Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.

21Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is 22 now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade 23 gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct 24 repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT 25 promoter hypermethylation is currently the only known biomarker for TMZ response in 26 glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic 27 rearrangements that lead to MGMT overexpression, independently from changes in its promoter 28 methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT 29 rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements 30 contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can 31 be detected in tumor-derived exosomes and could potentially represent an early detection marker 32 of tumor recurrence in a subset of patients treated with TMZ. Introduction 34The therapeutic benefits of TMZ depend on its ability to methylate DNA, which takes place at 35 the N-7 and O-6 positions of guanine and N-3 position of adenine. Although the minor product 36 O6-Methylguanine (O6-meG) accounts for less than 10% of total alkylation, it exerts the greatest 37 potential for apoptosis induction 1 . O6-meG pairs with thymine as opposed to cytosine during 38 DNA replication. The O6-meG:thymine mismatch can be recognized by the post-replication 39Mismatch Repair (MMR) system and, according to the futile repair hypothesis, ultimately 40 induces DNA double-strand breaks, cell cycle arrest and cell death 2 . The O-6-methylguanine-41 DNA methyltransferase (MGMT) is responsible for the direct repair of O6-meG lesion by 42 transferring the alkyl group from guanine to a cysteine residue. Epigenetic silencing, due to 43 promoter methylation, of the MGMT gene prevents the synthesis of this enzyme, and as a 44 consequence increases the tumours sensitivity to the cytotoxic effects induced by TMZ and other 45 alkylating compounds 3,4 . As today, MGMT promoter hypermethylation is the only known 46 biomarker for TMZ response 4 . However, the discordance between promoter methylation and 47 Nevertheless, the desired genomic rearrangements were further validated using a break-apart 132 fluorescence in situ hybridization (FISH) assay ( Supplementary Fig. 4). 133 134

Pancreatic ductal adenocarcinoma (PDAC) frequently metastasizes into the peritoneum, which contributes to poor prognosis. Metastatic spreading is promoted by cancer cell plasticity, yet its regulation by the microenvironment is incompletely understood. Here, we show that the presence of hyaluronan and proteoglycan link protein-1 (HAPLN1) in the extracellular matrix enhances tumor cell plasticity and PDAC metastasis. Bioinformatic analysis showed that HAPLN1 expression is enriched in the basal PDAC subtype and associated with worse overall patient survival. In a mouse model for peritoneal carcinomatosis, HAPLN1-induced immunomodulation favors a more permissive microenvironment, which accelerates the peritoneal spread of tumor cells. Mechanistically, HAPLN1, via upregulation of tumor necrosis factor receptor 2 (TNFR2), promotes TNF-mediated upregulation of Hyaluronan (HA) production, facilitating EMT, stemness, invasion and immunomodulation. Extracellular HAPLN1 modifies cancer cells and fibroblasts, rendering them more immunomodulatory. As such, we identify HAPLN1 as a prognostic marker and as a driver for peritoneal metastasis in PDAC.