KB, ask all the questions you want, my man. There is a very good chance you are asking questions other people are thinking, and if I can't explain it simply, I don't know it well enough.
1. The focus is on increasing m6A levels, which can be done by the inhibition of FTO (block the action of the protein) or by FTO knockdown (decrease the protein concentration). Lower levels of m6A drives cellular activity that promotes the hallmarks of cancer, while higher levels of m6A impair the hallmarks of cancer. FTO inhibition/knockdown increases m6A levels, whereas FTO overexpression decreases m6A levels. If a methyl group is present on the 6th Adenosine of mRNA strands, the strand is read and translates to particular proteins being expressed driving cellular function. If a methyl group is NOT present on the 6th Adenosine of mRNA strands, the strand is read and translates to particular proteins expressed driving cellular function. The expression of 1000s of proteins is regulated by whether a methyl group IS or IS NOT present on the 6th Adenosine of mRNA strands. Bisantrene is altering the levels of m6A in cancer cells through the inhibition of FTO, which - because m6A modifications are the most abundant internal modification - influences a broad range of cellular activity.
2. I think I understand what you are asking, and I don't think it is relevant. We need to appreciate that there is a big difference between the complete inhibition of FTO in cancer cells that depend on increased demethylase action for survival and the upregulation of writer complexes to increase m6A levels fighting the effects of FTO-driven hyperdemethylation. For example, the folllowing question highlights the difficulty in conceptualising this line of thinking: what fold increase in writers would need to be achieved to overcome the concentration of FTO to adjust m6A levels enough to sufficiently alter cellular activity sensitizing it to combination treatment?
For an analogy, if you're stuck in a cold room, inhibiting FTO is like turning off the power to the air conditioner, which stops the cooling immediately. On the other hand, overexpressing writer complexes is like adding more heaters to the room; however, you'd need to add enough heaters to counteract the continuous cooling effect of the still-running air conditioner.
3. FTO inhibition is measured via m6A levels. If there is intrinsic upregulation of proteins that adjusts m6A levels after Bisantrene treatment, it will not happen instantly, thus the changes in m6A levels following dosing will be a direct result of Bisantrene. STORM therapeutics have already proven a METTL3 inhibitor decreases m6A levels. I am confident we will see an increase in m6A levels following Bisantrene dosing.
Regarding anti-cancer efficacy, it's important to remember that a cancer cell has never had its FTO activity halted. If we think of a cancer cell as a city, FTO would be its power plant. The power plant runs the outer defense (immune checkpoint genes), controls the traffic in and out (influx and efflux transporters), supports growth (glycolytic activity), manages hospitals (internal repair systems and anti-apoptosis), and oversees many other key functions for the city. Bisantrene acts by cutting the power to the power plant, crippling the city. We've observed that cutting the power to the power plant once every three weeks isn't very effective, but when you cut the power for multiple consecutive days, chaos ensues. When you cut the power to the power plant AND send in the bombers (another chemotherapy), you get?
The sensitivity of FTO inhibitor/knockdown models in vitro, in vivo, and, in Bisantrenes case, in humans suggests cancer cells are not prepared to manage sudden ablation of FTO activity.
4. There will be a fair amount of testing done to establish an understanding of m6A levels and response to treatment. For example, does a larger change in m6A levels following Bisantrene (indicating patients with higher FTO levels) dosing correlate with better clinical efficacy, or does the change in m6A level not correlate with clinical responses, and it's moreso high m6A levels that matters. I think the latter is actually the most exciting part about Bisantrene in combination with other drugs, as this indicates high levels of m6A is the driver of synergy rather than the expression of FTO (much larger TAM). I suspect in vitro and in vivo testing of Bisantrene has demonstrated high m6A levels and not FTO expression (or m6A change) have correlated with efficacy, and this is why they are measuring this in patients. RAC do not do things without a very clear reason for doing so.
If there is another mechanism of anti-cancer efficacy or synergy, then we will have to explore that when the time comes.
Something I think you should spend some time on, KB, is reviewing the FTO-inhibition-associated synergy with cardiotoxic regimens in decreasing HCM viability. You have a heart cell with relatively normal levels of FTO and, as a result, m6A. When you inhibit FTO in these cells, there is an increase in m6A but no negative impact on HCM viability. However, when you inhibit FTO, increasing m6A, while also adding a cardiotoxic regimen, it synergises to decrease HCM viability. This is an example of where the difference in m6A levels prior to and following FTO inhibitor dosing may be small (since FTO is expressed at normal levels), but the increase in m6A is what drives the synergy with the cardiotoxic drug. Therefore, it is the level of m6A that correlates and not the change. I've added a figure below that highlights increasing m6A methylation levels in doxorubicin-treated heart cardiomyocytes. The question to ask is how many cancer patients with normal levels of FTO who were treated with Bisantrene did not respond, but would have been sensitive to combination therapy? We will see what translates to humans in time.
5. Daniel can definitely answer the measurement of m6A in cancer better than I can, but I have my ideas.
What I can say is FTO is expressed differently in the cells of the body, which influences m6A levels. It is a dynamic system that is always shifting and changing in response to stimuli.
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