Behind the Study: How ONC201 Kills Breast Cancer Cells

Scientists discuss their findings in a recently published paper in Oncotarget entitled, “ONC201 kills breast cancer cells in vitro by targeting mitochondria.” Oncotarget’s Behind The Study v1 The Behind the Study series transcribes videos of chosen researchers elaborating on their recent papers published in Oncotarget. Visit the Oncotarget YouTube channel for more insights from outstanding authors. Oncotarget Stan Lipkowitz Hello, I’m Stan Lipkowitz, the Branch Chief of the Women’s Malignancies Branch at the National Cancer Institute. Yoshimi Greer Hi, I’m Yoshimi Greer, Staff Scientist in Women’s Malignancies Branch, National Cancer Institute, NIH, Bethesda, Maryland. I’m the first author of the paper. Stan Lipkowitz So today we’d like to talk about our work with a novel class of drugs first described as a drug called ONC201 and a paper we published (in Oncotarget) on that demonstrating a novel mechanism of action, whereby the drugs target the mitochondria to kill breast cancer cells. Now, our interest in this drug really derives from a very longstanding interest that we’ve had in studying trail mediated apoptosis through death receptors in breast cancer cells. And so we read with a lot of interest, the paper published by Rafi Galderie’s group, which described a small molecule, which they called ONC201, which activated transcription of trail in the cancer cells. And then the cells would produce their own trail and kill themselves via the trail receptors and induction of apoptosis. So this, to us, was a novel way of inducing the trail system in breast cancer. But when we began studying it, one of the very first things we found, and this is described in both figure one and the supplementary figure one in our paper, was that the trail pathway didn’t seem to be involved at all. In fact, we don’t think that apoptosis is the mechanism of action of this drug. So in particular, what we found is that the drug did not activate cast bases. If you inhibited cast bases with a pan cast based inhibitor, the drug still killed cells, whereas trail activates cast bases and of course isn’t inhibited by cast base inhibitors. Facebook Oncotarget Furthermore, we knocked out the trail receptors and the drug still killed the cells. And again, trail of course can’t kill the cells if these receptors are gone. So this left us with a very interesting observation. We had a drug that killed breast cancer cells, and it seemed to kill all types of breast cancer cells, but we didn’t know the mechanism of action. And at this point, I’ll turn to Yoshimi and let her describe how we’ve discovered the mechanism of action for these drugs. Yoshimi Greer Okay, thank you, Stan. So as Stan mentioned, we did not observe any evidence that ONC201 kills breast cancer cells trail to receptor mechanisms. So we tested if other cells is that pathway such as necrosis, autophages are induced by ONC201. However, as shown in our supplementary figure, we did not detect any evidence of necrosis nor autophagy. This made us wonder what signaling pathway is dysregulated by ONC201. One at that time, two independent groups reported that stress markers, such as ATF4, CHOP are induced by ONC201 in multiple cancer cell lines with unknown mechanism. Consistent with these papers we observed that ATF4 and CHOP are induced by ONC201 in our breast cancer cell lines as shown in figure two. Then I wondered, what makes cancer cells so stressed out? Looking at the cells under a microscope every day, I thought these cells look literally tired. So then I wondered, maybe these cells are out of energy after ONC201 treatment. When it comes to energy and MPK, the MP activated protein kinase is known as an energy sensor. As you know, ATP is the energy source for every living cell. When ATP level is low in the cell, MP can sense this, that energy deficit, and gets activated. As shown in figure two, we detected AMPK phosphorylation was induced by ONC201 in parallel with induction of the stress markers ATF4, and CHOP. This data indicated that cells are facing low ATP level after over ONC201 treatment. I think this MPK activation finding was a critical starting point of this entire story. So that was good. We reconfirmed that cellular ATP level is depleted by ONC201 treatment in multiple breast cancer cell lines. At this point, it became clear that energy deficit seems to contribute to cell death in breast cancer cells. But the next question was: how ONC201 reduces the cellular ATP? In a cell, ATP is generated by two mechanisms. One is glycolysis, the other is mitochondrial respiration by electron transport chain. To dissect these two sources of ATP, we tested the effects of ONC201 in the presence or absence of glucose. In absence of glucose, cells can only rely on mitochondrial respiration as a source of cellular ATP. As a result, as shown in figure two cytotoxic effect was significantly enhanced in absence of glucose. This indicated that ONC201 targets mitochondria ATP production. Then we investigated that effect of ONC201 in mitochondria with multiple different approaches. First we used Seahorse XF analyzer, and confirmed that ONC201 inhibits mitochondrial respiration as shown in figure three. Interestingly, the inhibitory effect of ONC201 was not as rapid as aribamyosin that directly inhibits complex five in electron transport chains. It required a few hours to see the inhibitory effect of ONC201. This suggested that ONC201 does not directly target electron transport chains. Hospital We also applied isolated mitochondria and confirmed that ONC201 does not induce acute changes in mitochondrial respiration. We also looked at the impact of ONC201 on mitochondrial structures by multiple imaging analysis. As shown in figure four, we detected that ONC201 induces mitochondrial fragmentation and loss of mitochondrial membrane potential. Electron microscopy imaging revealed significant mitochondrial structure damages, such as matrix lysis, and necrocrystalysis, mitochondrial swelling. These mitochondrial damages were detected as early as three hours after ONC201 treatment. Interestingly, electron microscopy examination revealed that other cellular organelles such as endoplasmic reticulum, golgi nucleus remained intact until much later time point. This observation further suggested that ONC201 is specifically targeting mitochondria. To test his hypothesis, we looked at mitochondrial DNA as a specific marker of mitochondria. As shown in figure five, PicoGreen is a fluorescent molecule that visualized both nuclear and the mitochondrial DNA, but ONC201 treatment decreased PicoGreen signal only in mitochondria. Using the quantitative PCR, we confirmed that ONC201 depletes mitochondrial DNA copy number, and this effect was specific to ONC201 and it was not observed with other mitochondrial target drugs, such as aribamyosin, metformin. Next, we questioned, what about the effect of ONC201 on known cancer cells? He tested over the one on human foreskin fibroblasts HFF cells as shown in figure six. HFF cells were ONC201 resistant despite mitochondrial structural damages and the mitochondrial DNA depletion. Further studies showed that HFF cells are not highly dependent on mitochondrial respiration. Houston hospital Therefore, targeting mitochondria does not affect cell value viability HFF cells. This observation prompted the next step. Lastly, we hypothesized that cancer cells not dependent on mitochondrial respirations are ONC201 resistant. To test this, we used multiple models. One was renal cancer cell line UK262 FH negative. Exclusively, glycolysis dependence line due to genetic mutation of fumarate hydrotase, an enzyme in TCA cycle. Other model was breast cancer cells that lack mitochondrial DNA as shown in figure seven. These cell lines were not dependent on mitochondrial respiration and completely ONC201 resistant supporting our idea that ONC201 is specifically targeting mitochondria. In conclusion, we confirmed that ONC201 kills breast cancer cells by targeting mitochondria. Last year in 2019, two groups reported that ONC201 targets mitochondria as an agonist of mitochondrial matrix protease. These studies further solidified our discovery. With this, I would like to hand this over to Stan to summarize the remaining questions we have elected to address.