Long-Term Survival Outcomes With First-Line Nivolumab Plus Ipilimumab-Based Treatment in Patients With Metastatic Non-Small Cell Lung Cancer and Tumor PD-L1 <1%: a Pooled Analysis.

INTRODUCTION: Nivolumab plus ipilimumab-based treatment regimens have shown long-term, durable efficacy benefit in patients with metastatic non-small cell lung cancer (NSCLC). Here we report clinical outcomes from a pooled analysis of patients with metastatic NSCLC and tumor programmed death ligand 1 (PD-L1) <1% treated with first-line nivolumab plus ipilimumab with or without two cycles of chemotherapy versus up to four cycles of chemotherapy in the randomized phase 3 CheckMate 227 and CheckMate 9LA studies. METHODS: Patients were aged ≥18 years and had stage IV/recurrent NSCLC with no sensitizing EGFR/ALK alterations. Assessments included overall survival (OS), progression-free survival (PFS), objective response rate (ORR), duration of response (DOR), and safety. RESULTS: In patients with tumor PD-L1 <1% in the nivolumab plus ipilimumab with or without chemotherapy (n = 322) versus chemotherapy (n = 315) arms, median OS was 17.4 versus 11.3 months, respectively, (hazard ratio [HR] = 0.64; 95% confidence interval [CI]: 0.54-0.76; 5-year OS rate, 20% versus 7%) at a median follow-up of 73.7 months. OS benefit was observed across key subgroups, including difficult-to-treat populations such as those with baseline brain metastases (HR = 0.44; 95% CI: 0.26-0.75) or squamous NSCLC (HR = 0.51; 95% CI: 0.36-0.72). In the overall pooled population, median PFS was 5.4 versus 4.9 months (HR = 0.72; 95% CI: 0.60-0.87; 5-year PFS rate, 9% versus 2%), ORR was 29% versus 22%, and median DOR was 18.0 versus 4.6 months. No new safety signals were observed. CONCLUSION: Nivolumab plus ipilimumab with or without chemotherapy provides a long-term, durable clinical benefit in patients with metastatic NSCLC and tumor PD-L1 <1%, supporting the use of this strategy as a first-line treatment option in this population with high unmet need.

ALKTERNATE: A Pilot Study Alternating Lorlatinib With Crizotinib in ALK-Positive NSCLC With Prior ALK Inhibitor Resistance.

Introduction: ALK-positive lung cancers represent a molecularly diverse disease. With drug exposure, driving selection pressure, and resistance pathways, disease relapse will emerge. There is compelling rationale to investigate novel treatment strategies, informed by dynamic circulating tumor DNA (ctDNA) monitoring. Methods: The single-arm, pilot study ALKTERNATE investigated fixed alternating cycles of lorlatinib intercalated with crizotinib in individuals resistant to second-generation ALK inhibitors. Dynamic ctDNA explored the correlation with disease response and disease recurrence and defined disease resistance. The primary outcome was time-to-treatment failure, a composite of tolerability, feasibility, and efficacy. Secondary outcomes included standard survival measures, toxicity, pharmacokinetic analysis, and patient-reported outcomes. Tertiary outcomes were proteogenomic analyses of tissue and plasma. Results: A total of 15 individuals were enrolled; three encountered primary resistance to lorlatinib induction. There were 12 participants who received alternating therapy, and this approach revealed safety, feasibility, and effectiveness. Patient-reported outcomes were maintained or improved on therapy, and toxicity was consistent with previous reports. The pharmacokinetic measures were similar to the single-arm drug experience. Median time-to-treatment failure was 10 months; overall survival was 23 months. ctDNA profiles indicated inferior survival in those with preexistent TP53 mutations and those without clear or cleared ctDNA at trial induction. The study defined a vastly heterogeneous population with an abundance of ALK coexisting with non-ALK resistance variants. Conclusions: ALKTERNATE revealed feasibility with a novel alternating ALK inhibitor strategy in ALK-positive NSCLC. Results support progressing inquiry into this approach and propose a flexible design with drug(s) selected and alternating time frames, informed by real-time plasma profiling. Moving this concept to treatment naive may also optimize impact.

Patient Experience of Complex Genomic Sequencing Exploring Patient Preference, Barriers, and Enablers for Delivery.

PURPOSE: Despite increasing evidence of benefit supporting complex genomic sequencing (CGS) in personalizing cancer therapy, its widespread uptake remains limited. METHODS: This mixed-methods, prospective cross-institutional demonstration study was designed to evaluate implementation of CGS in the care of patients with advanced cancer. DNA sequencing was undertaken on formalin-fixed paraffin-embedded tumor and matched blood was completed with the Peter MacCallum Cancer Centre Comprehensive Cancer Panel; 391 genes via central laboratory. Oncologists performed consent and result delivery. Patients completed pre- and post-test surveys, including validated and study-specific questions and, if eligible, semistructured interviews. Qualitative interviews were undertaken with study clinicians to evaluate processes. RESULTS: One hundred ninety-nine (63%) had ≥1 finding with the potential to affect management, including 172 (55%) whose finding could affect their treatment options, 25 (8%) whose test led to the resolution of diagnostic ambiguity, and 49 (16%) with a pathogenic germline variant. In 6-month follow-up, 50 (16%) participants had their subsequent therapy changed on the basis of their CGS results. Two hundred ninety-three (88% of adult patients) completed surveys at three time points. At consent, patients cited multifaceted value in testing, showed good understanding of basic concepts, but most (69%) overestimated the likelihood of result-led change. Post-test patients remained consistently satisfied with accessing CGS. 21% struggled with understanding results but there were low levels of decisional regret after participation (89% had nil/mild regret). Clinicians cited collaboration and communication as critical to delivery. CONCLUSION: Patients undergoing CGS are generally satisfied and place value on its use beyond potential therapeutic benefit. Our results suggest that to improve test utility and delivery of CGS with value to patients and investing institutions, focus must be placed on addressing the additional barriers to its wider implications including efforts to improve process efficiencies, clinician genomic literacy, and decision-making support.

Lung cancer (internet-based) Delphi (LUCiD): A modified eDelphi consensus process to establish Australasian clinical quality indicators for thoracic cancer.

BACKGROUND AND OBJECTIVE: Approximately 16,000 new cases of lung cancer are diagnosed each year in Australia and Aotearoa New Zealand, and it is the leading cause of cancer death in the region. Unwarranted variation in lung cancer care and outcomes has been described for many years, although clinical quality indicators to facilitate benchmarking across Australasia have not been established. The purpose of this study was to establish clinical quality indicators applicable to lung and other thoracic cancers across Australia and Aotearoa New Zealand. METHODS: Following a literature review, a modified three round eDelphi consensus process was completed between October 2022 and June 2023. Participants included clinicians from all relevant disciplines, patient advocates, researchers and other stakeholders, with representatives from all Australian states and territories and Aotearoa New Zealand. Consensus was set at a threshold of 70%, with the first two rounds conducted as online surveys, and the final round held as a hybrid in person and virtual consensus meeting. RESULTS: The literature review identified 422 international thoracic oncology indicators, and a total of 71 indicators were evaluated over the course of the Delphi consensus. Ultimately, 27 clinical quality indicators reached consensus, covering the continuum of thoracic oncologic care from diagnosis to first line treatment. Indicators benchmarking supportive care were poorly represented. Attendant numeric quality standards were developed to facilitate benchmarking. CONCLUSION: Twenty-seven clinical quality indicators relevant to thoracic oncology care in Australasia were developed. Real world implementation will now be explored utilizing a prospective dataset collected across Australia.

Sotorasib (960 mg or 240 mg) once daily in patients with previously treated KRAS G12C-mutated advanced NSCLC.

BACKGROUND: Sotorasib 960 mg once daily is approved to treat KRAS G12C-mutated locally advanced or metastatic non-small cell lung cancer (NSCLC). Sotorasib exhibits non-dose proportional pharmacokinetics and clinical responses at lower doses; therefore, we evaluated the efficacy and safety of sotorasib 960 mg and 240 mg. METHODS: In this phase 2, randomized, open-label study, adults with KRAS G12C-mutated advanced NSCLC received sotorasib 960 mg or 240 mg once daily. Primary endpoints were objective response rate (ORR) and safety. Secondary endpoints included disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and pharmacokinetics. The study was not powered for formal statistical hypothesis testing. RESULTS: In the 960 mg group (n = 104), ORR was 32.7 % and DCR was 86.5 %. In the 240 mg group (n = 105), ORR was 24.8 % and DCR was 81.9 %. Median PFS was 5.4 months (960 mg) and 5.6 months (240 mg). At a median follow-up of 17.5 months, median OS was 13.0 months (960 mg) and 11.7 months (240 mg). AUC0-24 h and Cmax were 1.3-fold numerically higher with the 960 mg dose. Treatment-emergent adverse events (TEAEs, ≥10 %) for 960 mg and 240 mg doses, respectively, were diarrhea (39.4 %; 31.7 %), nausea (23.1 %; 19.2 %), increased alanine aminotransaminase (14.4 %; 17.3 %), and increased aspartate aminotransferase (13.5 %; 13.5 %). CONCLUSIONS: Patients treated with sotorasib 960 mg once daily had numerically higher ORR and DCR, and longer DOR and OS, than patients treated with 240 mg in this descriptive analysis. TEAEs were manageable with label-directed dose modifications. CLINICAL TRIAL REGISTRATION: NCT03600883.

Glycan Profiling in Small Extracellular Vesicles with a SERS Microfluidic Biosensor Identifies Early Malignant Development in Lung Cancer.

Glycosylation is the most common post-translational modification of proteins and regulates a myriad of fundamental biological processes under normal, and pathological conditions. Altered protein glycosylation is linked to malignant transformation, showing distinct glycopatterns that are associated with cancer initiation and progression by regulating tumor proliferation, invasion, metastasis, and therapeutic resistance. The glycopatterns of small extracellular vesicles (sEVs) released by cancer cells are promising candidates for cancer monitoring since they exhibit glycopatterns similar to their cell-of-origin. However, the clinical application of sEV glycans is challenging due to the limitations of current analytical technologies in tracking the trace amounts of sEVs specifically derived from tumors in circulation. Herein, a sEV GLYcan PHenotype (EV-GLYPH) assay that utilizes a microfluidic platform integrated with surface-enhanced Raman scattering for multiplex profiling of sEV glycans in non-small cell lung cancer is clinically validated. For the first time, the EV-GLYPH assay effectively identifies distinct sEV glycan signatures between non-transformed and malignantly transformed lung cells. In a clinical study evaluated on 40 patients, the EV-GLYPH assay successfully differentiates patients with early-stage malignant lung nodules from benign lung nodules. These results reveal the potential to profile sEV glycans for noninvasive diagnostics and prognostics, opening up promising avenues for clinical applications and understanding the role of sEV glycosylation in lung cancer.

A Mesoporous Gold Sensor Unveils Phospho PD-L1 in Extracellular Vesicles as a Proxy for PD-L1 Expression in Lung Cancer Tissue.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death ligand 1 (PD-L1), or its receptor, PD-1 have improved survival in patients with non-small-cell lung cancer (NSCLC). Assessment of PD-L1 expression requires tissue biopsy or fine needle aspiration that are currently used to identify patients most likely to respond to single agent anti-PD-1/PD-L1 therapy. However, obtaining sufficient tissue to generate a PD-L1 tissue proportion score (TPS) ≥ 50% using immunohistochemistry remains a challenge that potentially may be overcome by liquid biopsies. This study utilized a mesoporous gold sensor (MGS) assay to examine the phosphorylation status of PD-L1 in plasma extracellular vesicles (EV pPD-L1) and PD-L1 levels in plasma from NSCLC patient samples and their association with tumor PD-L1 TPS. The 3-dimensional mesoporous network of the electrodes provides a large surface area, high signal-to-noise ratio, and a superior electro-conductive framework, thereby significantly improving the detection sensitivity of PD-L1 nanosensing. Test (n = 20) (Pearson's r = 0.99) and validation (n = 45) (Pearson's r = 0.99) cohorts show that EV pPD-L1 status correlates linearly with the tumor PD-L1 TPS assessed by immunohistochemistry irrespective of the tumor stage, with 64% of patients overall showing detectable EV pPD-L1 levels in plasma. In contrast to the EV pPD-L1 results, plasma PD-L1 levels did not correlate with the tumor PD-L1 TPS score or EV pPD-L1 levels. These data demonstrate that EV pPD-L1 levels may be used to select patients for appropriate PD-1 and PD-L1 ICI therapy regimens in early, locally advanced, and advanced NSCLC and should be tested further in randomized controlled trials. Most importantly, the assay used has a less than 24h turnaround time, facilitating adoption of the test into the routine diagnostic evaluation of patients prior to therapy.

The expression and role of the Lem-D proteins Ankle2, Emerin, Lemd2, and TMPO in triple-negative breast cancer cell growth.

Background: Triple-negative breast cancer (TNBC) is a sub-classification of breast carcinomas, which leads to poor survival outcomes for patients. TNBCs do not possess the hormone receptors that are frequently targeted as a therapeutic in other cancer subtypes and, therefore, chemotherapy remains the standard treatment for TNBC. Nuclear envelope proteins are frequently dysregulated in cancer cells, supporting their potential as novel cancer therapy targets. The Lem-domain (Lem-D) (LAP2, Emerin, MAN1 domain, and Lem-D) proteins are a family of inner nuclear membrane proteins, which share a ~45-residue Lem-D. The Lem-D proteins, including Ankle2, Lemd2, TMPO, and Emerin, have been shown to be associated with many of the hallmarks of cancer. This study aimed to define the association between the Lem-D proteins and TNBC and determine whether these proteins could be promising therapeutic targets. Methods: GENT2, TCGA, and KM plotter were utilized to investigate the expression and prognostic implications of several Lem-D proteins: Ankle2, TMPO, Emerin, and Lemd2 in publicly available breast cancer patient data. Immunoblotting and immunofluorescent analysis of immortalized non-cancerous breast cells and a panel of TNBC cells were utilized to establish whether protein expression of the Lem-D proteins was significantly altered in TNBC. SiRNA was used to decrease individual Lem-D protein expression, and functional assays, including proliferation assays and apoptosis assays, were conducted. Results: The Lem-D proteins were generally overexpressed in TNBC patient samples at the mRNA level and showed variable expression at the protein level in TNBC cell lysates. Similarly, protein levels were generally negatively correlated with patient survival outcomes. siRNA-mediated depletion of the individual Lem-D proteins in TNBC cells induced aberrant nuclear morphology, decreased proliferation, and induced cell death. However, minimal effects on nuclear morphology or cell viability were observed following Lem-D depletion in non-cancerous MCF10A cells. Conclusion: There is evidence to suggest that Ankle2, TMPO, Emerin, and Lemd2 expressions are correlated with breast cancer patient outcomes, but larger patient sample numbers are required to confirm this. siRNA-mediated depletion of these proteins was shown to specifically impair TNBC cell growth, suggesting that the Lem-D proteins may be a specific anti-cancer target.

Pegargiminase Plus First-Line Chemotherapy in Patients With Nonepithelioid Pleural Mesothelioma: The ATOMIC-Meso Randomized Clinical Trial.

Importance: Arginine deprivation using ADI-PEG20 (pegargiminase) combined with chemotherapy is untested in a randomized study among patients with cancer. ATOMIC-Meso (ADI-PEG20 Targeting of Malignancies Induces Cytotoxicity-Mesothelioma) is a pivotal trial comparing standard first-line chemotherapy plus pegargiminase or placebo in patients with nonepithelioid pleural mesothelioma. Objective: To determine the effect of pegargiminase-based chemotherapy on survival in nonepithelioid pleural mesothelioma, an arginine-auxotrophic tumor. Design, Setting, and Participants: This was a phase 2-3, double-blind randomized clinical trial conducted at 43 centers in 5 countries that included patients with chemotherapy-naive nonepithelioid pleural mesothelioma from August 1, 2017, to August 15, 2021, with at least 12 months' follow-up. Final follow-up was on August 15, 2022. Data analysis was performed from March 2018 to June 2023. Intervention: Patients were randomly assigned (1:1) to receive weekly intramuscular pegargiminase (36.8 mg/m2) or placebo. All patients received intravenous pemetrexed (500 mg/m2) and platinum (75-mg/m2 cisplatin or carboplatin area under the curve 5) chemotherapy every 3 weeks up to 6 cycles. Pegargiminase or placebo was continued until progression, toxicity, or 24 months. Main Outcomes and Measures: The primary end point was overall survival, and secondary end points were progression-free survival and safety. Response rate by blinded independent central review was assessed in the phase 2 portion only. Results: Among 249 randomized patients (mean [SD] age, 69.5 [7.9] years; 43 female individuals [17.3%] and 206 male individuals [82.7%]), all were included in the analysis. The median overall survival was 9.3 months (95% CI, 7.9-11.8 months) with pegargiminase-chemotherapy as compared with 7.7 months (95% CI, 6.1-9.5 months) with placebo-chemotherapy (hazard ratio [HR] for death, 0.71; 95% CI, 0.55-0.93; P = .02). The median progression-free survival was 6.2 months (95% CI, 5.8-7.4 months) with pegargiminase-chemotherapy as compared with 5.6 months (95% CI, 4.1-5.9 months) with placebo-chemotherapy (HR, 0.65; 95% CI, 0.46-0.90; P = .02). Grade 3 to 4 adverse events with pegargiminase occurred in 36 patients (28.8%) and with placebo in 21 patients (16.9%); drug hypersensitivity and skin reactions occurred in the experimental arm in 3 patients (2.4%) and 2 patients (1.6%), respectively, and none in the placebo arm. Rates of poststudy treatments were comparable in both arms (57 patients [45.6%] with pegargiminase vs 58 patients [46.8%] with placebo). Conclusions and Relevance: In this randomized clinical trial of arginine depletion with pegargiminase plus chemotherapy, survival was extended beyond standard chemotherapy with a favorable safety profile in patients with nonepithelioid pleural mesothelioma. Pegargiminase-based chemotherapy as a novel antimetabolite strategy for mesothelioma validates wider clinical testing in oncology. Trial Registration: ClinicalTrials.gov Identifier: NCT02709512.

A Phase II trial of alternating osimertinib and gefitinib therapy in advanced EGFR-T790M positive non-small cell lung cancer: OSCILLATE.

In this phase II, single arm trial (ACTRN12617000720314), we investigate if alternating osimertinib and gefitinib would delay the development of resistance to osimertinib in advanced, non-small cell lung cancer (NSCLC) with the epidermal growth factor receptor (EGFR) T790M mutation (n = 47) by modulating selective pressure on resistant clones. The primary endpoint is progression free-survival (PFS) rate at 12 months, and secondary endpoints include: feasibility of alternating therapy, overall response rate (ORR), overall survival (OS), and safety. The 12-month PFS rate is 38% (95% CI 27.5-55), not meeting the pre-specified primary endpoint. Serial circulating tumor DNA (ctDNA) analysis reveals decrease and clearance of the original activating EGFR and EGFR-T790M mutations which are prognostic of clinical outcomes. In 73% of participants, loss of T790M ctDNA is observed at progression and no participants have evidence of the EGFR C797S resistance mutation following the alternating regimen. These findings highlight the challenges of treatment strategies designed to modulate clonal evolution and the clinical importance of resistance mechanisms beyond suppression of selected genetic mutations in driving therapeutic escape to highly potent targeted therapies.

Lorlatinib After Alectinib-Induced Pneumonitis: A Case Report.

ALK gene rearrangements are detected in approximately 3% to 5% of NSCLC. ALK tyrosine kinase inhibitors, such as third-generation lorlatinib, have exhibited remarkable efficacy in ALK-rearranged NSCLC; however, they have been associated with a low incidence of treatment-limiting and potentially fatal drug-induced interstitial lung disease (ILD). There is concern that this may represent a class effect, a theory that is supported by a number of case reports. Because of clinical trial exclusion criteria, there are limited prospective data to guide decision-making after ALK tyrosine kinase inhibitors-induced ILD. A systematic review of the literature was conducted and only identified four reported cases of lorlatinib safety in this context. Here, we report the successful sequencing of lorlatinib in a patient who discontinued alectinib secondary to grade 3 drug-induced ILD.

Inhibition of Aurora B kinase (AURKB) enhances the effectiveness of 5-fluorouracil chemotherapy against colorectal cancer cells.

BACKGROUND: 5-Fluorouracil (5-FU) remains a core component of systemic therapy for colorectal cancer (CRC). However, response rates remain low, and development of therapy resistance is a primary issue. Combinatorial strategies employing a second agent to augment the therapeutic effect of chemotherapy is predicted to reduce the incidence of treatment resistance and increase the durability of response to therapy. METHODS: Here, we employed quantitative proteomics approaches to identify novel druggable proteins and molecular pathways that are deregulated in response to 5-FU, which might serve as targets to improve sensitivity to chemotherapy. Drug combinations were evaluated using 2D and 3D CRC cell line models and an ex vivo culture model of a patient-derived tumour. RESULTS: Quantitative proteomics identified upregulation of the mitosis-associated protein Aurora B (AURKB), within a network of upregulated proteins, in response to a 24 h 5-FU treatment. In CRC cell lines, AURKB inhibition with the dihydrogen phosphate prodrug AZD1152, markedly improved the potency of 5-FU in 2D and 3D in vitro CRC models. Sequential treatment with 5-FU then AZD1152 also enhanced the response of a patient-derived CRC cells to 5-FU in ex vivo cultures. CONCLUSIONS: AURKB inhibition may be a rational approach to augment the effectiveness of 5-FU chemotherapy in CRC.

An Exploration of Small Molecules That Bind Human Single-Stranded DNA Binding Protein 1.

Human single-stranded DNA binding protein 1 (hSSB1) is critical to preserving genome stability, interacting with single-stranded DNA (ssDNA) through an oligonucleotide/oligosaccharide binding-fold. The depletion of hSSB1 in cell-line models leads to aberrant DNA repair and increased sensitivity to irradiation. hSSB1 is over-expressed in several types of cancers, suggesting that hSSB1 could be a novel therapeutic target in malignant disease. hSSB1 binding studies have focused on DNA; however, despite the availability of 3D structures, small molecules targeting hSSB1 have not been explored. Quinoline derivatives targeting hSSB1 were designed through a virtual fragment-based screening process, synthesizing them using AlphaLISA and EMSA to determine their affinity for hSSB1. In parallel, we further screened a structurally diverse compound library against hSSB1 using the same biochemical assays. Three compounds with nanomolar affinity for hSSB1 were identified, exhibiting cytotoxicity in an osteosarcoma cell line. To our knowledge, this is the first study to identify small molecules that modulate hSSB1 activity. Molecular dynamics simulations indicated that three of the compounds that were tested bound to the ssDNA-binding site of hSSB1, providing a framework for the further elucidation of inhibition mechanisms. These data suggest that small molecules can disrupt the interaction between hSSB1 and ssDNA, and may also affect the ability of cells to repair DNA damage. This test study of small molecules holds the potential to provide insights into fundamental biochemical questions regarding the OB-fold.

Targeting the COMMD4-H2B protein complex in lung cancer.

BACKGROUND: Lung cancer is the biggest cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 85-90% of all lung cancers. Identification of novel therapeutic targets are required as drug resistance impairs chemotherapy effectiveness. COMMD4 is a potential NSCLC therapeutic target. The aims of this study were to investigate the COMMD4-H2B binding pose and develop a short H2B peptide that disrupts the COMMD4-H2B interaction and mimics COMMD4 siRNA depletion. METHODS: Molecular modelling, in vitro binding and site-directed mutagenesis were used to identify the COMMD4-H2B binding pose and develop a H2B peptide to inhibit the COMMD4-H2B interaction. Cell viability, DNA repair and mitotic catastrophe assays were performed to determine whether this peptide can specially kill NSCLC cells. RESULTS: Based on the COMMD4-H2B binding pose, we have identified a H2B peptide that inhibits COMMD4-H2B by directly binding to COMMD4 on its H2B binding binding site, both in vitro and in vivo. Treatment of NSCLC cell lines with this peptide resulted in increased sensitivity to ionising radiation, increased DNA double-strand breaks and induction of mitotic catastrophe in NSCLC cell lines. CONCLUSIONS: Our data shows that COMMD4-H2B represents a novel potential NSCLC therapeutic target.

Mono-phosphorylation at Ser4 of barrier-to-autointegration factor (Banf1) significantly reduces its DNA binding capability by inducing critical changes in its local conformation and DNA binding surface.

Barrier-to-autointegration factor (Banf1) is a small DNA-bridging protein. The binding status of Banf1 to DNA is regulated by its N-terminal phosphorylation and dephosphorylation, which plays a critical role in cell proliferation. Banf1 can be phosphorylated at Ser4 into mono-phosphorylated Banf1, which is further phosphorylated at Thr3 to form di-phosphorylated Banf1. It was observed decades ago that mono-phosphorylated Banf1 cannot bind to DNA. However, the underlying molecular- and atomic-level mechanisms remain unclear. A clear understanding of these mechanisms will aid in interfering with the cell proliferation process for better global health. Herein, we explored the detailed atomic bases of unphosphorylated Banf1-DNA binding and how mono- and di-phosphorylation of Banf1 impair these atomic bases to eliminate its DNA-binding capability, followed by exploring the DNA-binding capability of mono- and di-phosphorylation Banf1, using comprehensive and systematic molecular modelling and molecular dynamics simulations. This work presented in detail the residue-level binding energies, hydrogen bonds and water bridges between Banf1 and DNA, some of which have not been reported. Moreover, we revealed that mono-phosphorylation of Banf1 causes its N-terminal secondary structure changes, which in turn induce significant changes in Banf1's DNA binding surface, thus eliminating its DNA-binding capability. At the atomic level, we also uncovered the alterations in interactions due to the induction of mono-phosphorylation that result in the N-terminal secondary structure changes of Banf1. Additionally, our modelling showed that phosphorylated Banf1 with their dominant N-terminal secondary structures bind to DNA with a significantly lower affinity and the docked binding pose are not stable in MD simulations. These findings help future studies in predicting effect of mutations in Banf1 on its DNA-binding capability and open a novel avenue for the development of therapeutics such as cancer drugs, targeting cell proliferation by inducing conformational changes in Banf1's N-terminal domain.

The fructose-bisphosphate, Aldolase A (ALDOA), facilitates DNA-PKcs and ATM kinase activity to regulate DNA double-strand break repair.

Glucose metabolism and DNA repair are fundamental cellular processes frequently dysregulated in cancer. In this study, we define a direct role for the glycolytic Aldolase A (ALDOA) protein in DNA double-strand break (DSB) repair. ALDOA is a fructose biphosphate Aldolase that catalyses fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), during glycolysis. Here, we show that upon DNA damage induced by ionising radiation (IR), ALDOA translocates from the cytoplasm into the nucleus, where it partially co-localises with the DNA DSB marker γ-H2AX. DNA damage was shown to be elevated in ALDOA-depleted cells prior to IR and following IR the damage was repaired more slowly. Consistent with this, cells depleted of ALDOA exhibited decreased DNA DSB repair via non-homologous end-joining and homologous recombination. In support of the defective repair observed in its absence, ALDOA was found to associate with the major DSB repair effector kinases, DNA-dependent Protein Kinase (DNA-PK) and Ataxia Telangiectasia Mutated (ATM) and their autophosphorylation was decreased when ALDOA was depleted. Together, these data establish a role for an essential metabolic protein, ALDOA in DNA DSB repair and suggests that targeting ALDOA may enable the concurrent targeting of cancer metabolism and DNA repair to induce tumour cell death.

Stochastic epithelial-mesenchymal transitions diversify non-cancerous lung cell behaviours.

Epithelial-mesenchymal plasticity (EMP) is a hallmark of cancer. By enabling cells to shift between different morphological and functional states, EMP promotes invasion, metastasis and therapy resistance. We report that near-diploid non-cancerous human epithelial lung cells spontaneously shift along the EMP spectrum without genetic changes. Strikingly, more than half of single cell-derived clones adopt a mesenchymal morphology. We independently characterise epithelial-like and mesenchymal-like clones. Mesenchymal clones lose epithelial markers, display larger cell aspect ratios and lower motility, with mostly unaltered proliferation rates. Stemness marker expression and metabolic rewiring diverge independently of phenotypes. In 3D culture, more epithelial clones become mesenchymal-like. Thus, non-cancerous epithelial cells may acquire cancer metastasis-associated features prior to genetic alterations and cancerous transformation.

Protein Tyrosine Phosphatase Non-Receptor 11 (PTPN11/Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC).

PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the mitogen-activated protein kinase (MAPK) pathway upstream of KRAS and MEK. PTPN11/Shp2 somatic mutations occur frequently in Juvenile myelomonocytic leukaemia (JMML); however, the role of mutated PTPN11 in lung cancer tumourigenesis and its utility as a therapeutic target has not been fully addressed. We applied mass-spectrometry-based genotyping to DNA extracted from the tumour and matched the normal tissue of 356 NSCLC patients (98 adenocarcinomas (LUAD) and 258 squamous cell carcinomas (LUSC)). Further, PTPN11 mutation cases were identified in additional cohorts, including TCGA, Broad, and MD Anderson datasets and the COSMIC database. PTPN11 constructs harbouring PTPN11 E76A, A72D and C459S mutations were stably expressed in IL-3 dependent BaF3 cells and NSCLC cell lines (NCI-H1703, NCI-H157, NCI-H1299). The MAPK and PI3K pathway activation was evaluated using Western blotting. PTPN11/Shp2 phosphatase activity was measured in whole-cell protein lysates using an Shp2 assay kit. The Shp2 inhibitor (SHPi) was assessed both in vitro and in vivo in a PTPN11-mutated cell line for improved responses to MAPK and PI3K targeting therapies. Somatic PTPN11 hotspot mutations occurred in 4/98 (4.1%) adenocarcinomas and 7/258 (2.7%) squamous cells of 356 NSCLC patients. Additional 26 PTPN11 hotspot mutations occurred in 23 and 3 adenocarcinomas and squamous cell carcinoma, respectively, across the additional cohorts. Mutant PTPN11 significantly increased the IL-3 independent survival of Ba/F3 cells compared to wildtype PTPN11 (p < 0.0001). Ba/F3, NCI-H1703, and NCI-H157 cells expressing mutant PTPN11 exhibited increased PTPN11/Shp2 phosphatase activity and phospho-ERK1/2 levels compared to cells expressing wildtype PTPN11. The transduction of the PTPN11 inactivating mutation C459S into NSCLC cell lines led to decreased phospho-ERK, as well as decreased phospho-AKT in the PTPN11-mutated NCI-H661 cell line. NCI-H661 cells (PTPN11-mutated, KRAS-wild type) were significantly more sensitive to growth inhibition by the PI3K inhibitor copanlisib (IC50: 13.9 ± 4.7 nM) compared to NCI-H1703 (PTPN11/KRAS-wild type) cells (IC50: >10,000 nM). The SHP2 inhibitor, in combination with the PI3K targeting therapy copanlisib, showed no significant difference in tumour development in vivo; however, this significantly prevented MAPK pathway induction in vitro (p < 0.0001). PTPN11/Shp2 demonstrated the in vitro features of a driver oncogene and could potentially sensitize NSCLC cells to PI3K inhibition and inhibit MAPK pathway activation following PI3K pathway targeting.

Phase III Study of Afatinib or Cisplatin Plus Pemetrexed in Patients With Metastatic Lung Adenocarcinoma With EGFR Mutations.

PURPOSE: The LUX-Lung 3 study investigated the efficacy of chemotherapy compared with afatinib, a selective, orally bioavailable ErbB family blocker that irreversibly blocks signaling from epidermal growth factor receptor (EGFR/ErbB1), human epidermal growth factor receptor 2 (HER2/ErbB2), and ErbB4 and has wide-spectrum preclinical activity against EGFR mutations. A phase II study of afatinib in EGFR mutation-positive lung adenocarcinoma demonstrated high response rates and progression-free survival (PFS). PATIENTS AND METHODS: In this phase III study, eligible patients with stage IIIB/IV lung adenocarcinoma were screened for EGFR mutations. Mutation-positive patients were stratified by mutation type (exon 19 deletion, L858R, or other) and race (Asian or non-Asian) before two-to-one random assignment to 40 mg afatinib per day or up to six cycles of cisplatin plus pemetrexed chemotherapy at standard doses every 21 days. The primary end point was PFS by independent review. Secondary end points included tumor response, overall survival, adverse events, and patient-reported outcomes (PROs). RESULTS: A total of 1,269 patients were screened, and 345 were randomly assigned to treatment. Median PFS was 11.1 months for afatinib and 6.9 months for chemotherapy (hazard ratio [HR], 0.58; 95% CI, 0.43 to 0.78; P = .001). Median PFS among those with exon 19 deletions and L858R EGFR mutations (n = 308) was 13.6 months for afatinib and 6.9 months for chemotherapy (HR, 0.47; 95% CI, 0.34 to 0.65; P = .001). The most common treatment-related adverse events were diarrhea, rash/acne, and stomatitis for afatinib and nausea, fatigue, and decreased appetite for chemotherapy. PROs favored afatinib, with better control of cough, dyspnea, and pain. CONCLUSION: Afatinib is associated with prolongation of PFS when compared with standard doublet chemotherapy in patients with advanced lung adenocarcinoma and EGFR mutations.

Systemic and Intracranial Outcomes With First-Line Nivolumab Plus Ipilimumab in Patients With Metastatic NSCLC and Baseline Brain Metastases From CheckMate 227 Part 1.

INTRODUCTION: In CheckMate 227 Part 1, nivolumab plus ipilimumab prolonged overall survival (OS) versus chemotherapy in patients with metastatic NSCLC, regardless of tumor programmed death-ligand 1 (PD-L1) expression. Here, we report post hoc exploratory systemic and intracranial efficacy outcomes and safety by baseline brain metastasis status at 5 years' minimum follow-up. METHODS: Treatment-naive adults with stage IV or recurrent NSCLC without EGFR or ALK alterations, including asymptomatic patients with treated brain metastases, were enrolled. Patients with tumor PD-L1 greater than or equal to 1% were randomized to nivolumab plus ipilimumab, nivolumab, or chemotherapy; patients with tumor PD-L1 less than 1% were randomized to nivolumab plus ipilimumab, nivolumab plus chemotherapy, or chemotherapy groups. Assessments included OS, systemic and intracranial progression-free survival per blinded independent central review, new brain lesion development, and safety. Brain imaging was performed at baseline (all randomized patients) and approximately every 12 weeks thereafter (patients with baseline brain metastases only). RESULTS: Overall, 202 of 1739 randomized patients had baseline brain metastases (nivolumab plus ipilimumab: 68; chemotherapy: 66). At 61.3 months' minimum follow-up, nivolumab plus ipilimumab prolonged OS versus chemotherapy in patients with baseline brain metastases (hazard ratio = 0.63; 95% confidence interval: 0.43-0.92) and in those without (hazard ratio = 0.76; 95% confidence interval: 0.66-0.87). In patients with baseline brain metastases, 5-year systemic and intracranial progression-free survival rates were higher with nivolumab plus ipilimumab (12% and 16%, respectively) than chemotherapy (0% and 6%). Fewer patients with baseline brain metastases developed new brain lesions with nivolumab plus ipilimumab (4%) versus chemotherapy (20%). No new safety signals were observed. CONCLUSIONS: With all patients off immunotherapy for more than or equal to 3 years, nivolumab plus ipilimumab continued to provide a long-term, durable survival benefit in patients with or without brain metastases. Intracranial efficacy outcomes favored nivolumab plus ipilimumab versus chemotherapy. These results further support nivolumab plus ipilimumab as an efficacious first-line treatment for patients with metastatic NSCLC, regardless of baseline brain metastasis status.