Purinergic Ca2+ Signaling as a Novel Mechanism of Drug Tolerance in BRAF-Mutant Melanoma

Simple Summary The treatment of BRAF-mutant melanoma with BRAF inhibitors is severely limited in clinical practice, in part due to the emergence of drug tolerance via non-genetic adaptation to therapies. Improving our understanding of the molecular mechanisms that underlie drug tolerance may lead to improved treatment strategies. Here, we describe a novel calcium-dependent signaling mechanism induced by BRAF inhibitor (BRAFi) treatment that provides compensatory mitogenic signaling to drug-tolerant persister cells in the form of MAPK reactivation. This calcium signaling mechanism has not previously been recognized in BRAFi-tolerant melanoma, may initiate a novel line of scientific inquiry, and presents a host of novel targets for therapeutic development in this field. Abstract Drug tolerance is a major cause of relapse after cancer treatment. Despite intensive efforts, its molecular basis remains poorly understood, hampering actionable intervention. We report a previously unrecognized signaling mechanism supporting drug tolerance in BRAF-mutant melanoma treated with BRAF inhibitors that could be of general relevance to other cancers. Its key features are cell-intrinsic intracellular Ca2+ signaling initiated by P2X7 receptors (purinergic ligand-gated cation channels) and an enhanced ability for these Ca2+ signals to reactivate ERK1/2 in the drug-tolerant state. Extracellular ATP, virtually ubiquitous in living systems, is the ligand that can initiate Ca2+ spikes via P2X7 channels. ATP is abundant in the tumor microenvironment and is released by dying cells, ironically implicating treatment-initiated cancer cell death as a source of trophic stimuli that leads to ERK reactivation and drug tolerance. Such a mechanism immediately offers an explanation of the inevitable relapse after BRAFi treatment in BRAF-mutant melanoma and points to actionable strategies to overcome it.

Figure S2.Spiking activity can be qualitatively grouped by spike pattern.Fura-2 Ca 2+ imaging (60 minutes) of A375 cells treated with 8 µM PLX4720 for 14 days.Four spike patterns became apparent upon close examination and are represented above.All pulsing cells with more than two spikes were manually gated into one of these four groups.Notably, some traces have mixes of spike patterns (i.e.high frequency bursts may repeat at regular frequencies and/or have single spikes interspersed).Cells with only one or two spikes were not grouped in the above and accounted for approximately 38% of the spiking population.Traces presented here are representative of the population, to the best of our ability.
Figure S3.Spiking activity in drug-sensitive cells is minimal.Proportion of spiking A375 cells after 60 minutes of BRAFi is approximately the same proportion found at 0 days BRAFi in Fig. 1C.Data was acquired using calbryte-520.Traces were manually analyzed for spiking activity.Bootstrapping was performed to generate proportions of spiking cells presented in the figure .Figure S4.Spiking activity in drug-tolerant cells treated for 8 days relies on extracellular Ca 2+ .A375 cells were treated with 8 µM PLX4720 for indicated periods of time and imaged with Fura-2 for 40 minutes.7 days of BRAFi treatment were taken from Fig. 1C for comparison to 8 days BRAFi without Ca 2+ ; since 10 days BRAFi has a similar proportion spiking as 7 days (Fig. 1C), we consider comparing the above conditions is appropriate.Ca 2+ -free HBSS was used as the imaging and wash buffer for the no Ca 2+ condition.
Figure S5.Single cell Ca 2+ traces in store operated Ca 2+ entry (SOCE) assays performed on BRAFi naive and 3 day BRAFi treated cells.Cells were loaded with the Ca 2+ dye Fura-2-AM and equilibrated in Ca 2+ -free buffer before the SERCA inhibitor, cyclopiazonic acid (CPA), was added to release free ER Ca 2+ into the cytoplasm.Quantification of ER Ca 2+ levels occurs during the phase labeled "CPA".Seven minutes after CPA treatment, extracellular Ca 2+ is added to measure SOCE activity.Quantification of SOCE activity levels occurs during the phase labeled "Ca2+ add".Cytoplasmic Ca 2+ clearance can also be calculated from the single-cell traces during the "CPA" phase.

Figure S7 .
Figure S7.Volcano plot of up-and down-regulated genes in drug-tolerant versus BRAFi-naive melanoma cells.Melanoma cells were treated with 8 µM BRAFi for 8 days (drug-tolerant) or left untreated (naive).Whole transcriptome analysis (bulk RNAseq) was performed on four BRAF mutant melanoma cell lines and analyzed together.Colors indicate distinct classes of genes, related to Ca 2+ transport and signaling, melanocyte differentiation, Jackpot cells, and a signature of MEK-dependent signaling.

Figure S8 .
Figure S8.Volcano plot of up-and down-regulated genes in melanoma cells under BRAFi conditions for 8 days, a treatment previously shown to induce an idling population of drug-tolerant cells.Data are from bulk RNAseq of four BRAF mutant melanoma cell lines that were tested.Highlighted are genes related to Ca 2+ and monoatomic cation channels (GO 5261).

Figure S9 .
Figure S9.TCGA data demonstrating high levels of P2RX7 expression in melanoma.Figures generated by the Human Protein Atlas.Top mRNA expression levels in different cancer types.Middle Antibody (HPA034968) detection of P2X7 in patients with different cancers.9 out of 11 patients showed medium to high expression, and all 11 patients showed at least low levels of expression.Bottom Survivorship curves of patients with high or low mRNA expression as determined by a cutoff value of 23.34 FPKM.

Figure S10 .
Figure S10.ATP stimulation generates spikes of Ca 2+ in drug tolerant cells.Representative traces from ATP stimulated (39 µM) Ca 2+ imaging in drug-tolerant A375 cells.Data traces are recorded over 5 minutes with an image recorded every 5 seconds.These cells were selected to be representative of those observed to experience obvious Ca 2+ spikes.

Figure S11 .
Figure S11.ATP stimulation of drug-tolerant idling cells.A375 cells treated with 8 µM PLX4720, or vehicle, for 8 days were imaged with Fura2.A background was recorded for 3 minutes before 10 mM ATP was added on top of cells (arrow).Average Ca 2+ trace with 95% confidence intervals are displayed for all cells imaged.

Figure S12 .
Figure S12.CPA induces ERK phosphorylation in drug-tolerant, but not drug-sensitive cells.Drug-sensitive (60 min BRAFi) (top) and drug-tolerant (3 days BRAFi) (bottom) A375 cells were treated with 50 µM CPA to induce a cytoplasmic Ca 2+ signal.Cells were incubated in CPA, fixed with 4% PFA, and stained for ppERK.Cells without detectable ppERK staining were given a value of -0.5 and the minimum ppERK level in the positive cells was set to 0. Drug-sensitive cells displayed little to no ppERK activation, whereas drug-tolerant cells did, as expected.

Figure S13 .
Figure S13.Cell lysate stimulates ppERK in drug-tolerant but not drug sensitive cells.Drug naive cells were trypsinized, washed, and boiled at 98 o C for 10 minutes, and centrifuged at 20,000xg for 5 minutes.Supernatant were diluted and incubated on drug-tolerant (3 days BRAFi) or drug-sensitive (60 min BRAFi) cells for 20 minutes before fixation and staining for ppERK.Quantification of the staining intensity (normalized to 0) are shown on the right, on each bipartite density distribution.

Figure S14 .
Figure S14.Drug-tolerant cells do not tolerate Ca 2+ free conditions.(Left) Drug-tolerant A375 cells (8 days BRAFi) immediately after being switched to Ca 2+ free imaging buffer (HBSS).(Right) Those same cells after 40 minutes in Ca 2+ free HBSS.Images were captured using Fura2 and are representative of the population.Cells do not ball up when imaged in Ca 2+ :HBSS, suggesting this effect is not related to imaging and/or phototoxicity.

Figure S15 .
Figure S15.Quantification of single cell SOCE Ca 2+ traces.Data represents quantification of max Ca 2+ levels subtracted from lowest during ER release phase.A375 cells were treated for indicated periods of time with 8 µM PLX4720.