By Manrose Singh, Danny MacKenzie, Sanket Desai, Noelle Batista, and Dong Zhang
Since the early 1990s, when telomerase was first cloned, targeting the telomere maintenance mechanism (TMM) to treat cancer has been considered to be a superior strategy to many, yet, almost three decades later, pursuing this strategy has led to no FDA approved oncology drugs. Targeting the TMM through telomerase is hypothesized to selectively kill the cancer cells while doing little harm to healthy cells, because normal somatic cells neither express telomerase nor do they require the TMM for their normal lifecycle. Recent in-depth understanding of another molecular mechanism of telomere maintenance, the Alternative Lengthening of Telomeres (ALT) pathway, provides insight into anti-telomerase therapy’s shortcomings and presents renewed opportunity for the development of efficacious TMM-based cancer therapies: both in improving clinical outcomes of the telomerase inhibitors, but also in targeting the many predominantly ALT-positive cancers. In this article, we summarize the recent progress in identifying novel molecular targets, such as FANCM, for treating the ALT-positive cancers. We also discuss the biomarkers for diagnosing ALT-positive cancers in the clinic. We hope these discussions will motivate and facilitate the development of targeted therapies based on TMM and other genetic profile of cancers.
Human telomeres are non-coding repetitive DNA at the end of each chromosome arm with many copies of the tandem DNA sequence, (TTAGGG) in which range approximately between 4 and 13 kilobases (kb)1,3. Because of the so-called DNA end replication problem, telomeres shorten with each cell division. When telomeres fall below a certain critical length, cells activate the DNA damage response (DDR) pathway to induce either permanent cell cycle arrest or cell death. Under normal physiological conditions, only a subset of cells in our body needs to constantly proliferate and express the telomerase, for example hematopoietic stem cells and stem cells in the gastrointestinal tract, whereas the vast majority of somatic cells do not. Following similar rationale, to maintain their immortality, the large majority of cancer cells thus evolve to adopt one of two TMM strategies to maintain their telomeres:1 the telomerase (TEL); or2 the Alternative Lengthening of Telomere (ALT) pathway. Approximately 85-90% of all cancers reactivate the expression of telomerase4,5, while the other 10-15% cancers primarily rely on the ALT pathway6. The biochemical property of telomerase is relatively straightforward: it copies off its RNA component, hTR, and adds one unit at a time of the hexa-nucleotide TTAGGG to any shortened telomeres7,8. In essence, telomerase is a simple reverse transcriptase with a built-in RNA template. In the past three decades, many telomerase inhibitors have been developed, however their clinical outcomes have been generally disappointing9. A likely explanation for this is that when the telomerase in cancers is inhibited, cancer cells are capable of switching to the ALT pathway to continue maintaining their telomeres and avoid the DDR-induced permanent cell cycle arrest or cell death10. Therefore, if targeting TMM is to become a viable anti-cancer strategy in the clinic, simultaneous co-inhibition with an ALT inhibitor may become essential.
Though ALT-positive (ALT+) cancers may account for only 10-15% of all cancers6, the ALT positivity in certain types of cancers, including osteosarcoma, anaplastic astrocytoma, oligoastrocytoma, and leiomyosarcoma, can be as high as over 60%11. Certain types of ALT+ cancers have a better overall prognosis than their counterparts with telomerase positivity (TEL+), but for others, including a small population of HER2+ breast cancers, they are worse-off (Figure 1). In the clinic, patients with these ALT+ cancers suffer from a lack of efficacious targeted therapies. Thereby, targeting the TMM via ALT should be a worthy strategy to consider for the numerous ALT-predominant cancers. In recent years, major advancements have been made in elucidating the ALT molecular mechanism and in identifying potential molecular targets for treating the ALT+ cancers12-14. At present, the most promising molecular target is the protein FANCM from the Fanconi Anemia family of proteins14,15. FANCM is a highly conserved DNA translocase that also manifests robust ATPase activity16,17. Therefore, it is quite feasible to develop a small molecular inhibitor targeting the ATPase activity of FANCM (FANCMi). FANCM was first implicated in the ALT pathway by a seminal publication from our group demonstrating that depletion of FANCM induces robust replication stress and DNA damage at telomeres in the ALT+ cells18. Most importantly, depletion of FANCM specifically affects the survival of ALT+ cancer cells but not the TEL+ cancer cells18-20. Moreover, by analyzing data from the Project Achilles (a genome database cataloging the essentiality of thousands of cancer genes for hundreds of cancer cell lines), Lu and Pickett found that most ALT+ cancer cell lines highly depends on the FANCM gene for their survival14. Given the recent success of targeting PARP in treating the BRCA1/2 mutant cancers in the clinic, the urgency of developing the FANCMi is warranted. Intriguingly, FANCM additionally manifests synthetic lethal interactions with BRCA1 and BLM in both ALT+ and TEL+ cells18,21,22. This suggests that FANCMi may also inhibit the growth TEL+ cancers, if they have particular genetic mutation backgrounds (such as with mutations in BRCA1 or BLM)! This also suggests that, treating TEL+ cancers simultaneously with a FANCMi and a BLM inhibitor (BLMi) may have beneficial anti-cancer effects. Therefore, FANCMi may be used: (1) as a single-agent therapy for treating the ALT+ cancers; (2) for treating TEL+ cancers with certain genetic mutations; or (3) in combination with other oncology drugs. Other potential molecular targets for treating the ALT+ cancers include ATR, SMARCAL1, and PPP1PCB, though they may not be as specific and/or potent as the FANCMi14,23. For the more detailed review of potential treatment strategies for ALT+ cancers, please read our recent article published in the journal Cancers11.
In addition to developing potent and efficacious ALT inhibitors, being able to clinically identify ALT+ cancers with high confidence is also crucial for their success. At present, two assays have been demonstrated to be feasible in identifying the ALT+ cancers using clinical tumor samples11. The easiest and most common method is Telo-FISH, which was developed by Heaphy and colleagues in 201124. After in situ hybridization with a fluorescent-labeled telomere peptide nuclei acid (PNA) probe, ALT+ cancers can be identified as manifesting large and intense nuclear puncta. Telo-FISH has since been used to survey thousands of clinical tumor samples. The second well-established method is the C-circle Assay (CCA); developed by Henson and colleagues in 2009, it is the most specific and quantifiable biomarkers for ALT+ cells11,25. CCA was recently modified by Idilli and colleagues to detect the amount of C-circle using the formalin-fixed paraffin-embedded tumor specimens26. However, its reliability in distinguishing ALT+ vs TEL+ cancers using patient tumor samples remains to be determined. Most recently, Chen and colleagues reported that C-circles can also be detected in the blood samples of the ALT+ cancer patients27. Therefore, the CCA could be easily optimized to become a less invasive biomarker to detect the ALT+ cancers–possibly even for use in early detection.
In summary, we believe that the time has come to develop potent and efficacious ALT inhibitors such as the FANCMi. ALT inhibitors not only have use as single-agent therapy in treating patients with ALT+ cancers, but when used together with telomerase inhibitors, they are likely to improve the treatment of TEL+ cancers as well. Failing to block the ALT pathway and allowing the TEL+ cancers to have an alternative escape route was likely the shortcoming of the past telomerase inhibitors. Potent ALT inhibitors will cut off that escape route and finally realize the dream of the TMM-based anti-cancer therapy.
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Dong Zhang, PhD, is an associate professor of cancer biology and the director of Center for Cancer Research at the College of Osteopathic Medicine, New York Institute of Technology (NYIT). Dr. Zhang is also the founder and CSO of ALT Therapeutics, a biotech company dedicated to develop drugs targeting the ALT cancers, which include some of the most deadly cancers, such as glioblastoma and pancreatic cancers. Previously, Dr. Zhang worked at Wyeth Pharmaceutical and Pfizer Pharmaceutical as a staff scientist. Dr. Zhang is also an associate editor of Animal Model and Experimental Medicine.