Wild-type Growth Research Articles

Imlunestrant is an Oral, Brain-Penetrant Selective Estrogen Receptor Degrader with Potent Antitumor Activity in ESR1 Wildtype and Mutant Breast Cancer.

Targeting of the estrogen receptor (ER) by anti-estrogens is the standard-of-care for patients with ER+ HER2- advanced/metastatic breast cancer. While anti-estrogens that degrade ERα (fulvestrant) or block estrogen production (aromatase inhibitors) have improved patient outcomes, clinically important challenges remain related to drug administration, limited bioavailability, lack of brain exposure, and acquired resistance due to ESR1 mutations. These limitations indicate a need for more robust ER-targeted therapies. Here, we discovered and characterized imlunestrant, a next-generation potent, brain-penetrant oral selective estrogen receptor degrader (SERD). Imlunestrant degraded ERα and decreased ERα-mediated gene expression both in vitro and in vivo. Cell proliferation and tumor growth in ESR1 wildtype and mutant models were significantly inhibited by imlunestrant. Combining imlunestrant with abemaciclib (CDK4/6 inhibitor), alpelisib (PI3K inhibitor), or everolimus (mTOR inhibitor) further enhanced tumor growth inhibition, regardless of ESR1 mutational status. In an ER+ breast cancer intracranial tumor model, imlunestrant prolonged survival compared to vehicle or alternative SERD therapies. Together, these finding support the potential of imlunestrant to degrade ERα and suppress the growth of ESR1 wildtype and mutant breast cancer, including brain metastatic tumors.

Vibrational spectroscopic characterization, quantum chemical, molecular docking and molecular dynamics investigations of cyclo(L-phenylalanyl-L-proline), an anticancer agent

The molecular structure and spectral properties of cyclo(L-Phenylalanyl-L-Proline) dipeptide, which has several biological activities, were investigated. Firstly, conformational preference of the cyclo(L-Phenylalanyl-L-Proline) was searched and obtained lowest energy conformer of the cyclic dipeptide was then optimized using Density Functional Theory with wb97xd/6-311++G(d,p) level of theory. A detailed vibrational spectral analysis has been carried out and assignments of the fundamental modes have been proposed. The experimental vibrational wavenumbers of the investigated compound display good agreement with the computed values. The Frontier Molecular Orbitals, Molecular Electrostatic Potential and electronic transitions of the investigated compound were discussed. In addition, the 1H and 13C NMR chemical shifts of the cyclic dipeptide were calculated and the results were compared with the experimental values. Also, to evaluate the anticancer potential, molecular docking studies of cyclo(L-Phenylalanyl-L-Proline) within the ATP-binding sites of both wild type (EGFRWT; ID: 4HJO) and mutant (EGFRT790M; ID: 3W2O) Epidermal Growth Factor Receptor were performed. The results indicated that cyclo(Phe-Pro) has high binding affinities toward both EGFRWT (−6.6 kcal/mol) and EGFRT790M (−7.7 kcal/mol) receptors, thus, has good anticancer activity and also has potential to overcome drug resistance in therapy. Molecular dynamics simulations on cyclo(L-Phenylalanyl-L-Proline)-wild type complex were conducted through a 50-nanosecond timed to investigate the ligand-receptor interactions in more detail, and to determine the binding free energy accurately. The binding free energy of the cyclo(L-Phenylalanyl-L-Proline)-wild type complex was calculated to be −27.18 kcal/mol.

Dairy liquid waste as substrate to obtain biotechnological chondroitin: A circular economy approach

Microbial polysaccharides have been gaining growing interest often as alternative to animal derived products or as sources of novel features for biotechnological applications. Process production costs, however, are still high. A possible solution to that exploits agri-food and dairy industrial byproducts as fermentation substrates. This approach also reduces the need for cost-intensive disposal treatments for these waste sources and supports green and circular economy policies. Therefore, as for other microbial glucuronic acid-based biopolymers (e.g. hyaluronic acid, alginate), in this perspective, wild type and engineered E. coli K4 were used in this work as cell factories to produce K4 capsular polysaccharide (CPS) from renewable sources. The backbone of the K4 CPS, chondroitin, is the precursor of chondroitin sulfate (CS), a glycosaminoglycan found in animal tissues that is extensively used for curing osteoarthritis and studied for several other emerging biomedical applications. Interestingly, also chondroitin showed promising bioactivity in vitro and in vivo. Due to its high availability from local companies, second cheese whey (SCW), a worldwide copious and polluting liquid waste, was used as fermentation substrate in this work. Results showed that SCW fully supports growth of wild type and recombinant E. coli K4 strains, and demonstrate, for the first time up to date, the production of K4 CPS from liquid waste as proof of principle. Batch processes in 3 L fermenters indicated a 100 % improvement of the polysaccharide yield and allowed the production of 1.1±0.1 g/L of product from the recombinant strain with very low accumulation of acetic acid, demonstrating that SCW by itself fully supports polysaccharide production.

AZD1775 synergizes with SLC7A11 inhibition to promote ferroptosis.

Tumor suppressor p53-mediated cell cycle arrest and DNA damage repair may exert cytoprotective effects against cancer therapies, including WEE1 inhibition. Considering that p53 activation can also lead to multiple types of cell death, the role of this tumor suppressor in WEE1 inhibitor-based therapies remains disputed. In this study, we reported that nucleolar stress-mediated p53 activation enhanced the WEE1 inhibitor AZD1775-induced ferroptosis to suppress lung cancer growth. Our findings showed that AZD1775 promoted ferroptosis by blocking cystine uptake, an action similar to that of Erastin. Meanwhile, inhibition of WEE1 by the WEE1 inhibitors or siRNAs induced compensatory upregulation of SLC7A11, which conferred resistance to ferroptosis. Mechanistically, AZD1775 prevented the enrichment of H3K9me3, a histone marker of transcriptional repression, on the SLC7A11 promoter by repressing the expression of the histone methyltransferase SETDB1, thereby enhancing NRF2-mediated SLC7A11 transcription. This finding was also validated using the H3K9me3 inhibitor BRD4770. Remarkably, we found that the nucleolar stress-inducing agent Actinomycin D (Act. D) inhibited SLC7A11 expression by activating p53, thus augmenting AZD1775-induced ferroptosis. Moreover, the combination of AZD1775 and Act. D synergistically suppressed wild-type p53-harboring lung cancer cell growth both in vitro and in vivo. Altogether, our study demonstrates that AZD1775 promotes ferroptosis by targeting cystine uptake but also mediates the adaptive activation of SLC7A11 through the WEE1-SETDB1 cascade and NRF2-induced transcription, and inhibition of SLC7A11 by Act. D boosts the anti-tumor efficacy of AZD1775 by enhancing ferroptosis in cancers with wild-type p53.

Calcium Rescues Streptococcus pneumoniae D39 ΔmntE Manganese-Sensitive Growth Phenotype.

Calcium (Ca2+) functions as a universal signal messenger in eukaryotes but in bacteria, the physiological roles for Ca2+ are limited. Here, we examine the role of Ca2+ in Streptococcus pneumoniae during manganese (Mn2+) intoxication. S. pneumoniae mntE mutants, lacking the Mn2+ efflux transporter, exhibit impaired growth due to accumulation of Mn2+ when exposed to elevated exogenous Mn2+. This Mn2+-sensitive growth defect is restored to wild-type growth level by exogenous Ca2+, in a Ca2+-dependent manner. Despite growth restoration of the mntE mutant to wild-type levels, cellular Mn2+ remains elevated in this strain. Bacterial capsule production is also increased for the mntE mutant, resulting in reduced adherence capacity to surfaces and poor biofilm formation, which is consistent with it experiencing Mn2+ intoxication. Ca2+ presence did not significantly impact bacterial capsule production or biofilm formation. Further analysis of the cell morphology demonstrates that Ca2+ contributes to cell division and reduces cell chain lengths. Together, these data describe the first role of Ca in S. pneumoniae that has potential implications in bacterial virulence since Ca affects cell division and likely Mn2+-associated cellular processes.

Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells.

The Warburg effect is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production, as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production via knockout have failed in mammalian bioprocessing since lactate dehydrogenase has proven essential. However, here we eliminated the Warburg effect in Chinese hamster ovary (CHO) and HEK293 cells by simultaneously knocking out lactate dehydrogenase and regulators involved in a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. In contrast to long-standing assumptions about the role of aerobic glycolysis, Warburg-null cells maintain wildtype growth rate while producing negligible lactate. Further characterization of Warburg-null CHO cells showed a compensatory increase in oxygen consumption, a near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. These cells remained amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titers and maintaining product glycosylation. Thus, the ability to eliminate the Warburg effect is an important development for biotherapeutic production and provides a tool for investigating a near-universal metabolic phenomenon.

CRP improves the survival and competitive fitness of Salmonella Typhimurium under starvation by controlling the cellular maintenance rate.

Catabolite repression is a mechanism of selectively utilizing preferred nutrient sources by redirecting the metabolic pathways. Therefore, it prevents non-essential energy expenditure by repressing the genes and proteins involved in the metabolism of other less favored nutrient sources. Catabolite repressor protein (CRP) is a chief mediator of catabolite repression in microorganisms. In this context, we investigated the role of CRP in starvation tolerance, at both cell physiology and molecular level, by comparing the growth, survival, competitive fitness, maintenance rate, and gene and protein expression of wild type (WT) and ∆crp of Salmonella Typhimurium, under nutrient-rich and minimal medium condition. The ∆crp shows slow growth upon the arrival of nutrient-limiting conditions, poor survival under prolong-starvation, and inability to compete with its counterpart WT strain in nutrient-rich [Luria broth (LB)] and glucose-supplemented M9 minimal medium. Surprisingly, we observed that the survival and competitive fitness of ∆crp are influenced by the composition of the growth medium. Consequently, compared to the glucose-supplemented M9 medium, ∆crp shows faster death and a higher maintenance rate in the LB medium. The comparative gene and protein expression studies of WT and ∆crp in LB medium show that ∆crp has partial or complete loss of repression from CRP-controlled genes, resulting in a high abundance of hundreds of proteins in ∆crp compared to WT. Subsequently, the addition of metabolizable sugar or fresh nutrients to the competing culture showed extended survival of ∆crp. Therefore, our results suggest that CRP-mediated gene repression improves starvation tolerance and competitive fitness of Salmonella Typhimurium by adapting its cellular maintenance rate to environmental conditions.IMPORTANCESalmonella Typhimurium is a master at adapting to chronic starvation conditions. However, the molecular mechanisms to adapt to such conditions are still unknown. In this context, we have evaluated the role of catabolite repressor protein (CRP), a dual transcriptional regulator, in providing survival and competitive fitness under starvation conditions. Also, it showed an association between CRP and nutrient composition. We observed that Δcrp growing on alternate carbon sources has lower survival and competitive fitness than Δcrp growing on glucose as a carbon source. We observed that this is due to the loss of repression from the glucose and CRP-controlled genes, resulting in elevated cellular metabolism (a high maintenance rate) of the Δcrp during growth in a medium having a carbon source other than glucose (e.g., Luria broth medium).

Therapeutic application of extracellular vesicular EGFR isoform D as a co-drug to target squamous cell cancers with tyrosine kinase inhibitors

Targeting wild-type epidermal growth factor receptor (EGFR) using tyrosine kinase inhibitors (TKIs) never achieved its purported success in cancers such as head and neck squamous cell carcinoma, which are largely EGFR-dependent. We had previously shown that exceptional responders to TKIs have a genetic aberration that results in overexpression of an EGFR splice variant, isoform D (IsoD). IsoD lacks an integral transmembrane and kinase domain and is secreted in extracellular vesicles (EVs) in TKI-sensitive patient-derived cultures. Remarkably, the exquisite sensitivity to TKIs could be transferred to TKI-resistant tumor cells, and IsoD protein in the EV is necessary and sufficient to transfer the phenotype invitro and invivo across multiple models and drugs. This drug response requires an intact endocytic mechanism, binding to full-length EGFR, and signaling through Src-phosphorylation within the endosomal compartment. We propose a therapeutic strategy using EVs containing EGFR IsoD as a co-drug to expand the use of TKI therapy to EGFR-driven cancers.

SIGLEC-3 (CD33) serves as an effective target in tumor progression

Abstract Human CD33 (Siglec-3) possesses an immune-receptor tyrosine-based inhibition motif (ITIM), which recruits Src homology 2 (SH2) containing phosphatases (SHP-1/2) and inositol phosphatase (SHIP) and exerts inhibitory effects. The structure and downstream signaling of CD33 bear similarity to programmed cell death protein-1 (PD-1), a promising target in cancer immune therapy. Previous studies indicate that CD33 serves as a therapeutic target in acute myeloid leukemia (AML), additionally, CD33 also acts as a prevalent surface marker of human myeloid-derived suppressor cells (MDSCs). According to the inhibitory signaling upon CD33 activation, CD33 may participate in tumor progression and the regulatory role of CD33 in solid tumors is still unclear. To investigate whether CD33 influences cancer progression, we established a melanoma tumor (B16F10) mouse model in monocyte/macrophage-specific human CD33 transgenic mice (hCD33 Tg mice, Cx3cr1cre/-hCD33M+/-). Compared to wild-type mice (WT), tumor growth and mortality were larger and higher in CD33 Tg mice. Moreover, an antagonistic anti-hCD33 monoclonal antibody (clone 4L3-46) treatment successfully attenuated tumor growth in hCD33 Tg mice and led to a restructuring of the infiltrating MDSCs population. These results indicate that human CD33 contributes to the advancement of tumors, and the blockade of CD33 represents a promising strategy in tumor therapy.

Quantitative proteomic analysis reveals unique Hsp90 cycle-dependent client interactions.

Hsp90 is an abundant and essential molecular chaperone that mediates the folding and activation of client proteins in a nucleotide-dependent cycle. Hsp90 inhibition directly or indirectly impacts the function of 10-15% of all proteins due to degradation of client proteins or indirect downstream effects. Due to its role in chaperoning oncogenic proteins, Hsp90 is an important drug target. However, compounds that occupy the ATP-binding pocket and broadly inhibit function have not achieved widespread use due to negative effects. More selective inhibitors are needed; however, it is unclear how to achieve selective inhibition. We conducted a quantitative proteomic analysis of soluble proteins in yeast strains expressing wild-type Hsp90 or mutants that disrupt different steps in the client folding pathway. Out of 2,482 proteins in our sample set (approximately 38% of yeast proteins), we observed statistically significant changes in abundance of 350 (14%) of those proteins (log2 fold change ≥ 1.5). Of these, 257/350 (∼73%) with the strongest differences in abundance were previously connected to Hsp90 function. Principal component analysis of the entire dataset revealed that the effects of the mutants could be separated into 3 primary clusters. As evidence that Hsp90 mutants affect different pools of clients, simultaneous co-expression of 2 mutants in different clusters restored wild-type growth. Our data suggest that the ability of Hsp90 to sample a wide range of conformations allows the chaperone to mediate folding of a broad array of clients and that disruption of conformational flexibility results in client defects dependent on those states.

Whole brain morphologic features improve the predictive accuracy of IDH status and VEGF expression levels in gliomas.

Glioma is a systemic disease that can induce micro and macro alternations of whole brain. Isocitrate dehydrogenase and vascular endothelial growth factor are proven prognostic markers and antiangiogenic therapy targets in glioma. The aim of this study was to determine the ability of whole brain morphologic features and radiomics to predict isocitrate dehydrogenase status and vascular endothelial growth factor expression levels. This study recruited 80 glioma patients with isocitrate dehydrogenase wildtype and high vascular endothelial growth factor expression levels, and 102 patients with isocitrate dehydrogenase mutation and low vascular endothelial growth factor expression levels. Virtual brain grafting, combined with Freesurfer, was used to compute morphologic features including cortical thickness, LGI, and subcortical volume in glioma patient. Radiomics features were extracted from multiregional tumor. Pycaret was used to construct the machine learning pipeline. Among the radiomics models, the whole tumor model achieved the best performance (accuracy 0.80, Area Under the Curve 0.86), while, after incorporating whole brain morphologic features, the model had a superior predictive performance (accuracy 0.82, Area Under the Curve 0.88). The features contributed most in predicting model including the right caudate volume, left middle temporal cortical thickness, first-order statistics, shape, and gray-level cooccurrence matrix. Pycaret, based on morphologic features, combined with radiomics, yielded highest accuracy in predicting isocitrate dehydrogenase mutation and vascular endothelial growth factor levels, indicating that morphologic abnormalities induced by glioma were associated with tumor biology.

Abstract 4526: Inhibition of RNA Pol I transcription and induction of nucleolar stress in cancer cells treated with the experimental alpha-adrenergic receptor antagonist JP-1302

Abstract The inhibition of ribosome biogenesis through pharmacological means has drawn attention as a potential strategy in cancer therapy. In our previous study we highlighted a novel property of the FDA-approved antimalarial drug amodiaquine, demonstrating its ability to dose-dependently inhibit ribosomal RNA (rRNA) transcription, a critical step in ribosome biogenesis (Espinoza JA, et al Cell Death Diff, 2020). Amodiaquine was found to induce the degradation of the catalytic subunit of RNA polymerase I, known as POLR1A (RPA194), and resulting in nucleolar stress and the stabilization of p53. Notably, RNA Pol I shutdown occurred independently of DNA damage. Motivated by these findings, we sought to find additional inhibitors of ribosome biogenesis. Acridine derivatives display anti-cancer properties. The activity of acridines is mainly attributed to the planarity of these structures, which can intercalate within the DNA structure, and inhibition of chromatin associated protein targets including topoisomerases. JP-1302 is an experimental drug, on a 9-aminoacridine structural scaffold, acting as a selective alpha-2 adrenergic receptor antagonist. It was recently shown that JP-1302 inhibits transcription, blocking RNA Pol II phosphorylation and inhibiting p21 expression at a concentration of 10 μM (Mitchell DC, et al Nat Biotech, 2023). Given JP-1302's structural similarity to other acridine derivatives such as aminacrine, ethacridine, and the experimental compound BMH-21, all known to inhibit RNA Pol I transcription, we hypothesized that JP-1302 might share similar properties. The purpose of our new study was to evaluate JP-1302´s effect on ribosome biogenesis, p53, and cancer cell growth. We show here that JP-1302 acts as an inhibitor of RNA Pol I transcription. Treatment with JP-1302 induced a rapid decrease in the levels of POLR1A, cessation of rRNA synthesis and nucleolar stress already within six hours and at a concentration of 0.5 μM. JP-1302 treatment stabilized wild type p53 and enhanced p21 expression in the osteosarcoma line U2OS. RNA Pol I shutdown occurred in the absence of DNA damage response, as determined by gH2A.X staining. JP-1302 impaired the growth of both p53 wild type and p53 null HCT116 cells with GI50 values of around 0.6 μM, as well as in U2OS cells at 0.8 μM, as determined after 48 hours. Paradoxically, at very low concentrations JP-1302 appeared to transiently stimulate RNA synthesis, a phenomenon that will require further investigation. In conclusion, we identified JP-1302 as a novel inhibitor of ribosomal RNA synthesis, exerting this effect at concentrations that do not induce DNA damage, and independently of p53. Given that JP-1302 passes the blood-brain barrier in rodents, and accumulating in different brain regions, it is of future interest to evaluate this drug in brain tumor models. Citation Format: Sheetanshu Saproo, Styliani Papadaki, Jaime A. Espinoza, Jiri Bartek, Mikael S. Lindström. Inhibition of RNA Pol I transcription and induction of nucleolar stress in cancer cells treated with the experimental alpha-adrenergic receptor antagonist JP-1302 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4526.

Abstract 5473: Combined inhibition of MDM2 and PARP lead to a synergistic anti-tumoral response in p53 wild-type rhabdomyosarcoma

Abstract Targeting MDM2-p53 interaction to enhance p53 activity represents an attractive antitumoral strategy for p53 wild-type tumors. However, early-phase clinical trials in hematological and solid cancers have shown limited efficacy of MDM2 inhibitors highlighting the necessity for exploring more effective combinations with additional agents. In this study, we provide results from a 22-drug combination screening demonstrating the therapeutic potential of combining Siremadlin (MDM2 inhibitor) and Olaparib (PARP inhibitor) for treating p53 wild-type rhabdomyosarcoma (RMS). Cell survival, cell death and apoptosis analysis revealed synergistic effects when combining Siremadlin and Olaparib in vitro. Combination of both drugs resulted in a significant increase of p53 activity as evidenced by p53 accumulation and K382 acetylation along with an increased expression of p53 targets. Finally, combination of both drugs resulted in significant reduction of tumor growth and increased overall survival outperforming individual treatments and control groups. In summary, our study demonstrates, for the first time, the synergistic effect of the combination between Siremadlin and Olaparib in the inhibition of p53 wild-type RMS tumor growth in vitro and in vivo. These findings advocate for the exploration of this drug combination in clinical trials and underscore the need for further investigation in tumors sharing similar molecular features. Citation Format: Guillem Pons, Gabriel Gallo-Oller, Lia García-Gilabert, Patricia Zarzosa, Júlia Sansa-Girona, Natalia Navarro, Paula Cabré-Fernández, Miguel F Segura, Josep Sánchez de Toledo, Soledad Gallego, Lucas Moreno, Josep Roma. Combined inhibition of MDM2 and PARP lead to a synergistic anti-tumoral response in p53 wild-type rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5473.

N6-isopentenyladenosine inhibits aerobic glycolysis in glioblastoma cells by targeting PKM2 expression and activity.

Glioblastoma (GBM) is a primary tumor in the central nervous system with poor prognosis. It exhibits elevated glucose uptake and lactate production. This metabolic state of aerobic glycolysis is known as the Warburg effect. N6-isopentenyladenosine (iPA), a natural cytokine modified with an isopentenyl moiety derived from the mevalonate pathway, has well-established anti-tumor activity. It inhibits cell proliferation in glioma cells, inducing cell death by apoptosis and/or necroptosis. In the present study, we found that iPA inhibits aerobic glycolysis in unmodified U87MG cells and in the same cell line engineered to over-express wild-type epidermal growth factor receptor (EGFR) or EGFR variant III (vIII), as well as in a primary GBM4 patient-derived cell line. The detection of glycolysis showed that iPA treatment suppressed ATP and lactate production. We also evaluated the response of iPA treatment in normal human astrocyte primary cells, healthy counterpart cells of the brain. Aerobic glycolysis in treated normal human astrocyte cells did not show significant changes compared to GBM cells. To determine the mechanism of iPA action on aerobic glycolysis, we investigated the expression of certain enzymes involved in this metabolic pathway. We observed that iPA reduced the expression of pyruvate kinase M2 (PKM2), which plays a key role in the regulation of aerobic glycolysis, promoting tumor cell proliferation. The reduction of PKM2 expression is a result of the inhibition of the inhibitor of nuclear factor kappa-B kinase subunit, beta/nuclear factor-kappa B pathway upon iPA treatment. In conclusion, these experimental results show that iPA may inhibit aerobic glycolysis of GBM in stabilized cell lines and primary GBM cells by targeting the expression and activity of PKM2.

In a novel autoimmune and high-pressure glaucoma model a complex immune response is induced.

The neurodegenerative processes leading to glaucoma are complex. In addition to elevated intraocular pressure (IOP), an involvement of immunological mechanisms is most likely. In the new multifactorial glaucoma model, a combination of high IOP and optic nerve antigen (ONA) immunization leads to an enhanced loss of retinal ganglion cells accompanied by a higher number of microglia/macrophages in the inner retina. Here, we aimed to evaluate the immune response in this new model, especially the complement activation and the number of T-cells, for the first time. Further, the microglia/macrophage response was examined in more detail. Six-week-old wildtype (WT+ONA) and βB1-connective tissue growth factor high-pressure mice (CTGF+ONA) were immunized with 1 mg ONA. A wildtype control (WT) and a CTGF group (CTGF) received NaCl instead. Six weeks after immunization, retinae from all four groups were processed for immunohistology, RT-qPCR, and flow cytometry, while serum was used for microarray analyses. We noticed elevated numbers of C1q+ cells (classical complement pathway) in CTGF and CTGF+ONA retinae as well as an upregulation of C1qa, C1qb, and C1qc mRNA levels in these groups. While the complement C3 was only increased in CTGF and CTGF+ONA retinae, enhanced numbers of the terminal membrane attack complex were noted in all three glaucoma groups. Flow cytometry and RT-qPCR analyses revealed an enhancement of different microglia/macrophages markers, including CD11b, especially in CTGF and CTGF+ONA retinae. Interestingly, increased retinal mRNA as well as serum levels of the tumor necrosis factor α were found throughout the different glaucoma groups. Lastly, more T-cells could be observed in the ganglion cell layer of the new CTGF+ONA model. These results emphasize an involvement of the complement system, microglia/macrophages, and T-cells in glaucomatous disease. Moreover, in the new multifactorial glaucoma model, increased IOP in combination with autoimmune processes seem to enforce an additional T-cell response, leading to a more persistent pathology. Hence, this new model mimics the pathomechanisms occurring in human glaucoma more accurately and could therefore be a helpful tool to find new therapeutic approaches for patients in the future.

Dominant NARS1 mutations causing axonal Charcot-Marie-Tooth disease expand NARS1-associated diseases.

Pathogenic variants in six aminoacyl-tRNA synthetase (ARS) genes are implicated in neurological disorders, most notably inherited peripheral neuropathies. ARSs are enzymes that charge tRNA molecules with cognate amino acids. Pathogenic variants in asparaginyl-tRNA synthetase (NARS1) cause a neurological phenotype combining developmental delay, ataxia and demyelinating peripheral neuropathy. NARS1 has not yet been linked to axonal Charcot-Marie-Tooth disease. Exome sequencing of patients with inherited peripheral neuropathies revealed three previously unreported heterozygous NARS1 variants in three families. Clinical and electrophysiological details were assessed. We further characterized all three variants in a yeast complementation model and used a knock-in mouse model to study variant p.Ser461Phe. All three variants (p.Met236del, p.Cys342Tyr and p.Ser461Phe) co-segregate with the sensorimotor axonal neuropathy phenotype. Yeast complementation assays show that none of the three NARS1 variants support wild-type yeast growth when tested in isolation (i.e. in the absence of a wild-type copy of NARS1), consistent with a loss-of-function effect. Similarly, the homozygous knock-in mouse model (p.Ser461Phe/Ser472Phe in mouse) also demonstrated loss-of-function characteristics. We present three previously unreported NARS1 variants segregating with a sensorimotor neuropathy phenotype in three families. Functional studies in yeast and mouse support variant pathogenicity. Thus, NARS1 is the seventh ARS implicated in dominant axonal Charcot-Marie-Tooth disease, further stressing that all dimeric ARSs should be evaluated for Charcot-Marie-Tooth disease.

PD-L1 mRNA derived from tumor-educated platelets as a potential immunotherapy biomarker in non-small cell lung cancer.

To date, the role of programmed death ligand-1 (PD-L1) messenger RNA (mRNA) derived from tumor-educated platelets (TEPs) has not been well investigated in patients with advanced non-small cell lung cancer (NSCLC). A few reports have examined whether mRNA in TEPs can predict the clinical responses of patients with advanced NSCLC following immunotherapy. This study aimed to identify novel biomarkers to improve the clinical benefits and outcomes of NSCLC patients. Advanced NSCLC patients receiving a combination of immunotherapy and chemotherapy, or immunotherapy alone as a first- or second-line treatment at the Fudan University Shanghai Cancer Center were enrolled in this study. All the patients had wild-type epidermal growth factor receptor/anaplastic lymphoma kinase. The patients were enrolled in clinical trials for immune checkpoint inhibitors (ICIs), including nivolumab, pembrolizumab, atezolizumab, durvalumab, tremelimumab, and camrelizumab. Tumoral PD-L1 expression was tested by immunohistochemistry (PD-L1 22C3 pharmDx kit, Agilent, Santa Clara, CA, USA) in archived tissue samples, when available, to calculate the tumor proportion scores (TPSs). RNA and exosomal RNA of blood were isolated before immunotherapy using the Yunying RNA extraction kit (Yunying Medicine, Shanghai, China). The concentration and quality of the RNA was determined using a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA). Finally, we analyzed the predictive value of TEP-derived PD-L1 mRNA expression and association with the level of the tumoral PD-L1 expression. In total, 72 patients were enrolled in this study. Most of the patients were male (n=54, 75.0%), had adenocarcinoma (n=49, 68.1%). We found there was no significant correlation between the TEP-derived mRNA of PD-L1 and tumoral PD-L1 expression based on the results of the Pearson Correlation test (r=-0.19, P=0.233). Based on the median of PD-L1 mRNA, 72 patients were divided into a high PD-L1 group and a low PD-L1 group. We found that 19 patients (44.4%) responded to immunotherapy [partial response or progression-free survival (PFS) >6 months] in the high PD-L1 group, but only five patients (13.9%) responded to immunotherapy in the low PD-L1 group (P<0.01). The median PFS of the low PD-L1 group was lower than that of the high PD-L1 group (2.8 vs. 8.3 months, P<0.001). For the patients who were treated with immunotherapy alone (n=64), a similar PFS advantage was observed in the high PD-L1 group (2.8 vs. 8.0 months, P=0.002). This article presented the first data on TEP-derived PD-L1 mRNA in advanced NSCLC patients following immunotherapy and showed the potential advantage of using it as the surrogate biomarker for predicting the PFS and overall survival of patients following immunotherapy.

Ninjurin 2, a Cell Adhesion Molecule and a Target of p53, Modulates Wild-Type p53 in Growth Suppression and Mutant p53 in Growth Promotion.

The nerve injury-induced protein 1 (NINJ1) and NINJ2 constitute a family of homophilic adhesion molecules and are involved in nerve regeneration. Previously, we showed that NINJ1 and p53 are mutually regulated and the NINJ1-p53 loop plays a critical role in p53-dependent tumor suppression. However, the biology of NINJ2 has not been well-explored. By using multiple in vitro cell lines and genetically engineered mouse embryo fibroblasts (MEFs), we showed that NINJ2 is induced by DNA damage in a p53-dependent manner. Moreover, we found that the loss of NINJ2 promotes p53 expression via mRNA translation and leads to growth suppression in wild-type p53-expressing MCF7 and Molt4 cells and premature senescence in MEFs in a wild-type p53-dependent manner. Interestingly, NINJ2 also regulates mutant p53 expression, and the loss of NINJ2 promotes cell growth and migration in mutant p53-expressing MIA-PaCa2 cells. Together, these data indicate that the mutual regulation between NINJ2 and p53 represents a negative feedback loop, and the NINJ2-p53 loop has opposing functions in wild-type p53-dependent growth suppression and mutant p53-dependent growth promotion.

A deep learning model integrating multisequence MRI to predict EGFR mutation subtype in brain metastases from non-small cell lung cancer.

To establish a predictive model based on multisequence magnetic resonance imaging (MRI) using deep learning to identify wild-type (WT) epidermal growth factor receptor (EGFR), EGFR exon 19 deletion (19Del), and EGFR exon 21-point mutation (21L858R) simultaneously. A total of 399 patients with proven brain metastases of non-small cell lung cancer (NSCLC) were retrospectively enrolled and divided into training (n = 306) and testing (n = 93) cohorts separately based on two timepoints. All patients underwent 3.0-T brain MRI including T2-weighted, T2-weighted fluid-attenuated inversion recovery, diffusion-weighted imaging, and contrast-enhanced T1-weighted sequences. Radiomics features were extracted from each lesion based on four sequences. An algorithm combining radiomics approach with graph convolutional networks architecture (Radio-GCN) was designed for the prediction of EGFR mutation status and subtype. The area under the curve (AUC) at receiver operating characteristic analysis was used to evaluate the predication capabilities of each model. We extracted 1,290 radiomics features from each MRI sequence. The AUCs of the Radio-GCN model for identifying EGFR 19Del, 21L858R, and WT for the lesion-wise analysis were 0.996 ± 0.004, 0.971 ± 0.013, and 1.000 ± 0.000 on the independent testing cohort separately. It also yielded AUCs of 1.000 ± 0.000, 0.991 ± 0.009, and 1.000 ± 0.000for predicting EGFR mutations respectively for the patient-wise analysis. The κ coefficients were 0.735 and 0.812, respectively. The constructed Radio-GCN model is a new potential tool to predict the EGFR mutation status and subtype in NSCLC patients with brain metastases. The study demonstrated that a deep learning approach based on multisequence MRI can help to predict the EGFR mutation status in NSCLC patients with brain metastases, which is beneficial to guide a personalized treatment. • This is the first study to predict the EGFR mutation subtype simultaneously. • The Radio-GCN model holds the potential to be used as a diagnostic tool. • This study provides an imaging surrogate for identifying the EGFR mutation subtype.

IMpower210: A phase III study of second-line atezolizumab vs. docetaxel in East Asian patients with non-small cell lung cancer.

IMpower210 (NCT02813785) explored the efficacy and safety of single-agent atezolizumab vs. docetaxel as second-line treatment for advanced non-small cell lung cancer (NSCLC) in East Asian patients. Key eligibility criteria for this phase III, open-label, randomized study included age ≥18 years; histologically documented advanced NSCLC per the Union for International Cancer Control/American Joint Committee on Cancer staging system (7th edition); Eastern Cooperative Oncology Group performance status of 0 or 1; and disease progression following platinum-based chemotherapy for advanced or metastatic NSCLC. Patients were randomized 2:1 to receive either atezolizumab (1,200 mg) or docetaxel (75 mg/m2). The primary study endpoint was overall survival (OS) in the intention-to-treat (ITT) population with wild-type epidermal growth factor receptor expression (ITT EGFR-WT) and in the overall ITT population. Median OS in the ITT EGFR-WT population (n=467) was 12.3 [95% confidence interval (95% CI), 10.3-13.8] months in the atezolizumab arm (n=312) and 9.9 (95% CI, 7.8-13.9) months in the docetaxel arm [n=155; stratified hazard ratio (HR), 0.82; 95% CI, 0.66-1.03]. Median OS in the overall ITT population was 12.5 (95% CI, 10.8-13.8) months with atezolizumab treatment and 11.1 (95% CI, 8.4-14.2) months (n=377) with docetaxel treatment (n=188; stratified HR, 0.87; 95% CI, 0.71-1.08). Grade 3/4 treatment-related adverse events (TRAEs) occurred in 18.4% of patients in the atezolizumab arm and 50.0% of patients in the docetaxel arm. IMpower210 did not meet its primary efficacy endpoint of OS in the ITT EGFR-WT or overall ITT populations. Atezolizumab was comparatively more tolerable than docetaxel, with a lower incidence of grade 3/4 TRAEs.