CDK7 Inhibition Suppresses Super-Enhancer-Linked Oncogenic Transcription in MYCN-Driven Cancer

The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, may be useful therapy for cancers that are driven by MYC family oncoproteins.United States. National Institutes of Health (R01CA148688)United States. National Institutes of Health (R01CA148688S1)United States. National Institutes of Health (R01CA179483-01)United States. National Institutes of Health (CA109901)United States. National Institutes of Health (HG002668)United States. National Institutes of Health (R21HG006778)American Cancer Society (RSG-12-247-TBG)United States. Department of Defense (PR120741A)Friends for Life Neuroblastoma Foundatio

Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas

RNA polymerase II mediates the transcription of all protein-coding genes in eukaryotic cells, a process that is fundamental to life. Genomic mutations altering this enzyme have not previously been linked to any pathology in humans, which is a testament to its indispensable role in cell biology. On the basis of a combination of next-generation genomic analyses of 775 meningiomas, we report that recurrent somatic p.Gln403Lys or p.Leu438_His439del mutations in POLR2A, which encodes the catalytic subunit of RNA polymerase II (ref. 1), hijack this essential enzyme and drive neoplasia. POLR2A mutant tumors show dysregulation of key meningeal identity genes including WNT6 and ZIC1/ZIC4. In addition to mutations in POLR2A, NF2, SMARCB1, TRAF7, KLF4, AKT1, PIK3CA, and SMO4 we also report somatic mutations in AKT3, PIK3R1, PRKAR1A, and SUFU in meningiomas. Our results identify a role for essential transcriptional machinery in driving tumorigenesis and define mutually exclusive meningioma subgroups with distinct clinical and pathological features

MiR-338-3p regulates neuronal maturation and suppresses glioblastoma proliferation

<div><p>Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral “sponge” to knock down <i>in vivo</i> expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously <i>in vivo</i>. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation <i>in vitro</i>. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.</p></div

<i>In vivo</i> verification of miR-338-3p sponge efficacy.

<p>(A) Design of lentiviral miR-338-3p sponge with a sensor cassette. The miR-338-3p sensor cassette contains 2 perfectly complementary miR-338-3p target sequences downstream of GFP driven by the pUbiquitin promoter and the sponge cassette consists of 6 targets downstream of both the H1 and U6 promoters for a total of 2 sensor targets to sense miR-338-3p activity and 12 sponge targets to sequester endogenous miR-338-3p. (B) Low magnification images of dentate gyrus show the mCherry control and the miR-338-3p sponge exhibit similarly high levels of expression. (C) Images from (B), but under high magnification. This demonstrates the ability of the sponge cassette to sequester ligand away from the miR-338-3p targets expressed in the sensor cassette.</p

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

Cdk12 is primarily involved in the regulation of DNA damage response (DDR) gene transcription as well as mRNA processing. Here, the authors demonstrate that CDK12 suppresses intronic polyadenylation, and that inhibition of this kinase primarily affects the expression of long genes with higher numbers of polyA sites, features common to many DDR genes

MiR-338-3p expression increases with maturity in dentate gyrus granule neurons.

<p>(A) Co-localization of nestin (arrowheads) with miR-338-3p sensor. (B) Co-localization of nestin (arrowheads) with miR-132-3p sensor. (C) Co-localization of NeuN (arrowheads) and miR-338-3p sensor. (D) Co-localization of NeuN (arrowheads) and miR-132-3p sensor. (E) Percentage of cells co-labeled with one of the sensors and nestin as compared to control mCherry virus. (F) Percentage of cells co-labeled with one of the sensors and doublecortin as compared to control mCherry virus. (G) Percentage of cells co-labeled with one of the sensors and NeuN as compared to control mCherry virus. (H) Ratio of cells co-labeled with either the miR-338-3p or miR-132-3p sensor and nestin as compared to control mCherry virus. (I) Ratio of cells co-labeled with either the miR-338-3p or miR-132-3p sensor and doublecortin as compared to control mCherry virus. (J) Ratio of cells co-labeled with either the miR-338-3p or miR-132-3p sensor and NeuN as compared to control mCherry virus. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA, analyzed post-hoc using Tukey’s range test. Results show mean ± SEM.</p