targeting telomeres in human disease: advances and ... · the maintenance of telomeres is essential...
TRANSCRIPT
1
Targeting Telomeres in Human Disease: Advances and Therapeutic Opportunities
30 April 2014 [0:00:00] Slide 1 Sean Sanders: Hello everyone and thank you for joining this Science/AAAS webinar. I'm
Sean Sanders, Editor for Custom Publishing at Science. Slide 2 The maintenance of telomeres is essential to the health of any cell while
dysfunction in telomere maintenance pathways plays an integral role in aging, cancer, and certain rare diseases. The molecular analysis of telomere lengths, telomerase levels, and activation of the alternative lengthening of telomere's pathway are currently being used as prognostic tools in human diseases and current research is targeting telomere lengthening mechanisms as a cancer therapy.
In this webinar, our experts will describe the advances in telomere
research as it applies to human health and how deep a characterization of the telomerase maintenance process can uncover targets for therapies. It's my pleasure to introduce our webinar panel to you now.
First, we have Dr. Roger Reddel from Children's Medical Research
Institute in Sydney, Australia. Our second speaker will be Dr. Suneet Agarwal from Boston Children's Hospital in Boston, Massachusetts. Thank you both very much for being here today. Before we get started, some important information for our viewers.
Slide 1 Note that you can resize or hide any of the windows in your viewing
console. The widgets at the bottom of the console control what you see. Click on these to see speaker bios, additional information about technologies related to today's discussion, or to download a PDF of the slides.
Each of our guests will give a short presentation followed by a Q&A
session, during which they will address the question submitted by our live
2
online viewers, so if you're joining us live, start thinking about some questions now and submit them at any time by typing them into the box on the bottom left of your viewing console and clicking the "submit" button.
If you can see this box, click the red Q&A widget at the bottom of the
screen. Please remember to keep your questions short and concise as this will give them the best chance of being put to the panel. You can also log in to your Facebook, Twitter, or LinkedIn accounts during the webinar to post updates or send tweets about the event. Just click the relevant widgets at the bottom of the screen. For tweets, you can add the hashtag #ScienceWebinar. Finally, thank you to PerkinElmer for sponsoring today's webinar.
Now, I'd like to introduce our first speaker, Dr. Roger Reddel. Dr. Reddel
is currently Director of Children's Medical Research Institute in Sydney, Australia where his studies have mostly focused on the role of p53 and retinoblastoma abnormalities, and on telomere length maintenance mechanisms. He is also the Lorimer Dods Professor in the Sydney Medical School at the University of Sydney.
Welcome, Dr. Reddel, and many thanks for being with us. Slide 3 Dr. Roger Reddel: Thanks very much for the introduction, Sean. I'd like to talk with you
today about the opportunities which may exist to target telomeres for cancer treatment.
There's a huge amount that still remains to be learned about telomere
structure and function, how this becomes abnormal in cancer cells. What I'll do today is give some background about telomere biology, make some comments about techniques for investigating telomere lengthening in cancers, and then outline some ideas about opportunities to apply some of what we do know about telomere biology to develop new cancer therapeutics.
Slide 4 The first point to note is that telomeres, the ends of chromosomes,
consist of repetitive DNA. In all vertebrate species, the repetitive unit is a hexanucleotide, TTAGGG repeated many times. In each of the diagrams that I've prepared for this webinar like the slide in front of you now, the bulk genomic DNA in the chromosome is drawn in blue with the
3
centromere being off the left of the slide and the repetitive DNA of the telomere is drawn in red.
Another point to note here is that the telomeric DNA is not double‐
stranded all the way to its end. The last part of it is single‐stranded. Slide 5 The repetitive DNA of the telomere is recognized by specific binding
proteins, especially shelterin. This is a complex of six different proteins which coat the telomere and protect it from being recognized as a DNA break, and therefore protect it from inappropriate DNA repair reactions.
Slide 6 Telomeres are able to adopt a higher order structure referred to as a
telomere loop or T‐loop where the telomere folds back on itself and the single‐stranded end burrows into a double‐stranded region forming a lasso or a lariat structure which protects the telomere's very end.
Slide 7 In addition, recent evidences confirm something that's long been
suspected that has to do with the G‐rich nature of the TTAGGG sequence. [0:05:03] Because telomeres are very G‐rich sequences, they can form G‐quartet
structures where four Gs bond to each other by Hoogsteen base pairing. When G‐quartets are stacked next to each other, the overall structure is referred to as the G‐quadruplex.
Slide 8 One of the most important pieces of background information to know
about telomeres is that they're not completely replicated right to their end when all the other DNA in the chromosomes is being replicated. So every time a cell replicates its DNA with every cell division, the telomere becomes a little shorter.
In normal cells, this continues until the telomere reaches a specific length
and causes a DNA damage response, which signals to the cell that it can no longer proliferate. The cell remains alive, but permanently unable to replicate in a state referred to as senescence. This provides a very potent
4
protection against cancer because if a cell is starting to accumulate pro‐oncogenic mutations, the telomere's shortening process eventually forces its progeny to stop dividing and become senescent before a clinically significant tumor forms.
Slide 9 This process of gradual telomere shortening is opposed by telomere
lengthening processes. One way in which telomeres can be lengthened is by the activity of an enzyme called telomerase, which synthesizes new telomeric DNA by reverse transcription. Telomerase contains a reverse transcriptase subunit (TERT) and an rRNA molecule which contains a template region for synthesis of the TTAGGG DNA sequence. The dyskerin subunit is an rRNA binding protein.
There are some normal cells which have low levels of telomerase which
slows down, but doesn't completely prevent the shortening process. In contrast however, in most cancers, telomerase is up‐regulated sufficiently to completely compensate for the normal shortening process so that telomere length in the cancer cells is maintained.
Slide 10 In a smaller subset of cancers, there's an alternative to telomerase called
ALT, Alternative Lengthening of the Telomeres. Slide 11 Unlike telomerase, which reverse transcribes an rRNA template, ALT
involves copying of a DNA template. Slide 12 What I've illustrated here is one telomere inviting another telomere and
using it as a template for extending itself by DNA replication. Slide 13 Slide 14 ALT involves the recombination step where the telomere invades other
telomeric DNA and the DNA replication step with the end resulting that there is synthesis of new telomeric DNA to counteract the normal telomere shortening process.
5
Slide 15 The great majority of cancers use one or other mechanism, telomerase or
ALT, to completely prevent telomere shortening, allowing them to bypass the senescence barrier and to go on dividing in unlimited number of times.
Slide 16 In addition to the normal, steady attrition of telomeric DNA that occurs
with each cell division, there is also a rapid shortening process called telomere trimming that occurs when telomeres get overlengthened for whatever reason.
Slide 17 If an overlengthened telomere occurs, the cell is able to correct this
rapidly by shedding a T‐circle, thereby shortening the telomere and restoring it to its former length.
Slide 18 Slide 19 If we're going to use cancer therapeutics that are based on telomere
biology, we need to have ways of detecting which telomere lengthening process is present in a tumor. For two decades now, the TRAP assay, the Telomere Repeat Amplification Protocol, has been used as the standard technique for detecting telomerase activity in cell lines and tumors.
The principle of this assay is that a cell lysate is presented with DNA
molecules which are subtrates for telomerase together with dNTPs. Extension of the substrate by telomerase results in a mixture of extension products with a range of lengths. Each of these DNA molecules can then be amplified by PCR in the presence of radiolabel or some other way of labeling the PCR products and visualized after gel electrophoresis.
As you can see from the panel on the right, an ALT cell line has no
detectable TRAP activity, whereas in a telomerase positive cell line or tumor, activity can be recognized by the presence of a six base pair ladder of extension products.
[0:10:07]
6
Slide 20 One of the known problems with a TRAP assay, especially for tumors, is
that there are TRAP inhibitors present in some tumor lysates. A potential way around this is to use an immunoprecipitation step first to clean up the lysate using an antibody against the TERT catalytic subunit of telomerase.
As illustrated here, telomerase can be pulled down with the antibody and
then released by supplying an excess of the peptide against which the antibody was originally raised. This cleaned up lysate is then presented with a telomerase substrate DNA molecule which gets extended in the presence of label as illustrated before, then the extension product is separated by gel electrophoresis and visualized as a six base pair ladder.
What you can see from the tumor panels on the right is that at the
highest amount of protein lysate from tumor A, the lane labeled 10, there is no detectable TRAP activity and it's only as the lysate is diluted that the TRAP activity begins to appear.
When immunoprecipitation is carried out first, there are no longer
problems with the inhibitors in the lysate, but if you compare the lanes labeled 0.1, the activity is clearly present in the total lysate, but there's no detectable activity of this deletion when IP clean‐up was done first, so there's a trade‐off here with the IP‐TRAP removing problems with inhibitors, but reducing the sensitivity of the assay, and the same effect is seen in tumor B in the panel below.
Slide 21 A more quantitative way of measuring telomerase activity is via a direct
assay and there are various protocols available. The one illustrated here was developed by Scott Cohen and you can see that the first step is an immunoprecipitation using an antibody against TERT.
Again, the telomerase is released from the antibody by adding an excess
of the peptide antigen against which the anti‐TERT antibody was generated, and then that cleaned up lysate is presented with a substrate molecule and nucleotides so the substrate can be extended by telomerase.
One of the nucleotides can be radiolabeled to allow visualization of the
extension products after gel electrophoresis. You can see from the
7
quadruplicate assay's reach of four telomerase positive cell lines that this method is highly reproducible.
Slide 22 We don't have an enzyme assay for alternative lengthening of telomeres
and we rely instead on various methods for detecting the phenotypic characteristics of cells in cancers that use ALT. One of these methods is terminal restriction fragment Southern blotting. The principle of this is that genomic DNA is first cut with restriction enzymes that don't recognize the TTAGGG repeat sequence, which generates terminal restriction fragments consisting mostly of the telomere plus a small amount of sub‐telomeric DNA down to the first restriction site.
These fragments have been separated by gel electrophoresis, Southern
blotted, and detected with a radiolabeled probe for telomeric DNA. What you can see here is the very stark contrast between the telomere smear pattern seen in ALT and telomerase positive cells. ALT cells typically have highly heterogeneous telomere lengths ranging from very short to very long with the main being much longer than in telomerase positive cells which have more homogeneous telomere lengths.
Slide 23 Another robust method of detecting ALT in tumors is to visualize ALT‐
associated PML bodies. PML bodies are structures found in the nuclei of most cells and what's unusual about ALT‐associated PML bodies is that they contain telomeric material, telomeric DNA, and telomeric binding proteins. They've been visualized here by immunostaining using antibodies against TRF2, one of the shelterin proteins, and the PML protein, which forms the outer shell of PML bodies.
As you can see, there's telomeric material inside the PML bodies in these
ALT cell nuclei. This technique is very useful for detecting ALT infections of formalin‐fixed, paraffin‐embedded tumors or in frozen sections of in cytology specimens.
Slide 24 And then the final technique for detecting ALT activity that I'll mention
today is the C‐Circle assay. ALT‐positive cell lines and tumors characteristically contain an unusual type of DNA molecule which consists of a circle of partially single‐stranded telomeric DNA where the C‐rich strand is intact and the G‐rich strand is incomplete.
8
We refer to these molecules as C‐Circles and they can be used as
substrates for rolling circle amplification by phi29 polymerase without the addition of any primer.
[0:15:07] The amplified material can be dot blotted and detected with a labeled
probe. What I've shown here is the amplification products from serially diluted genomic DNA from an ALT cell line IIICF/c and on the right, the phi29 negative controls, which are particularly important for tumor samples.
The C‐Circle assay is very useful for detecting ALT activity when genomic
DNA is available from tumors and if the amount of material available is very small, quantitative PCI can be used to detect the rolling circle amplification products instead of dot blotting and probing.
Slide 25 Tumors that predominantly use ALT include osteosarcomas, some types
of soft tissue sarcomas, and Grade 2 and 3 astrocytic brain tumors. ALT is also quite common in glioblastoma multiforme, liposarcomas, and neuroblastomas. It occurs much less commonly in some types of soft tissue sarcoma including rhabdomyosarcoma and most types of carcinomas. About 10% of human genomes are thought to use ALT and most of the remainder use telomerase.
Slide 26 Survival of patients with osteosarcomas was shown by a group at
Stanford that depended on whether the tumor had a detectable telomere lengthening mechanism at all. Those that had no detectable telomere lengthening mechanism did better than those that had either telomerase or ALT.
For liposarcomas, a group in Milan showed that those with no detectable
telomere lengthening mechanism did better than those with telomerase, which in turn did better than those with ALT. In other words, ALT is an indicator of poor prognosis in liposarcoma.
In contrast, in the case of glioblastoma multiforme, a group in Sheffield
showed that although all of the patients had a poor outcome, those with ALT‐positive tumors did significantly better than the others, so the
9
prognostic significance of the telomere lengthening mechanism depends very much on the tumor type.
Slide 27 When we turn our attention to what potentially can be done to target
telomeres for cancer therapy, I think there are many opportunities. Regarding telomerase, we need to be thinking not just about inhibiting its catalytic activity, but much more broadly about the many steps within its life cycle that could potentially be targeted.
Targeting the process of assembling the telomerase complex, the
telomerase biogenesis process has the potential to greatly reduce the amount of telomerase activity in the cancer cell. Once it's assembled, the telomerase molecule then needs to be transported or trafficked to the telomere, and this process could be inhibited.
When it arrives at the telomere, the telomerase needs to dock with the
telomeric DNA, a process which is intricately regulated by other proteins that are present at the telomere that could potentially be targeted.
The catalytic activity of telomerase can be inhibited in a number of ways.
An oligonucleotide molecule which does this by binding to the template region of telomerase's RNA subunit has been in clinical trials.
Finally, telomerase needs to move from one telomere to another by a
process of which we know very little, and this step could also potentially be targeted in cancer cells with therapeutic purposes.
Slide 28 Similarly, for ALT, we need to be thinking about all of the steps in the
process of telomere lengthening body's mechanism. For example, a telomere that undergoes a lengthening by ALT must first find and become juxtaposed to a copy template molecule such as another telomere and it may be possible to target this step.
Slide 29 It's known that there are peptide epitopes from the TERT catalytic
subunit of telomerase that are displayed on the surface of cancer cell that use telomerase and which can be recognized by T‐cells. There are clinical trials of treating cancer by vaccinating against these epitopes.
10
Slide 30 In addition, there may be aspects of the telomere architecture which are
different in cancer cells. We know in ALT cells that there are variant repeat DNA sequences present in unusually large quantities in the distal parts of their telomeres and this ALT is shelterin binding. This reduced protection of ALT telomeres by shelterin might create some vulnerabilities in cancer cells that depend on ALT, which could be exploited for therapy.
Slide 31 It's also possible that the telomeres of cells with up‐regulated telomere
lengthening mechanisms are more vulnerable to the binding of G4 ligands, molecules designed to bind to the G‐quadruplex structures that can form within telomeres.
Slide 32 The telomere lengthening processes are up‐regulated or switched on in
cancer cells by mechanisms which we don't yet fully understand, but the up‐regulation processes themselves may provide opportunities for new approaches to therapy.
[0:20:12] There have been attempts to target cancer cells in which expression of
TERT is up‐regulated by transactivation, using a gene therapy approach in which the TERT promoter is linked, for example, to a gene encoding a pro‐drug activating enzyme, which means that an anti‐cancer pro‐drug will be selectively activated in cells where telomerase is up‐regulated.
Slide 33 For ALT to be switched on, we know that there are repressor molecules
that need to be lost or inactivated and this may open up the possibility of synthetic lethal approaches to treating ALT cancers. For example, many ALT cancers lack expression of the ATRX protein and ATRX is involved in cellular resistance to various stresses, so it may be possible to find a stressor to which ATRX negative ALT cancers are highly susceptible.
Slide 34
11
Maybe it will be possible if we understand telomere trimming in more detail to selectively stimulate rapid telomere loss in cancer cells.
Slide 35 I think there are going to be a number of challenges that need to be faced
in developing cancer treatments based on what we're starting to learn about telomere biology. One of them is the length of time it may take for inhibitors of ALT or telomerase to act, which may depend in part on the pre‐existing telomere length in the cancer cells at the start of treatment.
Slide 36 Here's an example of a cell culture experiment where ALT was turned off
in a cell line using a genetic manipulation and the telomeres immediately began eroding. The telomeres appear as bands on this gel and are highlighted by the arrows on the left, whereas the arrows on the right highlight interstitial sequences that don't shorten.
The telomeres eroded at a rate of about 130 base pairs per cell division,
the sort of rate you'd expect if a cell had its telomere lengthening mechanisms completely inactivated, but the cells continued proliferating and the telomeres continued shortening for 60 to 80 population doublings before the effects in inhibiting ALT began to be seen.
This suggests, I think, that we're going to need to combine inhibitors of
telomere lengthening mechanisms with other treatments that rapidly debulk the tumor.
Slide 37 Other challenges may include side effects. As I mentioned earlier, some
types of normal cells require a level of telomere lengthening activity, which is not enough to allow them to permanently bypass senescence, but which is enough to allow them to continue proliferating throughout the human life span.
So inhibiting telomere lengthening activity may eventually have
consequences for normal somatic cells as well as for cells of the germ‐line.
Thirdly, there's evidence both in vitro and in vivo that tumor cells
respond to effective inhibition of one telomere lengthening mechanism
12
by switching on the other, which may mean that we'll need to learn how best to use a combination of ALT and telomerase inhibitors.
Finally, although it has become widely accepted that ALT cancer cells
have a telomere lengthening mechanism, there will be a subset of cancers that survive without any telomere lengthening mechanism at all. If this is correct, it'll be very important to identify these tumors and to avoid treating them with inhibitors of telomere lengthening, which are likely to be quite futile in this situation.
Slide 38 In concluding, I would especially like to acknowledge Cancer Council in
New South Wales and the other funding agencies shown here which have funded research in my lab.
Sean Sanders: Fantastic! Thank you so much, Dr. Reddel. We're going to move right on
to our second speaker today, and that is Dr. Suneet Agarwal. Slide 40 Dr. Agarwal is currently an assistant professor in Pediatrics at Harvard
Medical School, principal faculty at the Harvard Stem Cell Institute, and a staff physician in hematopoietic cell transplantation at Boston Children's Hospital and the Dana‐Farber Cancer Institute. He is interested in the mechanisms and therapy of genetic blood diseases with a focus on dyskeratosis congenita.
A warm welcome, Dr. Agarwal, and thanks for joining us today. Dr. Suneet Agarwal: Thank you very much. Thank you for the invitation to discuss human
telomere diseases today with you. A prototype of these diseases is dyskeratosis congenita and in the next 20 minutes, I'll describe dyskeratosis congenita or DC and give a historical view of genetic discoveries in the disease.
[0:24:59] I'll then discuss how genetics has led to an identification of a broad
spectrum of telomere diseases and the complex ways in which lesions and telomere biology genes are impacting clinical diagnosis and also impact clinical phenotype.
13
Finally, I will describe how this understanding of DC as a telomere disease is impacting therapy, in particular, in bone marrow transplantation.
Slide 41 Dyskeratosis congenita is a rare disorder first described almost a hundred
years ago and is characterized by a clinical triad of irregularities and skin pigment abnormalities in the nails and white plaque‐like lesions on mucosa surfaces such as the mouth.
These are the main and major findings, but even early on, it was
recognized that these signs were the outward manifestations of a broader systemic disease that cause life‐threatening dysfunction in other tissues.
Eighty percent of patients develop aplastic anemia or bone marrow
failure by age 30. There's a high incidence of cancer of the blood, skin, and mucosal surfaces, increased risk of liver and lung disease, and many other defects. In fact, any organ system can be involved leading to a significantly decreased overall survival. Over the course of several decades, despite a more refined clinical description, the ideology remained a mystery.
Slide 42 The first real inroads came from a recognition that there was a high
proportion of boys affected and in the '80s, this led to linkage studies implicating a region on the X‐chromosome.
The key was in the mid‐'90s when Inderjeet Dokal in London established
the dyskeratosis congenita registry. This was important in obtaining enough families and enough power and analysis to narrow down the region even further to an approximately 1.4 cM region on the X‐chromosome.
Dokal and colleagues analyzed the integrity of 28 genetic loci in this
interval in patient DNA samples and as shown here by Southern blot using a p32 radiolabeled cDNA probe.
We found a patient with a deletion and putative gene indicated by the
arrow. Sequencing of that gene led to the identification of missense mutations in five other patients. They dubbed this gene DKC1 and all that was really known about it was that it was a homolog of a yeast pseudouridine synthase that binds a class of small nucleolar RNAs via a
14
box H/ACA motif, and so, giving it a proposed role in ribosomal RNA modifications or ribosomal biogenesis, but the real pathophysiology was not understood by this finding.
Slide 43 The next major insight came from the work of Mitchell and Collins who
were studying the structure of vertebrate telomerase RNA component called TERC. They recognized that TERC contained a box H/ACA motif and proposed that perhaps there was a defect in this component in patients with DKC1 mutations.
The key experiment was this Northern blot in their Nature paper using a
p32 radiolabeled probe to quantify the TERC transcript and showing a defect in TERC levels in patients with DKC1 mutations. This led them to conclude that DC might actually be caused by deficiencies in telomerase. And very remarkably two years later, Vulliamy, et al. tied all of this together with a report of TERC mutations in a large family with autosomal dominant dyskeratosis congenita, which pointed firmly in the direction that indeed DC was likely to be a disorder of telomere biology.
Slide 44 Dr. Reddel has already introduced telomeres, but as way of brief
background, telomeres are the ends of chromosomes composed of hundreds of thousands of copies of hexanucleotide repeat. By forming a complex protein DNA structure, they protect chromosome ends and prevent unwanted chromosome fusions.
Because of the end replication problem, telomere repeats are lost with
each cell division and when enough copies are lost, somatic cells stop dividing as a protective measure and enter senescence. Therefore, telomere length is associated with cellular replicative capacity.
The ribonucleoprotein complex, telomerase, can extend telomeres and is
expressed in cells with self‐renewal capacities such as embryonic and adult stem cells and then cancer cells. It's composed of the RNA template, TERC, which we've already discussed; a reverse transcriptase, TERT, which is expressed only in the cells that have telomerase activity; and several constitutively expressed factors that are required for the stability of telomerase RNA component and also the subnuclear localization of the telomerase's whole enzyme.
Slide 45
15
Based on these findings, one would predict that if DC is a telomere
disease, then patients will have shorter telomere length in their tissues and decrease the replicative capacity of their cells, which may account for the disease findings, and both of these are true.
Here's a telomere Southern blot where the telomeres are detected from
the patient's peripheral blood DNA on the left. This can be done with a p32 radiolabeled probe against the telomere repeat, which has the advantages of providing not only a direct measure of telomere length distribution, but also a simple assessment of the quantity of overall telomere DNA.
Telomere length in the peripheral blood decreases as a function of age,
but the DC patient shown here has a shorter mean telomere length compared to his mother and father even though they are older.
[0:30:02] DC patient somatic cells also show impaired replicative capacity. There
are explants fibroblast growths in vitro showing the impaired proliferation capacity of the cells from the DC patient compared to the normal. This correlates what's shown below. The premature senescence is correlated with a decreased initial telomere length in the DC patient compared to the normal.
Slide 46 This finding of decreased telomere length can be exploited as a clinical
test. Lansdorp and colleagues have developed a fluorescence in situ hybridization base method using a fluorescent probe to hybridize quantitatively to telomere ends and peripheral bloods cells such that the fluorescence intensity correlates with the summation of lengths of all telomere ends in the cell.
By combining the FISH method with flow cytometry, the mean telomere
length and peripheral blood subsets are determined and plotted on a graph which has normalized for patient age.
Here, Alter and colleagues show that the detection of less than first
percentile telomere length age‐adjusted by flow‐FISH has a high sensitivity and positive predictive value for detecting patients with DC. This test is a change practice and we send it routinely on patients with
16
bone marrow failure as a screen for whether they may have a subclinical phenotype of dyskeratosis congenita.
Slide 47 In the past 15 years, ongoing genetic discovery has confirmed that DC is a
telomere disease and that mutations in nine genes in ALT and telomere biology have now been implicated and account for approximately 60% of patients with DC with 40% remaining unknown. Every inheritance swarm is represented. This includes mutations in telomerase components as well as telomere structural components and application machinery.
This diagram by Calado and Young makes a very important point
however. The groups of Young, Greider, Armanios, Garcia and others discovered that some patients who have isolated idiopathic aplastic anemia (bone marrow failure) or idiopathic pulmonary fibrosis (lung disease) alone without the classic findings of dyskeratosis congenita can also have mutations in TERC and TERT. This has led to a recognition that telomere diseases exist across a broad spectrum of phenotypes.
Slide 48 This is captured here in a report by Bessler, et al. where she shows that
some patients present with very severe forms of telomere diseases early in infancy such as the Revesz and Hoyeraal Hreidarsson syndromes, which are caused by DKC1 and TINF2 mutations.
Other patients can present in later decades of life with isolated aplastic
anemia, with cancer, with AML, and pulmonary fibrosis or lung fibrosis without plastic manifestations of DC. There is some genotype‐phenotype correlation, but it is imperfect. There are several factors that can account for some of this very presentation and I'd like to describe a few of them.
Slide 49 First, it was apparent from the early studies of Vulliamy and colleagues
that families with autosomal dominant forms of DC showed an anticipation, genetic anticipation, which means an earlier onset of symptoms in younger generations.
This can be explained by the theory that not only do affected individuals
and younger generations inherit the genetic lesion itself, but also that they are passed on shorter telomere length so that they have less of a
17
reserve or endowment of telomere length before they reach a critical stage where they're causing functional problems in tissues.
Slide 50 This raises an even more interesting issue of what happens if you inherit
only the short telomeres, but not the mutation itself, a term which is coined by Carol Greider called the telotype. Indeed, Garcia and colleagues have shown up here on the left that in families with TERT mutations, that in younger generations of people who have the mutation, telomere length is indeed compromised.
But even in those who don't inherit the mutation in subsequent
generations, telomere length is also shorter than in prior generations. Does that mean that the patients will actually have a problem just because they've inherited shorter telomere length?
The answer to that is not clear, but I'd like to share with you the pedigree
of a family that we've cared for on the right, which shows five children, all of whom were deemed clinically to actually have blood defects, but on mutational analysis, only three of the patients carry the mutation.
Interestingly, the other two siblings also had relatively short telomere
length around the first percentile. And for this reason, they were deemed unsuitable as bone marrow donors. So it may be that inheriting short telomeres does in fact cause tissue‐specific disorders.
Slide 51 Another complication is phenotypic variation, different manifestations
despite carrying the same mutations. I'd like to describe a case for you of a 15‐year‐old girl we cared for who had classic dyskeratosis congenita characterized by skin and oral changes and bone marrow failure.
She had very short telomere length as shown by the telomere plot on the
right, but she was negative for any known genes. In cases like this, we look for candidate genes to sequence or for new genetic discovery by exome sequencing.
Slide 52 When we're looking for candidate genes to sequence on this patient and
others, there's a very interesting report that came out from Anderson and colleagues in a seemingly unrelated syndrome called Coats plus.
18
[0:35:08] They were looking for the genetic basis of this disorder which is
characterized by brain cysts, bone lesions, and vascular abnormalities in the eyes and in the gut. By exome sequencing, they found mutations on a gene called CTC1, conserved telomere maintenance component.
Slide 53 Interestingly, when they were looking at these patients, they found that
some of them, not many of them, but some of them have sparse, gray hair, nail changes or low blood counts, none of which would qualify as bone marrow failure, but some abnormalities that seem to overlap with DC.
And because of the nature of this gene, some of their patients were
subjected to telomere length analysis and showed indeed first percentile or somewhat low telomere length compared to their parents. This led to the hypothesis that some individuals with DC might actually have mutations in the same gene called CTC1.
Slide 54 We sequence the genes on our 15‐year‐old patient and found compound
heterozygous mutations in CTC1 as well. One allele showed a frameshift, so predicting a truncated protein, and the other showed a single amino acid deletion. So it's possible that even though our patient didn't seem to have Coats plus but have DC that there could be allelic variation that would lead to different syndromes.
What was very interesting about this patient's alleles is that they were
also shared and found in the patients who actually had Coats plus, identical alleles causing the frameshift and also the single amino acid deletion.
Slide 55 We looked in this patient to see if she actually had manifestations of
Coats plus, which might not have been obvious. Slide 56
19
First, we looked at retinal eye vessels, which were subtly involved, but not in the same way that Coats plus patients are. She actually had subclinical calcifications and cysts in her brain and she had very insignificant vascular abnormalities in her gut, none of which would cause any significant problems, so she has some similarities, but important differences to the syndrome of Coats plus.
Slide 57 Subsequently, Dokal and colleagues using the UK registry also screened
for CTC1 mutations and found that 6 of 73 patients with classic DC had compound heterozygous CTC1 mutations. This is nicely summarized in this review by Sharon Savage. This basically proves that Coats plus and CTC1 mutations are along the spectrum of telomere diseases and that mutations in the same gene can cause very significantly different phenotypes even if they have similar alleles.
Slide 58 One other confounding factor about genetics in DC is the observation of
somatic reversion. In this very nice paper by Jongmans, et al. they showed that some patients with heterozygous TERC mutations had undergone reversion of the mutation in the blood. So on the lower chromatogram, you see that the patient has a heterozygous mutation and skin fibroblast, but only one pattern in the blood, and that is corresponding to the wild type allele.
This is corresponding to an event of uniparental disomy by loss of
heterozygosity in both myeloid and lymphoid cells in the patient, which indicated an early event in hematopoietic progenitors. This reminds us to look at somatic tissues when we're checking the genetics with patients with DC, but it also makes a strong statement about the potential selective advantage of cells with two functional copies of TERC in a tissue compartment such as the blood, which would reconstitute hematopoiesis if there was revertant mutation.
Slide 59 In summary, there's been huge progress in the genetics and
understanding of DC in the past 15 years. Patients are aided by a functional test called age‐adjusted mean telomere length testing and also by the identification of several genes that are implicated in DC and other related diseases, but there are several confounding factors which make it hard to diagnose patients, including an incomplete genetic
20
characterization, multiple modes of inheritance, genetic anticipation, variable penetrance and the telotype, and events of somatic reversion.
In practice, this means that we at this stage evaluate patients of any age
presenting with aplastic anemia, associated science, or a suspicious family history for DC or telomere disease. The diagnosis of DC or telomere disease has significant implications for management, therapy, and family counseling.
Slide 60 With regards to therapy, I'd like to describe to you now how progress in
understanding the telomere biology basis of DC is impacting care. In specific, I'd like to talk to you about allogeneic bone marrow transplantation because there are no cures for any of the defects in DC except for allogeneic bone marrow transplantation, which can cure the blood defects.
The difficulty has been that historically, there have been very poor
outcomes in DC patients undergoing a conventional form of bone marrow transplantation due to increased early and late complications. For this, I provide you a little bit of background about bone marrow transplantation in general.
Slide 61 There are two steps involved. [0:40:00] The first is conditioning and preparing the patient to receive the graft.
This is traditionally done with chemotherapy and radiation, which serves several purposes.
First, most bone marrow transplantations are done from cancer. And in
cases of trying to consolidate a cure for cancer, the chemotherapy and radiation does have some anti‐tumor effect. But in general, for bone marrow transplantation, what's being achieved with chemotherapy and radiation is providing strong immune suppression so that the recipient does not reject the graft, and also creating functional or physical space for the engraftment of hematopoietic stem cells in the recipient's niche.
The second aspect is the transplantation of hematopoietic progenitor
cells from the donor and this provides the advantage of replacing cells,
21
which are normal and not mutant, or enzyme replacement in the case of metabolic diseases, and in the case of malignancy because of some immune differences, providing a graft‐versus‐tumor effect.
Overall, the downsides of conditioning include significant collateral tissue
damage because of a non‐specificity of these agents and significant short and long‐term sequelae; the disadvantages in the case of engraftment across immune barriers or graft‐versus‐host disease, the immune suppression that's required to control that complication and the toxicities and risks.
Slide 62 Alter, et al. analyzed the outcome of BMT for DC in 2009 by reviewing 65
cases in literature and they documented that there was a very poor long‐term survival likely due to the previous position of these patients to pulmonary, hepatic, and vascular complications, and also long‐term secondary malignancies.
These transplants were done using conventional conditioning regimens
and have led to the recognition that perhaps decreasing the toxicity of the regimen would be necessary to achieve satisfactory outcomes in BMT for dyskeratosis congenita.
Slide 63 We and others have adopted reduced intensity conditioning regimens to
try to improve outcomes in DC and I'll tell you the rational for a trial that we opened recently, and that is that because DC is a non‐malignant disease, as long as engraftment is not compromised, the conditioning toxicity will improve outcomes because there's no desired anti‐tumor effect.
Second, eliminating alkylator and radiation exposure will decrease
toxicity to those same organs which DC patients are already predisposed for failure such as liver and lung, and also for the predisposition to cancer.
Third, based on the findings of telomere defects in patients from the
impaired replicative capacity of their cells, we believe that the telomere defect in DC will result in a defect in hematopoietic and immune response to the graft which may favor engraftment with a less toxic conditioning regimen.
22
Slide 64 This is schematized here where we are proposing to eliminate alkylating
agents and radiation in favor of noncytotoxic ‐‐ non‐generally cytotoxic immune suppression only and asking the question of whether engraftment will be successful in patients with dyskeratosis congenita.
Slide 65 This is a schema of the transplant protocol that we opened in July 2012
where we're using off‐label anti‐CD52 antibody as a lymphocyte‐depleting antibody for strong immune suppression, and fludarabine, which is a nucleocyte analog whose cytotoxic effects are limited to hematopoietic space. We use a bone marrow graft and conventional graft‐versus‐host disease prophylaxis.
This is an ongoing study and it's too early to draw conclusions, but we are
encouraged from this study and other recent studies using other reduced intensity conditioning regimens that there have been improved outcomes at least in the short‐term.
On this regimen, four patients have been treated so far and they have
achieved engraftment. The questions are whether they will sustain engraftment, whether this finding will be generalizable to various forms of DC, and whether there will be an improvement in the long‐term outcome.
Slide 66 In summary, DC has been firmly established as a disorder of telomere
biology and this was satisfying because it appears to affect the self‐renewal of cells in various tissues and explain the various manifestations in patients with DC and other related telomere diseases.
There are diagnostic advances, but also ongoing challenges based on
complex genetics and disease manifestations. The task in this area now is to apply new knowledge, to innovate therapies, and develop disease‐specific trials.
Slide 67 I'd like to acknowledge local collaborators and funding sources and
colleagues whose work I'd mentioned who actually made these
23
remarkable advances over the past 15 years in this disease, and also thanks to Science and PerkinElmer for sponsoring this webinar.
Sean Sanders: Fantastic! Thank you so much, Dr. Agarwal and many thanks to both of
our speakers for the wonderful presentations. Slide 68 We're going to move right on to questions submitted by our online
viewers. A quick reminder to those watching us live, you can still submit your questions by typing them into the textbox and clicking the "submit" button.
[0:45:04] We have received a lot of questions, so we're going to try to get through
as many as possible. The first question I'm going to put out to both speakers ‐‐ and I'll start
with you, Dr. Reddel ‐‐ is do you think telomere attrition per se is as significantly meaningful as a biomarker or is it better when combined with other biomarkers, and possibly if you could talk about which ones?
Dr. Roger Reddel: Thanks, Sean. Look, I think the answer to that question depends very
heavily on the context. In the case of the short telomere disease that we just heard about from Dr. Agarwal, it's critically important to know telomere length.
In many other contexts, it's a very interesting experimental subject, but it
doesn't really replace existing clinical markers. If you're studying cardiovascular disease, it doesn't replace ‐‐ it may be an interesting adjunct, but it doesn't replace lipid levels and blood pressure and so on. I got multiple examples.
One thing that's really interesting to keep in mind is that telomere length
really results from a lot of factors acting on it and it's just one number. So in many contexts, the value of its predictive ability is quite limited.
Sean Sanders: Dr. Agarwal, anything to add? Dr. Suneet Agarwal: I agree with Dr. Reddel. Certainly, it's being established that peripheral
blood telomere length can be helpful in the diagnosis of inherited telomere disorders, but there's still a conundrum in how to interpret it in other contexts. The test has to be used in a specific clinical context and
24
it's not really validated generally in other conditions yet, so I think the other biomarkers would be tissue, or to be specific, to take along with telomere length including the patient history to actually try to understand what's going on.
Sean Sanders: Now, a following to that, another question received is how accurate are
the current methods for determining telomere length? Maybe I'll give that to you, Dr. Reddel.
Dr. Roger Reddel: There is still a lot of work to be done. So in the context where it's really
important, and that is the DC and related syndromes, dyskeratosis congenita and other short telomere syndromes, usually the results are fairly unequivocal. It's possible to say with a degree of certainty that telomeres are very short.
In other contexts, the types of measurements that are being used, flow‐
FISH, qPCR, they're reproducible, but the value is often in epidemiological studies where there's a correlation between some parameter in telomere lengths and the value of an individual rating is not always clear.
Sean Sanders: Excellent! Now, Dr. Agarwal, I'm going to come to you with this question.
This is coming in a number of different ways, but essentially, what they're asking is when performing telomere therapy, is there a way to target certain cells or would all cells receive the therapy and would you not be promoting telomerase activity in both good and bad cells?
Dr. Suneet Agarwal: I think we have to speak about this in theory right now because there are
no definitive, proven telomere‐activating therapies that are really used. Androgens are thought to activate telomerase. Certainly, they're used in bone marrow failure for patients with DC, and there's one theory that they act in part by activating TERT, but that would be ‐‐ if the patient's taking that, it would presumably act on any cell that's expressing TERT.
I think the challenge of activating telomere lengthening mechanisms
were needed therapeutically or for whether or not someone was trying to use a rejuvenating therapy in general or for actual therapy would be the same as other challenges in creating drugs that target specific tissues.
If you manipulate TERC for instance, you could end up targeting cells that
were naturally expressing telomerase because the increase in TERC would only manifest as increased telomerase in the cells expressing TERT, but otherwise, I can't think of easy ways to target therapy specifically just to certain cells.
25
Sean Sanders: Dr. Reddel, coming back to you, is there a telomerase or ALT assay that might work on frozen whole blood samples?
[0:50:01] Dr. Roger Reddel: Telomerase and ALT on frozen ‐‐ so for telomerase, if the cells have been
froze, if they've been frozen in a viable state and telomerase activity assay can be done on those, then there's no problem there.
With ALT, there are a number of options. The short answer is "yes" and it
can be done by extracting DNA for C‐Circle assays or the frozen material could be processed for sectioning and immunofluorescence for ALT‐associated to PML bodies.
Sean Sanders: Excellent. Let me stay with you actually for another question, and that is
the G‐quartet that you talked about early on in your presentation, is this always present at telomeres or is it preferentially formed during a physiological process during the cell cycle? Do you have an answer for that?
Dr. Roger Reddel: It's really early days for that; the labs working on this have really only
developed the tools for studying it in cells in very recent years. Up until now, it's been known that telomeric DNA and other G‐rich DNA do form G‐quadruplexes in vitro and it's taken the development of some specific antibodies to show that they do occur at telomeres.
Beyond that, how many of the telomeres have G‐quadruplex formation
and for what percentage of the time, I think there's more work that needs to be done.
Sean Sanders: Dr. Agarwal, did you have any comments on that? Dr. Suneet Agarwal: No. I can't comment unfortunately on G‐quadruplex. Sean Sanders: Okay. No problem. Well, I have a question for you. What are other
common types of cancers among DC patients and have these been identified as telomerase or ALT cancers as described by Dr. Reddel, and is there a correlation?
Dr. Suneet Agarwal: I think that's a great and fundamental question. Why should patients with
DC who have a telomere defect have a predisposition to malignancy and then what is sustaining that malignancy's replicative state? They get myeloid cancers, blood cancers, and they typically can get also squamous cell cancers of the head, neck, and anogenital regions, so basically skin
26
surfaces and blood cells which are already degenerating as a process of these.
The real answer is that this actually just has not been studied adequately
and part of it has been a difficulty in a coordinated effort to get samples, but really once those samples are obtained as described by Dr. Reddel, there'll be several methods of actually determining what their state was in terms of telomerase sufficiency or ALT.
The patients have hypomorphic mutation, so it is possible that they are
still dependent on telomerase. It wouldn't have to be that they use an alternative mechanism, but it just has not been studied.
Sean Sanders: Next question I'm going to put to both of you, and I'll start with Dr.
Reddel ‐‐ I'm actually going to combine two questions ‐‐ is there a quantitative cellular response directly co‐relatable with telomere length? The other part of that is how do cells monitor telomere length? Do we know anything about that?
Dr. Roger Reddel: The answer to both of those questions pretty much is known. There's
more known about monitoring of telomere lengths. It appears to involve some of the proteins, which typically bind to telomeres. Beyond that, we have very little information.
Sean Sanders: Dr. Agarwal, anything to add to that one? Dr. Suneet Agarwal: I can speak to just a laboratory observation that's been there for a while,
and that's if you have patients who have dyskeratosis congenita or short telomeres, their explants such as fibroblasts show very obvious signs of senescence, various morphological and obviously cell cycle problems that are indicative.
So you can see morphologically abnormalities. They do express
senescence‐associated beta‐galactosidase, things that would be associated with senescence in general. Essentially, the converse is then true that if you restore their telomerase activity or overcome their problem in vitro, they take on a better, a more normal morphology and stop the senescence pathway, so that could be viewed, I suppose, as a correlate of a phenotype of telomere length.
Sean Sanders: All right. Related to that, have you seen any shelterin protein variations
or mutations in DC? [0:55:03]
27
Dr. Suneet Agarwal: Sharon Savage and colleagues and also subsequently Dokal and
colleagues described in a substantial proportion of DC patients both de novo and autosomal dominantly inherited mutations in TINF2, which encodes the TIN2 component of shelterin, and that's the main one.
In DC itself, Dr. Savage's group had also surveyed other shelterin
components and did not find any mutations in the registry samples they had. It's possible that there may be some that pop up in rare cases, but certainly shelterin mutations are associated with DC.
Sean Sanders: Great! We are almost out of time. I'm going to give you just one more
question that actually just came in and I thought was an interesting one. I think we'll start with you, Dr. Reddel. Has there been any attempt to link any signaling pathways to telomere maintenance specifically related to cell surface antigens?
Dr. Roger Reddel: They have, although that's a little bit controversial at the moment.
There's more known about the signaling pathway as a consequence of telomere dysfunction, so when the telomeres get very short, there are various signaling pathways that are activated and to do with DNA repair mediated by ATM or alternately by ATR DNA repair signaling pathway proteins.
Sean Sanders: Dr. Agarwal, last word to you. Dr. Suneet Agarwal: I don't have anything to add about the cell surface signaling pathways
leading to telomere extension. It certainly would be very interesting if there were some.
Sean Sanders: Excellent! Well, it sounds like there's still a lot of work to do, but
unfortunately we are out of time for this webinar. I want to thank both of our speakers very much for their wonderful presentations and the engaging discussion.
Slide 69 On behalf of myself and our viewing audience, thank you to Dr. Roger
Reddel from Children's Medical Research Institute and Dr. Suneet Agarwal from Boston Children's Hospital.
Please go to the URL now at the bottom of your slide view to learn more
about resources related to today's discussion and look out for more webinars from Science available at webinar.sciencemag.org. This
28
particular webinar will be made available to you again as an on‐demand presentation within about 48 hours from now.
We're interested to know what you thought of the webinar. Send us an
email at the address now up in your slide viewer, [email protected]. Again, thank you very much to our panel and to PerkinElmer for their
kind sponsorship of today's educational seminar. Goodbye. [0:57:45] End of Audio