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- All Subjects: Melanoma
- Creators: Wilson Sayres, Melissa
- Creators: Bertram, Jacobs
Description
While specific resistance mechanisms to targeted inhibitors in BRAF-mutant cutaneous melanoma have been identified, surprisingly little is known about the rate at which resistance develops under different treatment options. There is increasing evidence that resistance arises from pre-existing clones rather than from de novo mutations, but there remains the need for a better understanding of how different drugs affect the fitness of clones within a tumor population and promote or delay the emergence of resistance. To this end, we have developed an assay that defines the in vitro rate of adaptation by analyzing the progressive change in sensitivity of a melanoma cell line to different treatments. We performed a proof-of-theory experiment based on the hypothesis that drugs that cause cell death (cytotoxic) impose a higher selection pressure for drug-resistant clones than drugs that cause cell-cycle arrest (cytostatic drugs), thereby resulting in a faster rate of adaptation. We tested this hypothesis by continuously treating the BRAFV600E melanoma cell line A375 with the cytotoxic MEK inhibitor E6201 and the cytostatic MEK inhibitor trametinib, both of which are known to be effective in the setting of constitutive oncogenic signaling driven by the BRAF mutation. While the identification of confounding factors prevented the direct comparison between E6201-treated and trametinib-treated cells, we observed that E6201-treated cells demonstrate decreased drug sensitivity compared to vehicle-treated cells as early as 18 days after treatment begins. We were able to quantify this rate of divergence at 2.6% per passage by measuring the increase over time in average viability difference between drug-treated and vehicle-treated cells within a DDR analysis. We argue that this value correlates to the rate of adaptation. Furthermore, this study includes efforts to establish a barcoded cell line to allow for individual clonal tracking and efforts to identify synergistic and antagonist drug combinations for use in future experiments. Ultimately, we describe here a novel system capable of quantifying adaptation rate in cancer cells undergoing treatment, and we anticipate that this assay will prove helpful in identifying treatment options that circumvent or delay resistance through future hypothesis-driven experiments.
ContributorsDe Luca, Valerie Jean (Author) / Wilson Sayres, Melissa (Thesis director) / Trent, Jeff (Committee member) / Hendricks, William (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Unlike the autosomes, recombination on the sex chromosomes is limited to the pseudoautosomal regions (PARs) at each end of the chromosome. PAR1 spans approximately 2.7 Mb from the tip of the proximal arm of each sex chromosome, and a pseudoautosomal boundary between the PAR1 and non-PAR region is thought to have evolved from a Y-specific inversion that suppressed recombination across the boundary. In addition to the two PARs, there is also a human-specific X-transposed region (XTR) that was duplicated from the X to the Y chromosome. Genetic diversity is expected to be higher in recombining than nonrecombining regions, particularly because recombination reduces the effects of linked selection, allowing neutral variation to accumulate. We previously showed that diversity decreases linearly across the previously defined pseudoautosomal boundary (rather than drop suddenly at the boundary), suggesting that the pseudoautosomal boundary may not be as strict as previously thought. In this study, we analyzed data from 1271 genetic females to explore the extent to which the pseudoautosomal boundary varies among human populations (broadly, African, European, South Asian, East Asian, and the Americas). We found that, in all populations, genetic diversity was significantly higher in the PAR1 and XTR than in the non-PAR regions, and that diversity decreased linearly from the PAR1 to finally reach a non-PAR value well past the pseudoautosomal boundary in all populations. However, we also found that the location at which diversity changes from reflecting the higher PAR1 diversity to the lower nonPAR diversity varied by as much as 500 kb among populations. The lack of genetic evidence for a strict pseudoautosomal boundary and the variability in patterns of diversity across the pseudoautosomal boundary are consistent with two potential explanations: (1) the boundary itself may vary across populations, or (2) that population-specific demographic histories have shaped diversity across the pseudoautosomal boundary.
ContributorsCotter, Daniel Juetten (Author) / Wilson Sayres, Melissa (Thesis director) / Stone, Anne (Committee member) / Webster, Timothy (Committee member) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Melanoma is a type of skin cancer that can metastasize in advanced stages to other organs such as the brain, lymph nodes, lungs and liver. Current standard treatment options include surgery, radiation therapy, chemotherapy, and immunotherapy. More recently, oncolytic virotherapy is being studied as a new strategy to fight cancer. Specifically, for melanoma, a herpes virus (T-VEC) was approved by the U.S Food and Drug Administration in 2015 to treat advanced disease. Oncolytic viruses have the capacity to replicate mostly in cancer cells while leaving healthy somatic cells free from infection. Additionally, most of these viruses have the ability to induce an immune response against the cancer as well. Myxoma virus (MYXV) causes myxomatosis in European rabbits but not in any other mammal. In humans, MYXV can infect and kill cancer cells acting as an oncolytic virus. However, the mechanisms behind how myxoma kills cancerous cells are not completely known. To investigate this, we treated melanoma murine cancer cells (B16F10) in vitro with different genetically modified myxoma virus mutants, as well as with a novel second mitochondria-derived activator of caspase mimicking drug SMAC-LCL161, to understand the mechanisms by which MYXV induces cell death. In parallel, B16F10 lacZ cells were subcutaneously injected into mice to engraft melanoma tumors. These tumors were treated with intratumoral injections of different viral mutants or armed viruses derived from MYXV along with SMAC-L61. After a period of treatment, the tumors were isolated. Cell death pathways in both cell culture and in tumors obtained from subcutaneous pathways were identified using different techniques. The study showed an increase in activated caspase 3 and cleaved PARP-1 activity in B16F10 lacZ cells from cell culture when compared to cells in vivo however the two apoptosis markers did not track with each other consistently.
ContributorsKien, Cassandra T (Author) / McFadden, Grant (Thesis director) / Franco Achury, Lina (Committee member) / Bertram, Jacobs (Committee member) / Hugh Downs School of Human Communication (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-12