Mining cancer genomes for change-of-metabolic-function mutations.

Enzymes with novel functions are needed to enable new organic synthesis techniques. Drawing inspiration from gain-of-function cancer mutations that functionally alter proteins and affect cellular metabolism, we developed METIS (Mutated Enzymes from Tumors In silico Screen). METIS identifies metabolism-altering cancer mutations using mutation recurrence rates and protein structure. We used METIS to screen 298,517 cancer mutations and identify 48 candidate mutations, including those previously identified to alter enzymatic function. Unbiased metabolomic profiling of cells exogenously expressing a candidate mutant (OGDHLp.A400T) supports an altered phenotype that boosts in vitro production of xanthosine, a pharmacologically useful chemical that is currently produced using unsustainable, water-intensive methods. We then applied METIS to 49 million cancer mutations, yielding a refined set of candidates that may impart novel enzymatic functions or contribute to tumor progression. Thus, METIS can be used to identify and catalog potentially-useful cancer mutations for green chemistry and therapeutic applications.

Ataxia-telangiectasia mutated ( Atm ) disruption sensitizes spatially-directed H3.3K27M/TP53 diffuse midline gliomas to radiation therapy.

Diffuse midline gliomas (DMGs) are lethal brain tumors characterized by p53-inactivating mutations and oncohistone H3.3K27M mutations that rewire the cellular response to genotoxic stress, which presents therapeutic opportunities. We used RCAS/tv-a retroviruses and Cre recombinase to inactivate p53 and induce K27M in the native H3f3a allele in a lineage- and spatially-directed manner, yielding primary mouse DMGs. Genetic or pharmacologic disruption of the DNA damage response kinase Ataxia-telangiectasia mutated (ATM) enhanced the efficacy of focal brain irradiation, extending mouse survival. This finding suggests that targeting ATM will enhance the efficacy of radiation therapy for p53-mutant DMG but not p53-wildtype DMG. We used spatial in situ transcriptomics and an allelic series of primary murine DMG models with different p53 mutations to identify transactivation-independent p53 activity as a key mediator of such radiosensitivity. These studies deeply profile a genetically faithful and versatile model of a lethal brain tumor to identify resistance mechanisms for a therapeutic strategy currently in clinical trials.

Pooled genetic screens to identify vulnerabilities in TERT-promoter-mutant glioblastoma.

Pooled genetic screens represent a powerful approach to identify vulnerabilities in cancer. Here we used pooled CRISPR/Cas9-based approaches to identify vulnerabilities associated with telomerase reverse transcriptase (TERT) promoter mutations (TPMs) found in >80% of glioblastomas. We first developed a platform to detect perturbations that cause long-term growth defects in a TPM-mutated glioblastoma cell line. However, we could not detect dependencies on either TERT itself or on an E-twenty six transcription (ETS) factor known to activate TPMs. To explore this finding, we cataloged TPM status for 441 cell lines and correlated this with genome-wide screening data. We found that TPM status was not associated with differential dependency on TERT, but that E-twenty six (ETS) transcription factors represent key dependencies in both TPM+ and TPM- lines. Further, we found that TPMs are associated with expression of gene programs regulated by a wide array of ETS-factors in both cell lines and primary glioblastoma tissues. This work contributes a unique TPM cell line reagent, establishes TPM status for many deeply-profiled cell lines, and catalogs TPM-associated vulnerabilities. The results highlight challenges in executing genetic screens to detect TPM-specific vulnerabilities, and suggest redundancy in the genetic network that regulates TPM function with therapeutic implications.

Ganglioglioma deep transcriptomics reveals primitive neuroectoderm neural precursor-like population.

Gangliogliomas are brain tumors composed of neuron-like and macroglia-like components that occur in children and young adults. Gangliogliomas are often characterized by a rare population of immature astrocyte-appearing cells expressing CD34, a marker expressed in the neuroectoderm (neural precursor cells) during embryogenesis. New insights are needed to refine tumor classification and to identify therapeutic approaches. We evaluated five gangliogliomas with single nucleus RNA-seq, cellular indexing of transcriptomes and epitopes by sequencing, and/or spatially-resolved RNA-seq. We uncovered a population of CD34+ neoplastic cells with mixed neuroectodermal, immature astrocyte, and neuronal markers. Gene regulatory network interrogation in these neuroectoderm-like cells revealed control of transcriptional programming by TCF7L2/MEIS1-PAX6 and SOX2, similar to that found during neuroectodermal/neural development. Developmental trajectory analyses place neuroectoderm-like tumor cells as precursor cells that give rise to neuron-like and macroglia-like neoplastic cells. Spatially-resolved transcriptomics revealed a neuroectoderm-like tumor cell niche with relative lack of vascular and immune cells. We used these high resolution results to deconvolute clinically-annotated transcriptomic data, confirming that CD34+ cell-associated gene programs associate with gangliogliomas compared to other glial brain tumors. Together, these deep transcriptomic approaches characterized a ganglioglioma cellular hierarchy-confirming CD34+ neuroectoderm-like tumor precursor cells, controlling transcription programs, cell signaling, and associated immune cell states. These findings may guide tumor classification, diagnosis, prognostication, and therapeutic investigations.

The Effect of Atm Loss on Radiosensitivity of a Primary Mouse Model of Pten-Deleted Brainstem Glioma.

Diffuse midline gliomas arise in the brainstem and other midline brain structures and cause a large proportion of childhood brain tumor deaths. Radiation therapy is the most effective treatment option, but these tumors ultimately progress. Inhibition of the phosphoinositide-3-kinase (PI3K)-like kinase, ataxia-telangiectasia mutated (ATM), which orchestrates the cellular response to radiation-induced DNA damage, may enhance the efficacy of radiation therapy. Diffuse midline gliomas in the brainstem contain loss-of-function mutations in the tumor suppressor PTEN, or functionally similar alterations in the phosphoinositide-3-kinase (PI3K) pathway, at moderate frequency. Here, we sought to determine if ATM inactivation could radiosensitize a primary mouse model of brainstem glioma driven by Pten loss. Using Cre/loxP recombinase technology and the RCAS/TVA retroviral gene delivery system, we established a mouse model of brainstem glioma driven by Pten deletion. We find that Pten-null brainstem gliomas are relatively radiosensitive at baseline. In addition, we show that deletion of Atm in the tumor cells does not extend survival of mice bearing Pten-null brainstem gliomas after focal brain irradiation. These results characterize a novel primary mouse model of PTEN-mutated brainstem glioma and provide insights into the mechanism of radiosensitization by ATM deletion, which may guide the design of future clinical trials.

Reversible mislocalization of a disease-associated MRE11 splice variant product.

Ataxia-telangiectasia (AT) and related disorders feature cancer predisposition, neurodegeneration, and immunodeficiency resulting from failure to respond to DNA damage. Hypomorphic mutations in MRE11 cause an AT-like disorder (ATLD) with variable clinical presentation. We have sought to understand how diverse MRE11 mutations may provide unique therapeutic opportunities, and potentially correlate with clinical variability. Here we have undertaken studies of an MRE11 splice site mutation that was found in two ATLD siblings that died of pulmonary adenocarcinoma at the young ages of 9 and 16. The mutation, termed MRE11 alternative splice mutation (MRE11ASM), causes skipping of a highly conserved exon while preserving the protein's open reading frame. A new mouse model expressing Mre11ASM from the endogenous locus demonstrates that the protein is present at very low levels, a feature in common with the MRE11ATLD1 mutant found in other patients. However, the mechanisms causing low protein levels are distinct. MRE11ASM is mislocalized to the cytoplasm, in contrast to MRE11ATLD1, which remains nuclear. Strikingly, MRE11ASM mislocalization is corrected by inhibition of the proteasome, implying that the protein undergoes strict protein quality control in the nucleus. These findings raise the prospect that inhibition of poorly understood nuclear protein quality control mechanisms might have therapeutic benefit in genetic disorders causing cytoplasmic mislocalization.

Hematopoietic stem cells are acutely sensitive to Acd shelterin gene inactivation.

The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors.

Disease-associated MRE11 mutants impact ATM/ATR DNA damage signaling by distinct mechanisms.

DNA double-strand breaks (DSBs) can lead to instability of the genome if not repaired correctly. The MRE11/RAD50/NBS1 (MRN) complex binds DSBs and initiates damage-induced signaling cascades via activation of the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia- and rad3-related (ATR) kinases. Mutations throughout MRE11 cause ataxia-telangiectasia-like disorder (ATLD) featuring cerebellar degeneration, and cancer-predisposition in certain kindreds. Here, we have examined the impact on DNA damage signaling of several disease-associated MRE11A alleles to gain greater understanding of the mechanisms underlying the diverse disease sequelae of ATLD. To this end, we have designed a system whereby endogenous wild-type Mre11a is conditionally deleted and disease-associated MRE11 mutants are stably expressed at physiologic levels. We find that mutations in the highly conserved N-terminal domain impact ATM signaling by perturbing both MRE11 interaction with NBS1 and MRE11 homodimerization. In contrast, an inherited allele in the MRE11 C-terminus maintains MRN interactions and ATM/ATR kinase activation. These findings reveal that ATLD patients have reduced ATM activation resulting from at least two distinct mechanisms: (i) N-terminal mutations destabilize MRN interactions, and (ii) mutation of the extreme C-terminus maintains interactions but leads to low levels of the complex. The N-terminal mutations were found in ATLD patients with childhood cancer; thus, our studies suggest a clinically relevant dichotomy in MRE11A alleles. More broadly, these studies underscore the importance of understanding specific effects of hypomorphic disease-associated mutations to achieve accurate prognosis and appropriate long-term medical surveillance.

A spectrum of severe familial liver disorders associate with telomerase mutations.

BACKGROUND: Telomerase is an enzyme specialized in maintaining telomere lengths in highly proliferative cells. Loss-of-function mutations cause critical telomere shortening and are associated with the bone marrow failure syndromes dyskeratosis congenita and aplastic anemia and with idiopathic pulmonary fibrosis. Here, we sought to determine the spectrum of clinical manifestations associated with telomerase loss-of-function mutations. METHODOLOGY/PRINCIPAL FINDINGS: Sixty-nine individuals from five unrelated families with a variety of hematologic, hepatic, and autoimmune disorders were screened for telomerase complex gene mutations; leukocyte telomere length was measured by flow fluorescence in situ hybridization in mutation carriers and some non-carriers; the effects of the identified mutations on telomerase activity were determined; and genetic and clinical data were correlated. In six generations of a large family, a loss-of-function mutation in the telomerase enzyme gene TERT associated with severe telomere shortening and a range of hematologic manifestations, from macrocytosis to acute myeloid leukemia, with severe liver diseases marked by fibrosis and inflammation, and one case of idiopathic pulmonary fibrosis but not with autoimmune disorders. Additionally, we identified four unrelated families in which loss-of-function TERC or TERT gene mutations tracked with marrow failure, pulmonary fibrosis, and a spectrum of liver disorders. CONCLUSIONS/SIGNIFICANCE: These results indicate that heterozygous telomerase loss-of-function mutations associate with but are not determinant of a large spectrum of hematologic and liver abnormalities, with the latter sometimes occurring in the absence of marrow failure. Our findings, along with the link between pulmonary fibrosis and telomerase mutations, also suggest a common pathogenic mechanism for fibrotic diseases in which defective telomere repair plays important role.

Sex hormones, acting on the TERT gene, increase telomerase activity in human primary hematopoietic cells.

Androgens have been used in the treatment of bone marrow failure syndromes without a clear understanding of their mechanism of action. Blood counts of patients with dyskeratosis congenita or aplastic anemia with mutations in telomerase genes can improve with androgen therapy. Here we observed that exposure in vitro of normal peripheral blood lymphocytes and human bone marrow-derived CD34(+) cells to androgens increased telomerase activity, coincident with higher TERT mRNA levels. Cells from patients who were heterozygous for telomerase mutations had low baseline telomerase activity, which was restored to normal levels by exposure to androgens. Estradiol had an effect similar to androgens on TERT gene expression and telomerase enzymatic activity. Tamoxifen abolished the effects of both estradiol and androgens on telomerase function, and letrozole, an aromatase inhibitor, blocked androgen effects on telomerase activity. Conversely, flutamide, an androgen receptor antagonist, did not affect androgen stimulation of telomerase. Down-regulation by siRNA of estrogen receptor-alpha (ER alpha), but not ER beta, inhibited estrogen-stimulated telomerase function. Our results provide a mechanism for androgen therapy in bone marrow failure: androgens appear to regulate telomerase expression and activity mainly by aromatization and through ER alpha. These findings have potential implications for the choice of current androgenic compounds and the development of future agents for clinical use.

Constitutional hypomorphic telomerase mutations in patients with acute myeloid leukemia.

Loss-of-function mutations in telomerase complex genes can cause bone marrow failure, dyskeratosis congenita, and acquired aplastic anemia, both diseases that predispose to acute myeloid leukemia. Loss of telomerase function produces short telomeres, potentially resulting in chromosome recombination, end-to-end fusion, and recognition as damaged DNA. We investigated whether mutations in telomerase genes also occur in acute myeloid leukemia. We screened bone marrow samples from 133 consecutive patients with acute myeloid leukemia and 198 controls for variations in TERT and TERC genes. An additional 89 patients from a second cohort, selected based on cytogenetic status, and 528 controls were further examined for mutations. A third cohort of 372 patients and 384 controls were specifically tested for one TERT gene variant. In the first cohort, 11 patients carried missense TERT gene variants that were not present in controls (P < 0.0001); in the second cohort, TERT mutations were associated with trisomy 8 and inversion 16. Mutation germ-line origin was demonstrated in 5 patients from whom other tissues were available. Analysis of all 3 cohorts (n = 594) for the most common gene variant (A1062T) indicated a prevalence 3 times higher in patients than in controls (n = 1,110; P = 0.0009). Introduction of TERT mutants into telomerase-deficient cells resulted in loss of enzymatic activity by haploinsufficiency. Inherited mutations in TERT that reduce telomerase activity are risk factors for acute myeloid leukemia. We propose that short and dysfunctional telomeres limit normal stem cell proliferation and predispose for leukemia by selection of stem cells with defective DNA damage responses that are prone to genome instability.

Functional characterization of natural telomerase mutations found in patients with hematologic disorders.

Human telomerase hTERC RNA serves as a template for the catalytic hTERT protein to synthesize telomere repeats at chromosome ends. We have recently shown that some patients with bone marrow failure syndromes are heterozygous carriers for hTERC or hTERT mutations. These sequence variations usually lead to a compromised telomerase function by haploinsufficiency. Here, we provide functional characterization of an additional 8 distinct hTERT sequence variants and 5 hTERC variants that have recently been identified in patients with dyskeratosis congenita (DC) or aplastic anemia (AA). Among the mutations, 2 are novel telomerase variants that were identified in our cohort of patients. Whereas most of the sequence variants modulate telomerase function by haploinsufficiency, 2 hTERC variants with sequence changes located within the template region appear to act in a dominant-negative fashion. Inherited telomerase gene mutations, therefore, operate by various mechanisms to shorten telomere lengths, leading to limited marrow stem cell reserve and renewal capacity in patients with hematologic disorders.