Dissecting the oncogenic mechanisms of POT1 cancer mutations through deep scanning mutagenesis.

Mutations in the shelterin protein POT1 are associated with diverse cancers, but their role in cancer progression remains unclear. To resolve this, we performed deep scanning mutagenesis in POT1 locally haploid human stem cells to assess the impact of POT1 variants on cellular viability and cancer-associated telomeric phenotypes. Though POT1 is essential, frame-shift mutants are rescued by chemical ATR inhibition, indicating that POT1 is not required for telomere replication or lagging strand synthesis. In contrast, a substantial fraction of clinically-validated pathogenic mutations support normal cellular proliferation, but still drive ATR-dependent telomeric DNA damage signaling and ATR-independent telomere elongation. Moreover, this class of cancer-associated POT1 variants elongates telomeres more rapidly than POT1 frame-shifts, indicating they actively drive oncogenesis and are not simple loss-of-function mutations.

Distinct senescence mechanisms restrain progression of dysplastic nevi.

Telomerase reverse transcriptase (TERT) promoter mutations (TPMs) are frequently found in different cancer types, including ∼70% of sun-exposed skin melanomas. In melanoma, TPMs are among the earliest mutations and can be present during the transition from nevus to melanoma. However, the specific factors that contribute to the selection of TPMs in certain nevi subsets are not well understood. To investigate this, we analyzed a group of dysplastic nevi (DN) by sequencing genes commonly mutated in melanocytic neoplasms. We examined the relationship between the identified mutations, patient age, telomere length, histological features, and the expression of p16. Our findings reveal that TPMs are more prevalent in DN from older patients and are associated with shorter telomeres. Importantly, these TPMs were not found in nevi with BRAF V600E mutations. Conversely, DN with BRAF V600E mutations were observed in younger patients, had longer telomeres and a higher proportion of p16-positive cells. This suggests that these nevi arrest growth independently of telomere shortening through a mechanism known as oncogene-induced senescence (OIS). These characteristics extend to melanoma-sequencing datasets, where melanomas with BRAF V600E mutations were more likely to have a CDKN2A inactivation, overriding OIS. In contrast, melanomas without BRAF V600E mutations showed a higher frequency of TPMs. Our data imply that TPMs are selected to bypass replicative senescence (RS) in cells that were not arrested by OIS. Overall, our results indicate that a subset of melanocytic neoplasms face constraints from RS, while others encounter OIS and RS. The order in which these barriers are overcome during progression to melanoma depends on the mutational context.

Functional annotation of variants of the BRCA2 gene via locally haploid human pluripotent stem cells.

Mutations in the BRCA2 gene are associated with sporadic and familial cancer, cause genomic instability and sensitize cancer cells to inhibition by the poly(ADP-ribose) polymerase (PARP). Here we show that human pluripotent stem cells (hPSCs) with one copy of BRCA2 deleted can be used to annotate variants of this gene and to test their sensitivities to PARP inhibition. By using Cas9 to edit the functional BRCA2 allele in the locally haploid hPSCs and in fibroblasts differentiated from them, we characterized essential regions in the gene to identify permissive and loss-of-function mutations. We also used Cas9 to directly test the function of individual amino acids, including amino acids encoded by clinical BRCA2 variants of uncertain significance, and identified alleles that are sensitive to PARP inhibitors used as a standard of care in BRCA2-deficient cancers. Locally haploid human pluripotent stem cells can facilitate detailed structure-function analyses of genes and the rapid functional evaluation of clinically observed mutations.

Digital telomere measurement by long-read sequencing distinguishes healthy aging from disease.

Telomere length is an important biomarker of organismal aging and cellular replicative potential, but existing measurement methods are limited in resolution and accuracy. Here, we deploy digital telomere measurement by nanopore sequencing to understand how distributions of human telomere length change with age and disease. We measure telomere attrition and de novo elongation with unprecedented resolution in genetically defined populations of human cells, in blood cells from healthy donors and in blood cells from patients with genetic defects in telomere maintenance. We find that human aging is accompanied by a progressive loss of long telomeres and an accumulation of shorter telomeres. In patients with defects in telomere maintenance, the accumulation of short telomeres is more pronounced and correlates with phenotypic severity. We apply machine learning to train a binary classification model that distinguishes healthy individuals from those with telomere biology disorders. This sequencing and bioinformatic pipeline will advance our understanding of telomere maintenance mechanisms and the use of telomere length as a clinical biomarker of aging and disease.

Tracing cancer evolution and heterogeneity using Hi-C.

Chromosomal rearrangements can initiate and drive cancer progression, yet it has been challenging to evaluate their impact, especially in genetically heterogeneous solid cancers. To address this problem we developed HiDENSEC, a new computational framework for analyzing chromatin conformation capture in heterogeneous samples that can infer somatic copy number alterations, characterize large-scale chromosomal rearrangements, and estimate cancer cell fractions. After validating HiDENSEC with in silico and in vitro controls, we used it to characterize chromosome-scale evolution during melanoma progression in formalin-fixed tumor samples from three patients. The resulting comprehensive annotation of the genomic events includes copy number neutral translocations that disrupt tumor suppressor genes such as NF1, whole chromosome arm exchanges that result in loss of CDKN2A, and whole-arm copy-number neutral loss of homozygosity involving PTEN. These findings show that large-scale chromosomal rearrangements occur throughout cancer evolution and that characterizing these events yields insights into drivers of melanoma progression.

Distinct senescence mechanisms restrain progression of dysplastic nevi.

TERT promoter mutations (TPMs) are frequently found in different cancer types, including approximately 70% of sun-exposed skin melanomas. In melanoma, TPMs are among the earliest mutations and can be present during the transition from nevus to melanoma. However, the specific factors that contribute to the selection of TPMs in certain nevi subsets are not well understood. To investigate this, we analyzed a group of dysplastic nevi (DN) by sequencing genes commonly mutated in melanocytic neoplasms. We examined the relationship between the identified mutations, patient age, telomere length, histological features, and the expression of p16. Our findings reveal that TPMs are more prevalent in DN from older patients and are associated with shorter telomeres. Importantly, these TPMs were not found in nevi with BRAF V600E mutations. Conversely, DN with BRAF V600E mutations were observed in younger patients, had longer telomeres, and a higher proportion of p16-positive cells. This suggests that these nevi arrest growth independently of telomere shortening through a mechanism known as oncogene-induced senescence (OIS). These characteristics extend to melanoma sequencing data sets, where melanomas with BRAF V600E mutations were more likely to have CDKN2A inactivation, overriding OIS. In contrast, melanomas without BRAF V600E mutations showed a higher frequency of TPMs. Our data imply that TPMs are selected to bypass replicative senescence (RS) in cells that were not arrested by OIS. Overall, our results indicate that a subset of melanocytic neoplasms face constraints from RS, while others encounter OIS and RS. The order in which these barriers are overcome during progression to melanoma depends on the mutational context.

A non-genetic switch triggers alternative telomere lengthening and cellular immortalization in ATRX deficient cells.

Alternative Lengthening of Telomeres (ALT) is an aberrant DNA recombination pathway which grants replicative immortality to approximately 10% of all cancers. Despite this high prevalence of ALT in cancer, the mechanism and genetics by which cells activate this pathway remain incompletely understood. A major challenge in dissecting the events that initiate ALT is the extremely low frequency of ALT induction in human cell systems. Guided by the genetic lesions that have been associated with ALT from cancer sequencing studies, we genetically engineered primary human pluripotent stem cells to deterministically induce ALT upon differentiation. Using this genetically defined system, we demonstrate that disruption of the p53 and Rb pathways in combination with ATRX loss-of-function is sufficient to induce all hallmarks of ALT and results in functional immortalization in a cell type-specific manner. We further demonstrate that ALT can be induced in the presence of telomerase, is neither dependent on telomere shortening nor crisis, but is rather driven by continuous telomere instability triggered by the induction of differentiation in ATRX-deficient stem cells.

Highly efficient generation of isogenic pluripotent stem cell models using prime editing.

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.

From muscles to nerves, our body is formed of many kinds of cells which can each respond slightly differently to the same harmful genetic changes. Understanding the exact relationship between mutations and cell-type specific function is essential to better grasp how conditions such as Parkinson's disease or amyotrophic lateral sclerosis progress and can be treated. Stem cells could be an important tool in that effort, as they can be directed to mature into many cell types in the laboratory. Yet it remains difficult to precisely introduce disease-relevant mutations in these cells. To remove this obstacle, Li et al. focused on prime editing, a cutting-edge 'search and replace' approach which can introduce new genetic information into a specific DNA sequence. However, it was unclear whether this technique could be used to efficiently create stem cell models of human diseases. A first set of experiments showed that prime editing is superior to conventional approaches when generating mutated genes in stem cells. Li et al. then further improved the efficiency and precision of the method by tweaking how prime editing components are delivered into the cells. The refined approach could be harnessed to quickly generate large numbers of stem cells carrying mutations associated with Parkinson's disease; crucially, prime editing could then also be used to revert a mutated gene back to its healthy form. The improved prime editing approach developed by Li et al. removes a major hurdle for scientists hoping to use stem cells to study genetic diseases. This could potentially help to unlock progress in how we understand and ultimately treat these conditions.

Editing TINF2 as a potential therapeutic approach to restore telomere length in dyskeratosis congenita.

Mutations in the TINF2 gene, encoding the shelterin protein TIN2, cause telomere shortening and the inherited bone marrow (BM) failure syndrome dyskeratosis congenita (DC). A lack of suitable model systems limits the mechanistic understanding of telomere shortening in the stem cells and thus hinders the development of treatment options for BM failure. Here, we endogenously introduced TIN2-DC mutations in human embryonic stem cells (hESCs) and human hematopoietic stem and progenitor cells (HSPCs) to dissect the disease mechanism and identify a gene-editing strategy that rescued the disease phenotypes. The hESCs with the T284R disease mutation exhibited the short telomere phenotype observed in DC patients. Yet, telomeres in mutant hESCs did not trigger DNA damage responses at telomeres or show exacerbated telomere shortening when differentiated into telomerase-negative cells. Disruption of the mutant TINF2 allele by introducing a frameshift mutation in exon 2 restored telomere length in stem cells and the replicative potential of differentiated cells. Similarly, we introduced TIN2-DC disease variants in human HSPCs to assess the changes in telomere length and proliferative capacity. Lastly, we showed that editing at exon 2 of TINF2 that restored telomere length in hESCs could be generated in TINF2-DC patient HSPCs. Our study demonstrates a simple genetic intervention that rescues the TIN2-DC disease phenotype in stem cells and provides a versatile platform to assess the efficacy of potential therapeutic approaches in vivo.

Cancer-associated POT1 mutations lead to telomere elongation without induction of a DNA damage response.

Mutations in the shelterin protein POT1 are associated with chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, angiosarcoma, melanoma, and other cancers. These cancer-associated POT1 (caPOT1) mutations are generally heterozygous, missense, or nonsense mutations occurring throughout the POT1 reading frame. Cancers with caPOT1 mutations have elongated telomeres and show increased genomic instability, but which of the two phenotypes promotes tumorigenesis is unclear. We tested the effects of CAS9-engineered caPOT1 mutations in human embryonic and hematopoietic stem cells (hESCs and HSCs, respectively). HSCs with caPOT1 mutations did not show overt telomere damage. In vitro and in vivo competition experiments showed the caPOT1 mutations did not confer a selective disadvantage. Since DNA damage signaling is known to affect the fitness of HSCs, the data argue that caPOT1 mutations do not cause significant telomere damage. Furthermore, hESC lines with caPOT1 mutations showed no detectable telomere damage response while showing consistent telomere elongation. Thus, caPOT1 mutations are likely selected for during cancer progression because of their ability to elongate telomeres and extend the proliferative capacity of the incipient cancer cells.

TINF2 is a haploinsufficient tumor suppressor that limits telomere length.

Telomere shortening is a presumed tumor suppressor pathway that imposes a proliferative barrier (the Hayflick limit) during tumorigenesis. This model predicts that excessively long somatic telomeres predispose to cancer. Here, we describe cancer-prone families with two unique TINF2 mutations that truncate TIN2, a shelterin subunit that controls telomere length. Patient lymphocyte telomeres were unusually long. We show that the truncated TIN2 proteins do not localize to telomeres, suggesting that the mutations create loss-of-function alleles. Heterozygous knock-in of the mutations or deletion of one copy of TINF2 resulted in excessive telomere elongation in clonal lines, indicating that TINF2 is haploinsufficient for telomere length control. In contrast, telomere protection and genome stability were maintained in all heterozygous clones. The data establish that the TINF2 truncations predispose to a tumor syndrome. We conclude that TINF2 acts as a haploinsufficient tumor suppressor that limits telomere length to ensure a timely Hayflick limit.

Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells.

Genome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing.

TERT promoter mutations and telomeres during tumorigenesis.

Telomerase regulation and telomere shortening act as a strong tumor suppressor mechanism in human somatic cells. Point mutations in the promoter of telomerase reverse transcriptase (TERT) are the most frequent non-coding mutation in cancer. These TERT promoter mutations (TPMs) create de novo ETS factor binding sites upstream of the start codon of the gene, which can be bound by different ETS factors. TPMs can occur early during tumorigenesis and are thought to be among the first mutations in melanoma, glioblastoma and hepatocellular carcinoma. Despite their association with increased TERT levels, TPMs do not prohibit telomere shortening and TPM-harboring cancers present with short telomeres. Their short telomere length combined with their high prevalence and specificity for cancer makes TPMs an attractive target for future therapeutic exploitation of telomerase inhibition and telomere deprotection-induced cell death.

Genetically engineered human cortical spheroid models of tuberous sclerosis.

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder caused by mutations in the TSC1 or TSC2 genes, whose protein products are negative regulators of mechanistic target of rapamycin complex 1 signaling. Hallmark pathologies of TSC are cortical tubers-regions of dysmorphic, disorganized neurons and glia in the cortex that are linked to epileptogenesis. To determine the developmental origin of tuber cells, we established human cellular models of TSC by CRISPR-Cas9-mediated gene editing of TSC1 or TSC2 in human pluripotent stem cells (hPSCs). Using heterozygous TSC2 hPSCs with a conditional mutation in the functional allele, we show that mosaic biallelic inactivation during neural progenitor expansion is necessary for the formation of dysplastic cells and increased glia production in three-dimensional cortical spheroids. Our findings provide support for the second-hit model of cortical tuber formation and suggest that variable developmental timing of somatic mutations could contribute to the heterogeneity in the neurological presentation of TSC.

Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms.

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.

Mutations in the promoter of the telomerase gene TERT contribute to tumorigenesis by a two-step mechanism.

TERT promoter mutations (TPMs) are the most common noncoding mutations in cancer. The timing and consequences of TPMs have not been fully established. Here, we show that TPMs acquired at the transition from benign nevus to malignant melanoma do not support telomere maintenance. In vitro experiments revealed that TPMs do not prevent telomere attrition, resulting in cells with critically short and unprotected telomeres. Immortalization by TPMs requires a gradual up-regulation of telomerase, coinciding with telomere fusions. These data suggest that TPMs contribute to tumorigenesis by promoting immortalization and genomic instability in two phases. In an initial phase, TPMs do not prevent bulk telomere shortening but extend cellular life span by healing the shortest telomeres. In the second phase, the critically short telomeres lead to genome instability and telomerase is further up-regulated to sustain cell proliferation.

Defined and Scalable Differentiation of Human Oligodendrocyte Precursors from Pluripotent Stem Cells in a 3D Culture System.

Oligodendrocyte precursor cells (OPCs) offer considerable potential for the treatment of demyelinating diseases and injuries of the CNS. However, generating large quantities of high-quality OPCs remains a substantial challenge that impedes their therapeutic application. Here, we show that OPCs can be generated from human pluripotent stem cells (hPSCs) in a three-dimensional (3D), scalable, and fully defined thermoresponsive biomaterial system. We used CRISPR/Cas9 to create a NKX2.2-EGFP human embryonic stem cell reporter line that enabled fine-tuning of early OPC specification and identification of conditions that markedly increased the number of OLIG2+ and NKX2.2+ cells generated from hPSCs. Transplantation of 50-day-old OPCs into the brains of NOD/SCID mice revealed that progenitors generated in 3D without cell selection or purification subsequently engrafted, migrated, and matured into myelinating oligodendrocytes in vivo. These results demonstrate the potential of harnessing lineage reporter lines to develop 3D platforms for rapid and large-scale production of OPCs.

Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo.

Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo.

Cancer-associated TERT promoter mutations abrogate telomerase silencing.

Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells.

Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation.

Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis.