Change in the permeability of the mitochondrial membrane to proteins (cytochrome
c and Smac) and protons is a critical step in apoptosis. Although the time from the induction of apoptosis to the ...change of mitochondrial permeability is variable over a period of hours, the release of proteins is an “all or none” phenomenon that is completed in an individual cell within minutes. Here, using single-cell fluorescence microscopy, we show that the release of cytochrome
c from a single mitochondrion occurs in a single step. However, this increased permeability of the outer membrane to cytochrome
c propagates throughout the cell as a slower, spatially coordinated wave. The permeability of the outer membrane to Smac propagates with the same spatial pattern but lagging in time. This is followed by a wave of increased permeability of the inner membrane to protons. Only afterward do the mitochondria fission. The spatial dependence of the permeability wave was inhibited by thapsigargin, an inhibitor of the endoplasmic reticulum calcium pumps, but buffering cytosolic calcium had no effect. These results show that the trigger for apoptosis is spatially localized, initiating at one or only a few mitochondria preceding the loss of mitochondrial energetics, and the subsequent temporal propagation of mitochondrial membrane permeability is calcium-dependent.
Although the cellular decision to commit to apoptosis is important for organism homeostasis, there is considerable variability in the onset of apoptosis between cells, even in clonal populations. ...Using live single-cell imaging, we observed that the onset of apoptotic proteolytic activity was tightly synchronized between nearby cells. This synchrony was not a consequence of secreted factors and was not correlated to the cell cycle. The synchrony was only seen amongst related cells and was lost over successive generations. The times of apoptosis also diverged within a generation, but this was blocked by inhibiting protein synthesis before triggering apoptosis. These results suggest that the cell-cell variability of apoptosis times is due to the divergence of the molecular composition of the cell, and that the decision to commit to apoptosis at the time of drug addition is a deterministic decision.
BackgroundPersister tumor cells are a discrete and typically undetectable sub-population of cells that survive anti-cancer therapy and are thought to be a major cause of tumor recurrence. Persisters ...have been identified following both drug- and immunotherapy. The focus of this study is to examine any similarities or differences between immunotherapy persister cells (IPCs) and drug tolerant persister cells (DTPs). Pharmacological agents and immune cells often kill via apoptosis. Therefore, we investigated if reduced apoptotic sensitivity is a shared mechanism common to IPCs and DTPs and whether the use of BH3 mimetics to enhance apoptotic sensitivity could sensitize IPCs and DTPs. We also investigated if IPCs have an altered sensitivity profile to conventional chemotherapy, radiotherapy and epigenetic modulators. Similarly, we assessed the sensitivity of DTPs to T cell attack and radiotherapy.MethodsIPCs were generated by chronically co-culturing tumor cell lines (murine B16-OVA melanoma cells or human HeLa-EGFR cervical tumor cells) with T cells (OVA-T-1 murine T cells or human EGFR-CAR T cells) for 7 days. Six different DTP cell lines were generated by chronically treating tumor cells for 7 days with a high dose of drug that killed approximately 90% of the cells after 3 days (5-FU/oxaliplatin/docetaxel/etoposide/dinaciclib/panobinostat). Using a CellTiter-glo assay, we determined the sensitivity of IPCs and DTPs to drugs, radiation and T cell attack. Microscopy-based BH3 profiling was employed to measure the mitochondrial apoptotic sensitivity of persisters and to determine if there exists any anti-apoptotic dependencies that persisters might rely on to enhance their survival.ResultsIPCs acquired a reduced sensitivity to multiple drug classes and radiotherapy. Likewise, DTPs also developed a reduced sensitivity to multiple drug classes and radiotherapy but also acquired a reduced sensitivity to T cell killing. Depending on the cell line and therapy, some persisters developed either an increased or decreased sensitivity to apoptosis. Inhibition of specific anti-apoptotic dependencies using BH3 mimetics increased apoptotic sensitivity of persister cells.ConclusionsWe identified that reduced apoptotic sensitivity is a shared mechanism of survival common to many IPCs and DTPs. IPCs and DTPs have distinct anti-apoptotic dependencies compared to their parental cell lines wherein pharmacological inhibition of such dependencies enhanced apoptotic sensitivity. IPCs and DTPs also displayed a reduced sensitivity to other therapies that they were never exposed to suggesting that the acquired mechanisms of resistance to T cell killing or drug therapy also conferred a reduced sensitivity to many drug classes, cellular immunotherapies and radiation.
A potential cause of cancer relapse is pretreatment chemoresistant subpopulations. Identifying targetable features of subpopulations that are poorly primed for therapy-induced cell death may improve ...cancer therapy. Here, we develop and validate real-time BH3 profiling, a live and functional single-cell measurement of pretreatment apoptotic sensitivity that occurs upstream of apoptotic protease activation. On the same single cells, we perform cyclic immunofluorescence, which enables multiplexed immunofluorescence of more than 30 proteins on the same cell. Using cultured cells and rapid ex vivo cultures of colon cancer patient-derived xenograft (PDX) models, we identify Bak as a univariate correlate of apoptotic priming, find that poorly primed subpopulations can correspond to specific stages of the cell cycle, and, in some PDX models, identify increased expression of Bcl-XL, Mcl-1, or Her2 in subpopulations that are poorly primed for apoptosis. Last, we generate and validate mathematical models of single-cell priming that describe how targetable proteins contribute to apoptotic priming.
Activating mutations involving the PI3K pathway occur frequently in human cancers. However, PI3K inhibitors primarily induce cell cycle arrest, leaving a significant reservoir of tumor cells that may ...acquire or exhibit resistance. We searched for genes that are required for the survival of PI3K mutant cancer cells in the presence of PI3K inhibition by conducting a genome scale shRNA-based apoptosis screen in a
mutant human breast cancer cell. We identified 5 genes (
) whose suppression induced cell death upon PI3K inhibition. We showed that small molecule inhibitors of the PIM2 and ZAK kinases synergize with PI3K inhibition. In addition, using a microscale implementable device to deliver either siRNAs or small molecule inhibitors in vivo, we showed that suppressing these 5 genes with PI3K inhibition induced tumor regression. These observations identify targets whose inhibition synergizes with PI3K inhibitors and nominate potential combination therapies involving PI3K inhibition.
Directing stem cell fate requires knowledge of how signaling networks integrate temporally and spatially segregated stimuli. We developed and validated a computational model of signal transducer and ...activator of transcription-3 (Stat3) pathway kinetics, a signaling network involved in embryonic stem cell (ESC) self-renewal. Our analysis identified novel pathway responses; for example, overexpression of the receptor glycoprotein-130 results in reduced pathway activation and increased ESC differentiation. We used a systematic in silico screen to identify novel targets and protein interactions involved in Stat3 activation. Our analysis demonstrates that signaling activation and desensitization (the inability to respond to ligand restimulation) is regulated by balancing the activation state of a distributed set of parameters including nuclear export of Stat3, nuclear phosphatase activity, inhibition by suppressor of cytokine signaling, and receptor trafficking. This knowledge was used to devise a temporally modulated ligand delivery strategy that maximizes signaling activation and leads to enhanced ESC self-renewal.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Persistent myofibroblast activation distinguishes pathological fibrosis from physiological wound healing, suggesting that therapies selectively inducing myofibroblast apoptosis could prevent ...progression and potentially reverse established fibrosis in diseases such as scleroderma, a heterogeneous autoimmune disease characterized by multiorgan fibrosis. We demonstrate that fibroblast-to-myofibroblast differentiation driven by matrix stiffness increases the mitochondrial priming (proximity to the apoptotic threshold) of these activated cells. Mitochondria in activated myofibroblasts, but not quiescent fibroblasts, are primed by death signals such as the proapoptotic BH3-only protein BIM, which creates a requirement for tonic expression of the antiapoptotic protein BCL-X
to sequester BIM and ensure myofibroblast survival. Myofibroblasts become particularly susceptible to apoptosis induced by "BH3 mimetic" drugs inhibiting BCL-X
such as ABT-263. ABT-263 displaces BCL-X
binding to BIM, allowing BIM to activate apoptosis on stiffness-primed myofibroblasts. Therapeutic blockade of BCL-X
with ABT-263 (navitoclax) effectively treats established fibrosis in a mouse model of scleroderma dermal fibrosis by inducing myofibroblast apoptosis. Using a BH3 profiling assay to assess mitochondrial priming in dermal fibroblasts derived from patients with scleroderma, we demonstrate that the extent of apoptosis induced by BH3 mimetic drugs correlates with the extent of their mitochondrial priming, indicating that BH3 profiling could predict apoptotic responses of fibroblasts to BH3 mimetic drugs in patients with scleroderma. Together, our findings elucidate the potential efficacy of targeting myofibroblast antiapoptotic proteins with BH3 mimetic drugs in scleroderma and other fibrotic diseases.
Abstract
The use of imperfect models and ex vivo culture systems to try to predict patient drug response represents an enormous bottle neck in cancer treatment. Nonetheless, determining how effective ...an approved drug will be for an individual cancer patient, as well as identifying novel compounds that may be beneficial to a specific population often requires the use of primary tumor cells. Patient-derived organoids represent an intermediate between primary tumor cells, whose limited supply may hinder reliable drug testing, and cell lines, which often do not reflect what happens in vivo. Herein, we describe the development of a novel assay platform, termed 3D-DBP (3D dynamic BH3 profiling), to detect early apoptotic measurements in ovarian cancer patient-derived organoids and present evidence that this method can be used to predict patient response to therapy. We have optimized the use of patient-derived organoids from 16 individual tumors in a microscopy-based imaging assay. We image the BH3 peptide-induced release of cytochrome c from mitochondria, which indicates permeabilization of the outer mitochondrial membrane, in intact organoids. The less cytochrome c retained in each organoid, the more primed that organoid is for apoptosis. By comparing results of drug-treated and untreated cells, we can identify drugs that cause a significant increase in apoptotic priming in organoids. In the 16 patient-derived organoids investigated this 3D DBP technique was an effective means of predicting patient response to carboplatin therapy. In summary, we have not only created a means of visualizing drug response in intact organoids, but also have demonstrated its clinical utility.
Citation Format: Kelley E. McQueeney, Patrick Bhola, Sarah J. Hill, Anthony Letai. Early apoptotic measurements of patient-derived organoids predict patient response to therapy. abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4309.
Abstract
Given the rapid development of new small molecule cancer therapeutics, there is a growing need for predictive diagnostics to match cancer patients with optimal therapies. We previously ...developed a precision medicine technology with a functional phenotypic readout called dynamic BH3 profiling (DBP). DBP exposes cancer cells to drugs and measures induction of apoptotic cell death signaling after 24 hours ex vivo. Nonetheless, the application of DBP to core biopsies from metastatic tumors or other limited samples remains a technical challenge. Here, we adapt the DBP protocol for use on samples with small numbers of cells such as core biopsies. We maximize information returned per cell by imaging mitochondrial integrity in response to BH3 peptide exposure over time. We first show that the adapted protocol works in limited numbers of cancer cell lines, and in limited cells from the MMTV-PyMT genetically engineered mouse model of breast cancer. Specifically, we show that our ex vivo DBP predictions of the MMTV-PyMT mouse tumor matches known in vivo response. Finally, we apply our modified protocol to patient derived xenografts of colon cancer and primary patient colon tumors. We expect that our adapted protocol will find utility as a clinical biomarker, and as a method to optimize pre-clinical drug testing.
Citation Format: Rebecca German, Elizaveta Lavrova, Timothy Hagan, Otari Chipashvili, Ewa Sicinska, James Cleary, Kimmie Ng, Anthony Letai, Patrick Bhola. Identifying cancer drug sensitivity using live cell imaging dynamic BH3 profiling of solid tumor core biopsies abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 260.
Leiomyosarcoma (LMS) is a neoplasm characterized by smooth muscle differentiation, complex copy-number alterations, tumor suppressor loss, and the absence of recurrent driver mutations. Clinical ...management for advanced disease relies on the use of empiric cytotoxic chemotherapy with limited activity, and novel targeted therapies supported by preclinical research on LMS biology are urgently needed. A lack of fidelity of established LMS cell lines to their mesenchymal neoplasm of origin has limited translational understanding of this disease, and few other preclinical models have been established. Here, we characterize patient-derived xenograft (PDX) models of LMS, assessing fidelity to their tumors of origin and performing preclinical evaluation of candidate therapies.
We implanted 49 LMS surgical samples into immunocompromised mice. Engrafting tumors were characterized by histology, targeted next-generation sequencing, RNA sequencing, and ultra-low passage whole-genome sequencing. Candidate therapies were selected based on prior evidence of pathway activation or high-throughput dynamic BH3 profiling.
We show that LMS PDX maintain the histologic appearance, copy-number alterations, and transcriptional program of their parental tumors across multiple xenograft passages. Transcriptionally, LMS PDX cocluster with paired LMS patient-derived samples and differ primarily in host-related immunologic and microenvironment signatures. We identify susceptibility of LMS PDX to transcriptional cyclin-dependent kinase (CDK) inhibition, which disrupts an E2F-driven oncogenic transcriptional program and inhibits tumor growth.
Our results establish LMS PDX as valuable preclinical models and identify strategies to discover novel vulnerabilities in this disease. These data support the clinical assessment of transcriptional CDK inhibitors as a therapeutic strategy for patients with LMS.
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