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Creators: School of Molecular Sciences
Creators: Bond, Angela
Member of: Barrett, The Honors College Thesis/Creative Project Collection

Evading resistance: measuring melanoma's adaptation rate in different drug environments to identify the best course of treatment

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

Plasmodium Cost of Resistance and Life Stage Development within the Mosquito Vector

Description

Hundreds of thousands of people die annually from malaria; a protozoan of the genus Plasmodium is responsible for this mortality. The Plasmodium parasite undergoes several life stages within the mosquito vector, the transition between which require passage across the lumen of the mosquito midgut. It has been observed that in about 15% of parasites that develop ookinetes in the mosquito abdomen, sporozoites never develop in the salivary glands, indicating that passage across the midgut lumen is a significant barrier in parasite development (Gamage-Mendis et al., 1993). We aim to investigate a possible correlation between passage through the midgut lumen and drug-resistance trends in Plasmodium falciparum parasites. This study contains a total of 1024 Anopheles mosquitoes: 187 Anopheles gambiae and 837 Anopheles funestus samples collected in high malaria transmission areas of Mozambique between March and June of 2016. Sanger sequencing will be used to determine the prevalence of known resistance alleles for anti-malarial drugs: chloroquine resistance transporter (pfcrt), multidrug resistance (pfmdr1) gene, dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr). We compare prevalence of resistance between abdomen and head/thorax in order to determine whether drug resistant parasites are disproportionately hindered during their passage through the midgut lumen. A statistically significant difference between resistance alleles in the two studied body sections supports the efficacy of new anti-malarial gene surveillance strategies in areas of high malaria transmission.

ContributorsPhillips, Keeley Isabella (Author) / Huijben, Silvie (Thesis director) / Gile, Gillian (Committee member) / Young, Steven (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)

Created2021-05

Aedes aegypti Thermal Choice Experiment

Description

The non-native mosquito Aedes aegypti has become a common nuisance in Maricopa county. Associated with human settlement, Ae. aegypti is known to reproduce in standing water sources both indoors and outdoors, within vessels such as tires, flowerpots, and neglected swimming pools (Jansen & Beebe, 2010). Ae. aegypti and the related Ae. albopictus are the primary vectors of the arboviral diseases chikungunya, Zika, yellow fever and dengue. Ae. aegypti tends to blood feed multiple times per gonotrophic cycle (cycle of feeding and egg laying) which, alongside a preference for human blood and close association with human habitation, contributes to an increased risk of Ae. aegypti borne virus transmission (Scott & Takken, 2012). Between 2010-2017, 153 travel-associated cases of dengue were reported in the whole of Arizona (Rivera et al., 2020); while there have been no documented locally transmitted cases of Aedes borne diseases in Maricopa county, there are no apparent reasons why local transmission can’t occur in the future via local Aedes aegypti mosquitoes infected after feeding from travelling viremic hosts. Incidents of local dengue transmission in New York (Rivera et al., 2020) and Barcelona (European Center for Disease Control [ECDC], 2019) suggest that outbreaks of Aedes borne arbovirus’ can occur in regions more temperate than the current endemic range of Aedes borne diseases. Further, while the fact that Ae. aegypti eggs have a high mortality rate when exposed to cold temperatures limits the ability for Ae aegypti to establish stable breeding populations in temperate climates (Thomas, Obermayr, Fischer, Kreyling, & Beierkuhnlein, 2012), global increases in temperature will expand the possible ranges of Ae aegypti and Aedes borne diseases.

ContributorsHon, Ruiheng (Author) / Paaijmans, Krijn (Thesis director) / Bond, Angela (Committee member) / Angilletta, Michael (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)

Created2020-05