Baylin & Jones (CSH Perspectives, 2016

Q1. What are the three ways tumor suppressor genes (TSGs) can be inactivated in cancer?

1) Mutations (disabled function), 2) Loss of heterozygosity (gene completely lost), 3) Epigenetic silencing (switched off without DNA sequence change).

Q2. List the three major routes by which CpG methylation contributes to the oncogenic phenotype.

1) General hypomethylation of the cancer genome, 2) Focal hypermethylation at TSG promoters, 3) Direct mutagenesis of 5-methylcytosine-containing sequences (deamination, UV, carcinogens).

Q3. What percentage of CpG islands in gene promoters are typically methylated in normal cells vs. hypermethylated in cancer?

In normal cells, most CpG islands are unmethylated (active/open chromatin); in cancer, 5-10% of CpG island promoters become hypermethylated.

Q4. How does loss of imprinting (LOI) of IGF2 contribute to tumorigenesis?

Hypermethylation of a regulatory element upstream of H19 removes its insulator function, allowing IGF2 to be expressed biallelically → excess IGF2 growth-promoting protein → early colon cancer progression.

Q5. What is the "two-hit hypothesis" in TSG inactivation, and how can epigenetics serve as one hit?

Both alleles must be inactivated for full transformation. First hit = germline mutation; second hit = epigenetic silencing (promoter hypermethylation) of the second copy (e.g., MLH1 in colon cancer).

Q6. Name three classes of enzymes mutated in cancer that directly affect DNA methylation homeostasis, and one cancer type for each.

DNMT3A (AML), TET2 (myeloid malignancies), IDH1/2 (glioma, AML).

Q7. How do mutant IDH1/2 enzymes cause DNA and histone hypermethylation?

They produce 2-hydroxyglutarate (2-HG) from α-ketoglutarate, which inhibits TET enzymes (DNA demethylation) and lysine demethylases (histone demethylation) → hypermethylation.

Q8. What is the CpG island methylator phenotype (CIMP), and with which mutations is it associated?

A phenotype characterized by frequent promoter CpG island hypermethylation; associated with IDH1/2 mutations (gliomas, AML, colon cancer).

Q9. According to the paper, what is the typical proportion of CpG methylation in normal genome vs. cancer genome?

Normal: ~80% of CpGs (outside CpG islands) are methylated; Cancer: average 40-60% methylation (global hypomethylation).

Q10. What is the clinical utility of detecting O6-MGMT promoter methylation in gliomas?

Methylated O6-MGMT predicts better response to alkylating agents (temozolomide) because the repair gene is silenced, so guanosine adducts are not removed.

Q11. List the three FDA-approved epigenetic drug classes mentioned and one cancer indication for each.

DNMT inhibitors (azacitidine/decitabine for myelodysplastic syndrome), HDAC inhibitors (vorinostat/romidepsin for cutaneous T-cell lymphoma), (IDH inhibitors mentioned but not yet FDA-approved in 2016 paper).

Q12. What is the molecular mechanism of 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine)?

Nucleoside analogs incorporated into DNA (decitabine) or RNA (azacytidine); they inhibit DNMTs by forming covalent intermediates, cause DNMT protein degradation, and induce hypomethylation after replication.

Q13. Why were DNMT inhibitors initially toxic, and how was this overcome?

Initially used at very high doses; profoundly lowering doses reduced toxicity while retaining efficacy, leading to FDA approval for MDS.

Q14. What is the proposed mechanism for why genes with bivalent chromatin (H3K4me3 + H3K27me3) in stem cells are prone to hypermethylation in cancer?

In abnormally expanding adult stem cells, PcG-mediated repression at CpG island promoters can transition to more stable DNA methylation-associated silencing (epigenetic switching), locking genes in a repressed state.

Q15. Name one example of an oncogenic translocation that mistargets HDAC activity.

PML-RAR fusion in acute promyelocytic leukemia (APL) recruits HDAC and DNA methylation activity, causing transcriptional silencing and differentiation block.

Q16. What are hypomethylated blocks in the cancer genome, and what is their size range?

Megabase-scale regions (28 kb to 10 Mb) covering ~1/3 of the genome where DNA methylation drops from 80% to 40-60%; they often contain focal CpG island hypermethylation within them.

Q17. How do DNMT inhibitors and HDAC inhibitors synergize to reactivate silenced genes?

HDAC-mediated deacetylation collaborates with DNA methylation to silence genes; blocking HDAC after DNMT inhibition can additively lead to reexpression of hypermethylated genes.

Q18. What is the sensitivity of methylation-specific PCR (MSP) for detecting methylated alleles?

Can detect one methylated allele in a background of 1,000 to 50,000 unmethylated alleles, depending on assay design.

Q19. According to Table 1, give one example of an epigenetic alteration for each hallmark: (a) self-sufficiency in growth signals, (b) evading apoptosis, (c) DNA repair capacity.

(a) Methylation of RASSF1A, (b) Methylation of DAPK, ASC/TMS1, or HIC1, (c) Methylation of GSTP1, O6-MGMT, or MLH1.

Q20. What is the evidence that epigenetic changes are early events in tumor progression?

p16 silencing occurs in premalignant lung and breast lesions; O6-MGMT silencing in premalignant colon polyps before p53/RAS mutations; GST-Pi silencing in premalignant prostate lesions; age-related CpG island methylation parallels colon cancer risk