Committee Meeting Questions

Could the increase in stem-like features after cisplatin selection reflect selection of existing cells, induction of a new state, or both? How would you distinguish these possibilities?

Both are possibilities. Since tumors are heterogeneous, a subpopulation of cancer cells could be present that has the necessary features to resist cisplatin, and thus they are selected for when the drug is administered. However, it is also possible that the DNA damage induced by cisplatin generates new mutations that provide a fitness advantage to the cell, and allow them to survive in the presence of the drug, therefore inducing a new state. To distinguish between these two possibilities, confetti labeling maybe?

What are the strengths and limitations of using FaDu, CAL-33 and Detroit562 cells to study HPV-negative HNSCC?

Strengths: By using these 3 cell lines, we have cells from pharynx (FaDu) tongue (CAL-33) and a pleural effusion from a metastatic site (Detroit562) giving us representation from a range of anatomical sites.

Limitations: these cell lines have been cultured on plastic, and are therefore only representative of the subpopulation of tumor cells that could survive in culture conditions.

Are doing these experiments in cell lines sufficient? Is there other data necessary to show the protein/pathway you’re studying is actually relevant in patients?

No, we also utilize TCGA, GEPIA and DepMap to get a better sense of the relevance of our target(s) in vivo, and we will eventually validate our findings in our patient-derived organoid models.

How does cisplatin become reactive inside cells, and what are its principal molecular targets and DNA lesions?

Cisplatin becomes more reactive through a process called aquation, meaning that a water molecule replaces a chloride attached to the drug’s central platinum atom.This happens readily in cells because Cisplatin contains platinum attached to two ammonia groups and two chloride groups. In blood, the chloride concentration is relatively high, which favors the original, chloride-containing form. Inside many cells, chloride concentration is lower, favoring accumulation of water-containing, or aquated, forms. 

Replacing chloride with water makes the cisplatin reactive because the attached water can be displaced by electron-rich sites on biological molecules. In DNA, important binding sites are particular nitrogen atoms (N7 positions) on the bases guanine and adenine. These atoms can form strong bonds with platinum. Cisplatin first attaches to one DNA base and can subsequently attach to a second, creating a crosslink. It does not have to replace both chlorides with water before making its first DNA attachment.

Two types of crosslinks form from cisplatin: Intrastrand crosslinks, the predominant lesions, connect bases within the same DNA strand, especially adjacent guanines. Links between adjacent adenine and guanine also occur. Interstrand crosslinks, which are less common, connect the two opposite DNA strands.

How do cisplatin lesions lead to cell-cycle arrest and death, and must that death depend on functional p53?

Crosslinks distort DNA and obstruct the machinery that copies it during replication or reads it during transcription. Interstrand crosslinks also prevent the two strands from separating normally. If the damage cannot be adequately repaired or tolerated, it can trigger cell death.

While p53 is an important way that cells trigger apoptosis, cancer cells with mutant p53, like most HNSCC cell have, can still die through alternate mechanisms like Mitotic catastrophe, necroptosis, ferroptosis, etc. apoptosis can also still be activated if the signal is strong enough, as is the case with severe dna damage

How were the cisplatin resistant cell lines generated and validated?

Cells were cultured continuously in cisplatin in gradually increasing doses of the drug until a resistance factor greater than 4 was reached, and resistance was maintained after two weeks without the drug. Resistance was confirmed by monitoring colony formation and caspase-3 cleavage following cisplatin treatment in WT and Cis-R cells.

Why did you look at your protein (ATAD2) after just 24 hours in cisplatin? What is the significance of that experiment?

We have found that ATAD2 protein levels are increased in Cis-R cells. It is important to distinguish whether cisplatin treatment itself induces the expression of these genes, or, if their levels are increased by the cisplatin resistant cells specifically. By doing a 24 hour treatment with cisplatin, we were able to show that ATAD2 levels are not altered by acute cisplatin treatment.

How would you distinguish true cisplatin sensitization from an intervention that simply reduces baseline proliferation or survival?

Include untreated, cisplatin-only, intervention-only, and combination conditions. Normalize each genotype or intervention to its own appropriate untreated baseline and examine complete dose–response relationships. Quantify apoptosis and recovery after treatment, while measuring baseline growth and plating efficiency. Fewer colonies in the combination group alone cannot establish altered cisplatin sensitivity.

Why use both genetic depletion and pharmacologic inhibition of ATAD2? What additional evidence would establish inhibitor specificity?

Concordant results from distinct perturbations strengthen causal interpretation. Additionally, inhibition is different than depletion, and gives us additional information. If treatment with BAY-850 did not alter our phenotype, then that would suggest the bromodomain function of ATAD2 is not important for the phenotype, and likely ATAD2 was doing something other than binding to acetylated histones.

If you see reduced CX3CL1 mRNA following ATAD2 depletion or inhibition, how would you distinguish increased CX3CL1 transcription from increased CX3CL1 mRNA stability?

Can use a luciferase reporter to look at promoter activation with and without ATAD2 inhibition

What do recombinant CX3CL1 rescue, CX3CL1 neutralization, and CX3CR1 inhibition establish about pathway order? Why might rescue be incomplete?

We can add recombinant CX3CL1 to the ATAD2 depleted cells and show that the phenotype is restored. This would allow us to show that CX3CL1 is downstream of ATAD2 and necessary for the ATAD2 driven phenotype. We can also do the inverse experiment and inhibit CX3CL1 with a neutralizing antibody, or block the receptor in ATAD2-OE cells to show that if we block the ATAD2 driven CX3CL1 signaling, we lose the phenotype, and thus CX3CL1 is necessary for the ATAD2 mediated phenotype.

Could differences in cell proliferation rates (rather than invasion) account for changes in invaded cell counts, and how did you control for that?

Cells are serum-starved for 24 hours prior to the invasion assay being conducted to minimize the impact of proliferation on invasion counts (serum starvation slows/stops proliferation).

Why use clonogenic survival rather than relying only on a short-term viability assay? How should the  surviving fraction be calculated?

Clonogenic assays measure sustained reproductive capacity after treatment, capturing effects missed by short-term metabolic assays. Calculate plating efficiency as untreated colonies divided by cells seeded. Surviving fraction is treated colonies divided by cells seeded and the appropriate untreated plating efficiency.

How do transwell invasion and wound-closure assays differ?

Transwell invasion requires moving through the ECM, and can be facilitate by expression of MMPs that degrade matrix proteins. Migration/would-closure assays are 2d.

What controls are needed for co-immunoprecipitation?

Positive control to make sure your protein is in the sample (total extracts) and negative control using normal IgG antibody.

What constitutes a biological replicate?

A biological replicate is an independently established experimental unit, such as an independently prepared culture experiment or resistant lineage. Multiple wells, images, or cells within that unit are considered technical or nested observations.