Behavioral abnormalities and Parkinson’s-like histological changes resulting from Id2 inactivation in mice

SUMMARY Characterizing dopaminergic neuronal development and function in novel genetic animal models might uncover strategies for researchers to develop disease-modifying treatments for neurologic disorders. Id2 is a transcription factor expressed in the developing central nervous system. Id2−/− mice have fewer dopaminergic neurons in the olfactory bulb and reduced olfactory discrimination, a pre-clinical marker of Parkinson’s disease. Here, we summarize behavioral, histological and in vitro molecular biological analyses to determine whether midbrain dopaminergic neurons are affected by Id2 loss. Id2−/− mice were hyperactive at 1 and 3 months of age, but by 6 months showed reduced activity. Id2−/− mice showed age-dependent histological alterations in dopaminergic neurons of the substantia nigra pars compacta (SNpC) associated with changes in locomotor activity. Reduced dopamine transporter (DAT) expression was observed at early ages in Id2−/− mice and DAT expression was dependent on Id2 expression in an in vitro dopaminergic differentiation model. Evidence of neurodegeneration, including activated caspase-3 and glial infiltration, were noted in the SNpC of older Id2−/− mice. These findings document a novel role for Id2 in the maintenance of midbrain dopamine neurons. The Id2−/− mouse should provide unique opportunities to study the progression of neurodegenerative disorders involving the dopamine system

Targeting Cancer Gene Dependencies with Anthrax-Mediated Delivery of Peptide Nucleic Acids

Copyright © 2020 American Chemical Society. Antisense oligonucleotide therapies are important cancer treatments, which can suppress genes in cancer cells that are critical for cell survival. SF3B1 has recently emerged as a promising gene target that encodes a key splicing factor in the SF3B protein complex. Over 10% of cancers have lost one or more copies of the SF3B1 gene, rendering these cancers vulnerable after further suppression. SF3B1 is just one example of a CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) gene, but over 120 additional candidate CYCLOPS genes are known. Antisense oligonucleotide therapies for cancer offer the promise of effective suppression for CYCLOPS genes, but developing these treatments is difficult due to their limited permeability into cells and poor cytosolic stability. Here, we develop an effective approach to suppress CYCLOPS genes by delivering antisense peptide nucleic acids (PNAs) into the cytosol of cancer cells. We achieve efficient cytosolic PNA delivery with the two main nontoxic components of the anthrax toxin: protective antigen (PA) and the 263-residue N-terminal domain of lethal factor (LFN). Sortase-mediated ligation readily enables the conjugation of PNAs to the C terminus of the LFN protein. LFN and PA work together in concert to translocate PNAs into the cytosol of mammalian cells. Antisense SF3B1 PNAs delivered with the LFN/PA system suppress the SF3B1 gene and decrease cell viability, particularly of cancer cells with partial copy-number loss of SF3B1. Moreover, antisense SF3B1 PNAs delivered with a HER2-binding PA variant selectively target cancer cells that overexpress the HER2 cell receptor, demonstrating receptor-specific targeting of cancer cells. Taken together, our efforts illustrate how PA-mediated delivery of PNAs provides an effective and general approach for delivering antisense PNA therapeutics and for targeting gene dependencies in cancer

Partial gene suppression improves identification of cancer vulnerabilities when CRISPR-Cas9 knockout is pan-lethal

Abstract Background Hundreds of functional genomic screens have been performed across a diverse set of cancer contexts, as part of efforts such as the Cancer Dependency Map, to identify gene dependencies—genes whose loss of function reduces cell viability or fitness. Recently, large-scale screening efforts have shifted from RNAi to CRISPR-Cas9, due to superior efficacy and specificity. However, many effective oncology drugs only partially inhibit their protein targets, leading us to question whether partial suppression of genes using RNAi could reveal cancer vulnerabilities that are missed by complete knockout using CRISPR-Cas9. Here, we compare CRISPR-Cas9 and RNAi dependency profiles of genes across approximately 400 matched cancer cell lines. Results We find that CRISPR screens accurately identify more gene dependencies per cell line, but the majority of each cell line’s dependencies are part of a set of 1867 genes that are shared dependencies across the entire collection (pan-lethals). While RNAi knockdown of about 30% of these genes is also pan-lethal, approximately 50% have selective dependency patterns across cell lines, suggesting they could still be cancer vulnerabilities. The accuracy of the unique RNAi selectivity is supported by associations to multi-omics profiles, drug sensitivity, and other expected co-dependencies. Conclusions Incorporating RNAi data for genes that are pan-lethal knockouts facilitates the discovery of a wider range of gene targets than could be detected using the CRISPR dataset alone. This can aid in the interpretation of contrasting results obtained from CRISPR and RNAi screens and reinforce the importance of partial gene suppression methods in building a cancer dependency map

Multiplexed single-cell transcriptional response profiling to define cancer vulnerabilities and therapeutic mechanism of action

Large-scale screens of chemical and genetic vulnerabilities in cancer are typically limited to simple readouts of cell viability. Here, the authors develop a method for profiling post-perturbation transcriptional responses across large pools of cancer cell lines, enabling deep characterization of shared and context-specific responses

BET Bromodomain Inhibition of MYC-Amplified Medulloblastoma

PurposeMYC-amplified medulloblastomas are highly lethal tumors. Bromodomain and extraterminal (BET) bromodomain inhibition has recently been shown to suppress MYC-associated transcriptional activity in other cancers. The compound JQ1 inhibits BET bromodomain-containing proteins, including BRD4. Here, we investigate BET bromodomain targeting for the treatment of MYC-amplified medulloblastoma.Experimental designWe evaluated the effects of genetic and pharmacologic inhibition of BET bromodomains on proliferation, cell cycle, and apoptosis in established and newly generated patient- and genetically engineered mouse model (GEMM)-derived medulloblastoma cell lines and xenografts that harbored amplifications of MYC or MYCN. We also assessed the effect of JQ1 on MYC expression and global MYC-associated transcriptional activity. We assessed the in vivo efficacy of JQ1 in orthotopic xenografts established in immunocompromised mice.ResultsTreatment of MYC-amplified medulloblastoma cells with JQ1 decreased cell viability associated with arrest at G1 and apoptosis. We observed downregulation of MYC expression and confirmed the inhibition of MYC-associated transcriptional targets. The exogenous expression of MYC from a retroviral promoter reduced the effect of JQ1 on cell viability, suggesting that attenuated levels of MYC contribute to the functional effects of JQ1. JQ1 significantly prolonged the survival of orthotopic xenograft models of MYC-amplified medulloblastoma (P < 0.001). Xenografts harvested from mice after five doses of JQ1 had reduced the expression of MYC mRNA and a reduced proliferative index.ConclusionJQ1 suppresses MYC expression and MYC-associated transcriptional activity in medulloblastomas, resulting in an overall decrease in medulloblastoma cell viability. These preclinical findings highlight the promise of BET bromodomain inhibitors as novel agents for MYC-amplified medulloblastoma

Mitogenic and progenitor gene programmes in single pilocytic astrocytoma cells

Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma with a single driver BRAF rearrangement. Here, we perform scRNAseq in six PAs using methods that enabled detection of the rearrangement. When compared to higher-grade gliomas, a strikingly higher proportion of the PA cancer cells exhibit a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibit a progenitor-like phenotype with evidence of proliferation. These express a mitogen-activated protein kinase (MAPK) programme that was absent from higher-grade gliomas. Immune cells, especially microglia, comprise 40% of all cells in the PAs and account for differences in bulk expression profiles between tumor locations and subtypes. These data indicate that MAPK signaling is restricted to relatively undifferentiated cancer cells in PA, with implications for investigational therapies directed at this pathway