I’ve been away a while, but I’m here, don’t you worry.

Let’s continue on in our analyses, starting with another sensitisation study. Spoilers, FBH1 sensitises cells to WEE1 inhibition. Ring any bells?

IMAGE: Figure 3 from Jennings et al.’s 2024 study that we’ll summarise and discuss. HINT: take a look between the U2OS and FBH1 KO cell lines with and without WEE1 inhibition. Similar results, aren’t they… (Jennings et al., 2024).

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A quick note before we get into it: this paper was not as clear and plain as previous papers, so bear with me. It may be a bit more technical than others, but I am certainly trying to make it understandable (Jennings et al., 2024).

What is WEE1?

WEE1 is a kinase involved in the activation of the G2/M checkpoint via CDK activity. CDKs are cyclin-dependent kinases, which are a group of kinases involved in cell cycle signalling; they act on cyclin proteins. Together cyclins and CDKs regulate cell cycle progression and cell proliferation. We’ve previously discussed the G2 decatenation checkpoint, but here we’re talking about the G2 DNA damage checkpoint. 

The DNA damage checkpoint is often referred to as the G2/M checkpoint despite there being another checkpoint for decatenation. From what I’ve seen, the DNA damage checkpoint is more the focus, and there isn’t as much known about the decatenation checkpoint. The decatenation checkpoint is a newer discovery. 

DNA damage activates WEE1, which phosphorylates tyrosine 15 (Y15) in CDK1 and CDK2; this leads to cell cycle arrest at the G2/M checkpoint. If there is no DNA damage, Y15 phosphorylation is removed by CDC25 phosphatase to allow mitosis entry. WEE1 action controls replication in S phase via CDK2 activity.

Inhibition of WEE1 results in unscheduled replication initiation and depletes nucleotide pools. When WEE1 is inhibited, the G2/M checkpoint also would not activate when DNA damage is present, meaning that replication and mitotic catastrophe should occur (after which the cell dies). WEE1 inhibition (WEE1i) is an attractive chemotherapy target due to it’s role in G2/M and S phase. 

What is FBH1? 

FBH1 is a helicase important in apoptosis induction during replication stress and also has a role in replication fork reversal in response to hydroxyurea. 

FBH1 works with MUS81 to induce double-stranded breaks (DSBs) at stalled forks in order to start apoptosis during high replication stress. Previous studies have found that FBH1-deficient cells have decreased DSBs (less apoptosis initiation) and increased survival to hydroxyurea (a chemotherapy). FBH1 has a role in protecting melanocytes from UV-mediated transformation.  A loss of FBH1 has been found in 63% of melanomas. 

What is the link between FBH1 and WEE1? 

Well, we’ve outlined what WEE1 and FBH1 do, so why are they associated together in this study? 

WEE1 inhibition results in replication and mitotic catastrophe, as it’s role in S phase and G2/M phase are then not being performed. FBH1 is important for inducing apoptosis during replication stress and having tumour-suppressing functions. The study suggests that FBH1 knockout (KO) cell lines have a dependency on the G2/M checkpoint to repair damage before mitosis as a last resort. Removing both S phase and G2/M phase protections is then hypothesised to cause more damage, and therefore be more lethal to the cancer cells. The hypothesis of the study was that FBH1 would promote WEE1i sensitivity. 

Genome Instability:

FBH1 KO cells were produced and western blotting was performed to confirm that FBH1 was not present. The reason for this is: scientists often remove the protein of study in order to see what impact there is. With FBH1, they wanted to see what FBH1 was important for and what a loss of FBH1 would do. When results are obtained from a KO of a protein, you can then inference that the impacts it has on a certain signalling indicates that the presence of that protein is important for that process. E.g. FBH1 is important for apoptosis initiation as a response to replication stress to prevent replication catastrophe and DNA damage from entering G2/M and mitosis, so removal of FBH1 will see more genome instability and mistakes present. 

In this study, U2OS cells were used as a control for FBH1 compared to the FBH1 KO cell line. Both U2OS cells and FBH1 KO cells were treated with a WEE1i (adavosertib), and it was found that there was reduced CDK1 and CDK2 Y15 phosphorylation as expected. 

The researchers observed for any genome instability, particularly multinucleation. Genome instability is very frequent in cancer; cancerous cells have more mistakes and issues in their genomes that are constantly changing and vary between cells and tumours. For example, one cell may have 3 copies of a gene or even its entire genome whereas another may have 10 copies within the same tumour or different tumours! 

Imagine this: you’re in a bus in London filled with Londoners, and you’re all going to the same destination, but you are all wearing different clothes and eat different things and you look different even though you’re all from London and you are going to the same place in the same vehicle. The variation we see between people is a great example of the variation that can be seen between cancer cells, but it’s ALL in the wrong places. Yes, the genome is dynamic, it moves and vibrates and there are many epigenetic modifications, but the recipe should be the same. Cancer gets it all wrong, it’s why it’s cancer. 

Another example is baking; think of the genome with all it’s genes as a giant bookshelf filled with recipes. The recipes never change, you only take them off the shelf to use them exactly as the instructions say and then put them back. You may bookmark a page to refer to sometimes as well. One thing you don’t do is to make many copies of one recipe or to start shuffling the recipes. Now imagine everyone has this bookshelf and we all start shuffling them and replicating random recipes and putting them all out of order. Genome instability. 

Well, you get the idea, let’s continue. 

The researchers looked for multinucleation (multiple nuclei in one cell, when there should only be one – one bookshelf per person, people!) and they found that U2OS cells had increased multinucleated cells from 1.3% to 11.3% with WEE1i treatment. In comparison, FBH1 KO cells showed 3x increase in multinucleation after WEE1i compared to U2OS cells. There was more genomic instability with FBH1 loss AND WEE1i than with FBH1 presence and WEE1i. FBH1 prevents mitotic catastrophe with WEE1i compared to when FBH1 is lost. A lack of FBH1 resulted in more genomic instability and DNA damage being present; it can no longer initiate apoptosis in cells with replication stress. 

Single-strand DNA Accumulation:

Next up is the accumulation of single-stranded DNA (ssDNA); DNA is double-stranded unless replication occurs and the helix is opened. WEE1i treatment can lead to replication fork stalls and ssDNA accumulation, and FBH1 is important for hydroxyurea response. Hydroxyurea is a drug used for cancers, such as myeloid leukaemia, it’s been known to slow/stall replication forks. 

As WEE1i induces replication fork stalls and ssDNA accumulation, and FBH1 is important in the response to hydroxyurea which is involved in inducing replication fork stalls as well, the study wanted to see what the impact of FBH1 loss and WEE1i would have. They looked for replication stress markers and ssDNA accumulation. U2OS and FBH1 KO cells were grown in IdU with/without WEE1i; IdU is a nucleotide that is used to observe DNA replication; incorporated IdU indicates cells are in S phase. WEE1i treatment of U2OS cells showed increased IdU and increased S phase cells, and increased ssDNA. FBH1 KO cells had less IdU positive cells, less S phase cells, and less ssDNA accumulation when administered WEE1i. They also observed PCNA staining (used to stain for proliferating cells), finding that IdU differences are not due to increased S phase cells. By observing FBH1 loss, they stated that FBH1 promotes ssDNA accumulation in response to WEE1i. 

Double-Stranded Break Formations: 

WEE1i lead to unrestrained cleavage by MUS81 (an endonuclease); breaks in the DNA are uncontrolled when WEE1is are given. FBH1 also promotes MUS81-dependent cleavage of replication forks in response to hydroxyurea. 

The researchers wanted to observe if FBH1 promotes double-stranded break formation after WEE1i treatment. They performed a TUNEL assay; this is an assay used to observe DNA fragmentation, so this is a good method to see if DSBs are occurring in a population. TUNEL assays measure DNA breaks due to fragmentation that result from apoptosis or replicative fork breaks (due to replication catastrophe). TUNEL-positive cells show DNA breaks, however, the assay does not specifically inform whether this was from apoptosis or replicative catastrophe. The researchers combined the TUNEL assay with an apoptotic marker called cleaved Caspase 3, which would indicate if apoptosis was the cause of the breaks. There are two different outcomes: TUNEL-positive and cleaved Caspase 3-positive cells would indicate DNA fragmentation due to apoptosis; TUNEL-positive and cleaved Caspase 3-negative cells would indicate DNA breaks due to DSB formation. 

There was a significant reduction of TUNEL-positive cells in FBH1 KO cells compared to U2OS cells, therefore there were less DSBs.

Moreover, the researchers also wanted to observe for replication fork collapses specifically. When replication fork collapse occurs, Replication Protein A (RPA) is phosphorylated as well as a protein called KAP1. RPA is a ssDNA binding protein important for DNA replication, and KAP1 is a protein important for the formation of heterochromatin. Phosphorylated RPA (pRPA) and phosphorylated KAP1 (pKAP1) was observed via western blotting. U2OS cells had increased pRPA and pKAP1 after WEE1i, which indicates DSB formation due to replication fork collapse. FBH1 KO cells had reduced pRPA and pKAP1, indicating reduced DSB formation signalling. They concluded that FBH1 is required for replication stress reponse induced via WEE1i. 

Nuclear Aberrations:

Researchers use a certain staining, called H2AX staining, to observe for cells undergoing apoptosis or mitotic catastrophe. H2AX is a histone which is phosphorylated in response to DNA damage. Cells with damage will show intense H2AX staining, and this is referred to as pan-nuclear staining. 

Previous research has shown that FBH1-deficient cells have reduced pan-nuclear staining in response to replicative stress caused by hydroxyurea. The researchers of the current study measured H2AX staining in U2OS and FBH1 KO cells after WEE1i treatment. The U2OS cells had 30% positive cells, whereas the FBH1 KO cells had 33.7%; this is a non-significant finding. This seemed to indicate that FBH1 may not be important for H2AX induction in response to WEE1i.

Regardless, the investigators were stumped; there was reduced ssDNA and DSB formation in FBH1 KO cells, but no significant findings of the H2AX staining! They decided to investigate further to see specifically where the staining occurred. 

It was found that the nuclei differed between U2OS and FBH1 KO cells; 60% of the H2AX positive cells in the U2OS cell line had no abnormal nuclei and no aberrations. 70% of the H2AX positive cells in the FBH1 KO cell line occurred in multi-nucleated cells. There is a difference between the U2OS and FBH1 KO cells regarding the H2AX staining due to the differences in their nuclei and genomic instability. 

Reduced Mitotic Entry:

WEE1i results in premature mitotic entry, which leads to mitotic catastrophe in cells with DNA damage. The researchers wanted to observe if this premature mitotic entry was due to the WEE1i or if FBH1 loss also had an impact. 

Cells were treated with WEE1i and observed for phosphorylated H3 (an indicator of mitosis) via flow cytometry. In untreated cells, 2.3% of U2OS and 2.7% FBH1 KO cells were pH3 positive. With WEE1i treatment, there was a 5.4x increase in pH3 positive cells in the U2OS cell line and a 3.8x increase in the FBH1 KO cell line. More phosphorylation means more mitosis. Based off this, the fewer pH3 positive FBH1 KO cells indicates that fewer cells were in mitosis. An issue is that severe nuclear abnormalities, which are present in the FBH1 KO cell line, can impede the results after WEE1i treatment.

As a result, a co-staining for pH3 and EdU (a marker for cell proliferation) was done, after which no significant results were found. When WEE1i was applied, there was a significant increase of premature mitotic entry in U2OS cells but a 67% significant reduction in FBH1 KO cells. Less FBH1 KO cells were entering mitosis prematurely when WEE1is were applied. The researchers stated that they wanted to see if FBH1 KO cells had more DNA damage during mitosis than U2OS cells, so they examined for H2AX staining. After WEE1i treatment, 64.5% of pH3 positive cells stained for H2AX in U2OS, but only 49.5% in FBH1 KO cells. The fact that the loss of FBH1 did not significantly impact premature mitotic entry or DNA damage in mitotic cells indicates that FBH1 loss does not increase DNA damage in mitotic cells. The results indicate that increased mitotic catastrophe observed previously was not due to premature mitotic entry or mitotic DNA damage in FBH1 KO cells. 

Exhibited Growth:

In FBH1 KO cells, there is an observed increased mitotic catastrophe and decreased replication catastrophe; the researchers wanted to see the impact of cell survival on FBH1 KO cells with WEE1i treatment. They stained for an apoptotic marker, Annexin V, after a 24-hour WEE1i treatment. There was a significant difference in Annexin V positive populations in response to WEE1i in U2OS and in FBH1 KO cells, but no difference between the two cell lines. Similar results were found with cleavage Caspase 3. FBH1 is not necessary to initiate apoptosis in response to WEE1i.

With regard to recovery, they found that WEE1i treatment resulted in a significant reduction in cell growth compared to untreated; this applied to both U2OS and FBH1 KO cell lines. There was a 50% reduction in WEE1i-treated U2OS cells compared to untreated cells and 23.4% in WEE1i-treated FBH1 KO cells compared to untreated cells. They concluded that FBH1 KO cells treated with WEE1i have less survival and cell growth (Jennings et al., 2024).  

Summary:

1. WEE1 acts on CDK1 and CDK2 in response to DNA damage and also regulates replication in S phase.
2. It’s role in S phase and in the G2/M checkpoint is a chemotherapeutic target.
3. WEE1 inhibition leads to unscheduled DNA replication, nucleotide pool depletion, and replication catastrophe.
4. WEE1 inhibition effects need more investigation.
5. FBH1 has roles in apoptosis, double-stranded break formation and replication fork reversal.
6. Previous studies show reduced DSB formation and more resistance to replication stress agents.
7. Hypothesis: FBH1 may have a role in WEE1i due to its role in replication stress and DSB formation.
8. FBH1 KO cells with WEE1i show increased genome instability.
9. Increased mitotic catastrophe in FBH1 KO cells with WEE1i.
10. ssDNA accumulation reduced in FBH1 KO cells with WEE1i.
11. Reduced DSB breaks in FBH1 KO cells with WEE1i, meaning issues in DSB formation (DSB formation is sometimes needed for reponse to damage or replication stress).
12. FBH1 KO cells with WEE1i show multinucleation, indicating mitotic catastrophe.
13. FBH1 KO cells with WEE1i treatment showed premature mitotic entry, but this was less than the U2OS control.
14. There was no increased mitotic DNA damage in FBH1 KO cells with WEE1i treatment.
15. FBH1 KO cells with WEE1i treatment had reduced growth, suggesting more sensitivity to WEE1i treatment.
16. Conclusion: FBH1 deficiency reduces replication stress response, sensitises to mitotic catastrophe and sensitises to WEE1i treatment, but mitotic catastrophe is not due to excessive premature mitotic entry or mitotic DNA damage.

My Thoughts: 

1. First, the good things: this gives good insight into the roles of FBH1 in DNA replication, the replication stress response, and mitosis. We found that FBH1 is important for replication stress response (particularly DSB formation), and it is important for genome stability.
2. A lot of the results for the FBH1 KO cell lines apply to the U2OS cells, and often the cell lines have no significant difference. Regardless, the scientists state, for example, that an FBH1 loss results in less survival and cell growth. This statement applies to the U2OS cells as well. This, to me, indicates that FBH1 loss is not necessarily the condition needed to create this effect. They are very sly, they imply that FBH1 loss results in certain results – they do, but so do the U2OS cell line… This is not an effective or clear paper. I did not appreciate this. Though they reported their data, their explanation is a manipulation in it’s own way. I don’t see enough data to state that a loss of FBH1 makes more of an impact than when there is FBH1 present; the whole point of the study was to find a link between FBH1 loss and WEE1 inhibition.
3. The researchers said that the H2AX staining occurred in distinct subpopulations, but did not specify what they meant by this. 
4. This is hypothetical, and it’s more of a way for me to understand that impact that each thing may have in the cellular “ecosystem”: WEE1 inhibition results in depleted nucleotide pools. As we’ve covered in Fugger et al’s work previously, DNPH1 is a nucleotide sanitiser responsible for cleaving hmdUMP (a nucleotide) from DNA. hmdUMP causes DNA damage, and the study found that inhibition of DNPH1 and SMUG1 (leading to increasing hmdUMP and hmdU induced DNA damage) made cells more sensitive to PARPi. WEE1 inhibition depletes nucleotide pools, so could this actually reduce the amount of DNA damage if WEE1 and PARPi and DNPH1 inhibition was to occur? Therefore, would WEE1i application reduce PARPi sensitivity if given simultaneously? If so, could this be also a dose-dependent action, could there be a certain amount of both WEE1i and PARPi that would balance out the impacts? Then again, there also needs to be FBH1 deficiency, it seems there’s a lot of dependence of drug application sensitivity on many factors: WEE1 and FBH1, PARPis with DNPH1 and SMUG1. Regardless, the results were not the most compelling…
5. They also do not discuss any sensitivity in the main body of the paper really, only in the hypothesis. The experiments and their findings did not align very well with the question of whether sensitivity increased with FBH1 loss and WEE1i together. They rope in sensitivity in the discussion, but even this was unclear to me.
6. The researchers were not very clear, in my opinion, and could have been much more straightforward. For example, they stated that inhibition of WEE1 leads to unrestrained cleavage by MUS81 and that FBH1 also promotes MUS81-dependent cleavage of replication forks in response to hydroxyurea. They then wanted to see what the impact of FBH1 loss would be. I guess my point is: a lot of the functions of FBH1 seem to compliment WEE1i, so what benefit would FBH1 loss have exactly? 
7. To be honest, I did not like this paper very much, it was not very clear. I felt that I needed to read more around the paper just to get the point of it?? Yes, I understand primary literature, and other people reading primary literature also understand. The thing is, when you are in research, you also have to tailor your paper. What I mean by this is that: we all understand the detailed wording and all the experiments and results, but we are not all doing the same research or have the same experience. For example, my background is on the G2 decatenation checkpoint and p53 signalling (p53, p21, topo2a, PKCe, MDM2, etc), so although I have touched briefly on WEE1 and FBH1 in my experience, a little preface of them both and their relevance to each other and the EXACT intentions of the paper is important. Generally, a clear organised story is important for explaining your work, even to others in your field. This was a difficult read, despite the fact it’s shorter than other papers I’ve read over the years. It might just be me, I might have to re-read it, but I do think everyone should have their own opinions and reflect. We should reflect on the work of both our peers AND ourselves, we should reflect on our own work and our own opinions. Rant over lol, it’s important to read a variety of papers regardless. 

What next?

I was harsh, I know.

I do think this helped to find out more, though I think that the results were not enough to conclude that FBH1 ‘sensitises’ cells to WEE1i treatment compared to FBH1 presence. More research needs to be done.

They need to focus more on sensitivity, possibly looking at different doses to see if there could be any dose-dependent effects, looking at FBH1 loss and WEE1i specifically more regarding cell growth and survival. Looking at different cell lines or mouse models would be useful. They could even look at different WEE1 inhibitors. What about FBH1 chemical inhibition as well? There are a lot of possibilities, but I do feel that there needs to be more explanation and more data. There needs a follow-up.

References

Jennings, L., Walter, H. A., McCraw, T. J., Turner, J. L., & Mason, J. M. (2024). FBH1 deficiency sensitizes cells to WEE1 inhibition by promoting mitotic catastrophe. DNA Repair, [online] 133, 103611. Available at: https://www.sciencedirect.com/science/article/pii/S1568786423001659#ab0015