Mechanisms In Cell Lines Research Articles

Venetoclax resistance in acute lymphoblastic leukemia is characterized by increased mitochondrial activity and can be overcome by co-targeting oxidative phosphorylation

Deregulated apoptosis signaling is characteristic for many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN), a selective BCL-2 inhibitor has shown clinical activity in different lymphoid malignancies and is currently evaluated in first clinical trials in BCP-ALL. However, insensitivity to VEN has been described constituting a major clinical concern. Here, we addressed and modeled VEN-resistance in BCP-ALL, investigated the underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples and identified potential strategies to overcome VEN-insensitivity. Leukemia lines with VEN-specific resistance were generated in vitro and further characterized using RNA-seq analysis. Interestingly, gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain were significantly enriched and upregulated, indicating increased mitochondrial metabolism in VEN-resistant ALL. Metabolic profiling showed sustained high mitochondrial metabolism in VEN-resistant lines as compared to control lines. Accordingly, primary PDX-ALL samples with intrinsic VEN-insensitivity showed higher oxygen consumption and ATP production rates, further highlighting that increased mitochondrial activity is a characteristic feature of VEN-resistant ALL. VEN-resistant PDX-ALL showed significant higher mitochondrial DNA content and differed in mitochondria morphology with significantly larger and elongated structures, further corroborating our finding of augmented mitochondrial metabolism upon VEN-resistance. Using Oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic activity and apoptosis induction in VEN-resistant BCP-ALL cell lines and PDX samples, demonstrating that acquired and intrinsic VEN-insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. These findings of reprogrammed, high mitochondrial metabolism in VEN-resistance and synergistic activity upon co-targeting BCL-2 and oxidative phosphorylation strongly suggest further preclinical and potential clinical evaluation in VEN-resistant BCP-ALL.

Venetoclax Resistance in B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By Increased Mitochondrial Metabolism and Can be Overcome By Co-Targeting Oxidative Phosphorylation

Deregulated cell death pathways are a hallmark of many cancers and contribute to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules either inducing or counteracting cell death induction. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN) binds selectively to anti-apoptotic BCL-2 resulting in apoptosis induction and has shown clinical activity in lymphoid malignancies, but insensitivity and resistance have been described. In BCP-ALL, we and others have shown preclinical activity of VEN and VEN is evaluated in first clinical trials. In this study, we have addressed and modeled VEN resistance in BCP-ALL and investigated underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples in order to identify potential strategies to overcome VEN insensitivity. Starting from the BCP-ALL cell line RS4;11, five VEN insensitive lines were generated in parallel by exposure to increasing concentrations of VEN over time (49 passages, 8 months of continuous treatment) and five control lines were exposed to corresponding concentrations of solvent (DMSO). Measuring half maximal effective concentrations (EC50) showed increasing EC50 values from 4 nM to 26.2 µM over time in all VEN treated lines, indicating acquired resistance in our model. VEN insensitive leukemia lines displayed unchanged proliferation and same sensitivities to the prototypic apoptosis inducer staurosporine, the induction therapy drugs vincristine/dexamethasone/asparaginase or the anthracycline daunorubicin as compared to controls, indicating acquisition of VEN specific resistance. In order to further characterize the mechanism of VEN insensitivity, we performed RNA-Seq analysis comparing five RS4;11 VEN insensitive to five control lines. Interestingly, gene set enrichment analysis on significantly regulated genes showed a significant upregulation of gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain, pointing to increased mitochondrial metabolism in VEN resistant cells. We addressed mitochondrial activity and carried out metabolic profiling (Agilent Seahorse XF Cell Mito Stress Test) of VEN resistant and control lines (N=5+5). Most interestingly, in contrast to significant reduction of oxygen consumption rates (OCR) in VEN treated control lines, we found sustained high mitochondrial metabolism indicated by persistent high OCR in VEN resistant lines. To further validate our findings, we investigated a series of BCP-ALL PDX samples (N=30) and titrated VEN EC50 values identifying heterogenous VEN activities. Metabolic profiling (Mito Stress test) was performed on sensitive as compared to highly resistant leukemias (N=3+3). Most interestingly and in line with our findings in cells with acquired resistance, PDX samples with intrinsic VEN insensitivity showed higher oxygen consumption and ATP production rates, thus further highlighting that increased mitochondrial activity is a characteristic feature of VEN resistant ALL. Along this line, significant higher mitochondrial DNA content was found in highly insensitive PDX leukemias when compared to VEN sensitive samples. Moreover, we analyzed mitochondrial morphology by electron microscopy. Although numbers of mitochondria did not differ between VEN resistant and sensitive leukemias, the overall mitochondrial area and structure was found to be significantly larger and elongated, further corroborating our finding of augmented mitochondrial metabolism in VEN resistant ALL. Finally, we addressed whether VEN resistance in ALL can be overcome by directly targeting oxidative phosphorylation (OxPhos). Using oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic induction of apoptosis together with VEN in BCP-ALL cell lines and PDX samples, showing that acquired and intrinsic VEN insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. Taken together, we show that VEN resistance in BCP-ALL is characterized by high mitochondrial metabolic activity. Co-targeting of BCL-2 and oxidative phosphorylation results in synergistic anti-leukemia activity setting the basis for further preclinical and potential clinical evaluation.

Abstract 3209: Elucidating drug resistance mechanisms in melanoma with quantitative proteomics.

Abstract Introduction: Exciting breakthroughs have been made in targeted therapy for melanoma; however, the development of drug resistance remains a critical problem for patients due to the speed of onset and the numerous complex mechanisms. Parallel development of platform for quantitative protein expression enables biomarker monitoring to evaluate the multiple pathways through which tumor cells can acquire drug resistance. We have developed quantitative assays using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) to assess a diverse panel of >70 proteins involved in signaling pathways, including apoptosis, receptor tyrosine kinases, PI3K/Akt/MTOR, and MAP kinases. Assays quantify changes in response to targeted therapeutic agents and examine subsequent resistance mechanisms in cell lines, xenografts, and biopsies from patients. Experimental procedures: Naïve and BRAF V600E inhibitor-resistant melanoma cell lines were treated with vehicle control, BRAF V600E inhibitor (PLX4720), HSP90 inhibitor (XL-888, Exelixis), or MEK inhibitor (AZD6244). Immunoblotting and LC-MRM were used to evaluate protein expression levels. Heat shock proteins can be measured directly from whole cell lysates or tumor homogenates using a few micrograms of protein. For expression of cancer signaling proteins (many of which are HSP90 clients), 50 micrograms of total protein were fractionated by SDS-PAGE prior to digestion and LC-MRM quantification. Similar experiments were conducted with mouse xenograft tumors from treated and control mice; these measurements have also been applied to fine needle aspirates (FNAs) and core biopsies from patients in the context of a clinical trial. Data summary: LC-MRM quantification is effective in quantifying these proteins in cell lines, xenografts, and biopsies or FNAs. The LC-MRM panel assay was able to screen for a diverse range of proteins revealing previously undescribed cellular responses to treatment and elucidate potential mechanisms of resistance. As an example, both LC-MRM data and Westerns showed consistent responses to HSP90 inhibition, including increased expression of HSP71 and reduction of HSP90 client levels. Proteins were also modulated in response and resistance to BRAF and MEK inhibitors. Conclusion: LC-MRM quantifies biomarker panels and provides an assessment tool that can translated in a straightforward way from cell line models to patient specimens. This emerging technique holds great promise for biopsy-mandated clinical trials, particularly in diseases like melanoma that can be readily sampled. Measurements using this LC-MRM platform can be used to assess drug performance and patient response to evaluate treatment, detect drug resistance, and suggest rational combination therapies. Citation Format: Elizabeth Remily-Wood, Kim Paraiso, Eirik Haarberg, Y. Ann Chen, Vito Rebecca, Keiran Smalley, John M. Koomen. Elucidating drug resistance mechanisms in melanoma with quantitative proteomics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3209. doi:10.1158/1538-7445.AM2013-3209

Silibinin inhibits ultraviolet B radiation-induced DNA-damage and apoptosis by enhancing interleukin-12 expression in JB6 cells and SKH-1 hairless mouse skin

Recent studies have demonstrated silibinin efficacy against ultraviolet B (UVB)-induced skin carcinogenesis via different mechanisms in cell lines and animal models; however, its role in regulating interleukin-12 (IL-12), an immunomodulatory cytokine that reduces UVB-induced DNA damage and apoptosis, is not known. Here, we report that UVB irradiation causes caspase 3 and PARP cleavage and apoptosis, and addition of recombinant IL-12 or silibinin immediately after UVB significantly protects UVB-induced apoptosis in JB6 cells. IL-12 antibody-mediated blocking of IL-12 activity compromised the protective effects of both IL-12 and silibinin. Both silibinin and IL-12 also accelerated the repair of UVB-caused cyclobutane-pyrimidine dimers (CPDs) in JB6 cells. Additional studies confirmed that indeed silibinin causes a significant increase in IL-12 levels in UVB-irradiated JB6 cells as well as in mouse skin epidermis, and that similar to cell-culture findings, silibinin topical application immediately after UVB exposure causes a strong protection against UVB-induced TUNEL positive cells in epidermis possibly through a significantly accelerated repair of UVB-caused CPDs. Together, these findings for the first time provide an important insight regarding the pharmacological mechanism wherein silibinin induces endogenous IL-12 in its efficacy against UVB-caused skin damages. In view of the fact that an enhanced endogenous IL-12 level could effectively remove UVB-caused DNA damage and associated skin cancer, our findings suggest that the use of silibinin in UVB-damaged human skin would also be a practical and translational strategy to manage solar radiation-caused skin damages as well as skin cancer.

Synthesis and cytotoxic activity of diosgenyl saponin analogues

Diosgenyl saponins are steroidal glycosides that are often found as major components in many traditional oriental medicines. Recently, a number of naturally occurring diosgenyl saponins have been shown to exert cytotoxic activity against several strains of human cancer cells. Use of these saponin compounds for cancer treatment is hampered due to the lack of understanding of their action mechanism as well as limited access to such structurally complicated molecules. In the present paper, we have prepared a group of diosgenyl saponin analogues which contain a beta-D-2-amino-2-deoxy-glucopyranose residue having different substituents at the amino group. Moderate cytotoxic activity is found for most analogues against neuroblastoma (SK-N-SH) cells, breast cancer (MCF-7) cells, and cervical cancer (HeLa) cells. The analogue 13 that contains an alpha-lipoic acid residue exhibits the highest potency against all three cancer cell lines with IC(50) ranging from 4.8 microM in SK-N-SH cells to 7.3 microM in HeLa cells. Preliminary mechanistic investigation with one saponin analogue (10) shows that the compound induces cell cycle arrest at G(1) phase in SK-N-SH cells, but the same compound induces cell cycle arrest at G(2) phase in MCF-7 cells. This result suggests that the cytotoxic activity of these saponin analogues may involve different action mechanisms in cell lines derived from different cancer sites.

Simultaneous detection of l-glutamate and nitric oxide from adherently growing cells at known distance using disk shaped dual electrodes

An ex vivo system for simultaneous detection of nitric oxide (NO) and l-glutamate using integrated dual 250 μm platinum disk electrodes modified individually with suitable sensing chemistries has been developed. One of the sensors was coated with an electrocatalytic layer of Ni tetrasulfonate phthalocyanine tetrasodium salt (Ni-TSPc) covered by second layer of Nafion, which stabilises on the one hand the primary oxidation product NO + and prevents interferences from negatively charged compounds such as NO 2 −. For glutamate determination, the second electrode was modified with a crosslinked redox hydrogel consisting of Os complex modified poly(vinylimidazol), glutamate oxidase and peroxidase. A manual x– y– z micromanipulator on top of an inverted optical microscope was used to position the dual electrode sensor at a defined distance of 5 μm from a cell population under visual control. C6 glioma cells were stimulated simultaneously with bradykinin or VEGF to release NO while KCl was used to invoke glutamate release. For evaluation of the glutamate sensors, in some experiments HN10 cells were used. To investigate the sensitivity and reliability of the system, several drugs were applied to the cells, e.g. Ca 2+-channel inhibitors for testing Ca 2+-dependence of the release of NO and glutamate, rotenone for inducing oxidative stress and glutamate antagonists for analysing glutamate release. With these drugs the NO and glutamate release was modulated in a similar way then expected from previously described systems or even in-vivo measurements. We therefore conclude that our system is suitable to analyse stress-induced mechanisms in cell lines.

Histamine in brain development and tumors

Histamine is found in developing mammalian brain in both neurons and mast cells. Under normal conditions, histamine H1 and H2 receptors are found in neural, glial and endothelial cells, and H3 receptors at least on neurons. Experimental brain tumors display both H1 and H2 receptors, and histamine increases permeability in the tumors and in the neighboring areas. Many studies have addressed histaminergic signalling mechanisms in cell lines originating from brain tumors. However, the role of histamine in normal development of brain structures, proliferation and differentiation of neurons and glial cells, and growth of malignant tumors in situ is still poorly understood.