scholarly journals Therapeutic strategies for diffuse midline glioma from high-throughput combination drug screening

2019 ◽  
Vol 11 (519) ◽  
pp. eaaw0064 ◽  
Author(s):  
Grant L. Lin ◽  
Kelli M. Wilson ◽  
Michele Ceribelli ◽  
Benjamin Z. Stanton ◽  
Pamelyn J. Woo ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Agent ◽  
Proteasome Inhibition ◽  
Combination Drug ◽  
Drug Induced ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Nicotinamide Mononucleotide ◽  
Promising Therapeutic Approach ◽  
In Vivo Testing

Diffuse midline gliomas (DMGs) are universally lethal malignancies occurring chiefly during childhood and involving midline structures of the central nervous system, including thalamus, pons, and spinal cord. These molecularly related cancers are characterized by high prevalence of the histone H3K27M mutation. In search of effective therapeutic options, we examined multiple DMG cultures in sequential quantitative high-throughput screens (HTS) of 2706 approved and investigational drugs. This effort generated 19,936 single-agent dose responses that inspired a series of HTS-enabled drug combination assessments encompassing 9195 drug-drug examinations. Top combinations were validated across patient-derived cell cultures representing the major DMG genotypes. In vivo testing in patient-derived xenograft models validated the combination of the multi–histone deacetylase (HDAC) inhibitor panobinostat and the proteasome inhibitor marizomib as a promising therapeutic approach. Transcriptional and metabolomic surveys revealed substantial alterations to key metabolic processes and the cellular unfolded protein response after treatment with panobinostat and marizomib. Mitigation of drug-induced cytotoxicity and basal mitochondrial respiration with exogenous application of nicotinamide mononucleotide (NMN) or exacerbation of these phenotypes when blocking nicotinamide adenine dinucleotide (NAD+) production via nicotinamide phosphoribosyltransferase (NAMPT) inhibition demonstrated that metabolic catastrophe drives the combination-induced cytotoxicity. This study provides a comprehensive single-agent and combinatorial drug screen for DMG and identifies concomitant HDAC and proteasome inhibition as a promising therapeutic strategy that underscores underrecognized metabolic vulnerabilities in DMG.

scholarly journals Optimized 5-Fluorouridine Prodrug for Co-Loading with Doxorubicin in Clinically Relevant Liposomes

Pharmaceutics ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 107
Author(s):  
Debra Wu ◽  
Douglas Vogus ◽  
Vinu Krishnan ◽  
Marta Broto ◽  
Anusha Pusuluri ◽  
...  
Keyword(s):  
Single Agent ◽  
Molar Ratio ◽  
Control Group ◽  
Drug Tolerability ◽  
Breast Cancer Model ◽  
In Vivo Testing ◽  
Chemical Profiles ◽  
Hydrolysis Of ◽  
Doxorubicin Liposomes

Liposome-based drug delivery systems have allowed for better drug tolerability and longer circulation times but are often optimized for a single agent due to the inherent difficulty of co-encapsulating two drugs with differing chemical profiles. Here, we design and test a prodrug based on a ribosylated nucleoside form of 5-fluorouracil, 5-fluorouridine (5FUR), with the final purpose of co-encapsulation with doxorubicin (DOX) in liposomes. To improve the loading of 5FUR, we developed two 5FUR prodrugs that involved the conjugation of either one or three moieties of tryptophan (W) known respectively as, 5FUR−W and 5FUR−W3. 5FUR−W demonstrated greater chemical stability than 5FUR−W3 and allowed for improved loading with fewer possible byproducts from tryptophan hydrolysis. Varied drug ratios of 5FUR−W: DOX were encapsulated for in vivo testing in the highly aggressive 4T1 murine breast cancer model. A liposomal molar ratio of 2.5 5FUR−W: DOX achieved a 62.6% reduction in tumor size compared to the untreated control group and a 33% reduction compared to clinical doxorubicin liposomes in a proof-of-concept study to demonstrate the viability of the co-encapsulated liposomes. We believe that the new prodrug 5FUR−W demonstrates a prodrug design with clinical translatability by reducing the number of byproducts produced by the hydrolysis of tryptophan, while also allowing for loading flexibility.

IMMU-13. COMBINED MODULATION OF THE TGF-Β PATHWAY AND GITR IMMUNE CHECKPOINT SIGNALING PROMOTES ANTI-TUMOR IMMUNITY IN SYNGENEIC GLIOMA MODELS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi94-vi94
Author(s):  
Daniela Lorizio ◽  
Michael Weller ◽  
Manuela Silginer ◽  
Alan Epstein ◽  
Patrick Roth
Keyword(s):  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Transforming Growth Factor ◽  
Immune Cell ◽  
Single Agent ◽  
Underlying Mechanism ◽  
T Effector Cells ◽  
Promising Therapeutic Approach

Abstract The profound local immunosuppressive microenvironment is one hallmark of glioblastoma, which results in resistance to most immunotherapeutic strategies that have been explored so far. Reverting this condition in order to reinvigorate anti-glioma immunity might be a promising therapeutic approach. Transforming growth factor (TGF)-β signaling is deregulated in different cancer types and contributes to the malignant phenotype of glioma cells. Glioma-derived TGF-β is also a major immunosuppressive factor in the tumor microenvironment. Furthermore, intratumoral regulatory T (Treg) cells and activated T effector cells express high levels of the co-stimulatory immune checkpoint glucocorticoid-induced tumor necrosis factor receptor (GITR). Agonistic anti-GITR antibodies have been explored in preclinical tumor models and are under investigation in clinical trials for the treatment of solid tumors. We evaluated the effect of TGF-β and GITR targeting on anti-tumor immune responses in syngeneic mouse glioma models. In co-culture settings, GITR modulation with a GITR ligand (GITRL)-Fc fusion protein, given alone or in combination with a pharmacological TGF-β receptor inhibitor, led to increased T cell activation. Furthermore, the combined targeting of the two pathways resulted in significantly higher immune cell-mediated tumor cell killing than either treatment alone. In vivo, TGF-β inhibition and GITR signaling modulation resulted in a higher fraction of long-term surviving glioma-bearing mice than single-agent treatment. Surviving mice were resistant to tumor re-challenge, suggesting adaptive immunity as an underlying mechanism. These data support the assumption that combined immunotherapeutic strategies may represent a promising approach for the treatment of glioma.

The HIV-Protease Inhibitor Ritonavir(Norvir®) Is Active Against Human AML Blasts In Vitro and In Vivo and Has Synergistic Cytotoxic Activity with the Proteaseome Inhibitor Bortezomib (Velcade®).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2464-2464
Author(s):  
Christoph Driessen ◽  
Müller-Ide Hendrik ◽  
Gogel Jeanette ◽  
Kraus Marianne ◽  
Kanz Lothar ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cell Lines ◽  
Single Agent ◽  
Proteasome Inhibition ◽  
Hiv Protease ◽  
Malignant Cells ◽  
Short Period ◽  
Proteolytic Pathways

Abstract Upregulation of alternative proteolytic pathways characterizes malignant cells that overcome proteasome inhibition in vitro. The proteasome inhibitor Bortezomib (Velcade®), which selectively targets only one of the three active subunits of the proteasome, has shown limited activity in AML. Ritonavir (Norvir®) is an aspartate protease inhibitor used in intensive HIV-therapy, where therapeutic levels of 5–20μM are reached with an oral dose of 1200 mg/d. A cytotoxic effect of Ritonavir against malignant cells due to proteasome inhibition has been suggested (Gaedicke et al., Cancer Research 62, December 1, 2002). We have here tested the effect of Ritonavir on AML cells, both as single agent and in combination with Bortezomib. Ritonavir induced cytotoxic death in AML cell lines and primary AML blasts with an IC50 of 30–40 μM in vitro. The combination of Ritonavir and Bortezomib was synergistic in vitro, i.e. subtoxic concentrations of Ritonavir at 10 μM combined with subtoxic Bortezomib 5–10 nM induced robust cytotoxicity in AML cell lines and freshly isolated primary AML blasts. Using a novel chemical probe that for the first time allows to visualize the individual activity of proteasomal subunits in intact AML blasts, we show that Velcade selectively abrogates β5 proteasomal activity at 20 nM in AML cells, as expected. Ritonavir, by contrast, had no effect on active proteasomal subunits up to 50 μM. Thus, the synergistic effect of Ritonavir with Bortezomib on AML cells is not due to inhibition of the same proteasomal target by both drugs, but more likely mediated by blocking alternative proteolytic pathways. One individual patient aged 72 years with an early relaps of AML was treated with Ritonavir 400–600 mg/d p.o.. During treatment, the absolute leukocyte count dropped from 24000/μl to 8000/μl while the ANC raised from 185/μl to 1530/μl. Ritonavir was withdrawn due to diarrhoea and abdominal cramps, leading to a sharp increase in peripheral blood blasts and leukocytes. Retreatment with Ritonavir at a reduced dose of 200 mg/d combined with Velcade 1mg/sqm was tolerated and stabilized leukocyte counts for a short period of time. We conclude that Ritonavir has activity against chemotherapy-refractory AML in vitro and in vivo. The combination of Velcade and Ritonavir might allow to synergistically target the proteolytic machinery of AML blasts with tolerable toxicity.

scholarly journals Standard Morphologic Evaluation of the Heart in the Laboratory Dog and Monkey

2006 ◽  
Vol 34 (1) ◽  
pp. 67-74 ◽  
Author(s):  
Charlotte M. Keenan ◽  
Justin D. Vidal
Keyword(s):  
Morphological Changes ◽  
Small Sample Size ◽  
Cynomolgus Macaque ◽  
Small Sample ◽  
Heart Weight ◽  
Variable Degree ◽  
Drug Induced ◽  
Microscopic Evaluation ◽  
In Vivo Testing

The nonrodent species most commonly utilized in preclinical safety studies are the purpose-bred beagle dog and cynomolgus macaque ( Macaca fascicularis). Potential effects of a new chemical entity (NCE) on the heart pose serious concerns; consequently in vivo testing is focused on detection of functional alterations as well as morphological changes. Macroscopic and microscopic evaluation of the heart is based on a standard survey of key structures to properly assess presence of spontaneous and potential drug-induced lesions. Evaluation of historical controls to determine type and frequency of background change is valuable, as studies with non-rodent species generally have a small sample size. Archived control dog and monkey data were retrospectively reviewed, including terminal body weight (BW), heart weight (HW), and archival glass slides of heart. Control dogs had minimal background changes that included myxomatous or cartilagenous change in the cardiac skeleton and a variable degree of vacuolation in Purkinje fibers. Control monkey hearts commonly contained inflammatory cell infiltrates, myocyte anisokaryosis, and handling artifacts, while myocyte degeneration, squamous plaques, pigment, and intimal plaques were occasionally observed. These findings highlight the utility of consistently recorded and readily accessible archived control data when attempting to discern background spontaneous changes and artifacts from test-article induced changes.

scholarly journals Preclinical Evaluation of Carfilzomib for Infant KMT2A-Rearranged Acute Lymphoblastic Leukemia

2021 ◽  
Vol 11 ◽  
Author(s):  
Laurence C. Cheung ◽  
Rebecca de Kraa ◽  
Joyce Oommen ◽  
Grace-Alyssa Chua ◽  
Sajla Singh ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Drug Testing ◽  
Hematological Malignancies ◽  
Lymphoblastic Leukemia ◽  
Proteasome Inhibition ◽  
Chemotherapeutic Agents ◽  
Combination Drug

BackgroundInfants with KMT2A-rearranged B-cell precursor acute lymphoblastic leukemia (ALL) have poor outcomes. There is an urgent need to identify novel agents to improve survival. Proteasome inhibition has emerged as a promising therapeutic strategy for several hematological malignancies. The aim of this study was to determine the preclinical efficacy of the selective proteasome inhibitor carfilzomib, for infants with KMT2A-rearranged ALL.MethodsEight infant ALL cell lines were extensively characterized for immunophenotypic and cytogenetic features. In vitro cytotoxicity to carfilzomib was assessed using a modified Alamar Blue assay with cells in logarithmic growth. The Bliss Independence model was applied to determine synergy between carfilzomib and the nine conventional chemotherapeutic agents used to treat infants with ALL. Established xenograft models were used to identify the maximal tolerated dose of carfilzomib and determine in vivo efficacy.ResultsCarfilzomib demonstrated low IC50 concentrations within the nanomolar range (6.0–15.8 nm) across the panel of cell lines. Combination drug testing indicated in vitro synergy between carfilzomib and several conventional chemotherapeutic agents including vincristine, daunorubicin, dexamethasone, L-asparaginase, and 4-hydroperoxycyclophosphamide. In vivo assessment did not lead to a survival advantage for either carfilzomib monotherapy, when used to treat both low or high disease burden, or for carfilzomib in combination with multi-agent induction chemotherapy comprising of vincristine, dexamethasone, and L-asparaginase.ConclusionsOur study highlights that in vitro efficacy does not necessarily translate to benefit in vivo and emphasizes the importance of in vivo validation prior to suggesting an agent for clinical use. Whilst proteasome inhibitors have an important role to play in several hematological malignancies, our findings guard against prioritization of carfilzomib for treatment of KMT2A-rearranged infant ALL in the clinical setting.

AT-101, a Pan-Inhibitor of Bcl-2 Family Anti-Apoptotic Proteins Antagonizes the Protective Effect Conferred by Nurselike Cells on Primary Chronic Lymphocytic Leukemia (CLL) Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2100-2100 ◽  
Author(s):  
Danelle F. James ◽  
Rebecca M. Mervis ◽  
Ruzbeh Mosadeghi ◽  
Thomas J. Kipps
Keyword(s):  
B Cells ◽  
Cell Viability ◽  
Single Agent ◽  
Leukemia Cells ◽  
Drug Induced ◽  
Accessory Cells ◽  
The Mean ◽  
Apoptotic Proteins

Abstract Despite the longevity of CLL B cells in vivo, in vitro CLL cells readily undergo apoptosis under conditions that support the growth of normal B cells. However, when leukemia cells are co-cultured with non-neoplastic accessory cells, such as those found in the marrow stroma and nurselike cells (NLC) that can differentiate from blood mononuclear cells they become resistant to spontaneous and drug induced cell death. These findings suggest that the defective apoptosis of the CLL B cells is not only ascribed to intrinsic defects in the neoplastic cell, but also to extrinsic factors that influence their behavior provided by the tumor microenvironment. As such, identification of molecular targets that link malignant B cells to supportive cells in the microenvironment may lead to new therapeutic avenues for CLL patients. For example, we have found that co-culture with NLC induces expression of Mcl-1, an anti-apoptotic protein of the Bcl-2 family in CLL cells. [Nishio M 2005] Induction of such prosurvival proteins by accessory cells may play a role disease aggressiveness and leukemia cell resistance to chemotherapy in vivo. AT-101 is a small molecule that mimics the inhibitory BH3 domain of endogenous antagonists of the Bcl-2 family anti-apoptotic proteins negating their cytoprotective role. Fluorescence polarization assays demonstrate that AT -101 binds to Bcl-2, BcL-XL, Bcl-W, and Mcl-1. AT-101 is cytotoxic to primary CLL cells in vitro and has been shown to have single agent activity in high risk CLL patients. Therefore, we hypothesized that inhibition of Mcl-1 by AT-101 may diminish the protection of NLC on CLL cells and may render the leukemia cells more sensitive to spontaneous and drug induced apoptosis. Primary CLL cells from 20 different patients were plated with or without NLCs, and cell viability was assessed overtime. Co-culture of CLL cells with NLC protected the CLL cells from undergoing spontaneous apoptosis. After 48 hours the average viability (+/− SEM) of CLL cells with NLC was 75% (+/− 4%) whereas the mean viability of CLL cultured alone was significantly lower at 59% (+/−5%) (p<0.02). In addition, we found that co-culture of CLL cells with NLC protected the leukemia cells from the cytotoxic effects of fludarabine (F-ara-A). For instance, after 48 hours after treatment with 10 μM F-ara-A the mean viability of the CLL cells was 28% (+/− 4%) whereas the viability of CLL cells cultured with NLC was 51% (+/−4%), a difference that was highly significant (p<0.001). In contrast, NLC could not protect leukemia cells from apoptosis induced by AT-101. For example, treatment of CLL cells with AT-101 at 5 μM, with or without NLC, resulted in CLL cell viability at 48 hours of only 40% (+/−7%.) or 30% (+/−7%.), respectively, a difference that was not statistically significant. AT-101 and F-ara-A were titrated in the setting of NLC and appeared to act additively in inducing apoptosis of CLL cells. In fact, NLC protection of CLL cells from fludarabine mediated cytotoxicity was negated in the presence of AT-101. Together our results describe a new therapeutic mechanism for targeting CLL interaction with the microenvironment. Specifically, a pan-inhibitor of Bcl-2 family anti-apoptotic proteins AT-101 antagonizes the protection of CLL by NLC to both spontaneous and fludarabine mediated apoptosis.

Establishment of a chordoma xenograft and in vivo testing of compounds identified by high-throughput screening.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 10025-10025
Author(s):  
Matteo Maria Trucco ◽  
Breelyn A. Wilky ◽  
Ola Awad ◽  
Preeti Shah ◽  
Naheed Gul ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Xenograft Model ◽  
Dramatic Reduction ◽  
In Vivo Models ◽  
Drug Concentrations ◽  
In Vivo Testing

10025 Background: Chordoma is a rare primary bone malignancy that arises in the skull base, spine and sacrum and originates from remnants of the notochord. Therapy primarily consists of surgical resection and radiation. These tumors are typically resistant to conventional chemotherapy, and to date there are no FDA-approved agents for chordoma. The lack of in vivo models of chordoma has impeded the development of new therapies for this tumor. Methods: Primary tumor from a classic sacral chordoma was obtained, immediately processed into a single cell suspension and injected in to the parasacral area of a NOD/SCID/IL-2R gamma-null mouse, and tumor grew after 3 months. The NIH Chemical Genomics Center performed high-throughput screening of 2,816 compounds. Two established chordoma cell lines, U-CH1 and UCH2B, were treated and cell viability measured by CellTiter-Glo assay. Cells were incubated for 48 hours with drug concentrations ranging from 0.5nM to 46uM. The screen yielded several compounds that showed activity and two were tested in the xenograft. Results: We have established a xenograft model of dedifferentiated chordoma. High-throughput screening of compounds identified compounds that show activity against chordoma cell lines. In vivo testing of two identified compounds showed a dramatic reduction of tumor growth. Conclusions: We have established a xenograft model of dedifferentiated chordoma. High-throughput screening of compounds identified compounds that show activity against chordoma cell lines. In vivo testing of two identified compounds showed a dramatic reduction of tumor growth.

A precision medicine drug discovery pipeline to identify dual CDK2/9 inhibition as a novel treatment for colorectal cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e16056-e16056
Author(s):  
Roham Salman Roghani ◽  
Ali Sanjari moghaddam ◽  
Gabrielle Rupprecht ◽  
Erdem Altunel ◽  
So Young Kim ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Precision Medicine ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
High Throughput ◽  
Single Agent ◽  
Cdk Inhibitor ◽  
Cycle Arrest

e16056 Background: Colorectal cancer (CRC) is the 3rdmost common form of cancer in the US, responsible for over 50,000 death each year. Therapeutic options for advanced colorectal cancer are limited, and there remains an unmet clinical need to identify new therapies to treat this deadly disease. To address this need, we have developed a precision medicine pipeline that integrates high throughput chemical screens with matched patient-derived cell lines and patient-derived xenografts (PDXs) to identify new treatments for CRC. Methods: We used high-throughput chemical screens of 2,100 compounds across five low-passage, patient-derived CRC cell lines. These results were validated using dose-response IC50curves for CDK1, CDK2, CDK9 or CDK1/2/9 inhibitors and by siRNA-mediated knockdown of CDK9 with or without CDK2 inhibition. Cell cycle arrest analysis was performed by flow cytometry and anaphase catastrophe was analyzed by immunofluorescence staining. For in vivo studies, matched PDXs were treated with either CDK2, CDK9 or dual CDK2/9 inhibitors. Results: We identified the CDK inhibitor drug class as among the most effective cytotoxic compounds across all five CRC lines. Further analysis of the CDK inhibitor class revealed that combined targeting of CDK1, 2, and 9 was the most effective, with IC50 in the range of 110 nM to 1.2 μM. We further validated the efficacy of combined CDK2/9 inhibition using siRNA-mediated knockdown of CDK9 in the presence of a CDK2 inhibitor(CVT-313), and showed that CDK9 knockdown acted synergistically with CDK2 inhibition. Dual CDK2/9 inhibition led to significant G2/M cell cycle arrest and anaphase catastrophe. Finally, combined CDK2/9 inhibition in vivo synergistically inhibited PDX tumor growth as compared to single-agent CDK inhibitors. Conclusions: Our precision medicine pipeline revealed CDK2/9 dual inhibition as a combinatorial therapy to treat CRC and can also be used to identify new and novel therapies

scholarly journals Targeting NAMPT as a therapeutic strategy against stroke

2019 ◽  
Vol 4 (2) ◽  
pp. 83-89 ◽  
Author(s):  
Shu-Na Wang ◽  
Chao-Yu Miao
Keyword(s):  
Neuroprotective Effect ◽  
Haemorrhagic Stroke ◽  
Therapeutic Interventions ◽  
In Vivo Studies ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Nicotinamide Riboside ◽  
Nicotinamide Mononucleotide ◽  
Promising Target

Stroke is the second and the leading most common cause of death in the world and China, respectively, but with few effective therapies. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD) salvage synthesis in mammals, thereby influencing NAD-dependent enzymes and constituting a strong endogenous defence system against various stresses. Accumulating in-vitro and in-vivo studies have demonstrated the neuroprotective effect of NAMPT in stroke. Here, we review the direct evidence of NAMPT as a promising target against stroke from five potential therapeutic strategies, including NAMPT overexpression, recombinant NAMPT, NAMPT activators, NAMPT enzymatic product nicotinamide mononucleotide (NMN), and NMN precursors nicotinamide riboside and nicotinamide, and describe the relevant mechanisms and limitations, providing a promising choice for developing novel and effective therapeutic interventions against ischaemic and haemorrhagic stroke.

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