Biology subjects

Klima, R.

Publications and source records attributed to Klima, R..

7 recordsLinked to original sources

bioRxiv2026

Targeting hypoxia-responsive miR-155-5p and miR-210-3p restores alveolar regeneration and reverses pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an age-associated degenerative disease largely driven by failure of alveolar epithelial regeneration, yet current therapies slow fibrosis progression without restoring epithelial repair. Building on our previous unbiased microRNA screen in primary murine alveolar type II (ATII) cells, we identified miR-155-5p and miR-210-3p as previously unrecognized mediators of alveolar epithelial regenerative failure. Both microRNAs were markedly upregulated in ATII cells from patients with IPF and enriched within KRT17+/KRT5- aberrant transitional epithelial cells. Their expression also increased spontaneously with ageing in ATII cells from uninjured mice, linking these microRNAs to the age-dependent loss of ATII to ATI transdifferentiation capacity. Furthermore, we found that hypoxia-induced HIF signalling drives the expression of these microRNAs in ATII cells, locking them in a dysfunctional transitional state characterized by a profibrotic secretome that promotes paracrine myofibroblasts activation. Antisense oligonucleotide (ASO) inhibition of selected miRNAs restored ATII-to-ATI differentiation, eliminated aberrant transitional states, normalized epithelial-mesenchymal communication, promoted de novo alveolar regeneration and reversed fibrosis, including in both young and aged mice. Together, these findings identify the hypoxia-responsive miRNAs miR-155-5p and miR-210-3p as therapeutically actionable regulators of alveolar regenerative failure and establish their inhibition as a strategy to restore endogenous lung repair while disrupting pathological epithelial-mesenchymal crosstalk in pulmonary fibrosis.

cell biology↗

bioRxiv2026

AAV-miR-124 enhances endogenous alveolar epithelial regenerative plasticity and reverses bleomycin-induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by irreversible destruction of the alveolar epithelium and impaired regeneration. Although current therapies slow disease progression, they do not restore functional alveoli, highlighting the need for regenerative approaches that promote endogenous lung repair. Here, we performed the first unbiased functional screen of 2,042 human microRNA mimics in primary mouse alveolar type II (ATII) cells to identify regulators of ATII-to-alveolar type I (ATI) cell transdifferentiation. The screen identified miR-124-3p as the most effective promoter of ATI differentiation. In vitro, miR-124-3p promoted ATII-to-ATI transdifferentiation in healthy and bleomycin-injured ATII cells while also increasing the ATII cell pool, consistent with activity on epithelial progenitors. Using the engineered AAV6.2FF capsid, we generated a vector encoding miR-124-3p, which efficiently transduced ATII cells, MHC-II club distal progenitor cells, and injury-induced KRT8 epithelial intermediates. Therapeutic administration after fibrosis establishment reduced lung fibrosis, restored alveolar architecture, and showed greater efficacy than nintedanib in the bleomycin mouse model. Mechanistically, we propose a context-dependent model whereby miR-124-3p regulates epithelial cell states through the EZH2-C/EBP axis while attenuating epithelial transcriptional programs associated with IPF. Together, these findings support AAV-mediated delivery of miR-124 to promote alveolar repair in pulmonary fibrosis.

cell biology↗

bioRxiv2026

Molecular Profiling and High-Content Drug Screening of Metastatic Colorectal Cancer Organoids Reveal Evolutionary Mechanisms of Pan-KRAS Inhibitor Resistance

Metastatic colorectal cancer exhibits extensive intertumoral and intratumoral heterogeneity, which underlies variable treatment responses and the inevitable emergence of drug resistance. Preclinical models that accurately reflect this heterogeneity and enable the investigation of resistance mechanisms at the clonal level, while also providing sensitive and quantitative measurements of treatment response, remain limited. Here, we describe a living biobank of patient-derived organoids (PDOs), generated from metastatic colorectal cancer samples encompassing various genetic backgrounds, including different KRAS genotypes. These PDOs retain key phenotypic, genomic, and transcriptional features of their matched tumors, and, when coupled with a multiparametric high-content 3D drug-screening platform integrating volumetric growth, nuclear content, and proliferative readouts, enable sensitive detection of subtle responses that are not captured by conventional viability-based assays. Systematic phenotypic profiling identified KRAS mutational status as a major determinant of drug response, with KRAS wild-type organoids exhibiting predominant cytotoxic collapse, whereas KRAS-mutant PDOs displayed cytostatic growth arrest. Building on this phenotypic stratification, we investigated resistance to the pan-KRAS inhibitor BI-2865 by integrating longitudinal drug exposure with expressible DNA barcoding and single-cell RNA sequencing to resolve clonal and transcriptional dynamics. Resistance to BI-2865 arose primarily through transcriptional plasticity rather than widespread genomic alterations. Lineage tracing revealed genotype-dependent trajectories, with de novo emergent clones driving resistance in KRASWT PDO, whereas KRASG13D models were dominated by expansion of pre-existing clones. Single-cell analysis on KRASG13D demonstrated a shared transcriptional landscape comprising multiple resistant states rather than a single convergent identity. Together, our findings define resistance to pan-KRAS inhibition as a modular, state-resolved process driven by transcriptional plasticity and genotype-dependent clonal dynamics and establish PDO-based multiparametric phenotyping combined with lineage-resolved single-cell profiling as a robust framework for dissecting the evolutionary framework of therapeutic resistance in metastatic colorectal cancer.

cancer biology↗

bioRxiv2024

Drug-repurposing screen identifies thiostrepton as a novel regulator of the tumor suppressor DAB2IP

The tumor suppressor DAB2IP, a RasGAP and cytoplasmic adaptor protein, modulates signal transduction in response to several extracellular stimuli, negatively regulating multiple oncogenic pathways. Accordingly, the loss of DAB2IP in tumor cells fosters metastasis and enhances chemo- and radio-resistance. DAB2IP is rarely mutated in cancer but is frequently downregulated or inactivated by multiple mechanisms. Solid experimental evidence show that DAB2IP reactivation can reduce cancer aggressiveness in tumors driven by multiple different oncogenic mutations, making this protein an interesting target for anti-cancer therapy. Based on these premises, we screened a library of FDA-approved drugs to search for molecules that can increase DAB2IP protein levels. We exploited CRISPR/Cas9 gene editing to generate two prostate cancer cell models in which endogenous DAB2IP is fused to HiBiT, a peptide tag that enables luminescence-based detection of protein levels in a sensitive and quantitative manner. Using this approach, we identified drugs able to increase DAB2IP levels. We focus our attention on thiostrepton, a natural cyclic oligopeptide antibiotic that has been reported to inhibit survival of various cancer cell lines. Functional experiments revealed that the cancer inhibitory effect of thiostrepton is reduced in the absence of DAB2IP, suggesting that the observed upregulation contributes to its action. These findings encourage the further development of thiostrepton for the treatment of solid cancers, and unveil a novel molecular mechanism underlying its anti-tumoral action.

cancer biology↗

bioRxiv2021

TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila brains

Alterations in the function of the RNA-binding protein TDP-43 is largely associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease of the human motor system that leads to motoneurons degeneration and reduced life expectancy by molecular mechanisms not well known. Regarding to that, we found that the expression levels of the glutamic acid decarboxylase enzyme (GAD1), responsible to convert glutamate to {gamma}-aminobutyric acid (GABA), were downregulated in TBPH-null flies and motoneurons derived from ALS patients carrying mutations in TDP-43 suggesting that defects in the regulation of GAD1 may lead to neurodegeneration by affecting neurotransmitter balance. In this study, we observed that TBPH was required to regulate GAD1 pre-mRNA splicing and GABA levels in Drosophila brains. Interestingly, we discovered that pharmacological treatments aimed to modulate GABA neurotransmission were able to revert locomotion deficiencies in TBPH-minus flies revealing novel mechanisms and therapeutic strategies in ALS.

neuroscience↗

bioRxiv2019

TDP-43 regulates the expression levels of Disc-large in skeletal muscles to promote the assemble of the neuromuscular synapses in Drosophila

BackgroundAlterations in the intracellular distribution of TDP-43 were observed in the skeletal muscles of patients suffering from ALS. However, it is not clear whether these modifications play an active role in the disease or represent a physiological adaptation to muscles homeostasis.\n\nResultTo answer these questions, we modulated the activity of this protein in Drosophila muscles and observed that TDP-43 was required in these tissues to promote the formation and growth of the neuromuscular synapses. Moreover, we identified that TDP-43 regulates the expression levels of Disc-large (Dlg) and demonstrated that the modulation of Dlg activity, in skeletal muscles or motoneurons, was sufficient to recover the TDP-43-null locomotive and synaptic defects in flies. Additionally, we found that similar mechanisms are conserved in human cell lines and present in tissues derived from ALS patients.\n\nConclusionsOur results uncover the physiological role of TDP-43 in skeletal muscles as well as the mechanisms behind the autonomous and non-autonomous behavior of this protein in the organization of the neuromuscular synapses.

neuroscience↗

bioRxiv2019

Endogenous TDP-43 prevents retrotransposons activation through Dicer-2 activity and the RNA silencing machinery in Drosophila

The aberrant expression of retrotransposable elements (RTEs) was observed in different neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a terminal disorder characterized by functional alterations in the small RNA-binding protein TDP-43, suggesting that these events might be connected. Using genome wide gene expression profiles, we detected RTEs highly upregulated in TDP-43-null Drosophila heads while, the genetic rescue of TDP-43 function reverted these modifications. Furthermore, we found that TDP-43 modulates the small interfering RNA (siRNA) silencing machinery responsible for RTEs repression. Molecularly, we observed that TDP-43 regulates the expression levels of Dicer-2 by direct protein-mRNA interactions in vivo. Accordingly, the genetic or pharmacological recovery of Dicer-2 activity was sufficient to repress retrotransposons activation and revert the neurodegeneration in TDP-43-null Drosophila motoneurons. Our results, describe a novel physiological role of endogenous TDP-43 in the prevention of RTEs-induced neurodegeneration through the modulation of Dicer-2 activity and the siRNA pathway.

cell biology↗