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Background Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of non-small cell lung cancer (NSCLC). However, their low response rates and poor 5-year survivals indicate a need for improvement. One key factor in this resistance may be molecules that mediate immunosuppression within the tumor microenvironment (TME), such as adenosine. Combining therapies that mitigate the effects of adenosine with ICIs could potentially overcome these limitations. Methods We utilized the Lewis lung carcinoma (LLC) and CMT167 murine lung carcinoma models to investigate the combined use of the A2B receptor antagonist PBF1129 and anti-PD-1 ICI. The mechanisms underlying the efficacy of this combination therapy were explored using single-cell RNA sequencing (scRNA-seq). Results In both models, combination therapy improved tumor control. Our scRNA-seq analysis characterized alterations in malignant cells, macrophages, cancer-associated fibroblasts (CAFs), T cells, and endothelial cells in the tumor after treatment. Malignant cells treated with combination therapy exhibited reduced inflammatory, epithelial-to-mesenchymal transition (EMT) and angiogenic signatures. Malignant cells and M2-like macrophages showed high expression of TGFβ pathway genes, which were significantly reduced in the combination therapy group. We also observed decreased interactions between M2-like macrophages and CAFs as well as T cells, and dramatically increased Gzmb expression in M1-like macrophages with combination therapy. Combination therapy modulated TGFβ-mediated cellular crosstalk and extracellular matrix (ECM) remodeling. Conclusions Our findings suggest that inhibition of adenosine activity by blocking the A2B receptor reduces TGFβ signaling and enhances the efficacy of ICI therapy in NSCLC.

Histone deacetylase 3 (Hdac3) is an enzymatic component of transcriptional repression complexes recruited by the nuclear hormone receptors. Inactivation of Hdac3 in cancer cell lines triggered apoptosis, and removal of Hdac3 in the germ line of mice caused embryonic lethality. Therefore, we deleted Hdac3 in the postnatal mouse liver. These mice developed hepatomegaly, which was the result of hepatocyte hypertrophy, and these morphological changes coincided with significant imbalances between carbohydrate and lipid metabolism. Loss of Hdac3 triggered changes in gene expression consistent with inactivation of repression mediated by nuclear hormone receptors. Loss of Hdac3 also increased the levels of Ppar γ 2 , and treatment of these mice with a Pparγ antagonist partially reversed the lipid accumulation in the liver. In addition, gene expression analysis identified mammalian target of rapamycin signalling as being activated after deletion of Hdac3 , and inhibition by rapamycin affected the accumulation of neutral lipids in Hdac3 ‐null livers. Thus, Hdac3 regulates metabolism through multiple signalling pathways in the liver, and deletion of Hdac3 disrupts normal metabolic homeostasis.

Small cell lung cancer (SCLC) remains a deadly form of cancer, with a 5-year survival rate of less than 10 percent, necessitating novel therapies. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that is emerging as a therapeutic target and is co-expressed with BCL2 in multiple tumor types due to microRNA coregulation. We hypothesize that ROR1-targeted therapy is effective in small cell lung cancer and synergizes with therapeutic BCL2 inhibition. Tissue microarrays (TMAs) and formalin-fixed paraffin-embedded (FFPE) SCLC patient samples were utilized to determine the prevalence of ROR1 and BCL2 expression in SCLC. Eight SCLC-derived cell lines were used to determine the antitumor activity of a small molecule ROR1 inhibitor (KAN0441571C) alone and in combination with the BCL2 inhibitor venetoclax. The Chou-Talalay method was utilized to determine synergy with the drug combination. ROR1 and BCL2 protein expression was identified in 93% (52/56) and 86% (48/56) of SCLC patient samples, respectively. Similarly, ROR1 and BCL2 were shown by qRT-PCR to have elevated expression in 79% (22/28) and 100% (28/28) of SCLC patient samples, respectively. KAN0441571C displayed efficacy in 8 SCLC cell lines, with an IC50 of 500 nM or less. Synergy as defined by a combination index of <1 via the Chou-Talalay method between KAN0441571C and venetoclax was demonstrated in 8 SCLC cell lines. We have shown that ROR1 inhibition is synergistic with BCL2 inhibition in SCLC models and shows promise as a novel therapeutic target in SCLC.

EGFR tyrosine kinase inhibitors cause dramatic responses in EGFR-mutant lung cancer, but resistance universally develops. The involvement of β-catenin in EGFR TKI resistance has been previously reported, however, the precise mechanism by which β-catenin activation contributes to EGFR TKI resistance is not clear. Here, we show that EGFR inhibition results in the activation of β-catenin signaling in a Notch3-dependent manner, which facilitates the survival of a subset of cells that we call “adaptive persisters”. We previously reported that EGFR-TKI treatment rapidly activates Notch3, and here we describe the physical association of Notch3 with β-catenin, leading to increased stability and activation of β-catenin. We demonstrate that the combination of EGFR-TKI and a β-catenin inhibitor inhibits the development of these adaptive persisters, decreases tumor burden, improves recurrence free survival, and overall survival in xenograft models. These results supports combined EGFR-TKI and β-catenin inhibition in patients with EGFR mutant lung cancer. Treatment of EGFR mutant non-small cell lung cancer (NSCLC) often develops resistance to EGFR TKIs. In this study, the authors discover a non-canonical activation of β-catenin signaling through Notch3 as a mechanism of adaptation to and resistance to EGFR TKI treatment in NSCLC.

RANBP9 and RANBP10, also called Scorpins, are essential components of the C-terminal to LisH (CTLH) complex, an evolutionarily conserved poorly investigated multisubunit E3 ligase. Their role in non-small cell lung cancer (NSCLC) is unknown. In this study, first we used stable loss-of function and overexpression inducible cell lines to investigate the ability of either RANBP9 or RANBP10 to form their own functional CTLH complex. Then, we probed lysates from patient tumors and analyzed data from publicly available repositories to investigate the expression of RANBP9 and RANBP10. Finally, we used inducible cell lines in vitro to recapitulate the expression observed in patients and investigate the changes of the proteome and the ubiquitylome associated with either RANBP9 or RANBP10 in NSCLC. Here, we show that the two Scorpins are both expressed in NSCLC cells and that either of them can independently support the formation of the CTLH complex. Short-term experiments revealed that the RANBP9 and RANBP10 proteins balance each other in terms of expression, and the acute overexpression of one or the other results in significant reshaping of the NSCLC cell proteome and ubiquitylome. A higher RANBP9/RANBP10 ratio is associated with greater proliferation in both NSCLC cell lines and patients. Acute increased expression of RANBP10 slows NSCLC cell proliferation and decreases the level of proliferation-associated proteins, including key players in DNA replication. We present evidence that the Scorpins act as partial antagonists and work together as one sophisticated rheostat to modulate the CTLH complex ubiquitylation output, which regulates cell proliferation and other key biological processes in NSCLC. These results suggest that the two Scorpins can be considered as targets for the treatment of NSCLC.

Although limited by severe side effects and development of resistance, platinum-based therapies still represent the most common first-line treatment for non-small cell lung cancer (NSCLC). However, a crucial need in the clinical management of NSCLC is represented by the identification of cases sensitive to DNA damage response (DDR)-targeting drugs, such as cisplatin or PARP inhibitors. Here, we provide a molecular rationale for the stratification of NSCLC patients potentially benefitting from platinum compounds based on the expression levels of RANBP9, a recently identified player of the cellular DDR. RANBP9 was found overexpressed by immunohistochemistry (IHC) in NSCLC compared to normal adjacent tissues (NATs) ( n  = 147). Moreover, a retrospective analysis of 132 platinum-treated patients from the multi-centric TAILOR trial showed that RANBP9 overexpression levels are associated with clinical response to platinum compounds [Progression Free Survival Hazard Ratio (RANBP9 high vs low) 1.73, 95% CI 1.15–2.59, p  = 0.0084; Overall Survival HR (RANBP9 high vs low) 1.99, 95% CI 1.27–3.11, p  = 0.003]. Accordingly, RANBP9 KO cells showed higher sensitivity to cisplatin in comparison with WT controls both in vitro and in vivo models. NSCLC RANBP9 KO cells were also more sensitive than control cells to the PARP inhibitor olaparib alone and in combination with cisplatin, due to defective ATM-dependent and hyper-activated PARP-dependent DDR. The current investigation paves the way to prospective studies to assess the clinical value of RANBP9 protein levels as prognostic and predictive biomarker of response to DDR-targeting drugs, leading to the development of new tools for the management of NSCLC patients.

Conventional PD‐L1 immunohistochemical tissue biopsies only predict 20%–40% of non‐small cell lung cancer (NSCLC) patients that will respond positively to anti‐PD‐1/PD‐L1 immunotherapy. Herein, we present an immunogold biochip to quantify single extracellular vesicular RNA and protein (AuSERP) as a non‐invasive alternative. With only 20 μl of purified serum, PD‐1/PD‐L1 proteins on the surface of extracellular vesicles (EVs) and EV PD‐1/PD‐L1 messenger RNA (mRNA) cargo were detected at a single‐vesicle resolution and exceeded the sensitivities of their bulk‐analysis conventional counterparts, ELISA and qRT‐PCR, by 1000 times. By testing a cohort of 27 non‐responding and 27 responding NSCLC patients, AuSERP indicated that the single‐EV mRNA biomarkers surpass the single‐EV protein biomarkers in predicting patient responses to immunotherapy. Dual single‐EV PD‐1/PD‐L1 mRNA detection differentiated responders from non‐responders with an accuracy of 72.2% and achieved an NSCLC diagnosis accuracy of 93.2%, suggesting the potential for AuSERP to provide enhanced immunotherapy predictions and cancer diagnoses within the clinical setting.

The biological functions of the scaffold protein Ran Binding Protein 9 (RanBP9) remain elusive in macrophages or any other cell type where this protein is expressed together with its CTLH (C-terminal to LisH) complex partners. We have engineered a new mouse model, named RanBP9-TurnX, where RanBP9 fused to three copies of the HA tag (RanBP9-3xHA) can be turned into RanBP9-V5 tagged upon Cre-mediated recombination. We created this model to enable stringent biochemical studies at cell type specific level throughout the entire organism. Here, we have used this tool crossed with LysM-Cre transgenic mice to identify RanBP9 interactions in lung macrophages. We show that RanBP9-V5 and RanBP9-3xHA can be both co-immunoprecipitated with the known members of the CTLH complex from the same whole lung lysates. However, more than ninety percent of the proteins pulled down by RanBP9-V5 differ from those pulled-down by RanBP9-HA. The lung RanBP9-V5 associated proteome includes previously unknown interactions with macrophage-specific proteins as well as with players of the innate immune response, DNA damage response, metabolism, and mitochondrial function. This work provides the first lung specific RanBP9-associated interactome in physiological conditions and reveals that RanBP9 and the CTLH complex could be key regulators of macrophage bioenergetics and immune functions.

Tumor specimens are often preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks, the most common clinical source for DNA sequencing. Herein, we evaluated the effect of pre-sequencing parameters to guide proper sample selection for targeted gene sequencing. Data from 113 FFPE lung tumor specimens were collected, and targeted gene sequencing was performed. Libraries were constructed using custom probes and were paired-end sequenced on a next generation sequencing platform. A PCR-based quality control (QC) assay was utilized to determine DNA quality, and a ratio was generated in comparison to control DNA. We observed that FFPE storage time, PCR/QC ratio, and DNA input in the library preparation were significantly correlated to most parameters of sequencing efficiency including depth of coverage, alignment rate, insert size, and read quality. A combined score using the three parameters was generated and proved highly accurate to predict sequencing metrics. We also showed wide read count variability within the genome, with worse coverage in regions of low GC content like in KRAS. Sample quality and GC content had independent effects on sequencing depth, and the worst results were observed in regions of low GC content in samples with poor quality. Our data confirm that FFPE samples are a reliable source for targeted gene sequencing in cancer, provided adequate sample quality controls are exercised. Tissue quality should be routinely assessed for pre-analytical factors, and sequencing depth may be limited in genomic regions of low GC content if suboptimal samples are utilized.

Targeted cancer therapies often induce \"outlier\" responses in molecularly defined patient subsets. One patient with advanced-stage lung adenocarcinoma, who was treated with oral sorafenib, demonstrated a near-complete clinical and radiographic remission for 5 years. Whole-genome sequencing and RNA sequencing of primary tumor and normal samples from this patient identified a somatic mutation, ARAF S214C, present in the cancer genome and expressed at high levels. Additional mutations affecting this residue of ARAF and a nearby residue in the related kinase RAF1 were demonstrated across 1% of an independent cohort of lung adenocarcinoma cases. The ARAF mutations were shown to transform immortalized human airway epithelial cells in a sorafenib-sensitive manner. These results suggest that mutant ARAF is an oncogenic driver in lung adenocarcinoma and an indicator of sorafenib response.