Endothelial Rbpj is essential for the education of tumour-associated macrophages

Epithelial ovarian cancer (EOC) is one of the most lethal gynaecological cancers worldwide. EOC cells educate tumour-associated macrophages (TAMs) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumour microenvironment (TME). In addition, tumour cells frequently activate Notch1 receptors on endothelial cells (ECs) to facilitate metastasis. However, little is known whether the endothelium would also influence the education of recruited monocytes. Here, we report that canonical Notch signalling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature. This was associated with stronger cytotoxic activity of T cells and decreased tumour burden. Mechanistically, we identified CXCL2 as a novel Notch/RBPJ target gene. This angiocrine factor regulates the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. As such, EOC cells employ the tumour endothelium to secrete CXCL2 in order to facilitate an immunosuppressive microenvironment.

The minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers

Minor splicing is a second splicing system required for the correct expression of ∼700 human minor intron-containing genes (MIGs). Many MIGs are expressed in vigorously proliferating cells and are frequently dysregulated in cancer including BRAF, ERK, JNK and p38. Minor splicing is carried out by the minor spliceosome which comprises several unique components, including a 65kDa protein encoded by RNPC3. We show that Rnpc3 heterozygosity reduces tumour burden in a broad spectrum of in vivo cancer settings, without harming normal tissues. Using the collective power of zebrafish, mouse and human cancer models, we reveal a sequence of events connecting Rnpc3 deficiency and impaired splicing of MIGs to DNA damage and activation of a Tp53-dependent transcriptional program that restricts tumour burden by inducing cell cycle arrest and apoptosis. Interrogation of human liver and lung cancer transcriptomes curated in TCGA revealed that the expression of many of the genes encoding protein components of the minor spliceosome is upregulated in these cancers. This is accompanied by upregulation of the expression of MIGs that are enriched in cell cycle and DNA damage pathways. These findings suggest that cancer cells can invoke mechanisms to increase the efficiency of minor splicing to support their high proliferation rates. Finally, Kaplan Meier survival analysis shows that highly expressed MIGs are frequently associated with poor patient survival. Taken together, these results indicate that the minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers.

Development of a Hypoxia-Activated Trehalose Diester for the Treatment of Solid Tumours

<p><b>The potential of bacterial cell wall components in the treatment of various cancers was initially realised in the late 1800s during pioneering work with Coley’s toxins. Since this preliminary work, efforts have been concentrated on the isolation and identification of bacterial components that lead to tumour regression. Trehalose dimycolates (TDMs) are compounds isolated from the M. tuberculosis cell wall and are known to activate macrophages to give a polarised Th1 immune response resulting in reduced tumour burden. Consequently, TDMs have shown great promise in the treatment of solid tumours.</b></p> <p>In this thesis, work is presented towards the synthesis of trehalose glycolipid prodrugs that will be specifically activated inside the hypoxic tumour microenvironment, and thereby lead to a more selective form of cancer therapy. These hypoxia-activated trehalose glycolipids incorporate a nitroimidazole trigger that fragments upon enzymatic reduction (in the absence of oxygen) to give the active glycolipid. Throughout the course of this work, it was determined that the nitroimidazole trigger group could not be directly attached to the glycolipid and thus, an alternative carbonate-linker strategy was explored through the use of a reporter fluoroprobe. The validity of this approach was determined in various enzyme and cell-based assays.</p>

Development of a Hypoxia-Activated Trehalose Diester for the Treatment of Solid Tumours

<p><b>The potential of bacterial cell wall components in the treatment of various cancers was initially realised in the late 1800s during pioneering work with Coley’s toxins. Since this preliminary work, efforts have been concentrated on the isolation and identification of bacterial components that lead to tumour regression. Trehalose dimycolates (TDMs) are compounds isolated from the M. tuberculosis cell wall and are known to activate macrophages to give a polarised Th1 immune response resulting in reduced tumour burden. Consequently, TDMs have shown great promise in the treatment of solid tumours.</b></p> <p>In this thesis, work is presented towards the synthesis of trehalose glycolipid prodrugs that will be specifically activated inside the hypoxic tumour microenvironment, and thereby lead to a more selective form of cancer therapy. These hypoxia-activated trehalose glycolipids incorporate a nitroimidazole trigger that fragments upon enzymatic reduction (in the absence of oxygen) to give the active glycolipid. Throughout the course of this work, it was determined that the nitroimidazole trigger group could not be directly attached to the glycolipid and thus, an alternative carbonate-linker strategy was explored through the use of a reporter fluoroprobe. The validity of this approach was determined in various enzyme and cell-based assays.</p>

Diagnosing metastatic pheochromocytoma: Trick lies in attention to details!

Functional metastatic pheochromocytoma (PCC) is a very rare tumour and cytoreductive adrenalectomy with oligo metastatectomy is recommended in cases of low tumour burden. We report a rare case of metastatic PCC with an incidentally detected suspicious nodule seen on the anterior surface of the right lobe of the liver. The adrenal and the lesion were excised and sent for histopathology which was reported as a metastasis from PCC. This lesion was not visualised preoperatively on DOTA-PET-CT, highlighting the importance of keeping a low threshold for suspicion of metastasis in abnormal lesions and taking a biopsy during surgery. Inspection of the liver and rest of the abdomen for any abnormality should be done even when operating for any apparently benign lesions.

C.5 Musashi-1 is a master regulator of aberrant translation in MYC-amplified Group 3 medulloblastoma

Background: Medulloblastoma (MB) is the most common solid malignant pediatric brain neoplasm. Group 3 (G3) MB, particularly MYC amplified G3 MB, is the most aggressive subgroup with the highest frequency of children presenting with metastatic disease, and is associated with a poor prognosis. To further our understanding of the role of MSI1 in MYC amplified G3 MB, we performed an unbiased integrative analysis of eCLIP binding sites, with changes observed at the transcriptome, the translatome, and the proteome after shMSI1 inhibition. Methods: Primary human pediatric MBs, SU_MB002 and HD-MB03 were kind gifts from Dr. Yoon-Jae Cho (Harvard, MS) and Dr. Till Milde (Heidelberg) and cultured for in vitro and in vivo experiments. eCLIP, RNA-seq, Polysome-seq, and TMT-MS were completed as previously described. Results:MSI1 is overexpressed in G3 MB. shRNA Msi1 interference resulted in a reduction in tumour burden conferring a survival advantage to mice injected with shMSI1 G3MB cells. Robust ranked multiomic analysis (RRA) identified an unconventional gene set directly perturbed by MSI1 in G3 MB. Conclusions: Our robust unbiased integrative analysis revealed a distinct role for MSI1 in the maintenance of the stem cell state in G3 MB through post-transcriptional modification of multiple pathways including identification of unconventional targets such as HIPK1.