Search bioRxiv⌕ Search

Biology subjects

Dahl, N. A.

Publications and source records attributed to Dahl, N. A..

5 recordsLinked to original sources

Preclinical evaluation of ixazomib for high-risk pediatric brain tumors

Background: Many of the highest risk pediatric brain cancers continue to experience poor clinical outcomes despite intensification of current multimodal therapy. Proteasome inhibition has shown preclinical promise across a range of cancer models including diffuse midline glioma (DMG), medulloblastoma, and atypical teratoid / rhabdoid tumors (ATRT), though clinically viable agents have been limited. Recent studies in adults with glioblastoma suggest that ixazomib, a second-generation proteasome inhibitor, might achieve therapeutic concentrations in the CNS, presenting the opportunity that a CNS penetrant proteasome inhibitor might be similarly leveraged for benefit in childhood brain cancers. Methods: Ixazomib was tested against cell lines and orthotopic xenograft models of DMG, Myc-amplified medulloblastoma (Myc-MB), and ATRT. RNA sequencing and LC-MS based proteomics were utilized to define functional consequences of ixazomib treatment in these models. Proteasome activity readouts were used to assess ixazomib activity across brain regions and extracranial solid organs. Results: Ixazomib demonstrates consistent cytotoxic effect across high-risk brain tumor models at low nanomolar concentrations. Ixazomib treatment activates proteostatic stress response and apoptosis. Treatment with ixazomib does not demonstrate survival benefit in orthotopic models, however, and pharmacodynamic testing suggests insufficient inhibition of proteasome activity within the CNS compared to extracranial tissues. Conclusions: While many pediatric brain tumor models demonstrate susceptibility to proteasome inhibition, ixazomib may lack sufficient blood-brain barrier penetration to be a translationally viable means of exploiting this vulnerability.

cancer biology↗

Targeting processive transcription for Myc-driven circuitry in medulloblastoma

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSMedulloblastoma is the most common malignant brain tumor of childhood. The highest-risk tumors are driven by recurrent Myc amplifications (Myc-MB) and experience poorer outcomes despite intensive multimodal therapy. The Myc transcription factor defines core regulatory circuitry for these tumors and acts to broadly amplify downstream pro-survival transcriptional programs. Therapeutic targeting of Myc directly has proven elusive, but inhibiting transcriptional cofactors may present an indirect means of drugging the oncogenic transcriptional circuitry sustaining Myc-MB. MethodsIndependent CRISPR-Cas9 screens were pooled to identify conserved dependencies in Myc-MB. We performed chromatin conformation capture (Hi-C) from primary patient Myc-MB samples to map enhancer-promoter interactions. We then treated in vitro and xenograft models with CDK9/7 inhibitors to evaluate effect on Myc-driven programs and tumor growth. ResultsEight CRISPR-Cas9 screens performed across three independent labs identify CDK9 as a conserved dependency in Myc-MB. Myc-MB cells are susceptible to CDK9 inhibition, which is synergistic with concurrent inhibition of CDK7. Inhibition of transcriptional CDKs disrupts enhancer-promoter activity in Myc-MB and downregulates Myc-driven transcriptional programs, exerting potent anti-tumor effect. ConclusionsOur findings identify CDK9 inhibition as a translationally promising strategy for the treatment of Myc-MB. KO_SCPLOWEYC_SCPLOW PO_SCPLOWOINTSC_SCPLOWO_LICDK9 is an intrinsic dependency in Myc-driven medulloblastoma C_LIO_LIDual CDK9/7 inhibition disrupts Myc-driven transcriptional circuitry C_LIO_LICDK9 inhibitors should be developed as pharmaceutical agents for Myc-MB C_LI IO_SCPLOWMPORTANCEC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWOFC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWTHEC_SCPLOW SO_SCPLOWTUDYC_SCPLOWMedulloblastoma is the most common malignant brain tumor of childhood, and outcomes for high-risk subgroups remain unsatisfactory despite intensive multimodal therapy. In this study, we pool multiple independent CRISPR-Cas9 screens to identify transcriptional cofactors such as CDK9 as conserved dependencies in Myc-MB. Using Hi-C from primary patient samples, we map Myc enhancer-promoter interactions and show that they can be disrupted using inhibition of transcriptional CDKs. CDK9 inhibitor treatment depletes Myc-driven transcriptional programs, leading to potent anti-tumor effect in vitro and prolongation of xenograft survival in vivo. With a large number of CDK9 inhibitory compounds now in clinical development, this study highlights the opportunity for clinical translation of these for children diagnosed with Myc-MB.

cancer biology↗

H3K27me3 spreading organizes canonical PRC1 chromatin architecture to regulate developmental programs

Polycomb Repressive Complex 2 (PRC2)-mediated histone H3K27 tri-methylation (H3K27me3) recruits canonical PRC1 (cPRC1) to maintain heterochromatin. In early development, polycomb-regulated genes are connected through long-range 3D interactions which resolve upon differentiation. Here, we report that polycomb looping is controlled by H3K27me3 spreading and regulates target gene silencing and cell fate specification. Using glioma-derived H3 Lys-27-Met (H3K27M) mutations as tools to restrict H3K27me3 deposition, we show that H3K27me3 confinement concentrates the chromatin pool of cPRC1, resulting in heightened 3D interactions mirroring chromatin architecture of pluripotency, and stringent gene repression that maintains cells in progenitor states to facilitate tumor development. Conversely, H3K27me3 spread in pluripotent stem cells, following neural differentiation or loss of the H3K36 methyltransferase NSD1, dilutes cPRC1 concentration and dissolves polycomb loops. These results identify the regulatory principles and disease implications of polycomb looping and nominate histone modification-guided distribution of reader complexes as an important mechanism for nuclear compartment organization. Highlights{square} The confinement of H3K27me3 at PRC2 nucleation sites without its spreading correlates with increased 3D chromatin interactions. {square}The H3K27M oncohistone concentrates canonical PRC1 that anchors chromatin loop interactions in gliomas, silencing developmental programs. {square}Stem and progenitor cells require factors promoting H3K27me3 confinement, including H3K36me2, to maintain cPRC1 loop architecture. {square}The cPRC1-H3K27me3 interaction is a targetable driver of aberrant self-renewal in tumor cells.

molecular biology↗

Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy

AO_SCPLOWBSTRACTC_SCPLOWDynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates PTEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

cancer biology↗

Super elongation complex as a targetable dependency in diffuse midline glioma

Mutations in the histone 3 gene (H3K27M) are the eponymous drivers in diffuse intrinsic pontine gliomas (DIPGs) and other diffuse midline gliomas (DMGs), aggressive pediatric brain cancers for which no curative therapy currently exists. The salient molecular consequence of these recurrent oncohistones is a global loss of repressive H3K27me3 residues and broad epigenetic dysregulation. In order to identify specific, therapeutically targetable epigenetic dependencies within this disease context, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DMG cultures. This approach identified AFF4, the scaffold protein of the super elongation complex (SEC), as a previously-undescribed dependency in DMG. Interrogation of SEC function demonstrated a key role for maintaining DMG cell viability and clonogenic potential while promoting self-renewal of DMG tumor stem cells. Small-molecule inhibition of the SEC with the highly-specific, clinically relevant CDK9 inhibitors atuveciclib and AZD4573 restores regulatory RNA polymerase II pausing, promotes cellular differentiation, and leads to potent anti-tumor effect both in vitro and in patient-derived xenograft models. These studies present a biologic rationale for translational exploration of CDK9 inhibition as a promising therapeutic approach in a disease which currently has no effective medical therapies.

cancer biology↗