Bianchi, Giada

Introduction: Autosomal-dominant tubulointerstitial kidney disease (ADTKD) is characterized by chronic kidney disease (CKD) with an average age of end-stage renal disease (ESRD) of approximately 45 years, bland urinary sediment, the absence of proteinuria and autosomal dominant inheritance. While several causative genes have been found, there remain families in whom no molecular diagnosis has been identified (ADTKD-NMD). Methods: We identified BICC1 truncating variants in several families with ADTKD-NMD in the Wake Forest Rare Inherited Kidney Disease Registry and then screened families in our database and other referred families for BICC1 truncating variants. We performed segregation analysis and characterized affected individuals for clinical and histopathologic phenotypes. We analyzed oligomer formation of BICC1 mutants with wild-type BICC1-, ANKS3- and ANKS6 proteins through co-immunoprecipitation and Western blotting, and we tested for posttranscriptional regulation of the BICC1 target mRNA, Dand5, in a Luciferase reporter assay. Results: We found 6 heterozygous truncating mutations in BICC1 segregating with the ADTKD phenotype in 8 independent pedigrees worldwide. Affected individuals developed kidney failure in the 6th to 7th decade of life that was characterized pathologically by tubular atrophy and interstitial fibrosis. The truncated gene products localized to cytoplasmic bodies and demonstrated various degrees of self-association or binding to the known interaction partners, ANKS3 and ANKS6. While the wild-type BICC1 gene product acts as a posttranscriptional repressor of target mRNAs, all truncation variants exhibited increased expression of substrate mRNA. Conclusions: Truncating variants in BICC1 are a novel cause of ADTKD, segregating with the disease phenotype and upregulating BICC1 target gene expression through a dominant-negative- or a gain-of-function mode of action.

Abstract Multiple myeloma (MM) cells suffer from baseline proteotoxicity as the result of an imbalance between the load of misfolded proteins awaiting proteolysis and the capacity of the ubiquitin-proteasome system to degrade them. This intrinsic vulnerability is at the base of MM sensitivity to agents that perturb proteostasis, such as proteasome inhibitors (PIs), the mainstay of modern-day myeloma therapy. De novo and acquired PI resistance are important clinical limitations that adversely affect prognosis. The molecular mechanisms underpinning PI resistance are only partially understood, limiting the development of drugs that can overcome it. The transcription factor NRF1 is activated by the aspartic protease DNA damage inducible 1 homolog 2 (DDI2) upon proteasome insufficiency and governs proteasome biogenesis. In this article, we show that MM cells exhibit baseline NRF1 activation and are dependent upon DDI2 for survival. DDI2 knockout (KO) is cytotoxic for MM cells, both in vitro and in vivo. Protein structure-function studies show that DDI2 KO blocks NRF1 cleavage and nuclear translocation, causing impaired proteasome activity recovery upon irreversible proteasome inhibition and, thereby, increasing sensitivity to PIs. Add-back of wild-type, but not of catalytically dead DDI2, fully rescues these phenotypes. We propose that DDI2 is an unexplored promising molecular target in MM by disrupting the proteasome stress response and exacerbating proteotoxicity.

Abstract The bone marrow (BM) microenvironment actively promotes multiple myeloma pathogenesis, and therapies targeting both cancer cells and the niche are highly effective. We were interested in identifying novel signaling pathways supporting multiple myeloma–BM cross-talk. Mutations in the transmembrane receptor Roundabout 1 (ROBO1) were recently identified in patients with multiple myeloma; however, their functional consequences are uncertain. Through protein structure–function studies, we discovered that ROBO1 is necessary for multiple myeloma adhesion to BM stromal and endothelial cells and that ROBO1 knockout (KO) compromises BM homing and engraftment in a disseminated mouse model. ROBO1 KO significantly decreases multiple myeloma proliferation in vitro and intra- and extramedullary tumor growth in vivo. Mechanistically, the ROBO1 C-terminus is cleaved in a ligand-independent fashion and is sufficient to promote multiple myeloma proliferation. Vice versa, mutants lacking the cytoplasmic domain, including the human-derived G674* truncation, act dominantly negative. Interactomic and RNA-sequencing studies suggest that ROBO1 may be involved in RNA processing, supporting further studies. Significance: ROBO1 is highly expressed in t(4;14) multiple myeloma and supports homing and dissemination to the BM niche. ROBO1 knockout causes reduced tumor growth in intramedullary and extramedullary myeloma animal models, while the ROBO1 C-terminus is cleaved in multiple fragments and it is necessary and sufficient to sustain myeloma proliferation.