miRNA/TP53 Feedback Circuitry

[Guest guest]

Al --

This is fascinating and a bit elusive for someone who left chemistry behind many

moons ago. Please help me with this bit of terminology that seems to occur in

various discussions of DNA / RNA / genes / etc. " Codes for " : can you give me an

equivalent expression or translate this into lay terms? " Codes " is usually not

used as a verb. Does it mean " expresses " ? I don't think so. Would be

extremely helpful if you could parse and translate into lay terms this phrase:

" 13q14 DNA region codes for miRNAs, " . Perhaps, does it mean " produces " ? Clearly

it is part of the model for talking about these DNA/RNA/genetic bits, but would

be great if you could open us this window for us as I think what you have

provided is extremely valuable. I'd also love if you could provide a good

reference to get up to speed on the terminology. I did find a " DNA for dummies "

website, but it, too, immediately dropped into the " codes for " lingo with no

explanation of how that phrase fits into the model.

Thanks !!

Lynn

[Guest guest]

At 12:13 AM 1/14/2011, lynnb65 wrote:

>various discussions of DNA / RNA / genes / etc. " Codes for " : can you

>give me an equivalent expression or translate this into lay terms?

> " 13q14 DNA region codes for miRNAs, " . Perhaps, does it mean " produces " ?

Generally speaking, yes, DNA molecules (with the aid of enzymes and

other factors) do produce RNA molecules. Exactly what those RNA

molecules look like is dependent on what the DNA molecules (from

which they are produced) look like. The nature of what the DNA

molecules look like is described as a code, and, as a result,

specific DNA molecules with specific components are described as

coding for specific RNA molecules.

See:

http://en.wikipedia.org/wiki/DNA

SNIP............

" DNA is often compared to a set of blueprints, like a recipe or a

code, since it contains the instructions needed to construct other

components of cells, such as proteins and RNA molecules. "

Al Janski

[Guest guest]

Links to the " References " discussed are at the end of this post.

Re: [] Feed back loops in CLL ~ JAMA

At 06:59 PM 1/6/2011, cllcanada wrote:

>JAMA Scientific Paper:

><http://jama.ama-assn.org/content/305/1/59.short>http://jama.ama-assn.org/conte\

nt/305/1/59.short

> " The recurring deletion hot spots at 13q, 11q, and 17p actually

>represent nodes of a complex regulatory network in CLL that

>integrates the miR-15a/miR-16-1 and miR-34b/miR-34c clusters with

>TP53, " dorf and concluded in JAMA.

The new insights contained in this JAMA paper will likely enable new

understandings that could substantially accelerate progress for

better care for CLL. It is significant that the co-authors of this

research include many of the leading CLL specialists/researchers

(Drs. Calin, Croce, Keating, Kay, Rai, Kipps, etc.)

The detailed observations and analyses contained in this paper

'connect a lot of dots' [in the story of differential prognoses

associated with deletions in 13q vs. 11q vs. 17p chromosomal regions

in CLL cells] which had seemed biochemically unrelated.

Included (below) are some 'snips' from the paper and associated

editorial (summarizing the key observations of the

paper). Unfortunately, the full-text scientific paper and editorial

are not freely available.

A. Key Observations:

By making a list of the paper's key observations, one can create

(draw) lines between these previously unconnected dots associated

with the13q, 11q, and 17p nodes of the regulatory network taking

place 'within' CLL cells.

Below is list, organized by the gene (DNA) region at which RNAs are

coded, which, either, in the case of micro-RNAs (i.e. miRNAs),

directly have effects themselves or, in the case of messenger RNAs

(mRNAs), code for proteins that have effects.

1. 13q14 DNA region codes for miRNAs, miR-15a & miR-16-1 (abbreviated

as " miR15a/16-1 " ).

a. miR15a/16-1 inhibits production of TP53 protein (and thus inhibits

apoptosis of CLL cells) by inhibiting its expression from TP53 mRNA.

b. miR15a/16-1 inhibits production of Bcl-2 protein (and thus

increases apoptosis) by binding to Bcl-2 mRNA.

c. miR15a/16-1 inhibits production of Mcl-1 protein (and thus

increases apoptosis) by binding to Mcl-1 mRNA.

2. 17p13 DNA region codes for TP53 mRNA, which codes for TP53

protein, which promotes apoptosis

a. TP53 increases transcription of miR15a/16-1, which reduces TP53

(an example of a " feedback loop " , a common characteristic of most

biochemical pathways)

b. TP53 increases transcription of miR-34a, miR-34b & miR-34c

NOTE: In the JAMA paper 'italicized' TP53 depicts the TP53 gene,

whereas non-italicized TP53 refers to the TP53 protein. In this

posting, " TP53 " alone refers to TP53 protein.

3. 11q23 DNA region codes for the miRNAs miR-34a, miR-34b & miR-34c,

with the latter two (miR-34b/c) having similar effects.

a. miR-34b/c inhibits production of ZAP70 (and thus increases

apoptosis) as a result of its binding to ZAP70 mRNA

b. miR-34a increases activity of TP53 (and thus increases apoptosis)

by inhibiting SIRT-1 catalyzed deacetylation of TP53, increasing the

amount of the more active acetylated form of TP53.

4. 2q11 DNA region codes for ZAP70, which inhibits apoptosis

5. 18q21 DNA region codes for Bcl-2, which inhibits apoptosis

6. 1q21 DNA region codes for Mcl-1, which inhibits apoptosis

These observations (and their connections) were graphically displayed

in Fig. 6 of the JAMA paper, but I found that drawing my own picture

made this easier to visually grasp.

To better see the interconnections between these 6 gene loci, I drew

a hexagon, with each of the 6 above gene regions represented as one

side of the hexagon. Because each of the gene products can be

considered in the context of their ultimate effects (increase or

decrease) on apoptosis in CLL cells, in the center of this hexagon, I

drew a box around the word " APOPTOSIS " .

NOTE: Other processes, such as CLL cell proliferation, are probably

also affected by these mechanisms, but the model presented in Fig. 6

of the paper focused on apoptosis.

For example, one side of the hexagon is 13q14 DNA region, and from

that side I drew an arrow (within the hexagon) to its 'transcribed'

gene products, miR15a & miR16-1. Each of the other 5 sides are

represented by one of the remaining 5 gene regions (listed above) and

similar arrows from each gene to its specific transcribed mRNA. In

the case of chromosomal region 17p13, I drew an arrow to TP53 mRNA

and an arrow from there to TP53 protein (the product of transcription

of TP53 mRNA) and an arrow from TP53 to the APOPTOSIS box with the

word " increases " above the arrow. And so on.......

NOTE: A hexagon best depicts the discussion with this JAMA paper;

however, a complete picture of 'intracellular' CLL regulation would

require many more sides, with the additional sides representing other

important genetic loci, some of which are beginning to be understood,

some of which are yet to be discovered. A picture that includes both

intracellular regulators and extracellular regulators (e.g. via

stromal cells), would be even more complicated (see item F, below).

B. New Understandings from this Research about the 13q-11q-17p Interface:

Using such a diagram of interconnected effects of the 'active' gene

products (miRNAs or proteins), one can begin to visually understand

some the impacts of chromosomal deletions (in 13q, 11q & 17p

regions), or to ask better questions about impacts that can not yet

be understood.

For example, given that miR15a/16-1 decreases production of TP53

(which 'decreases' apoptosis) as well as decreases production of

Bcl-2 (which 'increases' apoptosis), is the ultimate overall effect

of miR15a/16-1 to increase or decrease apoptosis? The answer at

least partially depends on what's going on at 11q. And what's going

on at 11q (e.g. with or without a deletion) can vary for different

genetic clones of CLL cells within a given patient.

Specifically, TP53 increases production of miR-34b/miR-34c (coded by

11q), which in turn (1) inhibits production of ZAP70 protein (which

inhibits apoptosis), as well as (2) increasing the pro-apoptotic

'activity' of TP53 by increasing its acetylation (without changing

the amount of TP53 protein).

Patients having a 13q deletion (of the gene for miR15a/16-1), with no

other genetic abnormalities in CLL cells, is associated with better

prognoses (than with no 13q deletions), implying increased apoptosis

of CLL cells prevails in patients 'on average' in the 13q deletion

scenario. However, the mechanisms by which apoptosis prevails

involves what's going on at all three chomosomal regions.

The Editorial summarized this: " Although deletion of the

miR-15/miR-16 locus at 13q relieves BCL2 repression and promotes an

antiapoptotic phenotype, it additionally augments TP53 expression,

miR-34b/miR-34c cluster activation, and ultimately ZAP70 suppression.

To that end, the benefits experienced by patients with a 13q deletion

may reflect the capacity for greater restraint on ZAP70 expression

and its downstream targets, including the antiapoptotic Akt and ERK

pathways. Conversely, the poor outcomes associated with an 11q

deletion are likely a manifestation of dampening the TP53-mediated

miR-34b/miR-34c inhibition of ZAP70. "

In a related perspective on the biochemical nature of indolent CLL,

Wednesday's Science Daily article (link below) indicates: " With 13q

deletions, miR-15a and miR16-1 are reduced or absent, which allows

for increased expression of TP53. This further activates miR-34b and

miR-34a, resulting in greater inhibition of Zap70. That's the good

part. With miR-15a and 16-1 gone, expression of the two genes that

suppress programmed cell death, BCL2 and MCL1, increases, which

allows more aberrant cells to form and grow. While this effect causes

slow-growing CLL, the counterweight of suppressed ZAP70 keeps it from

getting worse, Calin said. "

From this perspective, one can better understand how there can be a

wide array of differences in whether and how fast disease progression

will occur in different patients with 13q deletions and indolent

CLL. The balance between increased Bcl-2/Mcl-1 vs. decreased ZAP70

can remain stable or be altered, depending on other factors (known or

unknown) affecting either side of that balance. Clearly, the

continued formation/growth of new CLL cells increases the potential

for development of new clones of CLL, some of which may contain more

aggressive traits (e.g. 17p or 11q deletions).

C. Homozygous vs. Heterozygous 13q Deletions:

One of the differences between patients with 13q deletions is whether

a 13q deletion is a deletion in one or both DNA strands of the 13q

region. When a deletion occurs in only one strand, miR-15/miR-16 can

continue to be expressed from the other strand. As such, one would

expect that levels of miR-15/miR-16 in CLL cells with no 13q

deletions (or any other deletions, i.e. " normal cytogenetics " ) would

be higher than its expression in CLL cells with deletions in one

strand ( " heterozygous " or " monoallelic " deletions), which, in turn,

would have higher levels of miR-15/miR-16 than CLL cells with

deletions in both strands ( " homozygous " or " biallelic " deletions) of

DNA in the 13q region. And that is what the authors observed and

reported in this JAMA paper.

Similarly, because decreased miR-15/miR-16 causes increased TP53, one

would expect that levels of TP53 would be lower in CLL cells with

normal cytogenetics than CLL cells with heterozygous 13q deletions,

which, in turn, would be expected to have lower levels of TP53 than

CLL cells with homozygous 13q deletions. And that is what the

authors observed.

Recently, it has been reported (H et al., Dec2010, reference

below) that CLL patients with 13q deletions outside of the minimally

deleted region (MDR) have a worse clinical outcome than patients with

deletions within the MDR region. The " minimally deleted region " is

defined as the smallest deleted region shared in all patients with

13q deletions. This JAMA paper did not report results for CLL cells

with deletions larger than MDR vs. CLL cells with deletions within

the MDR regions. However, that assessment would be an interesting

follow-up study.

D. CLL cells with 13q And 17p Deletions:

Although the negative effects of 17p deletions seem to dominate any

positive effects of 13q deletions, in some studies, patients having

both 13q and 17p deletions have been reported to have a better

prognosis than patients with only 17p deletions. In that context,

the authors state (in the eComments of the JAMA paper) that: " ....we

believe that in CLL patients with the concomitant 13q and 17p

deletions, the 13q deletion unlocks the residual TP53 from the

inhibitory effects of the miR-15a/16-1 cluster, which could explain

why their prognosis is better than that of patients with the 17p

deletion alone. "

E. Other Regulatory miRNAs:

The authors acknowledge that other, yet to be discovered, miRNAs

exist that are important in regulation of CLL cells, and additional

regulatory miRNAs could be coded for by chromosomal regions other

than (or including) the 6 chromosomal regions listed above.

F. Impacts of Stromal Microenvironments

Finally, " the Elephant waiting 'outside' of the room " , i.e.

regulation by microenvironment stromal cells:

To have achieved these important new understandings inherent in this

JAMA paper, I believe it was necessary to conduct the experiments as

they were done on isolated CLL cells, in the absence of the

complicating biochemical realities associated with the in vivo

pathogenic proliferation centers. In response to biochemical signals

from the CLL cells, stromal cells within these microenvironments of

proliferation are altered, which in turn stimulates (or destimulates)

the stromal cells to produce biochemical signals, the overall result

of which is to alter the tissue environment in ways that better

enable CLL cells to survive and proliferate.

As so often has happened with promising therapeutic ideas gleaned

from studies of isolated CLL cells, these CLL cell alterations of in

vivo stromal environments' have biochemically trumped and negated that promise.

Clearly, one of the next steps is to ask scientific questions about

how this miRNA/TP53 circuitry within CLL cells (indolent vs.

aggressive clones) is affected by factors, cells, environments (e.g.

oxygen levels, nutrient levels) that simulate those of in vivo

environments. When it comes time to draw a diagram that includes

these impacts of the stroma, it will be more important to add a box

enclosing the word " PROLIFERATION " .

G. New Kinase Inhibitors

The good news is that therapeutic agents are being reported to be

effective for CLL by over-riding the protective effects of the

stromal environments. These agents include R406 (a spleen tyrosine

kinase inhibitor, Syk inhibitor), CAL-101 (a phosphoinositide

3'-kinase inhibitor, PI3K inhibitor), PCI-32765 (a Bruton's tyrosine

kinase Inhibitor), etc. Links to the associated kinase inhibitor

research are below.

REFERENCES:

1. Science Daily News Article (Jan. 12, 2011):

http://www.sciencedaily.com/releases/2011/01/110107150604.htm

2. Medpage News Article (Jan. 5, 2011):

<http://www.medpagetoday.com/HematologyOncology/Leukemia/24186>http://www.medpag\

etoday.com/HematologyOncology/Leukemia/24186

3. JAMA Editorial: " Unraveling the Molecular Pathogenesis of Chronic

Lymphocytic Leukemia: Dissecting a MicroRNA Regulatory Network " ;

JAMA. 2011;305(1):95-97. doi: 10.1001/jama.2010.1940

http://jama.ama-assn.org/content/305/1/95.extract

4. JAMA Scientific Paper: Abstract

" Association of a MicroRNA/TP53 Feedback Circuitry With Pathogenesis

and Outcome of B-Cell Chronic Lymphocytic Leukemia " ; JAMA.

2011;305(1):59-67. doi: 10.1001/jama.2010.1919

<http://jama.ama-assn.org/content/305/1/59.short>http://jama.ama-assn.org/conten\

t/305/1/59.short

5. " 13q deletion anatomy and disease progression in patients with

chronic lymphocytic leukemia " ; H et al.; Leukemia advance

online publication, 10 December 2010; doi:10.1038/leu.2010.288

http://www.nature.com/leu/journal/vaop/ncurrent/abs/leu2010288a.html

6. " Phosphoinositide 3-Kinase (PI3K) Delta Inhibition with CAL-101

Blocks B-cell Receptor (BCR) Signaling and the Prosurvival Actions of

Nurselike cells (NLC), in Chronic Lymphocytic Leukemia " ; 52nd

American Society of Hematology (ASH) Annual Meeting, Dec. 4-7, 2010

View Presentation at: http://tinyurl.com/6yz6skh

7. " Brutons Tyrosine Kinase Inhibitor PCI-32765 Abrogates BCR- and

Nurselike Cell-Derived Activation of CLL Cells In Vitro and In Vivo "

; 52nd American Society of Hematology (ASH) Annual Meeting, Dec. 4-7, 2010

View Presentation at: http://tinyurl.com/46hxa2p

8. " Spleen tyrosine kinase inhibition prevents chemokine- and

integrin-mediated stromal protective effects in chronic lymphocytic

leukemia " ; Blood, 3 June 2010, Vol. 115, No. 22, pp. 4497-4506.

http://bloodjournal.hematologylibrary.org/cgi/content/abstract/115/22/4497

Al Janski