After decitabine failure in higher-risk MDS or AML, the only curative option is allogeneic stem cell transplantation (allo-SCT), which should be considered early in eligible patients. For those unfit for transplant, clinical trial enrollment is strongly recommended. Differentiating between primary resistance (no response after ≥6 cycles) and secondary resistance (relapse after initial response) is essential. Mutation profiling at relapse guides targeted therapies. Alternative epigenetic agents like guadecitabine, oral decitabine/cedazuridine, or CC-486 offer modest responses; sequential HMA use (e.g., azacitidine post-DAC) may yield temporary benefit. Targeted strategies using agents such as venetoclax, IDH1/2 inhibitors, FLT3 inhibitors, or investigational therapies (e.g., H3B-8800, checkpoint inhibitors) are best pursued in trials. Intensive chemotherapy may be used selectively in AML transformation to bridge to SCT. Given poor prognosis (median survival 4–6 months), a trial-based, mutation-informed approach is the preferred strategy.

I am attaching the document files here—they might be helpful.

Hi Chloe,

I have seen that you already a response from Shweta Dwivedi and I think in her document "Trouble shooting Decitabine" are some good recommendations. However I have some additional points .

First of all - Decitabine is also called 5-Aza-2'-5-aza-2'-deoxycytidine - if you search with this term you will find several publications were Burkitt lymphoma cells were treated with 5-Aza-2'-deoxycytidine.

"MiR-29 silencing modulates the expression of target genes related to proliferation, apoptosis and methylation in Burkitt lymphoma cells"

"Silencing of genes required for glycosylphosphatidylinositol anchor biosynthesis in Burkitt lymphoma"

"Viral driven epigenetic events alter the expression of cancer-related genes in Epstein-Barr-virus naturally infected Burkitt lymphoma cell lines"

"Inhibitor of DNA binding 4 functions as a tumor suppressor and is targetable by 5‑aza‑2'‑deoxycytosine with potential therapeutic significance in Burkitt's lymphoma" (I think this should be also 5-aza-2' deoxycytidine as deoxycytosine is most likely not correct, however I would nevertheless have a look at this publication)

In these publication the cells are treated mostly with higher concentration of 5-Aza-2'-deoxycytidine (from 1µmol to 10 µmol) - one reason for this might be that cells take up 5-Aza-2'-deoxycytidine differently and in the publication you have another cell line.

However, make sure that you don't use a concentration which is to high because at a certain level 5-Aza-2'-deoxycytidine simply become toxic for the cell.

To get some information how Decitabine/5-Aza-2'-deoxycytidine is act on a molecular level I would highly recommend to read this publication:

"Rewiring the Epigenetic Landscape of Cancer: The Role of 5-Azacytidine and 5-Aza-2′-Deoxycytidine"

I think you can find a lot more publications on the topic.

I think you can also disolve 5-Aza-2'-deoxycytidine in PBS - no need for DMSO (also you have very little DMSO in your medium - nevertheless if you use DMSO I would also recommend to do a DSMO only control).

Now regarding your problems: There are many thinks that can go wrong.

1. make sure you have a suitable concentration of 5-Aza-2′-Deoxycytidine (look in the literature)

2. make sure that your cells are dividing as 5-Aza-2′-Deoxycytidine is mainly acting on Dnmt1 during cell division - if the cells are not dividing/growing you might not see the effect you are wanting.

3. Check the genomic DNA on a gel before proceeding - if you don't have intact genomic DNA it might negativly influence your results.

4. Test your primer pairs before you start treatment genomic DNA before bisulfite conversion - your primer pair for methylated DNA should work on genomic DNA before bisulfite conservion (maybe this is clear to you but I have seen a lot of people mixing this up) as methylated DNA should not be affected by bisulfite conversion this primer pair should have the same sequence as the original DNA sequence.

Your primer pair for unmethylated DNA should show not not work on genomic DNA before bisulfite conversion as C should be replaced by a T on the forward primer and G for an A on the reverse primer. However, depending on your primer sequence and how many CpGs are in the sequence you might see a signal - if you see this try increasing the annealing temperature or order different primer.

5. Test both primer pairs on bisulfite converted DNA - also before you start treatment - here you might see a result for both primer depending on the degree of methyltion (even if a CpC is target of methylation it might not be methylated in all of the cells). If you see a strong signal for your methylated primers and only a faint for your unmethylated primer this can means two things - either your DNA is really methylated or your bisulfite conversion did not work properly (I think this is what you saw and unfortunatly you should consider that your bisulfite conversion did not work).

To distinguish between the two is not easy - I would look in the literature for a good working assay with nice results (for both methylated and unmethylated DNA) to verify that your bisulfite conversion worked - (I would not use the assay from the publication that you looked up - regarding the methylation analysis this was not convincing at all). You could use a good assay as a control for sucessfull bisulfite conversion and you should include this in all subsequent experiments were you do a new bisulfite conversion as this is a critical step.

If you see a strong signal for the unmethylated primer it is clear that the bisulfite conversion has worked and your DNA is mostly not methylated (especially when you have no signal from the primers for methylated DNA).

However, in this case you should consider if you want to proceed with treatment as I see no sense in treating the cells with a demethylating agent if the DNA-sequence you want to analyse is not methylated (as they have done in the publication you looked up) - for this reason I would do these tests before you start treatment.

Regarding your primers - an amplicon of 83 bp is pretty small - are you sure that you can distiguish your amplicon from primer Ddimers on the gel - depend on the lenght on your primers these things could be pretty close together

If it seems that your DNA is methylated (and you have verified that you beisulfite conversion worked - and check this whenever you don a new bisulfite conversion) you can start with the treatment and with subsequent analysis.

I know this is a lot and there a several things more to troubleshoot but these are the thing I would start with. THe efficiency of your bisulfite conversion, the efficiency of your priming and also the correct primer sequences for methylated and unmethylated primers are critically important.

If you are willing to share your first results (gel picture) and primer sequences it could be possible that I could give you some more concrete information.

Good luck