Small-molecule inhibition of the METTL3/METTL14 complex suppresses neuroblastoma tumor growth and promotes differentiation.

The N6-methyladenosine (m6A) RNA modification is an important regulator of gene expression. m6A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m6A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m6A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors in vivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.

Mesenchymal and adrenergic cell lineage states in neuroblastoma possess distinct immunogenic phenotypes.

Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage.

Synergistic Anti-Tumor Effect of Combining Selective CDK7 and BRD4 Inhibition in Neuroblastoma.

PURPOSE: Cyclin-dependent kinases (CDKs) that have critical roles in RNA polymerase II (Pol II)-mediated gene transcription are emerging as therapeutic targets in cancer. We have previously shown that THZ1, a covalent inhibitor of CDKs 7/12/13, leads to cytotoxicity in MYCN-amplified neuroblastoma through the downregulation of super-enhancer-associated transcriptional upregulation. Here we determined the effects of YKL-5-124, a novel covalent inhibitor with greater selectivity for CDK7 in neuroblastoma cells. EXPERIMENTAL DESIGN: We tested YKL-5-124 in MYCN-amplified and nonamplified neuroblastoma cells individually and in combination with other inhibitors in cell line and animal models. Cell viability, target validation, effects on cell cycle and transcription were analyzed. RESULTS: CDK7 inhibition with YKL-5-124 did not lead to significant cell death, but resulted in aberrant cell cycle progression especially in MYCN-amplified cells. Unlike THZ1, YKL-5-124 had minimal effects on Pol II C-terminal domain phosphorylation, but significantly inhibited that of the CDK1 and CDK2 cell cycle kinases. Combining YKL-5-124 with the BRD4 inhibitor JQ1 resulted in synergistic cytotoxicity. A distinct MYCN-gene expression signature associated with resistance to BRD4 inhibition was suppressed with the combination. The synergy between YKL-5-124 and JQ1 translated into significant tumor regression in cell line and patient-derived xenograft mouse models of neuroblastoma. CONCLUSIONS: The combination of CDK7 and BRD4 inhibition provides a therapeutic option for neuroblastoma and suggests that the addition of YKL-5-124 could improve the therapeutic efficacy of JQ1 and delay resistance to BRD4 inhibition.

Ex vivo screen identifies CDK12 as a metastatic vulnerability in osteosarcoma.

Despite progress in intensification of therapy, outcomes for patients with metastatic osteosarcoma (OS) have not improved in thirty years. We developed a system that enabled preclinical screening of compounds against metastatic OS cells in the context of the native lung microenvironment. Using this strategy to screen a library of epigenetically targeted compounds, we identified inhibitors of CDK12 to be most effective, reducing OS cell outgrowth in the lung by more than 90% at submicromolar doses. We found that knockout of CDK12 in an in vivo model of lung metastasis significantly decreased the ability of OS to colonize the lung. CDK12 inhibition led to defects in transcription elongation in a gene length- and expression-dependent manner. These effects were accompanied by defects in RNA processing and altered the expression of genes involved in transcription regulation and the DNA damage response. We further identified OS models that differ in their sensitivity to CDK12 inhibition in the lung and provided evidence that upregulated MYC levels may mediate these differences. Our studies provided a framework for rapid preclinical testing of compounds with antimetastatic activity and highlighted CDK12 as a potential therapeutic target in OS.

CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation.

Cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and selectively affects the expression of genes involved in the DNA damage response (DDR) and mRNA processing. Yet, the mechanisms underlying such selectivity remain unclear. Here we show that CDK12 inhibition in cancer cells lacking CDK12 mutations results in gene length-dependent elongation defects, inducing premature cleavage and polyadenylation (PCPA) and loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlates with an increased number of intronic polyadenylation sites, a feature especially prominent among DDR genes. Phosphoproteomic analysis indicated that CDK12 directly phosphorylates pre-mRNA processing factors, including those regulating PCPA. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition primarily due to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.

Anemia in Patients With Resistance to Thyroid Hormone α: A Role for Thyroid Hormone Receptor α in Human Erythropoiesis.

Context: Patients with resistance to thyroid hormone (TH) α (RTHα) are characterized by growth retardation, macrocephaly, constipation, and abnormal thyroid function tests. In addition, almost all RTHα patients have mild anemia, the pathogenesis of which is unknown. Animal studies suggest an important role for TH and TH receptor (TR)α in erythropoiesis. Objective: To investigate whether a defect in TRα affects the maturation of red blood cells in RTHα patients. Design, Setting, and Patients: Cultures of primary human erythroid progenitor cells (HEPs), from peripheral blood of RTHα patients (n = 11) harboring different inactivating mutations in TRα (P398R, F397fs406X, C392X, R384H, A382fs388X, A263V, A263S), were compared with healthy controls (n = 11). During differentiation, erythroid cells become smaller, accumulate hemoglobin, and express different cell surface markers. We assessed cell number and cell size, and used cell staining and fluorescence-activated cell sorter analysis to monitor maturation at different time points. Results: After ∼14 days of ex vivo expansion, both control and patient-derived progenitors differentiated spontaneously. However, RTHα-derived cells differentiated more slowly. During spontaneous differentiation, RTHα-derived HEPs were larger, more positive for c-Kit (a proliferation marker), and less positive for glycophorin A (a differentiation marker). The degree of abnormal spontaneous maturation of RTHα-derived progenitors did not correlate with severity of underlying TRα defect. Both control and RTHα-derived progenitors responded similarly when differentiation was induced. T3 exposure accelerated differentiation of both control- and RTHα patient-derived HEPs. Conclusions: Inactivating mutations in human TRα affect the balance between proliferation and differentiation of progenitor cells during erythropoiesis, which may contribute to the mild anemia seen in most RTHα patients.

Regulators of homologous recombination repair as novel targets for cancer treatment.

To cope with DNA damage, cells possess a complex signaling network called the 'DNA damage response', which coordinates cell cycle control with DNA repair. The importance of this network is underscored by the cancer predisposition that frequently goes along with hereditary mutations in DNA repair genes. One especially important DNA repair pathway in this respect is homologous recombination (HR) repair. Defects in HR repair are observed in various cancers, including hereditary breast, and ovarian cancer. Intriguingly, tumor cells with defective HR repair show increased sensitivity to chemotherapeutic reagents, including platinum-containing agents. These observations suggest that HR-proficient tumor cells might be sensitized to chemotherapeutics if HR repair could be therapeutically inactivated. HR repair is an extensively regulated process, which depends strongly on the activity of various other pathways, including cell cycle pathways, protein-control pathways, and growth factor-activated receptor signaling pathways. In this review, we discuss how the mechanistic wiring of HR is controlled by cell-intrinsic or extracellular pathways. Furthermore, we have performed a meta-analysis on available genome-wide RNA interference studies to identify additional pathways that control HR repair. Finally, we discuss how these HR-regulatory pathways may provide therapeutic targets in the context of radio/chemosensitization.

Gene expression analysis identifies global gene dosage sensitivity in cancer.

Many cancer-associated somatic copy number alterations (SCNAs) are known. Currently, one of the challenges is to identify the molecular downstream effects of these variants. Although several SCNAs are known to change gene expression levels, it is not clear whether each individual SCNA affects gene expression. We reanalyzed 77,840 expression profiles and observed a limited set of 'transcriptional components' that describe well-known biology, explain the vast majority of variation in gene expression and enable us to predict the biological function of genes. On correcting expression profiles for these components, we observed that the residual expression levels (in 'functional genomic mRNA' profiling) correlated strongly with copy number. DNA copy number correlated positively with expression levels for 99% of all abundantly expressed human genes, indicating global gene dosage sensitivity. By applying this method to 16,172 patient-derived tumor samples, we replicated many loci with aberrant copy numbers and identified recurrently disrupted genes in genomically unstable cancers.

The DNA damage response during mitosis.

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed.

[Timolol 0.5%/dorzolamide 2% fixed combination versus timolol 0.5%/pilocarpine 2% fixed combination in primary open-angle glaucoma or ocular hypertensive patients]. / Porównanie skutecznosci terapii skojarzonej 0.5% timololem i 2% dorzolamidem oraz 0.5% timololem i 2% pilokarpina w leczeniu jaskry pierwotnej otwartego kata i nadcisnienia ocznego.

PURPOSE: To establish the efficacy and safety of timolol 0.5%/dorzolamide 2% fixed combination versus timolol 0.5%/pilocarpine 2% fixed combination, each given twice daily, in primary open-angle glaucoma and ocular hypertensive patients. MATERIAL AND METHODS: In this prospective, multicenter, double-masked trial, 37 patients were treated twice daily with timolol for 4 weeks. Then, they were randomized to one of the treatment medications for 6 weeks, after which they were treated with timolol again for 2 weeks before placed on the opposite treatment medication for 6 weeks. RESULTS: A total of 36 patients completed the trial. The mean baseline intraocular pressure (IOP) was 22.3 +/- 3.7 mmHg. Following 6 weeks of treatment, the mean decrease of IOP was 18.0 +/- 2.2 mmHg for timolol/dorzolamide combination and 17.4 +/- 2.0 for timolol/pilocarpine combination. The mean diurnal curve IOP was 18.1 +/- 2.2 mmHg for timolol/dorzolamide group and 16.7 +/- 1.9 mmHg for timolol/pilocarpine group. These differences were statistically significant. Statistically more patients reported ocular pain and diminished vision during use of the timolol/pilocarpine combination. CONCLUSIONS: This study suggest that timolol 0.5%/pilocarpine 2% fixed combination can provide at least similar efficacious reduction in IOP as timolol 0.5 %/dorzolamide 2% fixed combination in patients with primary open-angle glaucoma and ocular hypertension.

Timolol 0.5%/dorzolamide 2% fixed combination versus timolol 0.5%/pilocarpine 2% fixed combination in primary open-angle glaucoma or ocular hypertensive patients.

PURPOSE: To establish the efficacy and safety of timolol maleate/dorzolamide fixed combination (TDFC) versus timolol maleate/pilocarpine fixed combination (TPFC), each given twice daily, in primary open-angle glaucoma or ocular hypertensive patients. METHODS: In this prospective, multicentred, double-masked trial, 37 patients were treated twice daily with timolol for 4 weeks. They were then randomized to one of the treatment medications for 6 weeks, after which they were treated with timolol again for 2 weeks before being placed on the opposite treatment medication for 6 weeks. RESULTS: A total of 36 patients completed the trial. Their mean baseline intraocular pressure (IOP) was 22.3 +/- 3.7 mmHg. Following 6 weeks of treatment, the mean trough (08.00 hours) IOP was 18.0 +/- 2.2 mmHg for TDFC and 17.4 +/- 2.0 mmHg for TPFC (p = 0.22). The mean diurnal curve IOP was 18.1 +/- 2.2 mmHg for TDFC and 16.7 +/- 1.9 mmHg for TPFC (p = 0.0007). At the remaining time-points (10.00, 18.00 and 20.00 hours), TPFC IOPs were statistically lower than TDFC IOPs (p < 0.03). There were statistically more unsolicited reports of vision change and ocular pain associated with TPFC (p = 0.04). Six patients were discontinued early from TPFC therapy (17%) versus two from TDFC (6%) (p = 0.13). CONCLUSIONS: This study suggests that TPFC can provide at least a similar efficacious reduction in IOP as TDFC in patients with primary open-angle glaucoma or ocular hypertension.