Abstract B32: Single-cell non-genetic heterogeneity confers drug resistance in melanoma
Authors: Shaffer Sydney M. , Biaesch Andrew G. , Torborg Stefan , Krepler Clemens , Herlyn Meenhard , Raj Arjun 
Published: May 2016

Melanoma is the most aggressive and deadliest form of skin cancer. Historically, treatment has consisted of harsh chemotherapeutics that kill all rapidly proliferating cells in the body. In 2011, however, the first targeted therapy, named vemurafenib, was approved for metastatic melanoma. While this drug had an unprecedented success at shrinking tumor mass, drug resistance arose in the majority of patients. Importantly, the fact that patients initially respond positively to this drug suggests that the majority of the tumor is drug sensitive (and therefore killed by the drug); however, the fact that the tumor returns suggests that a few cells survive the drug treatment and give rise to resistance.

Generally, researchers believe that drug resistance has a genetic basis, in which cells survive drug treatment because they have a secondary mutation to overcome the drug effects. In this framework, resistance is a Darwinian trait, passed on during each cell division. An alternative hypothesis is that drug resistance is Lamarckian and comes from a non-heritable or reversible phenotype, such as a transient cell state. Using patient derived melanoma cell lines, we tested these hypotheses to determine if resistance to vemurafenib is Darwinian or Lamarckian. Drawing experimental inspiration from Luria and Delbruck with E. coli and resistance to T1 phage, we found that resistance to vemurafenib is not heritable prior to treatment with drug, consistent with a Lamarckian hypothesis. This result suggests that resistance is not mediated by a genetic change, but rather that vemurafenib resistance is transient and not heritable.

With these phenomenological results in hand, we then sought to characterize the molecular underpinnings of resistance. First, we performed high throughput RNA sequencing on 38 resistant melanoma colonies and 20 non-resistant clones to identify alterations in gene expression that are associated with drug resistance. Next, we used high throughput imaging and iterative cycles of RNA FISH in situ hybridizations to measure the resistance marker transcriptome for &gt;10,000 individual melanoma cells at multiple time points after drug treatment. In drug naive cells, we find that there is a sub-population of individual cells that already express these resistance markers and that there is tremendous single-cell heterogeneity in expression for these resistance markers. For example, if the mean expression level for a population is 0-2 RNA per cell, we also find a few cells (1:100 to 1:2000) that have hundreds of the same RNA. Surprisingly, we find that some of these cells have many of the resistance markers suggesting that the resistance state is coordinated, while other cells in the same population can have high levels of only one marker. These experiments lead us to EGFR as a marker of pre-existing resistance and a known vemurafenib resistance mechanism in literature. Taken together, our results suggest that random, but coordinated fluctuations in gene expression allow melanoma cells to overcome vemurafenib treatment, potentially giving rise to disease relapse in vivo.

Citation Format: Sydney M. Shaffer, Andrew G. Biaesch, Stefan Torborg, Clemens Krepler, Meenhard Herlyn, Arjun Raj. Single-cell non-genetic heterogeneity confers drug resistance in melanoma. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B32.