Diffuse midline gliomas (DMGs) are invariably fatal pediatric brain tumours that are inherently resistant to conventional therapy. In recent years our understanding of the underlying molecular mechanisms of DMG tumorigenicity has resulted in the identification of novel targets and the development of a range of potential therapies, with multiple agents now being progressed to clinical translation to test their therapeutic efficacy. Here, we provide an overview of the current therapies aimed at epigenetic and mutational drivers, cellular pathway aberrations and tumor microenvironment mechanisms in DMGs in order to aid therapy development and facilitate a holistic approach to patient treatment.

Differentiation of stem cells into highly specialised cells requires gene expression changes brought about by remodelling of the chromatin architecture. During this lineage-commitment process, the majority of DNA needs to be packaged into inactive heterochromatin, allowing only a subset of regulatory elements to remain open and functionally required genes to be expressed. Epigenetic mechanisms such as DNA methylation, post-translational modifications to histone tails, and nucleosome positioning all potentially contribute to the changes in higher order chromatin structure during differentiation. The mammary gland is a particularly useful model to study these complex epigenetic processes since the majority of its development is postnatal, the gland is easily accessible, and development occurs in a highly reproducible manner. Inappropriate epigenetic remodelling can also drive tumourigenesis; thus, insights into epigenetic remodelling during mammary gland development advance our understanding of breast cancer aetiology. We review the current literature surrounding DNA methylation and histone modifications in the developing mammary gland and its implications for breast cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC ( Pdx1-Cre , LSL-Kras ^G12D/+ , LSL-Trp53 ^R172H/+ ) and poorly metastatic KP ^fl C ( Pdx1-Cre , LSL-Kras ^G12D/+ , Trp53 ^fl/+ ) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KP ^fl C, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.

The basic helix-loop-helix (bHLH) transcription factors inhibitor of differentiation 1 (Id1) and inhibitor of differentiation 3 (Id3) (referred to as Id) have an important role in maintaining the cancer stem cell (CSC) phenotype in the triple-negative breast cancer (TNBC) subtype. In this study, we aimed to understand the molecular mechanism underlying Id control of CSC phenotype and exploit it for therapeutic purposes. We used two different TNBC tumor models marked by either Id depletion or Id1 expression in order to identify Id targets using a combinatorial analysis of RNA sequencing and microarray data. Phenotypically, Id protein depletion leads to cell cycle arrest in the G0/G1 phase, which we demonstrate is reversible. In order to understand the molecular underpinning of Id proteins on the cell cycle phenotype, we carried out a large-scale small interfering RNA (siRNA) screen of 61 putative targets identified by using genomic analysis of two Id TNBC tumor models. Kinesin Family Member 11 (Kif11) and Aurora Kinase A (Aurka), which are critical cell cycle regulators, were further validated as Id targets. Interestingly, unlike in Id depletion conditions, Kif11 and Aurka knockdown leads to a G2/M arrest, suggesting a novel Id cell cycle mechanism, which we will explore in further studies. Therapeutic targeting of Kif11 to block the Id1–Kif11 axis was carried out using small molecular inhibitor ispinesib. We finally leveraged our findings to target the Id/Kif11 pathway using the small molecule inhibitor ispinesib in the Id+ CSC results combined with chemotherapy for better response in TNBC subtypes. This work opens up exciting new possibilities of targeting Id targets such as Kif11 in the TNBC subtype, which is currently refractory to chemotherapy. Targeting the Id1–Kif11 molecular pathway in the Id1+ CSCs in combination with chemotherapy and small molecular inhibitor results in more effective debulking of TNBC.

Inhibitor of differentiation (ID) proteins dimerize with basic HLH (bHLH) transcription factors, repressing transcription of lineage-specification genes across diverse cellular lineages. ID4 is a key regulator of mammary stem cells; however, the mechanism by which it achieves this is unclear. Here, we show that ID4 has a cell autonomous role in preventing myoepithelial differentiation of basal cells in mammary organoids and in vivo. ID4 positively regulates proliferative genes and negatively regulates genes involved in myoepithelial function. Mass spectrometry reveals that ID4 interacts with the bHLH protein HEB, which binds to E-box motifs in regulatory elements of basal developmental genes involved in extracellular matrix and the contractile cytoskeleton. We conclude that high ID4 expression in mammary basal stem cells antagonizes HEB transcriptional activity, preventing myoepithelial differentiation and allowing for appropriate tissue morphogenesis. Downregulation of ID4 during pregnancy modulates gene regulated by HEB, promoting specialization of basal cells into myoepithelial cells.