Rocco Piazza

Bladder cancer is one of the most common malignancies worldwide, impacting public health systems due to its high rate of recurrence. Metabolic alterations enable the identification of novel, needed prognostic markers and therapeutic vulnerabilities for precision oncology. Here, we use a systems metabolomics approach that integrates metabolic functional data with multi-omics, transcriptomics and metabolomics, through biostatistics and mathematical modelling to characterise metabolic rearrangements associated with FGFR3 oncogenic activation, one of the most frequent lesions in bladder cancer. The analyses conducted on a panel of five human bladder cancer cell lines reveal a significant correlation between FGFR3 alterations and the acquisition of a predominantly oxidative, poorly migratory phenotype, regardless of tumour progression. These preclinical results, validated through FGFR3 and oxidative phosphorylation pharmacological inhibition, and computational analysis on bladder cancer cell line and patient publicly available datasets, support the therapeutic potential of targeting oxidative metabolism in FGFR3-altered tumours, including more aggressive subtypes.

The RNA-binding protein Musashi-2 (MSI2) is emerging as key regulator of cancer biology. However, its functional role in high-risk pediatric B-cell Acute Lymphoblastic Leukemia (B-ALL) is not fully elucidated. In this study, we demonstrate that MSI2 promotes leukemic cell growth in vitro and sustains the leukemogenic potential of KMT2A-rearranged infant ALL in vivo. Mechanistically, MSI2 is involved in Glucocorticoids resistance by regulating mitochondria metabolism. Targeting MSI2 impairs the mitochondrial function in leukemic cells upon Glucocorticoids exposure and increases their susceptibility to metabolic perturbations, highlighting a novel role of MSI2 in cell metabolism. Targeting the mitochondria activity in KMT2Ar infant ALL cells with OxPhos inhibitors may therefore represent a promising strategy to improve treatment response in patients. Overall, our findings identify MSI2 as a critical regulator of leukemic maintenance and drug resistance in KMT2Ar infant ALL, providing mechanistic insight into therapy failure and supporting the targeting of metabolic vulnerabilities as a promising therapeutic strategy for this high-risk pediatric leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries, and B-cell receptor (BCR) pathway inhibitors such as idelalisib and ibrutinib are currently established therapies for CLL. Although effective, these drugs frequently lead to resistance, but the mechanisms are still not fully understood. Activation-induced cytidine deaminase (AID) is a B-cell enzyme essential for antibody diversification. However, it can also introduce off-target mutations, leading to genomic instability. This study investigates whether treatment with BCR pathway inhibitors increases AID activity in CLL and whether this activity contributes to the development of drug resistance. Peripheral blood samples from CLL patients were collected before and after treatment with idelalisib or ibrutinib. Targeted sequencing was used to identify mutations in known AID off-target genes. Concurrently, AID-wild type (AID-WT) and AID-knockout (AID-KO) CLL cell lines were established and subsequently exposed to escalating doses of BCR pathway inhibitors to develop drug-resistant models. In patient samples, treatment with BCR pathway inhibitors was associated with an increase in AID-dependent mutations in off-target genes, including BCL2, MYC, and IRF8. The in vitro models efficiently recapitulated the patients’ data, as only AID-WT CLL cells accumulated mutations in the same AID off-target genes after drug exposure. However, no mutations were detected in genes that could mediate drug resistance. We conclude that BCR pathway inhibitors enhance AID mutational activity in CLL, but this does not appear to be directly involved in driving drug resistance. AID-targeted loci may nonetheless serve as biomarkers for monitoring genomic instability during treatment and inform further study.

Supplementary Table 3. List of significant genes (FDR-adjusted p-value < 0.05, and |Log2-FoldChange| > 0.25, detected in not less than 10% of cells) identified from the comparison Liver vs Colon within the inflammatory or immunoregulatory TAMs. In green are indicated the genes up-regulated in the liver, while in orange are indicated the colon-associated ones. Table related to Fig. 5A.

Supplementary Figure 5. Differences between inflammatory and immunoregulatory TAMs in colon and liver

Supplementary Figure 2. Transcriptomic characterization of healthy liver and Kupffer cells

Supplementary Figure 4. Pseudotime trajectory analysis shows macrophage blood derivation

Supplementary Table S2: List of genes used to compute the gene module scores related to the antigen-presentation (n genes = 24), lipid-associated (n genes = 32), angiogenic (n genes = 31), and cytotoxic-inflammatory (n genes = 21) macrophage scores. Table related to Fig. 2E, 3E-G, supplementary figure 2F, and supplementary figure 3E.

Supplementary Figure 1. scRNA-sequencing analysis of early phases synchronous CRLM patients