scholarly journals Efficacy of Targeted Therapy in Novel Pre-Clinical in Vitro and In Vivo Models of Richter Transformation-Diffuse Large B-Cell Lymphoma (RT-DLBCL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3961-3961
Author(s):  
Vrajesh Karkhanis ◽  
Warren Fiskus ◽  
Christopher Peter Mill ◽  
Abhishek Maiti ◽  
Bernardo H Lara ◽  
...  
Keyword(s):  
Research Funding ◽  
Clonal Evolution ◽  
Disease Free Survival ◽  
B Cell Lymphoma ◽  
Fish Analysis ◽  
In Vivo Evaluation ◽  
In Vivo Models ◽  
Induced Apoptosis

Richter Transformation (RT) is defined as the development of aggressive DLBCL (mostly ABC-type) in up to ~15% of patients with antecedent or concurrent diagnosis of CLL. Based on the comparison of immunoglobin gene rearrangements, approximately 80% of RT-DLBCL arise due to a direct clonal evolution of the underlying CLL clone, i.e., clonally related (CLR) RT-DLBCL, which exhibit poor median survival (MS) of one year. Approximately 20% of RT-DLBCLs are clonally unrelated (CUR) to the underlying CLL, arising most likely due to branched clonal evolution from a common pre-CLL progenitor. CUR RT-DLBCLs exhibit a better MS of 5 years. Although chemo-immunotherapy and treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib or anti-apoptotic BCL2 inhibitor venetoclax can induce remissions, they fail to induce prolonged disease-free survival in RT-DLBCL. Majority of patients relapse with therapy-refractory disease. Lack of availability of in vitro cultured RT-DLBCL cells or PD xenograft models has prevented pre-clinical testing and development of novel targeted agents against RT-DLBCL. Here, we report the establishment of 3 patient-derived xenograft (PDX) models of RT-DLBCL. Based on immunoglobulin heavy chain (IGH) clonality testing by NGS, the RT-DLBCL RT17 was CLR, RT15 was CUR and RT5 was of indeterminate clonality. The PDXs were generated by tail-vein infusion and engraftment of luciferase-transduced CD19+ RT-DLBCL cells in NSG mice. The RT-DLBCL PDXs grew in the bone marrow and spleen, causing marked splenomegaly, requiring euthanasia 4 to 6 weeks after engraftment. All three RT-DLBCL PDX cells were EBV-negative by genomic and EBNA2 protein expression analyses. NextGen DNA sequencing of RT17, RT15, and RT5 cells showed large numbers of genetic mutations, including mutations in TP53, ATM, NOTCH2, TET2 and MLL3 genes with a high variant allelic frequencies. Array-CGH showed DNA copy gains or losses in multiple chromosomes, including 3, 8, 9, 11, 12, 17 and 18. A 5'-MYC amplification was detected by FISH analysis in RT5 DLBCL cells. ATAC-Seq showed increased signal intensity representing increased chromatin accessibility in the RT-DLBCL cells compared to CD34+ normal progenitors. High peak numbers were detected in specific loci, including TCF4, PAX5, IRF4, MYB, MYC, BCL2L1 and BCL-2. Anti-H3K27Ac ChIP-Seq analysis showed increased average, normalized read-densities at super-enhancers/enhancers (SEs/Es), including those of TCF4, PAX5, IRF4, BCL2 (RT17 and RT15) and MYC (RT5). Western analyses showed that all three RT-DLBCL PDX cells expressed TCF4, c-Myc, and BRD4, with highest expression in RT5 cells. Accordingly, RT5 cells were more sensitive than cells RT17 and RT15 cells to the BET protein inhibitor (BETi) OTX015-induced apoptosis. This was associated with greater, OTX015-mediated, depletion of c-Myc in RT5 cells. RT17 and RT15 expressed high levels of BCL2, Bcl-xL and MCL1, whereas RT5 lacked BCL2 expression. Consistent with this, RT17 and RT15 cells were significantly more sensitive than RT5 cells to venetoclax-induced apoptosis (p < 0.01). RT17 and RT15, but not RT5 cells, expressed NFkB2 (p52), consistent with activation of non-canonical NFkB signaling. This was associated with resistance of RT17 and RT15 cells to ibrutinib-induced apoptosis (p < 0.001). However, co-treatment with OTX015 and ibrutinib or venetoclax induced synergistic lethality in all RT-DLBCL cells (combination indices < 1.0). BET-PROTAC ARV-825 and ARV-771 treatment depleted BRD4, leading to marked reduction in c-Myc levels and apoptosis of all RT-DLBCL cells. Treatment with the ATP-competitive, CDK9 inhibitor NVP2 also dose-dependently induced apoptosis in RT-DLBCL cells associated with depletion of c-Myc, Bcl-xL, and MCL1 protein levels. These findings highlight the activity and support further in vitro and in vivo evaluation of BETi, BET-PROTAC or CDK9i-based combinations with ibrutinib or venetoclax against genetically-profiled RT-DLBCL cells that are clonally-related or clonally-unrelated to the antecedent CLL. Disclosures Maiti: Celgene: Other: research funding. Bhalla:Beta Cat Pharmaceuticals: Consultancy. Khoury:Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding.

scholarly journals In Vitro and In Vivo Inhibitory Effect of Citrus Junos Tanaka Peel Extract against Oxidative Stress-Induced Apoptotic Death of Lung Cells

Antioxidants ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1231
Author(s):  
Jin Woo Kim ◽  
Eun Hee Jo ◽  
Ji Eun Moon ◽  
Hanvit Cha ◽  
Moon Han Chang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protective Effect ◽  
Inhibitory Effect ◽  
Lung Cells ◽  
In Vivo Models ◽  
Citrus Junos ◽  
Induced Apoptosis ◽  
Peel Extract

Various stresses derived from both internal and external oxidative environments lead to the excessive production of reactive oxygen species (ROS) causing progressive intracellular oxidative damage and ultimately cell death. The objective of this study was to evaluate the protective effects of Citrus junos Tanaka peel extract (CE) against oxidative-stress induced the apoptosis of lung cells and the associated mechanisms of action using in vitro and in vivo models. The protective effect of CE was evaluated in vitro in NCI-H460 human lung cells exposed to pro-oxidant H2O2. The preventive effect of CE (200 mg/kg/day, 10 days) against pulmonary injuries following acrolein inhalation (10 ppm for 12 h) was investigated using an in vivo mouse model. Herein, we demonstrated the inhibitory effect of CE against the oxidative stress-induced apoptosis of lung cells under a highly oxidative environment. The function of CE is linked with its ability to suppress ROS-dependent, p53-mediated apoptotic signaling. Furthermore, we evaluated the protective role of CE against apoptotic pulmonary injuries associated with the inhalation of acrolein, a ubiquitous and highly oxidizing environmental respiratory pollutant, through the attenuation of oxidative stress. The results indicated that CE exhibits a protective effect against the oxidative stress-induced apoptosis of lung cells in both in vitro and in vivo models.

Pralatrexate Has Potent Activity Against Multiple Myeloma In Vitro and In Vivo, and Activity Correlates with Tumor RFC-1 and DHFR Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1831-1831 ◽  
Author(s):  
Michael Mangone ◽  
Luigi Scotto ◽  
Enrica Marchi ◽  
Owen A. O'Connor ◽  
Hearn J. Cho
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Research Funding ◽  
Folate Metabolism ◽  
Nog Mice ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Functional Biomarkers

Abstract Abstract 1831 Multiple myeloma (MM) is the second most common hematologic malignancy. Although there are effective new agents that can induce remission, relapse is inevitable and the disease is currently incurable. Progress in the treatment of this disease demands development of novel therapeutics and identification of functional biomarkers that may be used to distinguish tumors that are susceptible to specific targeted agents, creating a “personalized” therapeutic strategy for individual patients. We investigated these principles with anti-folates, which are not commonly used in MM but have demonstrated activity in this disease. Pralatrexate (PDX, 10-propargyl 10-deazaaminopterin) is a folate analogue that was rationally designed to have high affinity for Reduced Folate Carrier (RFC)-1, an oncofetal protein expressed in many cancers that actively transports folates into cells. PDX induced dose-dependent apoptotic cell death in a subset of human myeloma cell lines (HMCL) and CD138+ MM cells isolated from a clinical specimen. In sensitive cell lines, PDX exhibited 10-fold greater potency compared to the structurally related drug methotrexate (MTX). PDX induced dose-dependent, intrinsic apoptosis in sensitive HMCLs, characterized by cleavage of caspase-3 and -9 and accompanied by the loss of full-length Mcl-1, a Bcl-2 family protein that plays a critical role in drug-induced apoptosis in MM. Furthermore, the activity of PDX is not abrogated by the presence of exogenous interleukin-6 or by co-culture with HS-5 bone marrow stromal cells, both of which exert powerful survival effects on MM cells and can antagonize apoptosis in response to some cytotoxic chemotherapy drugs. Sensitivity to PDX-induced apoptosis correlated with higher relative levels of RFC-1 mRNA in sensitive compared to resistant HMCL. Resistant HMCL also exhibited a dose-dependent up-regulation of dihydrofolate reductase (DHFR) protein, a primary molecular target for anti-folates, in response to PDX exposure, whereas sensitive HMCL did not. These changes in functional folate metabolism biomarkers, high baseline RFC-1 expression and upregulation of DHFR in response to PDX, appeared to be mutually exclusive to sensitive or resistant HMCL, respectively. Importantly, PDX was also effective against sensitive HMCL in vivo in a novel mouse xenograft model. NOD/Shi-scid/IL-2Rγnull (NOG) mice were inoculated with MM.1s HMCL stably transduced to express both GFP and luciferase (GFP-luc). GFP-luc MM.1s cells engrafted into the long bones, pelvis, and vertebral column of NOG mice within 4–7 days after injection of cells, as assessed by in vivo bioluminescent imaging. Treatment with PDX resulted in a significant reduction in tumor burden after two doses. These results demonstrate that PDX has potent anti-myeloma activity in vitro and in vivo, and that RFC-1 expression and DHFR upregulation are robust functional biomarkers that may identify patients who are likely to benefit from PDX therapy. These data support further exploration of PDX therapy in clinical trials for MM and investigation of folate metabolism biomarkers as indices for treatment with this class of drugs. Improved anti-folates such as PDX are a promising class of agents that may be a valuable addition to the arsenal against MM. Disclosures: O'Connor: Celgene: Consultancy, Research Funding; Merck: Research Funding; Novartis: Research Funding; Spectrum: Research Funding.

Somatic Mutational Landscape of AML with Inv(16) and t(8;21) Identifies Two Distinct Patterns in Relapse Tumors

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2362-2362
Author(s):  
Raman B. Sood ◽  
Nancy F Hansen ◽  
Frank X Donovan ◽  
Blake Carrington ◽  
Baishali Maskeri ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Clonal Evolution ◽  
Treatment Strategies ◽  
Gene Mutations ◽  
Driver Gene ◽  
Intrinsic Resistance ◽  
Driver Genes ◽  
In Vivo Models

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease with a wide prognostic spectrum ranging from poor to good depending upon the underlying mutations and/or cytogenetic abnormalities. Although AMLs with inv(16)/t(16:16) or t(8,21), collectively referred to as core binding factor leukemias (CBF-AMLs), are classified as prognostically favorable, such patients often succumb to their disease following relapse after an initial response to cytarabine/anthracyclin-based treatment regimens. Thus, to develop successful treatment strategies, it is critical to understand the mechanisms leading to disease relapse and target them with novel therapeutic approaches. To pursue this goal, we applied genomic approaches (whole exome sequencing and single nucleotide polymorphism arrays) on DNA from samples collected at sequential time points (i.e., diagnosis, complete remission and relapse) in seven patients with inv(16) and six patients with t(8;21). We identified mutations in several previously identified AML driver genes, such as KIT, FLT3, DNMT3A, EZH2, SMC1A, SMC3, WT1 and NRAS. Three relapse samples showed mosaicism for monosomy/disomy of the region of chromosome 3 containing GATA2. Overall, our data revealed two distinct profiles that support different mechanisms of relapse: 1) diagnosis and relapse blasts harbor the same driver gene mutations, indicating the intrinsic resistance of the major clones present at diagnosis to treatment regimen used; 2) diagnosis and relapse tumors have different driver gene mutations, indicating disease clonal evolution possibly through treatment selective pressure. Furthermore, our data has identified previously unreported putative driver genes for AML. Among these, we identified same somatic variant (R222G) in DHX15, an RNA helicase involved in splicing, in two patients at diagnosis. The variant was also detected at relapse in one of these patients. Functional validation of the mechanistic roles of wild type and mutated DHX15 in hematopoiesis and leukemogenesis, respectively, is ongoing in in vitro and in vivo models. Disclosures No relevant conflicts of interest to declare.

Fabrication of a pH responsive DOX conjugated PEGylated palladium nanoparticle mediated drug delivery system: an in vitro and in vivo evaluation

RSC Advances ◽  
2015 ◽  
Vol 5 (56) ◽  
pp. 44998-45014 ◽  
Author(s):  
Krishnamurthy Shanthi ◽  
Karuppaiya Vimala ◽  
Dhanaraj Gopi ◽  
Soundarapandian Kannan
Keyword(s):  
Drug Delivery ◽  
Drug Delivery System ◽  
Delivery System ◽  
Hela Cells ◽  
Ph Responsive ◽  
Palladium Nanoparticle ◽  
In Vivo Evaluation ◽  
Induced Apoptosis

Schematic illustration of the possible mechanism of pH based drug delivery system of DOX conjugated PEGylated PdNPs induced apoptosis in HeLa cells.

scholarly journals TNFR2 As a Target to Improve CD19-Directed CART Cell Fitness and Antitumor Activity in Large B Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 901-901
Author(s):  
Claudia Manriquez Roman ◽  
Michelle J. Cox ◽  
Reona Sakemura ◽  
Kun Yun ◽  
Mohamad M. Adada ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Cell Activation ◽  
B Cell Lymphoma ◽  
Death Receptors ◽  
Target Cells ◽  
Advisory Committees ◽  
Anti Tumor Activity

Abstract Introduction: It has become increasingly apparent that chimeric antigen receptor T (CART) cell activation and differentiation level is an important determinant of CART cell fate and response to therapy. In this study, we aimed to 1) measure levels of activation-induced surface death receptors and ligands on CART cells; 2) investigate how CART cell activation could impact their fitness and clinical responses, and 3) identify cell-based targets to modulate CART cell activation, apoptosis, and cytotoxicity to improve anti-tumor activity. Methods: We performed flow cytometric studies on ex-vivo stimulated, clinically annotated CART products of patients with large B cell lymphoma from the pivotal ZUMA-1 clinical trial that led to FDA-approved Axicabtagene ciloleucel (Axi-Cel). We investigated possible correlations of a number of surface death receptors and ligands with T cell differentiation status and post-infusion CART cell expansion, utilizing samples from ZUMA-1 patients who achieved a complete response as a best outcome ('responders') vs patients who achieved stable or progressive disease('non-responders'). CART cell effector functions in vitro were measured, and CART apoptosis was assessed using Annexin V. For in vitro and in vivo functional studies, we used CART19 generated from healthy donors (HD CART19) as indicated in the specific experiment. CRISPR/Cas9 was employed during CART cell production to disrupt specific genes. A xenograft model of lymphoma was used to investigate the in vivo antitumor activity of CART19. Results: Following an ex vivo stimulation of Axi-Cel products with CD19 + target cells, we observed upregulation of death receptors and ligands in CART19 from non-responders, compared to responders. We also observed a possible association between such upregulated surface markers with CART cell differentiation as measured by CCR7 expression. In an extended in vitro co-culture assay, where HD CART19 cells were repeatedly stimulated through the CAR, we found that tumor necrosis factor α receptor 2 (TNFR2), unlike other death receptors and ligands, was persistently elevated, suggesting a possible role for TNFR2 in long-term antigen-dependent CART19 dysfunction (Figure 1A). We further found that HD CART19 upregulate TNFR2, but not TNFR1, upon CAR stimulation (Figure 1B). While non-specific TCR activation (CD3 stimulation) of HD CART19 cells protected them from activation-induced apoptosis, antigen-specific activation through the CAR resulted in significant initiation of apoptosis within 2 hours of stimulation (Figure 1C). Having identified a possible association between TNFR2 and CART19 dysfunction, we aimed to study the impact of TNFR2 knockout on HD CART19 functions. Using CRISPR/Cas9 during CART cell manufacturing, we generated TNFR2 k/o HD CART19 cells with a knockout efficiency of around 50%, where the expression levels of TNFR2 in activated CART19 cells were reduced, compared to control CART19 cells (with non-targeting gRNA CRISPR/Cas9, Figure 1D). TNFR2 k/o CART19 cells demonstrated reduced early activation surface markers compared to control CART19, as measured by CD25 and CD69 surface expression (Figure 1E), reduced apoptosis initiation as measured by the Annexin V assay (Figure 1F), and enhanced antigen-specific proliferation and cytotoxicity (Figure 1G). Finally, in an in vivo xenograft model of CD19 + lymphoma, TNFR2 k/o CART19 resulted in enhanced CART cell expansion and anti-tumor activity (Figure 1H). Conclusions: Our results indicate that TNFR2 plays a role in early activation and apoptosis initiation of CART19 following CAR stimulation with CD19 + target cells and present TNFR2 knockout as a strategy to enhance CART19 anti-tumor activity. Figure 1 Figure 1. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura: Humanigen: Patents & Royalties. Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Genentech: Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Research Funding; Tolero Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees. Scholler: Kite: Current Employment. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Mattie: Kite: Current Employment. Kim: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment. Filosto: Kite, a Gilead Company: Current Employment; Tusk Therapeutics: Patents & Royalties: or other intellecular property; Gilead Sciences: Other: stock or other ownership . Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

scholarly journals The Nek2 Genetic Knockout Mouse Model Provides Novel Treatment Strategies in Myeloma and Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5612-5612
Author(s):  
Li Can ◽  
Kalyan Nadiminti ◽  
Yuqi Zhu ◽  
Yogesh Jethava ◽  
Ivana Frech ◽  
...  
Keyword(s):  
B Cell ◽  
Burkitt Lymphoma ◽  
Cell Lymphoma ◽  
Tumor Development ◽  
B Cell Lymphoma ◽  
Cell Tumor ◽  
High Dose ◽  
In Vivo Models

Abstract Background; Major progress in the treatment of B cell tumors has been made in the past decades. Nevertheless, relapses and refractoriness to currently available chemotherapy and even to high dose therapy with stem cell transplantation still cause significant mortality. NEK2, NEver in Mitosis Gene A (NIMA)-Related Kinase 2, is a serine/threonine kinase. High expression of NEK2 increases cell survival and drug resistance, resulting in poor clinical outcome in multiple cancers including multiple myeloma and lymphoma. In this study, we used genetic mouse models to evaluate whether NEK2 is a druggable target in the treatment of B cell tumors including myeloma and diffuse large B-cell lymphoma (DLBCL). Materials and Methods: We have generated Nek2 knockout mice and crossed these with Eµ-Myc mice. RNA-sequencing was performed to determine signaling pathways related to Nek2 inhibition. Both NEK2 and USP7 (a protein interacting with NEK2) inhibitors were applied to treat myeloma and DLBCL in vitro and in vivo. Results: Mouse studies showed that Nek2 played a critical role in B cell tumor development and progression. Specifically, in genetic Eμ-MYC transgenic mice, which spontaneously develop DLBCL and Burkitt lymphoma, knockout of Nek2 prevented B cell tumor development and significantly extended mouse survival. Further, immunohistochemistry analyses showed that Nek2 was highly detected in biopsies from aggressive Burkitt lymphoma patients. Our data also indicate that both NEK2 and USP7 inhibitors significantly inhibited myeloma cell and lymphoma cell growth in vitro and in vivo models and without apparent toxicity to normal tissues. Intriguingly, the combination of USP7 inhibitor P5091 with doxorubicin blocked B cell lymphoma development and extended lymphoma mouse survival. Conclusions: Our studies demonstrate the importance of Nek2 function in tumorigenesis and progression in B cell lineage malignancies. Both NEK2 and USP7 inhibitors showed excellent efficacy in the treatment of myeloma and B-cell lymphoma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mcl-1/CDK9 Targeting By AZD5991/AZD4573 Overcomes Intrinsic and Acquired Venetoclax Resistance in Vitro and In Vivo in PDX Model of AML through Modulation of Cell Death and Metabolic Functions

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 768-768 ◽  
Author(s):  
Bing Z Carter ◽  
Po Yee Mak ◽  
Wenjing Tao ◽  
Mark Warmoes ◽  
Philip L Lorenzi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Research Funding ◽  
Single Agent ◽  
O2 Consumption ◽  
Mitochondrial Oxidative Phosphorylation ◽  
Metabolic Functions ◽  
Pdx Model ◽  
Induced Apoptosis

Abstract Mcl-1 and Bcl-2 are two major anti-apoptotic Bcl-2 proteins frequently overexpressed in malignant cells. They cooperatively support cell survival and are associated with therapy resistance. ABT-199 (venetoclax), a highly selective Bcl-2 inhibitor, showed potent preclinical activity but limited clinical efficacy in AML as a single agent. Mcl-1 is induced by and a major resistance factor to ABT-199. Mcl-1 was recently found to also positively regulate mitochondrial oxidative phosphorylation that induces cancer stem cells and promotes chemoresistance. Mcl-1 is essential for the development of AML and for the survival of AML cells and stem cells. Increased mitochondrial oxidative phosphorylation has been demonstrated in these cells. First we found that Mcl-1 overexpressing (OE)/knockdown (KD) AML cells were markedly more resistant/sensitive to ABT-199 than were corresponding control cells, supporting the notion of Mcl-1 as a resistance factor to ABT-199. Inhibition of Mcl-1 by the selective Mcl-1 inhibitor AZD5991 or the CDK9 inhibitor AZD4573, which down-regulates short-lived proteins such as Mcl-1, induced apoptosis and showed strong synergy when combined with ABT-199 in AML cell lines, primary AML blasts, and stem/progenitor cells from patients. Importantly, combinations of AZD5991 or AZD4573 with ABT-199 synergistically induced apoptosis in OCI-AML3 and Mcl-1 OE cells intrinsically resistant to ABT-199 and in AML cell lines and primary patient cells with acquired resistance to ABT-199. Although OE/KD Mcl-1 in AML cells did not show obvious alterations in baseline cell viability, NSGS mice harboring Mcl-1 OE/KD OCI-AML3 cells survived significantly shorter/longer than those transplanted with control cells, supporting additional, non-apoptogenic functions of Mcl-1 in AML. We observed that genetic modulation of Mcl-1 alters cellular mitochondrial respiration and ROS levels. AML cells with Mcl-1 OE/KD increased/decreased O2 consumption and mitochondrial ATP and ROS generation. Consistent with this finding, inhibition of Mcl-1 by AZD5991 or AZD4573 decreased O2 consumption and ATP generation in AML cells and also in MV4-11 cells with acquired ABT-199 resistance. Mass spectrometry-based stable isotope tracing experiments using 1,2-13C-glucose showed that both genetic and pharmacological inhibition of Mcl-1 decreased flux of glucose carbon through glycolysis, the TCA cycle, and the pentose phosphate pathway, suggesting a role for Mcl-1 in cellular respiration and redox metabolism. To further assess the efficacy of combined Mcl-1 and Bcl-2 inhibition in primary AML cells resistant to ABT-199, we developed a PDX model using cells from an AML patient who initially responded to ABT-199/demethylating agent and then relapsed. NSGS mice engrafted with these PDX cells were treated with ABT-199 (50 mg/kg, oval gavage qd), AZD5991 (60 mg/kg, i.v. weekly), AZD4573 (15 mg/kg, i.p. bid with 2 h interval for two consequent days/week), ABT-199+AZD5991, or ABT-199+AZD4573 for 6 wks. Flow cytometric analysis of circulating human CD45+ cells on day 18 of therapy showed that each agent significantly decreased leukemia burden and that the combinations were significantly more effective (P<0.01) than each single agent. CyTOF analysis of BM cells (day 25) showed that both combinations markedly reduced (P<0.001) human CD45+ cells and, more importantly, human CD34+CD38+/CD38- and CD34+CD38+/CD38-CD123+ cells. Those combination treatments also decreased Mcl-1, Bcl-2, b-catenin, c-Myc, and FAK protein expression in CD34+CD38-CD123+ cells. Interestingly, AZD5991, AZD4573, or their combinations with ABT-199 greatly decreased CXCR4 in all cell populations. Ultimately, each single agent only marginally prolonged survival, whereas ABT-199+AZD4573 and even more so ABT-199+AZD5991 markedly improved survival in this highly ABT-199 resistant PDX model (Fig). Conclusion: we demonstrate that Mcl-1 has metabolic functions in AML and that inhibition of Mcl-1 enhances ABT-199 apoptogenic activity and overcomes intrinsic and acquired ABT-199 resistance in vitro and in vivo in a PDX murine model of AML, suggesting that inhibition of Mcl-1 improves the efficacy of ABT-199, and overcomes established resistance to Bcl-2 inhibition. Suppressing metabolic activity and CXCR4 inhibition may also contribute to the efficacy of this combination against AML stem cells in the BM microenvironment. Figure. Figure. Disclosures Carter: AstraZeneca: Research Funding; novartis: Research Funding. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties. Cidado:AstraZeneca: Employment, Equity Ownership. Drew:AstraZeneca: Employment. Andreeff:AstraZeneca: Research Funding.

scholarly journals The SMAC mimetic LCL-161 displays antitumor activity in preclinical models of rituximab-resistant B-cell lymphoma

2018 ◽  
Vol 2 (23) ◽  
pp. 3516-3525 ◽  
Author(s):  
Kyle Runckel ◽  
Matthew J. Barth ◽  
Cory Mavis ◽  
Juan J. Gu ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
Antitumor Activity ◽  
Cell Lines ◽  
Antitumor Effect ◽  
Ex Vivo ◽  
B Cell Lymphoma ◽  
Sensitive Cell ◽  
In Vivo Models ◽  
Synergistic Enhancement

Abstract Clinical observations suggest the existence of shared resistance pathways between rituximab and chemotherapy agents. To explore the mechanisms of rituximab resistance, our group created rituximab-resistant cell lines (RRCLs), which display altered expression of several inhibitor of apoptosis (IAP) family proteins. Here, we provide evidence to support pharmacologically targeting IAPs in lymphoma with LCL-161, a small molecule mimetic of the second mitochondria-derived activator of caspases (SMAC). The antitumor effect of LCL-161 was determined using luminescent adenosine triphosphate assays, flow cytometry, SCID mouse xenografts, and ex vivo patient biopsy sample studies. In vitro exposure to LCL-161 also resulted in a dose-dependent decrease in IAP levels, along with synergistic enhancement of the antitumor effect of cytotoxic chemotherapy, in rituximab-sensitive cell lines and RRCLs. In addition, LCL-161 increased the cytotoxic effect of the proteasome inhibitor carfilzomib in ex vivo lymphoma patient samples. The combination of LCL-161 with the chemotherapy regimen rituximab, gemcitabine, and vinorelbine (RGV) improved in vivo survival compared with RGV alone in severe combined immunodeficient mice implanted with RRCLs but not in animals implanted with rituximab-sensitive cell lines. In summary, LCL-161 exhibits synergistic antitumor activity in both in vitro and in vivo models of resistant lymphoma. Our data support further preclinical investigation of LCL-161 as a novel antilymphoma agent.

scholarly journals ROS-mediated inactivation of the PI3K/AKT pathway is involved in the antigastric cancer effects of thioredoxin reductase-1 inhibitor chaetocin

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Chuangyu Wen ◽  
Huihui Wang ◽  
Xiaobin Wu ◽  
Lu He ◽  
Qian Zhou ◽  
...  
Keyword(s):  
Critical Role ◽  
Thioredoxin Reductase ◽  
Akt Pathway ◽  
Ros Generation ◽  
In Vivo Models ◽  
M Phase ◽  
Tumorigenesis And Progression ◽  
Induced Apoptosis

Abstract Novel drugs are urgently needed for gastric cancer (GC) treatment. The thioredoxin-thioredoxin reductase (TRX-TRXR) system has been found to play a critical role in GC tumorigenesis and progression. Thus, agents that target the TRX-TRXR system may be highly efficacious as GC treatments. In this study, we showed that chaetocin, a natural product isolated from the Chaetomium species of fungi, inhibited proliferation, induced G2/M phase arrest and caspase-dependent apoptosis in both in vitro and in vivo models (cell xenografts and patient-derived xenografts) of GC. Chaetocin inactivated TRXR-1, resulting in the accumulation of reactive oxygen species (ROS) in GC cells; overexpression of TRX-1 as well as cotreatment of GC cells with the ROS scavenger N-acetyl-L-cysteine attenuated chaetocin-induced apoptosis; chaetocin-induced apoptosis was significantly increased when GC cells were cotreated with auranofin. Moreover, chaetocin was shown to inactivate the PI3K/AKT pathway by inducing ROS generation; AKT-1 overexpression also attenuated chaetocin-induced apoptosis. Taken together, these results reveal that chaetocin induces the excessive accumulation of ROS via inhibition of TRXR-1. This is followed by PI3K/AKT pathway inactivation, which ultimately inhibits proliferation and induces caspase-dependent apoptosis in GC cells. Chaetocin therefore may be a potential agent for GC treatment.

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