Hematologic Malignancies II

Question 1

What is a method for determining the genotype of any abnormal cells? Method 1

Answer 1

Routine cytogenetics

Question 2

How do cytogenetic studies work?

Answer 2

Routine cytogenetic studies identify and enumerate the chromosomes present in dividing (metaphase) cells.

put cells in growth medium, arrest mitosis, squash cells onto a slide, stain, image

Question 3

Chronic myelogenous leukemia, or CML, is consistent with what chromosomal abnormality?

Answer 3

Philadelphia chromosome- a 9;22 translocation between a region on the long (q) arm of 9 and the long (q) arm of 22).

see CMOD for explanation

Question 4

It’s not always easy to visualize chromosomes in metaphase cells. A method to do so in cells with intact nuclei (“interphase” cells) would be useful. How we do this shit?

Answer 4

FISH (fluorescent in situ hybridization)

Question 5

Example of how a FISH would work for CML.

Answer 5

the probes for chromosome 9q34 and 22q11.2 overlap at two locations, indicating that the 9;22 translocation (and its reciprocal 22;9 translocation) are present.

Question 6

What is a key advantage to using FISH to ID a malignancy?

Answer 6

cells don’t have to be growing (as they do in conventional cytogenetic studies).

Question 7

Cytogenetic studies are“low resolution” scans of the genome. Significance?

Answer 7

They can only detect relatively large abnormalities involving loss or translocation of parts of chromosomes.

Acute leukemias in fact show normal cytogenetic findings quite often – about half of the time in AML, for example. FISH studies can detect smaller abnormalities, but they are almost never able to detect point mutations.

Question 8

It turns out that looking for particular sets of point mutations, and other types of small scale genetic variants, can help us to accurately diagnose, prognose, and treat some subsets of hematologic malignancies. So targeted sequencing studies are now standard of care in cases for which the morphology and immunophenotype leads in the direction of those subtypes.

Answer 8

usually done with PCR with appropriate primers for selected target genes

Question 9

What is the ultimate method for genetically characterizing malignancies?

Answer 9

is to sequence them entirely – either the entire genome (in the range of 3 billion nucleotides) or the exome (1-2% of the genome).

This requires comparison, of course, to the presumably unmutated DNA in each patient’s unaffected genome – usually obtained via a skin biopsy sample.

This type of study is currently a research tool, not a routine clinical one. It could, however, become cheap enough to become standard of care in the not too distant future.

Question 10

Translocations that contribute mechanistically to malignancies generally do one of two things:

Answer 10

1) they activate transcription of an oncogene (by moving it next to an active promoter) or
2) they generate an abnormal fusion protein. The 9;22 translocation is in the second category.

Question 11

How does the Philadelphia fusion protein cause CML?

Answer 11

BCL:ABL fusion can activate targets such as transcription factors or other kinases; it can increase its own activity by autophosphorylating at the tyrosine (Y) residue; and its other binding domains (for actin, DNA, and a multipurpose signal transduction component called Rho) enable it to migrate to multiple different locations in the cell.

Question 12

Excess activation of the STAT family of transcription factors by another mechanisms (a mutated Jak-2 tyrosine kinase) can result in what?

Answer 12

unregulated activity of erythropoiesis or thrombopoiesis (platelet production)

Question 13

The BCR-Abl1 fusion protein activates some of the Stats, but it also kicks a number of other regulatory pathways into high gear. What else can it cause besides CML?

Answer 13

ALL.

Why you need to know this: The tyrosine kinase function necessarily requires ATP to provide that phosphate group. This domain therefore must contain an ATP-binding pocket specifically designed to facilitate this particular kind of phosphorylation. Drugs that compete with ATP for just that pocket can, and do, inhibit the activity of this fusion protein.

Question 14

What gene translocation is common in acute promyelocytic leukemia (APL)?

Answer 14

PML gene (chrom 15) and RARa gene (chrom 17)

Question 15

What does the PML gene do?

Answer 15

Facilitates nuclear localization of binding partners

Question 16

What does the RARa gene do?

Answer 16

RARA alters transcription patterns in a way that results in impairment of granulocyte differentiation

Question 17

How does the PML-RARa fusion protein change the function of RARa?

Answer 17

fusion of RARa to PML apparently augments that function enormously. Proliferation, though, is not impaired by PML-RARa. The end result is a proliferation of immature granulocyte precursors (promyelocytes).

Question 18

How is PML degraded normally?

Answer 18

a built-in kill switch, a site (K160) which is recognized in the first step of a process that ends with ubiquitination of the protein

Question 19

What can PML degradation be induced by?

Answer 19

arsenic trioxide (drug)

Retinoic acid regulates RARa in the same way, by inducing its degradation. In combination, these two targets (K160 and AF2) provide an effective way to treat the form of acute leukemia induced by this fusion protein.

Question 20

Since a number of chemotherapeutic agents act by inducing severe DNA damage, loss of p53 can induce resistance to them while allowing mutations to accumulate. So knocking out both copies of p53 both increases the number of mutant clones and makes it harder to kill them.

Answer 20

Since a number of chemotherapeutic agents act by inducing severe DNA damage, loss of p53 can induce resistance to them while allowing mutations to accumulate. So knocking out both copies of p53 both increases the number of mutant clones and makes it harder to kill them.

Question 21

Where is p53 located in the genome?

Answer 21

p53 is a major negative prognostic marker in a number of malignancies. It’s location is 17p.

Question 22

What is Core binding factor (CBF)?

Answer 22

a heterodimeric “transcription activator” that plays key roles in the growth and differentiation of both myeloid and lymphoid precursors.

Question 23

Describe the composition of CBF.

Answer 23

It consists of two subunits named Runx1 (the alpha subunit of the complex, previously called AML1) and CBFβ.

Question 24

What happens when CBF is activated?

Answer 24

• binds DNA (at enhancer sequences)

- binds a complex of several proteins that possess histone acetyl transferase (HAT) activity.

Question 25

What are the target genes of CBF?

Answer 25

MPO, M-CSF receptor

Question 26

What does acetylation of histone cause? Inhibition?

Answer 26

loosens the structure of the nucleosomes, allowing transcription factors to reach their targets in these areas. This allows expression of genes associated with DIFFERENTIATION, in both myeloid and lymphoid lineages.

This process can be reversed by histone deacetylases (abbreviated HDACs), and there are systems in place to move such enzymes into position to do so.

For purists: CBF can in fact bind deacetylases and other co-repressors as well as HAT enzymes.

Question 27

There are a large number of fusion proteins that are generated, in a number of malignancies, from the CBF subunits. The prototype is the Runx1/Runx1T1 fusion protein (previously termed AML-1/ETO), generated by a translocation between a gene called RunxT1 on chromosome 8 and Runx1 on chromosome 21. What does this result in?

Answer 27

• The fusion protein heterodimerizes with CBFβ and binds the same DNA enhancer elements as Runx1.
• Instead of recruiting a series of HAT proteins, it engages with a series of HDACs. The result is the opposite of what CBF normally does; the fusion protein shuts down transcription of its targets and inhibits DIFFERENTIATION of precursor cells resulting in accumulation of myeloid precursor cells= AML.

Question 28

AML 2. The other subunit of CBF (CBFβ) is fused to a protein capable of binding co-repressors. The other component of the fusion in this case is called MYH11 (also called SMMHC).

Once bound (via Runx1) to its enhancer target, it can recruit co-repressors to the site and impair transcription. The result is, again, an accumulation of undifferentiated myeloid precursors in the form of a type of AML.

The MYH11 gene is located on the same chromosome (16) as CBFβ, but the two genes are on opposite arms (one on the short or “p” arm, one on the long or “q” arm).

Answer 28

In the example shown here, the other subunit of CBF (CBFβ) is fused to a protein capable of binding co-repressors. The other component of the fusion in this case is called MYH11 (also called SMMHC).

Once bound (via Runx1) to its enhancer target, it can recruit co-repressors to the site and impair transcription. The result is, again, an accumulation of undifferentiated myeloid precursors in the form of a type of AML.

The MYH11 gene is located on the same chromosome (16) as CBFβ, but the two genes are on opposite arms (one on the short or “p” arm, one on the long or “q” arm).

Question 29

ALL 1. Fusion of Runx1 (AML-1) (21) to a target called ETV6 (TEL) (12). The order is reversed (Runx1 is on the carboxyl side of this fusion) but the effect is the same: recruitment of repressors such as HDAC-3, impairment of differentiation, acute leukemia.

In this case, for reasons that are not clear, the process takes place in lymphocyte precursors, and the outcome is a type of acute lymphoblastic leukemia.

Answer 29

The last example we’ll cover is fusion of Runx1 to a target called ETV6. The order is reversed (Runx1 is on the carboxyl side of this fusion) but the effect is the same: recruitment of repressors such as HDAC-3, impairment of differentiation, acute leukemia.

In this case, for reasons that are not clear, the process takes place in lymphocyte precursors, and the outcome is a type of acute lymphoblastic leukemia.

Question 30

T or F. AMLs caused by CBF fusions, like those involving PML-RARA, tend to be very treatable

Answer 30

T. (as implied by the low cytogenetic risk score). That’s also the case for ALL cases involving CBF fusions.

Question 31

What is DNMT3A?

Answer 31

DNMT3A (DNA methyl transferase 3A) normally functions to methylate cytosine residues

Question 32

Where are the targets of DNMT3A?

Answer 32

Usually adjacent to G residues, and are referred to as CpG islands. Methylation of these islands often inhibits transcription.

Question 33

In at least some cell lineages, DNMT3A activity maintains precursor cells in a quiescent state. How?

Answer 33

It does so by inhibiting transcription of PROLIFERATION-associated genes, particularly those encoding certain tyrosine kinases such as FLT3.

Question 34

What is the effect of mutations that inactivate DNMT3A?

Answer 34

the net result is a push toward more cell PROLIFERATION.

Mutations that inactivate DNMT3A are found in a number or leukemias.

Question 35

DNMT3A mutations, loss of both alleles, and occasional mutations in its paralog (DNMT3B) are very frequent in ____.

Answer 35

AML.

Mutations in other genes involved in DNA methylation tend to occur in the same patients.

Question 36

What are Tet1 and Tet2?

Answer 36

enzymes that initiate demethylation of cytosine residues in and near target genes.

Question 37

How do Tet1/2 demethylate cytosine residues?

Answer 37

• oxidizing the methyl group

- something has to be reduced in the process.

Question 38

What is reduced in the Tet1/2 demethylation reaction?

Answer 38

Alpha keto glutarate, an abundant intermediate in the Krebs cycle, is the cofactor.

Question 39

T or F. The demethylating process of Tet1/2 is a targeted to a different set of genes than those targeted by DNMT3.

Answer 39

T. Instead of targeting genes involved in proliferation, Tet1 and Tet2 target genes involved in DIFFERENTIATION.

So the activity of Tet1 and/or Tet2 is necessary for that normal function. Mutations that inhibit these enzymes would cause undifferentiated cells to accumulate*

Question 40

What enzyme is needed to keep this process supplied with alpha keto glutarate?

Answer 40

• Isocitrate dehydrogenase (IDH; two types- IDH1 and IDH2)

- This part of the process can fail as well.

Question 41

What would a gain of function mutants in IDH1 cause?

Answer 41

alteration the activity of the enzyme in such a way that instead of producing alpha-kg, it produces a slightly less oxidized product, alpha hydroxy glutarate.

Question 42

What can do alpha hydroxy glutarate do?

Answer 42

inhibit Tet1 and Tet2. The net result is the same as a loss of function mutation in Tet1 or Tet2: impaired differentiation and accumulation of undifferentiated cells.