ◽

Gunnar Cario ◽

...

Keyword(s):

Cell Death ◽

De Novo ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Short Circuit ◽

Large Set ◽

Ex Vivo Model ◽

Resistant Disease

Abstract Dysregulation of apoptotic pathways provides an indiscriminate mechanism for refractory acute lymphoblastic leukemia (ALL) to escape cell death induced by many chemotherapeutic compounds. Here we have assessed the potential of SMAC mimetic (SM) compounds to short circuit cell death resistance by blocking the pro-survival cellular inhibitor of apoptosis (cIAP) proteins. By screening a large set of patient-derived precursor B-cell ALL samples in an ex vivo model of the leukemia microenvironment we detect exquisite sensitivity to two different SM compounds, Birinapant and LCL161, in about one third of ALL samples. Strong ex vivo SM activity correlated with potent in vivo anti-leukemic efficacy against de novo refractory and relapsed ALL xenografts. Intriguingly, we find that although SM-sensitivity is independent of TNF and TNFR1 levels, expression of TNFR2 is highly predictive of response to SM in two separate cohorts of ALL samples, suggesting that TNFR2 expression may represent a promising biomarker for identifying SM-sensitive cells. Downstream, we employ a novel and powerful multi-colour Lenti-CRISPR approach to show that simultaneous disruption of both apoptotic and necroptotic genes is necessary to block SM-induced death. In contrast, disruption of RIP1 alone was adequate to block SM-induced apoptosis and necroptosis. Surprisingly, RIP1 loss had no significant impact on response to standard anti-leukemic therapies, supporting a view that the RIP1-dependent death pathway is not likely to be selected against in leukemia cells that have failed to respond to front line therapy. These results provide the first evidence that SM compounds can circumvent apoptotic escape in drug-refractory ALL through parallel activation of both RIP1-dependent apoptosis and necroptosis. Furthermore, our data strongly support further development of SM as anti-leukemic agents for treatment in resistant disease. Disclosures No relevant conflicts of interest to declare.

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Magnitude of thromboxane receptor antagonism necessary for antithrombotic activity in monkeys

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1989.256.3.h726 ◽

1989 ◽

Vol 256 (3) ◽

pp. H726-H734

Author(s):

W. A. Schumacher ◽

C. L. Heran ◽

H. J. Goldenberg ◽

D. N. Harris ◽

M. L. Ogletree

Keyword(s):

Ex Vivo ◽

Shape Change ◽

Concentration Effect ◽

Antithrombotic Activity ◽

Thromboxane Receptor ◽

Effect Curve ◽

Thromboxane Receptors ◽

The Right

SQ 30741 was characterized as a competitive antagonist of thromboxane receptor-mediated platelet activation in vitro that does not inhibit the activity of enzymes involved in prostaglandin, prostacyclin, and thromboxane biosynthesis. The threshold intravenous dose for antithrombotic activity was measured in anesthetized monkeys as the minimum amount of SQ 30741 required to inhibit thrombotic cyclic blood flow reductions in stenotic renal arteries. Platelet responsiveness was measured ex vivo before and during inhibition of thrombosis by the shape-change response to a thromboxane mimetic, U 46619. The threshold antithrombotic SQ 30741 dose (0.32 +/- 0.04 mg/kg; n = 5) was accompanied by an 8.5 +/- 1.1-fold shift to the right of the U 46619 concentration-effect curve, implying antagonism of 87 +/- 3% of platelet thromboxane receptors. The antithrombotic activity of SQ 30741 persisted for 109 +/- 10 min and was not reversed by indomethacin. However, in two out of seven monkeys SQ 30741 (7 mg/kg iv) did not interrupt the cyclical flow reductions. Vehicle treatment did not impede thrombosis and caused a 1.4 +/- 0.3-fold shift of the U 46619 concentration-effect curve (n = 4). In separate monkeys, SQ 30741 (0.33 mg/kg iv) produced identical dose ratios (8.6 +/- 0.7, n = 8) for inhibition of U 46619-induced mesenteric vasoconstriction. Thus the threshold antithrombotic dose of SQ 30741 caused the same magnitude of antagonism of platelet (ex vivo) and vascular (in vivo) thromboxane receptors.

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A Dendritic Cell Based Vaccination Strategy Geared towards Eradication of Leukemic Stem Cells.

Blood ◽

10.1182/blood.v114.22.2046.2046 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2046-2046

Author(s):

Hetty J Bontkes ◽

Jurjen Ruben ◽

Willemijn van den Ancker ◽

Theresia M Westers ◽

G. Ossenkoppele ◽

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Keyword(s):

Stem Cells ◽

T Cells ◽

Tumor Antigens ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Vaccination Strategy ◽

Leukemic Stem Cells ◽

Cell Immunity

Abstract Abstract 2046 Poster Board II-23 Introduction: In the majority of cases, initial remission of acute myeloid leukemia (AML) is reached but unfortunately relapse rates remain high and therefore novel treatments are needed. It is thought that recurrent AML originates from chemotherapy resistant quiescent leukemic stem cells (LSC). The application of immunotherapeutic approaches to eradicate LSC remaining after first line chemotherapy may contribute to improved disease outcome. Vaccination strategies have often used dendritic cells (DC) ex vivo pulsed with tumor-derived whole lysates or peptides as modalities to present a broad range of tumor antigens to T cells to stimulate effective anti-tumor T-cell immunity in vivo. It is likely that certain proteins expressed by LSC have a distinct antigenicity as compared to more mature AML blasts and thus provide targets for specific T-cells. Even without identification of specific antigens, LSC can be a useful source of tumor antigens in DC vaccination-based immunotherapy. CD34+CD38- LSC can be identified using malignant stem cell associated cell surface markers including CLL-1 and lineage markers such as CD7, CD19 and CD56. However, the low frequency of these cells precludes the use of LSC derived apoptotic cells or lysates for DC loading. Alternatively, mRNA isolated from LSC can be amplified and subsequently transfected into DC. Materials and Methods: We have made use of the CD38- AML derived cell line MUTZ-3 which contains a subpopulation of CD34+CLL1+ cells which resembles the phenotype of a putative LSC. CLL1+CD34+ and CLL1-CD34- cells were isolated by FACS sorting and total RNA was isolated. mRNA was converted to cDNA and amplified by PCR using the SMART system. Subsequently, mRNA was in vitro transcribed from the amplified cDNA. Mature monocyte derived DC (MoDC) were generated from healthy donor blood and transfected with amplified CLL1+CD34+ derived mRNA and used to stimulate autologous CD8β+ T-cells. After three weekly re-stimulations with CLL1+CD34+ mRNA transfected DC, specificity of the T-cells was analyzed by intracellular IFNγ staining upon 5 hour stimulation with autologous immature MoDC transfected with GFP mRNA, mRNA amplified from unsorted, CLL1+CD34+ or CLL1-CD34- MUTZ-3 subpopulations. Results: Amplification of CLL1 and survivin (also expressed by MUTZ-3) transcripts was confirmed by RT-PCR. After 3 weekly re-stimulations with CLL1+CD34+ amplified RNA transfected DC, 0.04% (range 0.01-0.12%) of the T-cells were positive for IFNγ upon a 5 hr re-stimulation with GFP transfected DC. 0.44% (range 0.04-0.69%) of the T-cells responded to DC transfected with unsorted MUTZ-3 amplified mRNA (p<0.00005 versus GFP control, 2-sided student's T-test), 0.51% (range 0.24-1.35%) responded to DC transfected with CLL1+CD34+ amplified mRNA (p<0.005 versus GFP control) and 0.46% (range 0.24-0.94%) responded to DC transfected with CLL1-CD34- amplified mRNA (p<0.0001 versus GFP control). Conclusion: We show that MoDC transfected with RNA amplified from one MUTZ-3 sub-population resembling the phenotype of LCS cells are capable of inducing T-cells which recognize both cells transfected with mRNA from the LSC resembling MUTZ-3 subset as well as the CLL1-CD34- subset. We are currently testing the efficacy and feasibility of this approach in an autologous setting in vitro. CD8β+ T-cells are stimulated with autologous MoDC from AML patients transfected with amplified mRNA isolated from their own LSC enriched populations. The capacity of these T-cells to kill autologous AML blasts and LSC is subsequently analysed in a 6-colour FACS based cytotoxicity assay. Disclosures: No relevant conflicts of interest to declare.

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