Therapeutic Targeting of Telomere Diseases Via Novel Small Molecule Modulators of Non-Coding RNA Biogenesis

Germline mutations that impair telomere maintenance cause dyskeratosis congenita (DC), and predispose to hematologic diseases including bone marrow failure, myelodysplastic syndrome, and acute myeloid leukemia. Mutations in several DC-associated genes (DKC1, NOP10, NHP2, NAF1, PARN, TERC) act by disrupting steady-state levels of the essential non-coding RNA component of telomerase (TERC), which is limiting for telomerase activity and consequently self-renewal capacity in human stem cells. Ectopic expression of TERC RNA restores telomere length in cellular models of DC, but this approach poses major challenges for clinical translation. Based on insights from genetic discoveries in DC, we hypothesized that manipulating non-coding RNA pathways could be therapeutically useful to augment TERC in patients with telomere diseases. We and others recently demonstrated that PARN mutations cause DC via destabilization of nascent TERC RNA. Specifically, PARN is an RNA exonuclease that is required to remove post-transcriptionally added, non-genomically encoded adenosine residues - "oligo(A) tails" - that target TERC for destruction by the nuclear exosome (Fig. A). We subsequently identified the non-canonical poly(A) polymerase PAPD5 as the enzyme responsible for TERC oligo-adenylation and destabilization (Fig. A), and demonstrated that RNA interference of PAPD5 restored telomere length in DC patient cells. To further address this, we disrupted PAPD5 by CRISPR/Cas9-mediated genome editing in DC patient induced pluripotent stem cells (iPSCs) and heterologous human cell lines. We found that graded deficiencies in PAPD5 resulted in dose-dependent increases in TERC RNA and telomere length, and that PAPD5-null clones could be isolated and propagated indefinitely. These results provide genetic evidence of an unanticipated therapeutic window for PAPD5 inhibition, as a potential strategy to modulate TERC RNA levels.

Pharmacological inhibitors of PAPD5 have not been identified. We therefore developed and executed a high-throughput functional screen to discover novel small molecule PAPD5 inhibitors. Of 100,055 compounds tested, we identified several hits that inhibited recombinant PAPD5 (rPAPD5), and triaged based on potency, specificity, and activity in orthogonal in vitro assays. Here we report on one of these novel compounds studied most extensively, BCH001. In vitro, BCH001 inhibited rPAPD5-mediated oligonucleotide extension, and was specific for rPAPD5 compared to other canonical and non-canonical poly-nucleotide polymerases. Differential scanning fluorimetry indicated direct binding of BCH001 to rPAPD5 that was ATP- and dose-dependent. We next used human cell-based assays to determine the impact of BCH001 on TERC RNA processing, telomere dynamics and the transcriptome. In PARN-mutant iPSC clones derived from patients with telomere diseases, we found that treatment with BCH001 in the 100 nM - 1 µM range reduced TERC RNA oligo-adenylation and increased steady state TERC RNA levels. Strikingly and consistently, treatment with BCH001 led to telomere elongation across multiple patient iPSCs tested (Fig. B), establishing a normalized telomere length set-point over weeks that was reversible with drug removal. RNA-Seq analysis demonstrated that only the non-coding small Cajal body RNA 13 (scaRNA13), which is known to be dependent on PARN for stability, was significantly differentially expressed between drug-treated and untreated PARN-mutant patient iPSCs. These data indicate minimal changes across the transcriptome at doses of BCH001 that are sufficient to alter TERC RNA levels and restore telomere length in DC patient iPSCs. We further found that BCH001 could shift the balance in favor of productive TERC RNA 3' end maturation across a range of human cell types, including primary fibroblasts, transformed cell lines, normal iPSCs, and iPSCs from patients with specific DC-associated DKC1 and TERC mutations.

Taken together, these data provide genetic and pharmacological evidence that TERC RNA and telomere length can be controlled in human cells by modulation of the PARN/PAPD5 post-transcriptional regulatory axis. This has important therapeutic implications for the hematologic and non-hematologic manifestations of DC and related disorders, and possibly a broad range of human degenerative diseases.

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