Precision oncology approaches for patients with colorectal cancer (CRC) continue to lag behind other solid cancers. Functional precision oncology—a strategy that is based on perturbing primary... Show morePrecision oncology approaches for patients with colorectal cancer (CRC) continue to lag behind other solid cancers. Functional precision oncology—a strategy that is based on perturbing primary tumor cells from cancer patients—could provide a road forward to personalize treatment. We extend this paradigm to measuring proteome activity landscapes by acquiring quantitative phosphoproteomic data from patient-derived organoids (PDOs). We show that kinase inhibitors induce inhibitor- and patient-specific off-target effects and pathway crosstalk. Reconstruction of the kinase networks revealed that the signaling rewiring is modestly affected by mutations. We show non-genetic heterogeneity of the PDOs and upregulation of stemness and differentiation genes by kinase inhibitors. Using imaging mass-cytometry-based profiling of the primary tumors, we characterize the tumor microenvironment (TME) and determine spatial heterocellular crosstalk and tumor-immune cell interactions. Collectively, we provide a framework for inferring tumor cell intrinsic signaling and external signaling from the TME to inform precision (immuno-) oncology in CRC. Show less
Sambri, I.; Ferniani, M.; Campostrini, G.; Testa, M.; Meraviglia, V.; Araujo, M.E.G. de; ... ; Ballabio, A. 2023
Heterozygous mutations in the gene encoding RagD GTPase were shown to cause a novel autosomal dominant condition characterized by kidney tubulopathy and cardiomyopathy. We previously demonstrated... Show moreHeterozygous mutations in the gene encoding RagD GTPase were shown to cause a novel autosomal dominant condition characterized by kidney tubulopathy and cardiomyopathy. We previously demonstrated that RagD, and its paralogue RagC, mediate a non-canonical mTORC1 signaling pathway that inhibits the activity of TFEB and TFE3, transcription factors of the MiT/TFE family and master regulators of lysosomal biogenesis and autophagy. Here we show that RagD mutations causing kidney tubulopathy and cardiomyopathy are "auto- activating", even in the absence of Folliculin, the GAP responsible for RagC/D activation, and cause constitutive phosphorylation of TFEB and TFE3 by mTORC1, without affecting the phosphorylation of "canonical" mTORC1 substrates, such as S6K. By using HeLa and HK-2 cell lines, human induced pluripotent stem cell-derived cardiomyocytes and patient-derived primary fibroblasts, we show that RRAGD auto-activating mutations lead to inhibition of TFEB and TFE3 nuclear translocation and transcriptional activity, which impairs the response to lysosomal and mitochondrial injury. These data suggest that inhibition of MiT/TFE factors plays a key role in kidney tubulopathy and cardiomyopathy syndrome.Mutations in the RRAGD gene are causative of an autosomal dominant disorder characterized by kidney tubulopathy and cardiomyopathy. Here, the authors identify a new RRAGD P88L mutation, demonstrating that all the identified RRAGD mutations inhibit the nuclear translocation of MiT/TFE transcription factors, resulting in defective responses to lysosomal or mitochondrial damage. Show less
