Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c+ dendritic cells.

Inflammasomes are multiprotein platforms responsible for the release of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18. Mouse studies have identified inflammasome activation within dendritic cells (DC) as pivotal for driving tubulointerstitial fibrosis and inflammation, the hallmarks of chronic kidney disease (CKD). However, translation of this work to human CKD remains limited. Here, we examined the complex tubular cell death pathways mediating inflammasome activation in human kidney DC and, thus, CKD progression. Ex vivo patient-derived proximal tubular epithelial cells (PTEC) cultured under hypoxic (1% O2) conditions modelling the CKD microenvironment showed characteristics of ferroptotic cell death, including mitochondrial dysfunction, reductions in the lipid repair enzyme glutathione peroxidase 4 (GPX4) and increases in lipid peroxidation by-product 4-hydroxynonenal (4-HNE) compared with normoxic PTEC. The addition of ferroptosis inhibitor, ferrostatin-1, significantly reduced hypoxic PTEC death. Human CD1c+ DC activated in the presence of hypoxic PTEC displayed significantly increased production of inflammasome-dependent cytokines IL-1ß and IL-18. Treatment of co-cultures with VX-765 (caspase-1/4 inhibitor) and MCC950 (NLRP3 inflammasome inhibitor) significantly attenuated IL-1ß/IL-18 levels, supporting an NLRP3 inflammasome-dependent DC response. In line with these in vitro findings, in situ immunolabelling of human fibrotic kidney tissue revealed a significant accumulation of tubulointerstitial CD1c+ DC containing active inflammasome (ASC) specks adjacent to ferroptotic PTEC. These data establish ferroptosis as the primary pattern of PTEC necrosis under the hypoxic conditions of CKD. Moreover, this study identifies NLRP3 inflammasome signalling driven by complex tubulointerstitial PTEC-DC interactions as a key checkpoint for therapeutic targeting in human CKD.

Adenine overload induces ferroptosis in human primary proximal tubular epithelial cells.

The pathogenesis of crystal nephropathy involves deposition of intratubular crystals, tubular obstruction and cell death. The deposition of 8-dihydroxyadenine (DHA) crystals within kidney tubules, for instance, is caused by a hereditary deficiency of adenine phosphoribosyl transferase in humans or adenine overload in preclinical models. However, the downstream pathobiological patterns of tubular cell attrition in adenine/DHA-induced nephropathy remain poorly understood. In this study, we investigated: (i) the modes of adenine-induced tubular cell death in an experimental rat model and in human primary proximal tubular epithelial cells (PTEC); and (ii) the therapeutic effect of the flavonoid baicalein as a novel cell death inhibitor. In a rat model of adenine diet-induced crystal nephropathy, significantly elevated levels of tubular iron deposition and lipid peroxidation (4-hydroxynonenal; 4-HNE) were detected. This phenotype is indicative of ferroptosis, a novel form of regulated necrosis. In cultures of human primary PTEC, adenine overload-induced significantly increased mitochondrial superoxide levels, mitochondrial depolarisation, DNA damage and necrotic cell death compared with untreated PTEC. Molecular interrogation of adenine-stimulated PTEC revealed a significant reduction in the lipid repair enzyme glutathione peroxidase 4 (GPX4) and the significant increase in 4-HNE compared with untreated PTEC, supporting the concept of ferroptotic cell death. Moreover, baicalein treatment inhibited ferroptosis in adenine-stimulated PTEC by selectively modulating the mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) and thus, suppressing mitochondrial superoxide production and DNA damage. These data identify ferroptosis as the primary pattern of PTEC necrosis in adenine-induced nephropathy and establish baicalein as a potential therapeutic tool for the clinical management of ferroptosis-associated crystal nephropathies (e.g., DHA nephropathy, oxalate nephropathy).

Cep55 overexpression promotes genomic instability and tumorigenesis in mice.

High expression of centrosomal protein CEP55 has been correlated with clinico-pathological parameters across multiple human cancers. Despite significant in vitro studies and association of aberrantly overexpressed CEP55 with worse prognosis, its causal role in vivo tumorigenesis remains elusive. Here, using a ubiquitously overexpressing transgenic mouse model, we show that Cep55 overexpression causes spontaneous tumorigenesis and accelerates Trp53+/- induced tumours in vivo. At the cellular level, using mouse embryonic fibroblasts (MEFs), we demonstrate that Cep55 overexpression induces proliferation advantage by modulating multiple cellular signalling networks including the hyperactivation of the Pi3k/Akt pathway. Notably, Cep55 overexpressing MEFs have a compromised Chk1-dependent S-phase checkpoint, causing increased replication speed and DNA damage, resulting in a prolonged aberrant mitotic division. Importantly, this phenotype was rescued by pharmacological inhibition of Pi3k/Akt or expression of mutant Chk1 (S280A) protein, which is insensitive to regulation by active Akt, in Cep55 overexpressing MEFs. Moreover, we report that Cep55 overexpression causes stabilized microtubules. Collectively, our data demonstrates causative effects of deregulated Cep55 on genome stability and tumorigenesis which have potential implications for tumour initiation and therapy development.

CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer.

Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.

Underlying Histopathology Determines Response to Oxidative Stress in Cultured Human Primary Proximal Tubular Epithelial Cells.

Proximal tubular epithelial cells (PTEC) are key players in the progression of kidney diseases. PTEC studies to date have primarily used mouse models and transformed human PTEC lines. However, the translatability of these models to human kidney disease has been questioned. In this study, we investigated the phenotypic and functional response of human primary PTEC to oxidative stress, an established driver of kidney disease. Furthermore, we examined the functional contribution of the underlying histopathology of the cortical tissue used to generate our PTEC. We demonstrated that human primary PTEC from both histologically 'normal' and 'diseased' cortical tissue responded to H2O2-induced oxidative stress with significantly elevated mitochondrial superoxide levels, DNA damage, and significantly decreased proliferation. The functional response of 'normal' PTEC to oxidative stress mirrored the reported pathogenesis of human kidney disease, with significantly attenuated mitochondrial function and increased cell death. In contrast, 'diseased' PTEC were functionally resistant to oxidative stress, with maintenance of mitochondrial function and cell viability. This selective survival of 'diseased' PTEC under oxidizing conditions is reminiscent of the in vivo persistence of maladaptive PTEC following kidney injury. We are now exploring the impact that these differential PTEC responses have in the therapeutic targeting of oxidative stress pathways.

Blockade of PDGFRß circumvents resistance to MEK-JAK inhibition via intratumoral CD8+ T-cells infiltration in triple-negative breast cancer.

BACKGROUND: Despite the increasing progress in targeted and immune based-directed therapies for other solid organ malignancies, currently there is no targeted therapy available for TNBCs. A number of mechanisms have been reported both in pre-clinical and clinical settings that involve inherent, acquired and adaptive resistance to small molecule inhibitors. Here, we demonstrated a novel resistance mechanism in TNBC cells mediated by PDGFRß in response to JAK2 inhibition. METHODS: Multiple in vitro (subG1, western blotting, immunofluorescence, RT-PCR, Immunoprecipitation), in vivo and publically available datasets were used. RESULTS: We showed that TNBC cells exposed to MEK1/2-JAK2 inhibitors exhibit resistant colonies in anchorage-independent growth assays. Moreover, cells treated with various small molecule inhibitors including JAK2 promote PDGFRß upregulation. Using publically available databases, we showed that patients expressing high PDGFRß or its ligand PDGFB exhibit poor relapse-free survival upon chemotherapeutic treatment. Mechanistically we found that JAK2 expression controls steady state levels of PDGFRß. Thus, co-blockade of PDGFRß with JAK2 and MEK1/2 inhibitors completely eradicated resistant colonies in vitro. We found that triple-combined treatment had a significant impact on CD44+/CD24- stem-cell-like cells. Likewise, we found a significant tumor growth inhibition in vivo through intratumoral CD8+ T cells infiltration in a manner that is reversed by anti-CD8 antibody treatment. CONCLUSION: These findings reveal a novel regulatory role of JAK2-mediated PDGFRß proteolysis and provide an example of a PDGFRß-mediated resistance mechanism upon specific target inhibition in TNBC.

The 2-µm plasmid encoded protein Raf1 regulates both stability and copy number of the plasmid by blocking the formation of the Rep1-Rep2 repressor complex.

The 2-µm plasmid of the budding yeast Saccharomyces cerevisiae achieves a high chromosome-like stability with the help of four plasmid-encoded (Rep1, Rep2, Raf1 and Flp) and several host-encoded proteins. Rep1 and Rep2 and the DNA locus STB form the partitioning system ensuring equal segregation of the plasmid. The Flp recombinase and its target sites FRTs form the amplification system which is responsible for the steady state plasmid copy number. In this work we show that the absence of Raf1 can affect both the plasmid stability and the steady sate copy number. We also show that the Rep proteins do bind to the promoter regions of the 2-µm encoded genes, as predicted by earlier models and Raf1 indeed blocks the formation of the Rep1-Rep2 repressor complex not by blocking the transcription of the REP1 and REP2 genes but by physically associating with the Rep proteins and negating their interactions. This explains the role of Raf1 in both the partitioning and the amplification systems as the Rep1-Rep2 complex is believed to modulate both these systems. Based on this study, we have provided, from a systems biology perspective, a model for the mechanism of the 2-µm plasmid maintenance.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.

Enhanced dependency of KRAS-mutant colorectal cancer cells on RAD51-dependent homologous recombination repair identified from genetic interactions in Saccharomyces cerevisiae.

Activating KRAS mutations drive colorectal cancer tumorigenesis and influence response to anti-EGFR-targeted therapy. Despite recent advances in understanding Ras signaling biology and the revolution in therapies for melanoma using BRAF inhibitors, no targeted agents have been effective in KRAS-mutant cancers, mainly due to activation of compensatory pathways. Here, by leveraging the largest synthetic lethal genetic interactome in yeast, we identify that KRAS-mutated colorectal cancer cells have augmented homologous recombination repair (HRR) signaling. We found that KRAS mutation resulted in slowing and stalling of the replication fork and accumulation of DNA damage. Moreover, we found that KRAS-mutant HCT116 cells have an increase in MYC-mediated RAD51 expression with a corresponding increase in RAD51 recruitment to irradiation-induced DNA double-strand breaks (DSBs) compared to genetically complemented isogenic cells. MYC depletion using RNA interference significantly reduced IR-induced RAD51 foci formation and HRR. On the contrary, overexpression of either HA-tagged wild-type (WT) MYC or phospho-mutant S62A increased RAD51 protein levels and hence IR-induced RAD51 foci. Likewise, depletion of RAD51 selectively induced apoptosis in HCT116-mutant cells by increasing DSBs. Pharmacological inhibition targeting HRR signaling combined with PARP inhibition selectivity killed KRAS-mutant cells. Interestingly, these differences were not seen in a second isogenic pair of KRAS WT and mutant cells (DLD-1), likely due to their nondependency on the KRAS mutation for survival. Our data thus highlight a possible mechanism by which KRAS-mutant-dependent cells drive HRR in vitro by upregulating MYC-RAD51 expression. These data may offer a promising therapeutic vulnerability in colorectal cancer cells harboring otherwise nondruggable KRAS mutations, which warrants further investigation in vivo.