Project description:Treatment of HIV-infected individuals with antiretroviral agents selects for drug-resistant mutants, resulting in frequent treatment failures. Although the major antiretroviral resistance mutations are routinely characterized by DNA sequencing, treatment failures are still common, probably in part because undetected rare resistance mutations facilitate viral escape. Here we combined DNA bar coding and massively parallel pyrosequencing to quantify rare drug resistance mutations. Using DNA bar coding, we were able to analyze seven viral populations in parallel, overall characterizing 118 093 sequence reads of average length 103 bp. Analysis of a control HIV mixture showed that resistance mutations present as 5% of the population could be readily detected without false positive calls. In three samples of multidrug-resistant HIV populations from patients, all the drug-resistant mutations called by conventional analysis were identified, as well as four additional low abundance drug resistance mutations, some of which would be expected to influence the response to antiretroviral therapy. Methods for sensitive characterization of HIV resistance alleles have been reported, but only the pyrosequencing method allows all the positions at risk for drug resistance mutations to be interrogated deeply for many HIV populations in a single experiment.
Project description:Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). We optimized and leveraged this property to identify drug resistance mutations, a technique we term LentiMutate. After validating this technique by identifying clinically relevant EGFR resistance mutations, we applied this technique to two additional anti-cancer drugs, imatinib and AMG 510. We find novel deletions in BCR-ABL1 that confer resistance to BCR-ABL1 inhibitors and point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations, in KRAS-G12C or wild-type KRAS, respectively, that confer resistance to AMG 510. LentiMutate may prove highly valuable to clinical and preclinical cancer drug development.
Project description:Recently, we presented evidence that high mitochondrial ATP production is a new therapeutic target for cancer treatment. Using ATP as a biomarker, we isolated the "metabolically fittest" cancer cells from the total cell population. Importantly, ATP-high cancer cells were phenotypically the most aggressive, with enhanced stem-like properties, showing multi-drug resistance and an increased capacity for cell migration, invasion and spontaneous metastasis. In support of these observations, ATP-high cells demonstrated the up-regulation of both mitochondrial proteins and other protein biomarkers, specifically associated with stemness and metastasis. Therefore, we propose that the "energetically fittest" cancer cells would be better able to resist the selection pressure provided by i) a hostile micro-environment and/or ii) conventional chemotherapy, allowing them to be naturally-selected for survival, based on their high ATP content, ultimately driving tumor recurrence and distant metastasis. In accordance with this energetic hypothesis, ATP-high MDA-MB-231 breast cancer cells showed a dramatic increase in their ability to metastasize in a pre-clinical model in vivo. Conversely, metastasis was largely prevented by treatment with an FDA-approved drug (Bedaquiline), which binds to and inhibits the mitochondrial ATP-synthase, leading to ATP depletion. Clinically, these new therapeutic approaches could have important implications for preventing treatment failure and avoiding cancer cell dormancy, by employing ATP-depletion therapy, to target even the fittest cancer cells.
Project description:Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). Here we optimized and leveraged this property to identify drug resistance mutations, developing a technique we term LentiMutate. This technique was validated by identifying clinically relevant EGFR resistance mutations, then applied to two additional clinical anticancer drugs: imatinib, a BCR-ABL inhibitor, and AMG 510, a KRAS G12C inhibitor. Novel deletions in BCR-ABL1 conferred resistance to imatinib. In KRAS-G12C or wild-type KRAS, point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations conferred resistance to AMG 510. LentiMutate should prove highly valuable for clinical and preclinical cancer-drug development. SIGNIFICANCE: LentiMutate can evaluate a drug's on-target activity and can nominate resistance mutations before they occur in patients, which could accelerate and refine drug development to increase the survival of patients with cancer.
Project description:Therapies targeting signaling molecules mutated in cancers can often have striking short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures. Resistance can sometimes result from a secondary mutations in rare cells, but other times, there is no clear genetic cause, raising leaving the possibility of non-genetic rare cell variability. Here, we show that melanoma cells can display profound transcriptional variability at the single cell level that predicts which cells will ultimately resist drug treatment. This variability involves semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces an epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming begins withis a progressive process consisting of a loss of SOX10-mediated differentiation followed by activation of new signaling pathways, partially mediated by activity of Jun-AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics. We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general rare-cell expression program.
Project description:Therapies targeting signaling molecules mutated in cancers can often have striking short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures. Resistance can sometimes result from a secondary mutations in rare cells, but other times, there is no clear genetic cause, raising leaving the possibility of non-genetic rare cell variability. Here, we show that melanoma cells can display profound transcriptional variability at the single cell level that predicts which cells will ultimately resist drug treatment. This variability involves semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces an epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming begins with a progressive process consisting of a loss of SOX10-mediated differentiation followed by activation of new signaling pathways, partially mediated by activity of Jun-AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics. We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general rare-cell expression program.
Project description:Drug resistance has remained an important issue in the treatment and prevention of various diseases, including cancer. Herein, we found that USP24 not only repressed DNA-damage repair (DDR) activity by decreasing Rad51 expression to cause the tumor genomic instability and cancer stemness, but also increased the levels of the ATP-binding cassette (ABC) transporters P-gp, ABCG2, and ezrin to enhance the pumping out of Taxol from cancer cells, thus resulted in drug resistance during cancer therapy. A novel USP24 inhibitor, NCI677397, was screened for specific inhibiting the catalytic activity of USP24. This inhibitor was identified to suppress drug resistance via decreasing genomic instability, cancer stemness, and the pumping out of drugs from cancer cells. Understanding the role and molecular mechanisms of USP24 in drug resistance will be beneficial for the future development of a novel USP24 inhibitor. Our studies provide a new insight of USP24 inhibitor for clinically implication of blocking drug resistance during chemotherapy.
Project description:Gastric cancer is the fourth most common malignancy and the third leading cause of cancer-related deaths worldwide. Advanced gastric cancer patients can notably benefit from chemotherapy including adriamycin, platinum drugs, 5-fluorouracil, vincristine, and paclitaxel as well as targeted therapy drugs. Nevertheless, primary drug resistance or acquisition drug resistance eventually lead to treatment failure and poor outcomes of the gastric cancer patients. The detailed mechanisms involved in gastric cancer drug resistance have been revealed. Interestingly, different noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), are critically involved in gastric cancer development. Multiple lines of evidences demonstrated that ncRNAs play a vital role in gastric cancer resistance to chemotherapy reagents and targeted therapy drugs. In this review, we systematically summarized the emerging role and detailed molecular mechanisms of ncRNAs impact drug resistance of gastric cancer. Additionally, we propose the potential clinical implications of ncRNAs as novel therapeutic targets and prognostic biomarkers for gastric cancer.
Project description:Therapies that target signalling molecules that are mutated in cancers can often have substantial short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures. Resistance can result from secondary mutations, but in other cases there is no clear genetic cause, raising the possibility of non-genetic rare cell variability. Here we show that human melanoma cells can display profound transcriptional variability at the single-cell level that predicts which cells will ultimately resist drug treatment. This variability involves infrequent, semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming begins with a loss of SOX10-mediated differentiation followed by activation of new signalling pathways, partially mediated by the activity of the transcription factors JUN and/or AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics in single cells. We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general program in which expression is displayed in rare subpopulations of cells.