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ALT Associated Mutations in Histone H3, ATRX and DAXX

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Besides the contribution of DNA sequence and the state of shelterin binding in telomeric recombination and ALT activity, telomere architecture, and specially histone modifications, are responsible for some phenotypic characteristics of ALT. Recent studies of ALT tumors correlate telomere maintenance mechanism of ALT with activity of the complex ATRX/DAXX.

ATRX and DAXX form a chromatin remodeling complex that moderates chromatin changes. ATRX is responsible for encoding a member of the switch/sucrose non-fermentable (SWI/SNF) family ATP-dependent helicases that have chromatin-remodeling activity namely α-thalassemia mental retardation X-linked protein. DAXX encodes another component of ATRX that is a binding partner death-associated protein 6. ATRX/DAXX complex is needed for chromatin deposition of a histone associated with transcriptionally active open chromatin namely H3.3. It has been reported that the inhibition of this complex suppresses the recombinogenic nature of repetitive telomeric DNA, resulting in loss of hetrochromatic marks. Limitation of expression or loss of the complex ATRX/DAXX show to limit the H3.3 incorporation in telomeric chromatin with a positive effect in telomere recombination. The recruitment of such proteins in detriment of others may affect the construction of ALT telomeres. An example of that are the mutations in H3F3A, the encoding gene of H3.3 that has demonstrated alterations in histone incorporation in the genome with alterations at chromatin remodeling and gene expression.

Loss of ATRX/DAXX activity can originate altered gene expression that can promote ALT activity. This complex has been related with ALT, being associated or not with p53, and the majority of cancers with prevalence of ALT have ATRX mutations. The first correlation between ALT and epigenetic and chromatin dysfunction was found in PanNET and GBM tumors. In these tumors there were observed remains of ALT-associated PML bodies (APBs) and loss of the ATRX (and often) DAXX expression.

It has been described a linkage between ATRX and DNA methylation, chromatin conformation, replication and transcriptional repression, particularly in tandem repeated sections. Loss of ATRX affect telomeres very similarly to ALT, and possibly contributes to ALT activation. The majority of ALT cells exhibit deregulated ATRX expression, however, the sequence is present in the genome, suggesting a role of ATRX protein in ALT suppression, after specific changes in protein maturation.

In addition to ATRX, DAXX and p53 mutations, some tumors exhibit heterozygous mutations in the histone H3.3 gene, H3F3A. It is suggest that these alterations facilitate the activation of TMM by ALT mechanism through the epigenetic changes in the telomeric chromatin. However, some ALT tumors have functional histone H3, ATRX and DAXX expression, which indicates that other fails may contribute to the expression of some ALT hallmarks.

Cells with different TMM have significant differences in telomeric chromatin construction; in ALT cells it is possible to observe more compact telomeric chromatin, relatively with telomerase-dependent cells, that is consistent with most interactions between telomeres observed in these cells allowing HR-dependent telomere maintenance and promoting telomere transcription.

In the majority of tumors with ATRX, DAXX or H3F3A mutations it is also observed p53 mutation, which is indicative of a possible interaction between the mutations.

Non-determined telomere maintenance mechanism

Telomere maintenance mechanism in tumor cells are usually classified in two groups: telomerase-dependent TMM or ALT-dependent TMM. However, this classification is not consensual and there are some studies that identified some groups of tumors with characteristics of both mechanisms or neither.

There are some evidences that ALT pathway may be activated when there is a loss or inhibition of telomerase function. According to the results obtained by Chen et al. ALT telomere elongation is present following telomerase inhibition. In a similar manner, in a study with ALT active cells, the fusion with normal somatic cells or telomerase-positive cancer cells leaded to ALT activity suppression. Unlike to the expected, in a research in which there were used keratinocytes, normal human cells showed the presence of ALT pathway active, but repression of this mechanism in cells telomerase-immortalized. The cells were able to maintain telomeres by both ALT and telomerase and when there is dominance of one mechanism there is competition and further elongation of telomeres. In the same way it has been documented in a research of human sarcomas that the two mechanisms were not mutually exclusive in some tumors being observed in the same tumor some cells having ALT-associated PML bodies, characteristic of ALT, and other expressing telomerase.

The immortalization of cells is considered a general feature of cancer, however, in some conditions the acquirement of limitless growth through the activation of the telomere maintenance mechanisms is not required. Some tumors have neither evidences of the presence of telomerase activity nor ALT. There are some hypothesis to justify that specific condition, that are the fact of the tumor rise in cells that start out with long telomeres; the absence of adverse tumor factors, increasing the cell surviving and the presence of genetic mutations.

ALT-target therapies

At this time, there are no therapies specifically targeting ALT. In some cancer types, particularly some with difficult prognostic, the frequency of ALT mechanisms has increased and the somatic mutations in the ATRX, DAXX and in the histone variant H3.3 found in some tumors can be used as ALT hallmarks. One possibility to target ALT positive cells is through the development of synthetic lethal approaches to kill ATL cells deficient in ATRX or DAXX producing stress for which the genes will act in a protective function.

There are evidences that ALT-dependent cancers use homologous recombination to maintain their telomere length, and this fault in specific cancers can be a promising target to therapy. An efficient therapeutic targeting of TMM in cancers can include the development of ALT inhibitors, since telomerase inhibitors are not effective for ALT tumors. The recent results obtained by Flynn et al. shows hypersensitivity of ATL positive cells to ATR (a critical regulator of recombination) inhibitors and their application in vitro disturb and selectively kill the ALT positive cells. Despite being necessary further research, the application of ATR inhibitors to ALT-positive cancer cells is a promising discovery to clinical studies.

As previously referred, some tumors exhibit telomerase and ALT activity in the same cell and both mechanisms can co-exist. It has been reported in mouse models experiments that in mosaic tumors their survival was promoted by ALT pathway when telomerase was inhibited. In order to obtain an efficient therapeutic; the tumor cells have to be evaluated in what concern the TMM present and in the case of mosaicism, the tumor may be targeted in each type of cell through combination therapies.

In tumors with a single mechanism of telomere maintenance, the treatment with the corresponding inhibitor may lead to the appearance of drug resistance or can treat just the relevant TMM, can be applied selection pressure and activation of the other TMM. Similarly to the treatment of mosaicism tumors, either use of both telomerase and ALT inhibitors is required.

Final remarks and future perspectives:

The unlimited proliferation capacity is one of the hallmarks of cancer and the activation of telomerase maintenance mechanisms (TMM) is essential in tumorigenesis to perform replicative immortality and replication-induced senescence resultant of telomere shortening. The most common mechanism to extend critically short telomeres is the activation of telomerase, but fewer yet significant numbers of tumors extend their telomeres through alternative recombination-based methods- ALT.

In order to develop anticancer therapies targeting ALT mechanisms of TMM, it is needed to intensify the study and the exploration of these alternative mechanisms. There are a number of issues that must be solved, focusing mostly in the epigenetic mechanisms present in ALT telomeres it is necessary to understand the epigenetic landscape and the correlation of the mutations in ALT tumors. For this purpose, the effect of re-introduction the expression of ATRX and the effect of chromatin disruption in ALT cells are experiments that can be performed. It can be further studied the repetitive structure of ALT telomeres and the behavior of each shelterin protein in these cells, specially the TRF2 that seems to have great influence. Furthermore, the specific characteristics of ALT cells like the function of PML bodies and the dynamics of APB formation can contribute to understand the induction of these alternative mechanisms. Complemented with in vivo trials and the research of pharmacological inhibition of ALT tumors it is possible the development of preclinical and clinical studies to treatments and consequently maximize cancer therapeutic efficacy.


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