(419d) Systematic Analysis of Adaptive Resistance and Fractional Responses of Melanoma Cancer Cells to RAF/MEK Inhibition

Activation of BRAF via a V600E mutation is the most prevalent genetic change in human melanoma (the deadliest form of skin cencaer), found in >50% of tumors, and induces constitutive activation of pro-mitogenic RAF/MEK/ERK signaling. Treatment of BRAF^V600E melanomas with drugs, such as vemurafenib, that inhibit RAF/MEK signaling is effective in the short term, but remission is not durable. Drug resistance appears to involve short-term adaptive responses that compensate for RAF/MEK inhibition via up-regulation of other pro-growth mechanisms. Understanding and ultimately preventing adaptive responses is key to durable therapy. Systematic data comparing BRAF^V600E tumor cells is generally lacking and it is not known whether adaptation is fundamentally similar across cell types or among individual cells within a cell population.

We apply a systematic approach to studying the responses of human melanoma cell lines to five RAF and MEK inhibitors, with the overall goal of (i) characterizing variability in adaptation with time, dose, cell type and across individual cells, (ii) discovering new or poorly characterized adaptive mechanisms, and (iii) demonstrating the effectiveness of a high-throughput approach involving multiplex measurement, single-cell analysis and computational modeling. The data involves time-course measurement of total level and activity of signaling proteins and cell state markers using array-based methods and single-cell immunofluorescence assays as well as measurement of apoptosis and cell viability under the same conditions. Statistical modeling using partial least squares regression (PLSR) revealed which of the changes in the ~200,000 point dataset were phenotypically consequential.

We found that responses to RAF inhibitors are remarkably diverse and involve multiple pathways that can be up or down-regulated over time, with significant variability across cell types and individual cells. We identified a role for JNK/c-Jun signaling in altering the cell-cycle distribution of melanoma cells, causing apoptosis-resistant cells to accumulate and drug maximal effect (Emax) to fall; co-drugging with RAF and JNK inhibitors or JUN knockdown reverse this effect. Our study shows that a systems-level approach (combining high density time-dependent measurements, quantitative modeling and single-cell analysis) may provide a general framework for evaluating new drugs with adaptive and paradoxical response, and identifying potentially useful combination therapies.